Combined Stresses in Plants

Combined Stresses in Plants
Ramamurthy Mahalingam
Editor
Combined Stresses in Plants

Physiological, Molecular, and Biochemical
Aspects
1 3
Editor
Department of Biochemistry
and Molecular Biology
Oklahoma State University
Stillwater, OK
USA
ISBN 978-3-319-07898-4 ISBN 978-3-319-07899-1 (eBook)

DOI 10.1007/978-3-319-07899-1
Library of Congress Control Number: 2014956105
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Preface
The renowned Greek philosopher Heraclitus’ famous quote “Change is the only
constant in life” seems very apt in a plant’s life. Plants, being sessile in nature,
are exposed to a wide variety of environmental perturbations from seed germina-
tion to senescence. These environmental changes can be caused due to abiotic and
biotic factors. Abiotic factors includes physical aspects of a plant’s environment
such as soil moisture conditions, soil nutrients, and climatic components such as
light, temperature extremes, air pollutants, UV-radiation, and wind. Biotic factors
encompass pathogens, pests, parasites, animals, and humans. It is also apparent that
the various biotic and abiotic factors are constantly changing during the life cycle
of a plant. Furthermore, these external factors co-occur in nature. Plants have to
make decisions about fine-tuning their responses to allocate resources efficiently
for responding to the more serious threats at any given point in time. Paradoxically,
most studies of stress responses in plants focus on a single inciting agent. From the
point of view of conducting a well-controlled experiment it is the most ideal strat-
egy. However, the results from such studies may not necessarily mimic the response
that a plant would elicit under realistic field conditions where multiple factors are
simultaneously operating. In recent years several research groups working on dif-
ferent stress combinations and in different plant species have shown that plants
evoke a “unique response” to combined stresses. In other words, combined stress
response is not just an additive effect of the responses elicited when the stresses are
imposed singly.
The unique responses to combined stresses in plants have been observed at the
physiological, biochemical, and molecular levels. The chapters in this book address
all the three levels of change in various plants in response to various combinations
of stresses.
Chapter 1 provides a general review of the combined stress paradigm.
Chapters 2 through 4 focus on the impact of higher CO2 levels in combination with
other stresses (temperature, salinity, and soil contaminants). In Chapters 5 through
8 drought stress is examined in conjunction with other abiotic factors (salinity, heat,
and ozone) in different crop plants. Chapters 9 and 10 examine the combination of
biotic and abiotic factors. The impact of combined stresses in forest ecosystems are
discussed in Chapters 11 and 12.
v
vi Preface
It is my sincere appeal that the plant stress community embraces the concept of
combined stress in their future research. A much-needed second green revolution
can become a reality when we incorporate the concept of combined stresses in plant
stress research.
This book would not have been possible without the contributions of the experts
who were willing to share their knowledge in various stress combinations, and my
heartiest thanks to each of them. I would like to convey my thanks to Mr. Eric
Stannard of Springer Science+Business Media for broaching the theme of com-
bined stress in plants for a book. I would also like to extend my thanks to my
production editor, Mr. Joseph Quatela, along with the entire production team for
their efforts in bringing out this book. I would like to convey my sincere thanks
to Dr. John Gustafson, professor and head of the department of Biochemistry and
Molecular Biology at Oklahoma State University for his encouragement and sup-
port for taking up this book project.
Contents
1 Consideration of Combined Stress: A Crucial Paradigm

for Improving Multiple Stress Tolerance in Plants............................... 1
2 The Impact of Enhanced Atmospheric CO2 Concentrations

on the Responses of Maize and Soybean to Elevated Growth
Temperatures............................................................................................ 27
Richard C. Sicher and James A. Bunce
3 Investigating the Effect of Elevated CO2 in the Growth

Environment of Salt-Stressed Plants Using Integrated
Omic Analyses.......................................................................................... 49
Matthaios-Emmanouil P. Papadimitropoulos and Maria I. Klapa
4 Combination of Elevated CO2 Levels and Soil Contaminants’

Stress in Wheat and Rice......................................................................... 71
Hongyan Guo, Hui Zhou, Yaodan Zhang, Wenchao Du,
Yuanyuan Sun, Ying Yin, Daping Pei, Rong Ji, Jichun Wu,
Xiaorong Wang and Jianguo Zhu
5 Tolerance to Combined Stress of Drought and Salinity in Barley....... 93

Imrul Mosaddek Ahmed, Umme Aktari Nadira,
Noreen Bibi, Guoping Zhang and Feibo Wu
6 Combined Abiotic Stress in Legumes..................................................... 123

Santiago Signorelli, Esteban Casaretto, Jorge Monza and
Omar Borsani
7 Interactive Effects Between Ozone and Drought: Sorrow or Joy?..... 147

Sacha Bohler, Ann Cuypers and Jaco Vangronsveld
vii
viii Contents
8 Effect of High Temperature and Water Stress

on Groundnuts Under Field Conditions.............................................. 159
Vijaya Gopal Kakani, Timothy R. Wheeler,
Peter Q. Craufurd and Rao C. N. Rachaputi
9 The Response of Plants to Simultaneous Biotic

and Abiotic Stress................................................................................... 181
Nicky J Atkinson, Ritushree Jain and Peter E Urwin
10 Impact of Concurrent Drought Stress and Pathogen

Infection on Plants................................................................................. 203
Prachi Pandey, Ranjita Sinha, Kirankumar S. Mysore
and Muthappa Senthil-Kumar
11 Combined Stresses in Forests................................................................ 223

Patrick Mitchell, Tim Wardlaw and Libby Pinkard
12 The Interactive Effects of Drought and Herbivory

on Ecophysiology of Trees..................................................................... 245
Sheel Bansal
Index................................................................................................................ 261
Contributors
Imrul Mosaddek Ahmed Department of Agronomy, College of Agriculture and

Biotechnology, Zijingang Campus, Zhejiang University, Hangzhou, China
Nicky J Atkinson Faculty of Biological Sciences, Centre for Plant Sciences,
University of Leeds, Leeds, UK
Sheel Bansal USDA-Forest Service, Pacific Northwest Research Station,
Olympia, WA, USA
Noreen Bibi Department of Agronomy, College of Agriculture and Biotechnology,
Zijingang Campus, Zhejiang University, Hangzhou, China
Sacha Bohler Centre for Environmental Sciences, Environmental Biology,
Hasselt University, Diepenbeek, Belgium
Omar Borsani Laboratorio de Bioquímica, Departamento de Biología Vegetal,
Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay
James A. Bunce Crop Systems and Global Change Laboratory, Beltsville
Agricultural Research Center, Beltsville, MD, USA
Esteban Casaretto Laboratorio de Bioquímica, Departamento de Biología
Vegetal, Facultad de Agronomía, Universidad de la República, Montevideo,
Uruguay
Peter Q. Craufurd Global Conservation Agriculture Program, CIMMYT,
Nairobi, Kenya
Ann Cuypers Centre for Environmental Sciences, Environmental Biology,
Wenchao Du State Key Laboratory of Pollution Control and Resource Reuse,
School of Environment, Nanjing University, Nanjing, China
Hongyan Guo State Key Laboratory of Pollution Control and Resource Reuse,
Institute for Climate and Global Change Research (ICGCR), Nanjing University,
Nanjing, China
ix
x Contributors
Ritushree Jain Faculty of Biological Sciences, Centre for Plant Sciences,

Rong Ji State Key Laboratory of Pollution Control and Resource Reuse, School
of Environment, Nanjing University, Nanjing, China
Vijaya Gopal Kakani Department of Plant and Soil Sciences, Oklahoma State
University, Stillwater, OK, USA
Maria I. Klapa Metabolic Engineering and Systems Biology Laboratory, Institute
of Chemical Engineering Sciences, Foundation for Research & Technology-Hellas
(FORTH/ICE-HT), Patras, Greece
Departments of Chemical & Biomolecular Engineering & Bioengineering,
University of Maryland, College Park, MD, USA
Ramamurthy Mahalingam Department of Biochemistry and Molecular Biology,
Oklahoma State University, Stillwater, OK, USA
Patrick Mitchell CSIRO Land and Water Flagship, Tasmania, Australia
Jorge Monza Laboratorio de Bioquímica, Departamento de Biología Vegetal,
Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay
Kirankumar S. Mysore Plant Biology Division, The Samuel Roberts Noble
Foundation, Ardmore, OK, USA
Umme Aktari Nadira Department of Agronomy, College of Agriculture and
Prachi Pandey National Institute of Plant Genome Research, New Delhi, India
Matthaios-Emmanouil P. Papadimitropoulos Metabolic Engineering and
Systems Biology Laboratory, Institute of Chemical Engineering Sciences,
Foundation for Research & Technology-Hellas (FORTH/ICE-HT), Patras, Greece
Division of Genetics, Cell Biology & Developmental Biology, Department of
Biology, University of Patras, Patras, Greece
Daping Pei State Key Laboratory of Pollution Control and Resource Reuse,
Libby Pinkard CSIRO Land and Water Flagship, Tasmania, Australia
Rao C.N. Rachaputi Center for Plant Science, University of Queensland,
Kingaroy, Australia
Muthappa Senthil-Kumar National Institute of Plant Genome Research, New
Delhi, India
Richard C. Sicher Crop Systems and Global Change Laboratory, Beltsville
Agricultural Research Center, Beltsville, MD, USA
Contributors xi
Santiago Signorelli Laboratorio de Bioquímica, Departamento de Biología

Vegetal, Facultad de Agronomía, Universidad de la República, Montevideo,
Uruguay
Ranjita Sinha National Institute of Plant Genome Research, New Delhi, India
Yuanyuan Sun State Key Laboratory of Pollution Control and Resource Reuse,
School of Earth Sciences and Engineering, Hydrosciences Department, Nanjing
University, Nanjing, China
Peter E Urwin Faculty of Biological Sciences, Centre for Plant Sciences,
Jaco Vangronsveld Centre for Environmental Sciences, Environmental Biology,
Xiaorong Wang State Key Laboratory of Pollution Control and Resource Reuse,
Tim Wardlaw Forestry Tasmania, Hobart, Tasmania, Australia
Timothy R. Wheeler Department of Agriculture, University of Reading, Reading,
UK
Feibo Wu Department of Agronomy, College of Agriculture and Biotechnology,
Zijingang Campus, Zhejiang University, Hangzhou, China
Jichun Wu State Key Laboratory of Pollution Control and Resource Reuse,
School of Earth Sciences and Engineering, Hydrosciences Department, Nanjing
University, Nanjing, China
Ying Yin State Key Laboratory of Pollution Control and Resource Reuse, School
Guoping Zhang Department of Agronomy, College of Agriculture and
Yaodan Zhang State Key Laboratory of Pollution Control and Resource Reuse,
Hui Zhou State Key Laboratory of Pollution Control and Resource Reuse, School
Jianguo Zhu State Key Laboratory of Soil and Sustainable Agriculture, Institute
of Soil Science, Chinese Academy of Science, Nanjing, Jiangsu, China
Abbreviations
A Net photosynthetic rate

ABA Abscisic acid
ABF ABRE binding factor
ABRE ABA response element
AB-QTL Advanced backcross—quantitative trait locus
AOC Allene oxide cyclase
AOS Allene oxide synthase
APX Ascorbate peroxidase
AREB ABA response element binding protein
AsA Ascorbate
ASM Available soil moisture
ATAF1 Arabidopsis thaliana activating factor 1
ATL4 Arabidopsis Toxicos En Levadura
BABA Beta-amino-butyric acid
BCAA Branched chain amino acids
BM Biomass
CAT Catalase
CBF C-repeat binding factor
CCA1 Circadian clock associated 1
CDPK Calcium-dependent protein kinase
CE-MS Capillary electrophoresis-mass spectrometry
CH4 Methane
Chl Chlorophyll
Chl* Excited chlorophyll
CN Cyst nematode
CO2 Carbon dioxide
COI1 Coronatine insensitive 1
Cyt Cytochrome
DAF Days after flowering
DAS Days after sowing
DH Doubled haploid
DMNT ( E)-4,8-dimethyl-1,3,7-nonatriene
xiii
xiv Abbreviations
DREB Dehydration response element binding protein

Es Soil evaporation
EREB Ethylene responsive element binding protein
ET Ethylene
ET Evapotranspiration
ETc Crop evapotranspiration
FAC Fatty acid chains
FACE Free-air CO2 enrichment
FC Field capacity of soil
FDR False discovery rate
FLN Flower number
FT-ICR-MS Fourier transformation cyclotron resonance mass
spectrometry
GABA Gamma-aminobutyric acid
GC-MS Gas chromatography-mass spectrometry
GLV Green leaf volatiles
GR Glutathione reductase
GSH Glutathione
H2O2 Hydrogen peroxide
HCL Hierarchical clustering analysis
HI Harvest index
HSF Heat shock factor
HSP Heat shock protein
IPCC Intergovernmental Panel on Climate Change
JA Jasmonic acid
JAZ Jasmonate ZIM-domain
LC-MS Liquid chromatography-mass spectrometry
LD Linkage disequilibrium
LEA proteins Late embryogenesis abundant proteins
LOX Lipoxygenase
LTP Lipid transfer protein
MCP 1-Methylcyclopropene
MDMV Maize dwarf mosaic virus
MEOX derivative Methoxime derivative
MQTL Meta-quantitative trait locus
MV Methyl viologen
NMR Spectroscopy Nuclear magnetic resonance spectroscopy
NO Nitric oxide
NO2 Nitrogen dioxide
NOx Oxides of nitrogen
NOA NO-associated protein
NR Nitrate reductase
O●− Activated oxygen atom
O2●− Superoxide radical
O2 Atmospheric oxygen
O3 Ozone
●
OH Hydroxyl radical
Abbreviations xv
OPDA 12-Oxo-phytodienoic acid

OPR3 OPDA reductase 3
OTC Open top chambers
PAMP Pathogen-associated molecular patterns
P5CS Delta 1-pyrroline-5-carboxylate synthetase
PC Plastocyanin
PCA Principal component analysis
PCR Polymerase chain reaction
PMWaV-1 Pineapple mealybug wilt-associated virus-1
POD Peroxidase
PPN Plant parasitic nematodes
PR Pathogenesis-related
PSI Photosystem I
PSII Photosystem II
PQ Plastoquinone
QTL Quantitative trait loci
RH Relative humidity
RKN Root knot nematode
RPF1 Ribosome production factor 1
ROS Reactive oxygen species
RuBisCO Ribulose-1,5-bisphosphate carboxylase
SACC Screen aided CO2 control
SAM analysis Significant analysis of microarrays
SAR Systemic acquired resistance
SAT Semi-arid tropics
SCF Skp, Cullin, F-box
SIPK Salicylic acid-induced protein kinase
SLA Specific leaf area
SOx Oxides of sulphur
SOD Superoxide dismutase
SOS Salt overly sensitive
T Transpiration
Tcalc Calculated transpiration
Tsla Specific leaf area based transpiration
Tsim Simulated transpiration
TBARS Thiobarbituric acid reactive substances
TC Thermocouple
TCA cycle Tricarboxylic acid cycle
TE Transpiration efficiency
TMS Trimethylsilyl derivative
TMTT ( E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene
TMV Tobacco mosaic virus
TSI1 Tobacco stress-induced1
TuMV Turnip mosaic virus
VOC Volatile organic compounds
VPD Vapour pressure deficit
WIPK Wound-induced protein kinase
Chapter 1
Consideration of Combined Stress: A Crucial
Paradigm for Improving Multiple Stress
Tolerance in Plants
1.1 Introduction
Food security is a major issue in the global policy agenda. In the next 40 years,
demand for cereal production is predicted to increase by 60 % as the population
rises from the current 6.6 to 8.7 billion by the year 2050 (Bengtsson et al. 2006).
In a world where population growth exceeds food supply (Malthus 1817), a second
green revolution is necessary. But the challenges in overcoming the constraints in
food production are complex. The ongoing change in climate mostly due to anthro-
pogenic activities causes increases in carbon dioxide (CO2) emissions (Peters et al.
2011), further exacerbating the agricultural land deterioration due to increasing
temperature (Kissoudis et al. 2014). Increasing temperature in turn leads to higher
evapotranspiration, drought intensification, and increasing soil salinization (Munns
and Tester 2008; Zhao and Running 2010). Though the existing data on the impact
of climate change on pathogen spread are inconclusive, evidence points to increased
reproductive potential and geographic expansion leading to interactions with more
hosts and new virulent pathogenic strains (Garrett et al. 2006). An analysis of the
natural disasters that resulted in more than a billion dollars in the USA in the past
three decades clearly shows that both the frequency and intensity of these events are
increasing (Fig. 1.1). Hence, the chances of plants encountering new combination
of stresses in the future are likely to be higher. It thus behooves upon plant scientists
working on stress resistance to consider the combination of stresses that are likely
to co-occur under field conditions.
R. Mahalingam ()
246 Noble Research Center, Department of Biochemistry and Molecular Biology,
Oklahoma State University, Stillwater, OK 74078, USA
e-mail: ramamurthy.mahalingam@okstate.edu
© Springer International Publishing Switzerland 2015 1

R. Mahalingam (ed.), Combined Stresses in Plants, DOI 10.1007/978-3-319-07899-1_1
2 R. Mahalingam
Fig. 1.1 The US billion-dollar weather and climate disaster time series from 1980 to 2011.
(Adapted from reference Smith and Katz 2013)
1.2 Importance of Combined Stress
Literature is replete with studies on plant responses to stresses. PubMed search us-
ing keywords “stress” and “plants” in title and abstract field alone identified nearly
15,300 citations while “combined stress” and “plants” retrieved 480 citations. A
closer inspection of the latter search revealed only around 180 original articles that
actually dealt with the combination of two or more stresses in plants. A listing of
primary research articles on combined stress in various plant species is given in
Table 1.1.
The combined occurrence of drought and heat in the USA from 1980 to 2012 was
shown to cause fivefold more damage when compared to drought alone (Fig. 1.2).
Increase in global surface temperature is a major indicator of global warming (Van
Vuuren et al. 2008). This rise in mean global temperature is attributed to increases
in the greenhouse gases such as CO2 and air pollutants such as ozone (O3) that
have been brought about by anthropogenic activities. For the first time in recorded
history, the average level of CO2 has topped 400 parts per million (ppm) for an
entire month in April 2014 according to the Scripps Institution of Oceanography.
Efforts to control CO2 emissions on a global scale will be difficult to enforce given
the political and economic implications surrounding such legislations. More than
400 ppm of CO2 may thus be the new reality for crop plants in the future.
1 Consideration of Combined Stress 3
Table 1.1 Primary research studies of combined stresses in various plant species
Stress combination Plant species (references)
Drought + heat Arabidopsis (Koussevitzky et al. 2008; Rizhsky et al. 2004; Vile
et al. 2012; Wolfe and Tonsor 2014), tobacco (Cvikrova et al.
2013; Rizhsky et al. 2002), wheat (Keles and Oncel 2002; Prasad
et al. 2011; Rampino et al. 2012; Szucs et al. 2010; Yang et al.
2011), Sorghum (Johnson et al. 2014), Carissa spinarum (Zhang
et al. 2010), lotus (Sainz et al. 2010), soybeans (Simon-Sarkadi
et al. 2005), Jatropha (Silva et al. 2010); barley (Rollins et al.
2013); poplar (Centritto et al. 2011); prosopis (Delatorre et al.
2008)
Drought + chilling Sugarcane (Sales et al. 2013), maize (Aroca 2003)
Drought + high light Arabidopsis (Estavillo et al. 2011; Giraud et al. 2008), Haberlea
rhodopensis (Georgieva et al. 2010), rice (Zhou et al. 2007),
watermelon (Nanasato et al. 2005), pearl millet and Sorghum
(Masojidek et al. 1991); Nerium oleander (Demmig et al. 1988)
Drought + heavy metals Red maple (de Silva et al. 2012); Populus cathayana (Han et al.
2013); oak (Sardans and Penuelas 2007); Stackhousia tryonii
(Bhatia et al. 2005)
Drought + ozone Birch (Paakkonen et al. 1998), beech (Nunn et al. 2007), Medi-
cago truncatula (Iyer et al. 2013), Quercus (Alonso et al. 2014),
poplar (Bohler et al. 2013), Dactylis glomerata, and Ranunculus
acris (Wagg et al. 2012); wheat (Biswas and Jiang 2011; Herbin-
ger et al. 2002), spruce (Karlsson et al. 1997; Kivimaenpaa et al.
2003); Pinus halepensis (Manes et al. 2001; Fontaine et al. 2003)
Drought + salinity Barley (Ahmed et al. 2013a, b, c); Sesuvium portulacastrum
(Slama et al. 2008)
Drought + soil compaction Tobacco (Alameda et al. 2012)
Drought + nutrients Maize (Kandianis et al. 2013; Makumburage and Stapleton
2011); wheat (Wei et al. 2013); potato (Germ et al. 2007)
Drought + UV Maize (Makumburage et al. 2013); wheat (Feng et al. 2007; Zhao
et al. 2009); Arabidopsis (Comont et al. 2012; Schmidt et al.
2000); barley (Bandurska et al. 2012); peas (Nogues et al. 1998);
Populus cathayana (Lu 2009); willows (Turtola 2006); soybeans
(Sullivan and Teramura 1990)
Drought + high CO2 Potato (Barnaby et al. 2014); maize (Sicher and Barnaby 2012);
Phaseolus vulgaris (Medeiros and Ward 2013); Viguiera discolor
(Oliveira et al. 2013); eucalyptus (Crous et al. 2012; Duursma
et al. 2011; Lewis et al. 2013; Zeppel et al. 2011); maize and sor-
ghum (Allen et al. 2011; Kakani et al. 2011; Leakey et al. 2006);
pepper (del Amor et al. 2010); populus (Bobich et al. 2010);
cucumber (Li et al. 2008); oak and pine (Schwanz et al. 1996)
Drought + pathogens/pest Arabidopsis (Atkinson et al. 2013; Anderson et al. 2004); tobacco
(Ramegowda et al. 2013); rice (Campo et al. 2012); Alnus fruti-
cosa (Rohrs-Richey et al. 2011); beet and rice (Xu et al. 2008)
Salinity + heat Tomato (Rivero et al. 2014); poplar (Behnke et al. 2013); Arte-
misia (Wen et al. 2005) Swietenia macrophylla (Rahman et al.
2013)
4 R. Mahalingam
Table 1.1 (continued)

Salinity + ozone Alfalfa (Maggio et al. 2009); chickpea (Welfare et al. 2002); rice
(Welfare et al. 1996); wheat (Zheng et al. 2012)
Salinity + pathogens Rice (Xiong and Yang 2003)
Salinity + nutrients Barley (Talbi Zribi et al. 2011); Hordeum maritimum (Talbi
Zribi et al. 2012); spinach (Kaya et al. 2001); soybeans (Grat-
tan and Maas 1988); peanuts (Silberbush and Ben-Asher 1989);
Crithmum maritimum (Labidi et al. 2011); broccoli (del Carmen
Martinez-Ballesta 2008)
Salinity + high CO2 Arabidopsis (Kanani et al. 2010); Spartina maritima (Mateos-
Naranjo et al. 2010a); barley (Perez-Lopez et al. 2009, 2012);
pepino (Chen et al. 1999); melon (Mavrogianopoulos et al. 1999);
citrus (Garcia-Sanchez et al. 2006); olive (Melgar et al. 2008);
aster (Geissler et al. 2009, 2010); tomato (Takagi et al. 2009);
Spartina densiflora (Mateos-Naranjo et al. 2010b)
Heat + ozone Birch (Kasurinen et al. 2012; Maenpaa et al. 2011; Riikonen et al.
2009, 2013); spruce (Riikonen et al. 2012); populus (Hartikainen
et al. 2009); bean (Albertine and Manning 2009); radish (Kleier
et al. 2001)
Heat + light Sunflower (Hewezi et al. 2008); Brassica (Diaz et al. 2007), oats
(Quiles 2006); seagrass (York et al. 2013); apple (Chen et al.
2008); grapes (Greer and Weedon 2012); Arabidopsis (Burgos
et al. 2011; Lokhande et al. 2003; Vasseur et al. 2011); Dunaliella
salina (Haghjou et al. 2009); Phragmites australis (Loreto et al.
2006); wheat (Monneveux et al. 2003); spruce (Mahoney et al.
1998)
Heat + UV Wheat (Zheng et al. 2011); cucumber (Caldwell 1994)
Heat + high CO2 Tomato (Li et al. 2014b); Kentucky bluegrass (Song et al. 2014);
aspen (Sun et al. 2013); soybeans (Sicher 2013); rice (Madan
et al. 2012); eucalyptus (Loveys et al. 2006); bell pepper (Aloni
et al. 2001; Karni and Aloni 2002); Abutilon theophrasti (Ziska
2001); cotton and tobacco (Crafts-Brandner and Salvucci 2000)
Temperature + pathogens Arabidopsis (Szittya et al. 2003; Yang and Hau 2004; Zhu et al.
2010); tomato (de Jong et al. 2002)
Ozone + high CO2 Soybeans (Ainsworth et al. 2008; Gillespie et al. 2012); populus
(Kets et al. 2010); wheat (Mishra et al. 2013)
Ozone + UV Linseed (Tripathi and Agrawal 2013a, b); birch (Pliura et al.
2008); Elymus athericus (van de Staaij et al. 1997)
Ozone + pathogens Tobacco (Ye et al. 2012); soybeans (Bilgin et al. 2008); Beech
and spruce (Luedemann et al. 2005)
Chilling + high light Tomato (Wang et al. 2008); cotton (Kornyeyev et al. 2001; Pay-
ton et al. 2001)
Chilling + pathogens Arabidopsis (Yang et al. 2010)
UV + heavy metals Brassica campestris (Shukla et al. 2008); Pisum sativum (Srivas-
tava et al. 2012)
UV + pathogens Arabidopsis (Kunz et al. 2006); tea (Gunasekera et al. 1997); cab-
bage (Brown et al. 2001); tobacco (Yalpani et al. 1994)

High CO2 + high light Chlorella (Kozlowska-Szerenos et al. 2004)
Nutrient + pathogens Arabidopsis (Amtmann et al. 2008)
Drought + heat + high Hibiscus (Munoz and Quiles 2013); Rosa meillandina (Paredes
light and Quiles 2013); wheat (Sharma and Singhal 1993)
Drought + high light + UV Arabidopsis (Poulson et al. 2006)
Drought + heat + virus Arabidopsis (Prasch and Sonnewald 2013)
CO2 + temperature + UV Cowpea (Singh et al. 2010); soybeans (Koti et al. 2005); birch
(Lavola et al. 2013)
Ozone + light Trifolium subterraneum (Vollsnes et al. 2009)
CO2 + temperature + Eucalyptus (Roden and Ball 1996)
drought
UV + nutrients Vigna radiata (Agrawal et al. 2006); wheat (Shukla et al. 2002)
CO2 + ozone + insects Soybeans (Casteel et al. 2008)
CO2 + temperature + Soybeans (Niziolek et al. 2013)
insects
250
200
Billion US dollarrs
150
100
B
50
0
Drought/heat Drought Flooding Freeze/Cold
Fig. 1.2 A meta-analysis of naturally occurring disasters in the USA. Losses due to weather-
related disasters (excluding tornadoes, hurricanes, and wildfires) occurring between 1980 and
2011 that exceeded more than a billion dollars were included in this analysis. Damage costs were
normalized to the 2013 US dollar value. Raw data for this analysis were from reference (Smith
and Katz 2013)
Here is an example to illustrate the importance of considering more than a single

stress. A recent study indicated that rising CO2 levels increased the estimated yield
levels of soybeans during 2002–2006 by 4.34, 7.57, and 5.10 %, in the USA, Brazil,
and China, respectively (Sakurai et al. 2014). However, there are other studies using
the free-air concentration enrichment (FACE) technology that consider the increas-
ing levels of ozone, the most abundant air pollutant that will negate the fertilizing
effects of CO2 and predict a less-than-expected yield due to the increasing levels of
CO2 (Long et al. 2005, 2006).
6 R. Mahalingam
1.3 Which Combination of Stresses to Study?
This begs the question which combination of stresses to study. As indicated earlier,
plants are continually challenged by diverse array of biotic and abiotic agents from
seed germination to senescence. We can envisage considering those stresses that
are most likely to co-occur under field conditions and whose combined impact can
adversely affect the final yield.
Stress Matrix Approach Mittler and coworkers have advocated the use of a stress
matrix showing different combinations of potential environmental stresses that can
affect crops. The use of colors to indicate potential positive and negative interactions
provides a visually appealing schema for depicting combined stresses (Fig. 1.3). It
Fig. 1.3 The stress matrix. Different combinations of potential environmental stresses that can
affect crops in the field are shown in the form of a matrix. The matrix is color-coded to indicate
stress combinations that were studied with a range of crops and their overall effect on plant growth
and yield. References for these studies are given in the text and in Table 1.1. (Adapted from Suzuki
et al. (2014) and modified from Mittler (2006))
Fig. 1.4 Combined ozone and drought stress in two soybean cultivars. Cultivar Forrest (sensitive
to ozone, sensitive to heat) and Essex (tolerant to ozone, tolerant to heat) were simultaneously
exposed to 75–100 ppb of ozone and higher temperature of 37 °C for 3 h a day for 3 consecutive
days. Photographs were taken at the end of the combined ozone and heat treatment on day 1 (A),
day 2 (B), day 3 (C), 24 h after the end of the treatment (E), and 10 days of post-recovery in a
growth chamber (F)
should be noted that this is a vast oversimplification of the complexity involved in

combined stress scenarios. For example, Medicago truncatula cultivar Jemalong is
sensitive to ozone and drought when the stresses were applied singly (Puckette et al.
2007). The combined application of drought and ozone in Jemalong evoked a very
different transcriptome and metabolic response that manifested as a stress-tolerant
phenotype (Iyer 2013). To test whether this observation can be extended to other
legumes, we used the soybean cultivar Forrest that is sensitive to drought and ozone
when applied singly. The combined application of drought and ozone for a period of
3 days was detrimental for Forrest plants (Fig. 1.4). Thus, based on single-case stud-
ies, it is naïve to interpret the interactions between stress combinations as positive
or negative. Biswas and Jiang (2011) reported that, under conditions of combined
ozone and drought stress, the ozone-sensitive modern winter wheat cultivar ( Triti-
cum aestivum L. cv. Xiaoyan 22) improved its tolerance against ozone, while the
ozone-tolerant primitive wheat ( Turgidum ssp. durum) lost ozone tolerance. Crops
show wide variability in their phenotypic responses to stresses and this includes
both the intra- and inter-specific variation (Biswas et al. 2008; Brosche et al. 2010).
8 R. Mahalingam
Furthermore, it has been shown that the order in which the stress combinations
are applied may evoke a different response. An early drought could lead to a de-
crease in stomatal conductance and a subsequent protection against a later ozone ex-
posure while the appearance of drought during preexisting ozone stress would suffer
under the appearing sluggishness of stomata, initially caused by ozone (Paoletti and
Grulke 2010).
Plants can show varied responses to stresses depending on their developmental
stage. This adds an additional layer of complexity in the analysis of plant stress
studies. If a field is affected by stress at a very early stage of development (e.g.,
seedling stage), a farmer may be able to undertake second planting and still recover
his losses. On the other hand, a severe stress in field during the reproductive stage of
development will not be amenable for such amends. It has been reported that most
plants of agronomic importance are gullible to abiotic stresses during reproductive
stages with detrimental consequences to the yield (Barnabas et al. 2008). Interest-
ingly, the consequences to yield in response to stresses are not considered in most
studies involving model plants like Arabidopsis. The usefulness of model plants for
understanding plant stress responses can be greatly increased by assessing impact
of stress on seed yield and seed quality. From an agronomic perspective, the most
important aspect of plant stress interactions will be to understand its impact on the
final yield.
1.4 Omics of Combined Stress
A detailed review of the transcriptome studies on combined stresses in plants has

been reported (Jambunathan et al. 2010). A few proteomic studies on the combined
stresses have been reported. This includes drought and ozone in poplar (Bohler
et al. 2013), drought, and heat in Arabidopsis, barley, Carissa spinarum (Koussev-
itzky et al. 2008; Rollins et al. 2013; Zhang et al. 2010), toxic compounds like mer-
cury and salinity in Suaeda salsa (Liu et al. 2013), high temperature and humidity in
Portulaca oleracea (Yang et al. 2012). Interestingly, transcriptomic and proteomic
analysis of several different combined stresses in several different plant species
converges on the antioxidant defense machinery as a key pathway. The observed
higher antioxidant capacity and/or lower accumulation of the reactive oxygen
species (ROS) seems to be a mechanism operative in plants tolerant to combined
stresses (Iyer et al. 2013; Koussevitzky et al. 2008; Ahmed et al. 2013b; Perez-
Lopez et al. 2009; Rivero et al. 2014; Sales et al. 2013). Omics approaches have
also shown that there are unique transcription factors, hormone-responsive genes
and osmolytes that are differentially expressed in response to different combined
stresses (Iyer et al. 2013; Atkinson et al. 2013; Rasmussen et al. 2013; Rizhsky et al.
2004). An apparent gap in the knowledge is the lack of information on posttran-
scriptional gene regulation by microRNAs in response to combined stresses. In fact,
a comprehensive analysis of transcriptome, proteome, metabolome, and miRNome
even in response to a single stress has not been reported. Such integrated omics
studies of combined stresses imposed during reproductive stages of crop develop-

ment are warranted.
DNA cytosine methylation and histone modifications such as methylation and
acetylation affect transcription especially in response to changes in environment
(Mirouze and Paszkowski 2011). Epigenetic modifications involving chromatin-
regulated gene activation govern priming responses (Conrath 2011) and widespread
alterations in DNA methylation have been reported in response to biotic and abiotic
stresses (Bilichak et al. 2012; Dowen et al. 2012). The knowledge of epigenetic
modifications in the wake of combined stresses is relatively unknown and is worthy
of further investigations. It has been speculated that epigenetic modifications in re-
sponse to a stress may predispose plants to a subsequent stress by either sensitizing
or desensitizing. Such acclimation/predisposition may provide a novel avenue for
preparing seeds for stressful environments (Kissoudis et al. 2014).
1.5 Phenotypic Responses to Stresses
From an agronomic point of view, the definition of plant sensitivity to stresses can
be misleading. For example, crops can be sensitive to ozone with reference to vis-
ible foliar damage at early stages of growth but may not have a net impact on the
grain yield during harvest. In rice and wheat, plants with least visible foliar symp-
toms showed maximum yield losses (Picchi et al. 2010; Sawada and Kohno 2009)
and this was explained on the basis of stomatal closure response. Cultivars in which
ozone causes stomatal closure prevent the influx of ozone and reduce the extent of
foliar injury. Thus, based on the damage to leaves, these cultivars are resistant to
ozone. However, prolonged stomatal closure affects carbon fixation and in turn the
amount of assimilates required for grain filling. Thus, with reference to yield these
cultivars are ozone sensitive. Other mechanisms for the negative effect of ozone
could be due to the reduction of new growth (McKee and Long 2001), reduced root
biomass (Grantz et al. 2006), reduced phloem translocation efficiency, or reduced
carbon portioning to grains over synthesis of protective chemicals (Betzelberger
et al. 2010).
It is important to understand the differences between sensitive and resistant re-
sponses that can differ depending on the stress. Let us consider the example of
ozone exposure. The visible injury symptoms due to ozone are mostly assessed by
damage to foliage. In sensitive plants, they appear as small chlorotic or necrotic le-
sions on leaves that can coalesce into larger patches of injured area, and such leaves
usually senesce early. This reduces the effective biomass that in turn will take a toll
on crop yields (Wilkinson et al. 2012). The same necrotic lesions on the foliage
in response to avirulent pathogen infections are termed as hypersensitive response
and the plant is considered to be resistant to the pathogen. The characterization of
the same phenotype as being resistant with respect to one stress and as sensitive
response to another stress is important to bear in mind while considering the com-
bination of biotic and abiotic stresses.
10 R. Mahalingam
1.6 Contrasts Between Laboratory and Field Studies
In several recent reviews, the limitations of single stress studies in controlled con-
ditions compared to field conditions have been examined (Mittler and Blumwald
2010; Suzuki et al. 2014). The study of combined stresses in the laboratory is ad-
vocated so that the molecular pathways for tolerance to stresses that prevail under
field condition can be identified. Most of the studies on combined stresses so far
have been conducted under growth chamber or greenhouse conditions (Suzuki et al.
2014). Here, we have contrasted the combined stress studies in laboratory condi-
tions versus the field conditions (Fig. 1.5). Combined stresses dealing with edaphic
factors can be conducted effectively in greenhouse conditions. This includes the
combinations of drought and nutrients, drought and salinity, drought and soil patho-
gen/pests such as nematodes. Combined stress experiments that involve interactions
between climate change factors including CO2, ozone, and temperature extremes
(heat or cold) are ideal for growth-chamber studies. But the main constraint here
is the number of large-sized plants that can be accommodated in such chambers. If
greenhouse space and infrastructure for regulating gaseous mixtures (for example,
CO2 and ozone) are available, it provides an ideal platform for conducting controlled
combined stress analysis of climate change variables and edaphic factors. Several
reviews have examined the advantages and disadvantages of open-top chambers
(OTCs), FACE systems, and screen-aided CO2 control (SACC; Ainsworth et al.
2008; Li et al. 2007). Though FACE and OTCs provides an opportunity to examine
the impact of climate change factors in actual field environment, it will be hard to
use these facilities in combined stress scenarios such as drought or temperature
stress. Rainout shelters can be constructed for studying drought in combination with
other climate change factors in a FACE but may be expensive.
'ƌŽǁƚŚ ĐŚĂŵďĞƌ 'ƌĞĞŶ ŚŽƵƐĞ &ŝĞůĚ ƐƚƵĚŝĞƐ

ŽŶƚƌŽůůĞĚ ĞŶǀŝƌŽŶŵĞŶƚĂů ĐŽŶĚŝ ŽŶƐ͘ ŽŶƚƌŽůůĞĚ ĞŶǀŝƌŽŶŵĞŶƚĂů ĐŽŶĚŝ ŽŶƐ͘ ,ĞƚĞƌŽŐĞŶĞŽƵƐ ĞŶǀŝƌŽŶŵĞŶƚĂů ĐŽŶĚŝ ŽŶƐ
^ƚƵĚŝĞƐ ŽŶ ƐƚƌĞƐƐ ƚƌĞĂƚŵĞŶƚ ĐŽŵďŝŶĂ ŽŶƐ ĂƌĞ KŶůǇ ƐŽŵĞ ƐƚƌĞƐƐ ƚƌĞĂƚŵĞŶƚ ĐŽŵďŝŶĂ ŽŶƐ ĂƌĞ WůĂŶƚƐ ĂƌĞ ĞǆƉŽƐĞĚ ƚŽ ŵƵů ƉůĞ ƐƚƌĞƐƐĞƐ
ĞĂƐǇ ƚŽ ĐŽŶĚƵĐƚ͘ ĞĂƐǇ ƚŽ ĐŽŶĚƵĐƚ͘ ĨƌŽŵ ŐĞƌŵŝŶĂ ŽŶ ƚŽ ŵĂƚƵƌŝƚǇ
EŽ ŝŶƚĞƌĂĐ ŽŶƐ ǁŝƚŚ ŽƚŚĞƌ ǁĞĞĚ ƉůĂŶƚƐ͕ EŽ ŝŶƚĞƌĂĐ ŽŶƐ ǁŝƚŚ ŽƚŚĞƌ ǁĞĞĚ ƉůĂŶƚƐ͕ ŽŶƐƚĂŶƚ ŝŶƚĞƌĂĐ ŽŶƐ ǁŝƚŚ ǁĞĞĚƐ͕ ƉĞƐƚƐ͕ ĂŶĚ
Žƌ ŝŶƐĞĐƚ ƉĞƐƚƐ ƐŽŵĞ ŝŶƐĞĐƚ ƉĞƐƚƐ ƉŽƐƐŝďůĞ ǀĂŐĂƌŝĞƐ ŽĨ ŶĂƚƵƌĞ
hŶŝĨŽƌŵ ƐŽŝů ďƵƚ ůŝŵŝƚĞĚ ƐŽŝů ǀŽůƵŵĞ hŶŝĨŽƌŵ ƐŽŝů ďƵƚ ůŝŵŝƚĞĚ ƐŽŝů ǀŽůƵŵĞ ʹ ĐĂŶ ^Žŝů ƉŚǇƐŝĐĂů ƉƌŽƉĞƌƟĞƐ ŵĂǇďĞ
ďĞ ŝŵƉƌŽǀĞĚ ďǇ ƵƐŝŶŐ ůĂƌŐĞ ƐŝǌĞĚ ƉŽƚƐ ŚĞƚĞƌŽŐĞŶĞŽƵƐ ďƵƚ ƐŽŝů ǀŽůƵŵĞ ŝƐ ŶŽƚ ůŝŵŝ ŶŐ
DŽƐƚ ƐƵŝƚĂďůĞ ĨŽƌ ƐŵĂůů ƐŝǌĞĚ ƉůĂŶƚƐ ůŝŬĞ ĂŶ ĂĐĐŽŵŵŽĚĂƚĞ ŵŽƌĞ ŶƵŵďĞƌ ŽĨ ƉůĂŶƚƐ ĂŶ ĂĐĐŽŵŵŽĚĂƚĞ ůĂƌŐĞ ŶƵŵďĞƌ ŽĨ ƉůĂŶƚƐ ĨŽƌ
ƌĂďŝĚŽƉƐŝƐ͕ ďƌĂĐŚǇƉŽĚŝƵŵ͕ ĨŽǆƚĂŝů ŵŝůůĞƚ͘ ĐŽŵƉĂƌĞĚ ƚŽ Ă ŐƌŽǁƚŚ ĐŚĂŵďĞƌ͘ ^ƵŝƚĂďůĞ ĨŽƌ ƉƌŽǀŝĚŝŶŐ ƌŽďƵƐƚ ĂƐƐĞƐƐŵĞŶƚ ŽĨ ƉŚĞŶŽƚǇƉĞƐ
ůĂƌŐĞƌ ƉůĂŶƚƐ ůŝŬĞ ƐŽǇďĞĂŶƐ͕ ǁŚĞĂƚ͕ ĐŽƌŶ ŝŶ ĂŶǇ ĐƌŽƉ ƉůĂŶƚƐ
Fig. 1.5 Comparisons between growth chamber, green house, and field studies for analyzing the
effects of combined stresses in plants
1.7 Advances in Phenomics
Following the enormous advances in the sequencing technologies, it has now be-
come routine to sequence large collections of accessions or mapping populations
in a plant species (Lam et al. 2010; Li et al. 2014a; Weigel and Mott 2009). The
major bottleneck currently in utilizing the genome sequence deluge is the ability to
procure reliable phenotype data. Over the past decade field, phenotyping has made
rapid strides by utilizing remote-sensing technologies for crop monitoring (Furbank
and Tester 2011). The field of phenomics described as a “high-throughput plant
physiology” makes use of noninvasive imaging, infrared thermography, spectrosco-
py, robotics, image analysis, and high-performance computing. Several successful
phenotyping screens for single stresses such as drought, UVB have been reported in
model plant systems (Jansen et al. 2010; Woo et al. 2008) as well as in crop plants
(Chapuis et al. 2012; Honsdorf et al. 2014; Sirault et al. 2009).
For UV stress and temperature extremes, the photosynthetic light-harvesting ap-
paratus is often the first site of damage. UV stress can result in oxidative damage to
the photosystems, perceived as a loss of efficiency of light harvesting, that can be
exploited as a screening tool for tolerance to UVB exposure (Jansen et al. 2010). In
the case of temperature extremes, the effects on photosynthesis and even changes
in membrane lipid properties can lead to immediate effects on chlorophyll fluores-
cence (Armond et al. 1980).
Digital imaging in visible wavelength regions provides information on plant size,
and also on the color of the plants. This information enables the quantification of
senescence arising from nutrient deficiencies or toxicities, or pathogen infections.
Germanium, a toxic analog of boron, was tested in a mapping population of barley
to identify a Quantitative Trait Loci (QTL) at the same locus as previously identi-
fied for boron tolerance using a visual score of symptoms (Schnurbusch et al. 2010).
Near-surface reflectance spectroscopy was used to monitor the leaf nitrogen and
chlorophyll content and epoxidation state of xanthophyll cycle pigments in field-
grown soybean plants exposed to ozone (Ainsworth et al. 2014). This study shows
that the leaf optical properties can be monitored using remote-sensing techniques to
assess ozone damage and provide a promising tool for elucidating ozone tolerance
in plants.
The examples mentioned above demonstrate the utility of the phenomics tools for
precisely monitoring the physiological impacts of single stresses such as drought,
salinity, nutrient deficiency, and air pollutants. It is conceivable that these tools will
be harnessed for the analysis of combined stresses in the future.
1.8 Strategies for Improving Tolerance to Combined

Stresses
Two major strategies can be envisaged for improving the tolerance to combined
stresses (Fig. 1.6). First strategy involves the meta-analysis of whole genome
expression studies in response to various biotic and abiotic stresses that can be
12 R. Mahalingam
Meta-analysis of omics data from single or combined stress Genome wide association studies of combined stresses
Phenotyping Genotyping
Manipulation of key Pyramiding
regulatory factors
Landraces SNP chips
Transcription factors Defense Abiotic stress Elite germplasm Sequencing

Receptor like kinases R-genes Hormonal signaling Inbred lines Public database
Flavonoid metabolism RLKs Antioxidants Mapping populations
Redox homeostasis Pre-invasion barriers Ion homeostasis

Chromatin remodeling
Non-host resistance Identify novel lines with tolerance to combined stress
Incorporate into cultivars by marker assisted breeding

Cisgenesis or transgenesis of desired genes or gene cassettes
into commercial cultivars and advanced breeding lines
Fig. 1.6 Strategies for building tolerance to combined stresses in plants. A compendium approach
for identifying key regulatory factors or by pyramiding key genes important in co-occurring stress
scenarios that can be transferred into desired cultivars by genetic engineering. Another strategy
will be to use genome-wide association mapping to identify novel germplasm containing alleles
favorable for imparting tolerance to combined stresses and use naturally occurring variation for
developing cultivars with improved resistance to multiple stresses via marker-assisted breeding
accessed through programs like Genevestigator (Zimmermann et al. 2004). Re-

cent advances in computational tools such as co-expression modules and machine-
learning approaches provide novel means for identifying the candidate genes for
engineering broad-spectrum resistance based on gene expression data (Shaik and
Ramakrishna 2013, 2014). Genetic components that potentially regulate the resis-
tance to multiple stresses will be utilized for developing transgenic crops. Examples
of genes for this strategy include stress-inducible transcription factors, receptor-like
kinases, flavonoid metabolism, redox homeostasis, and chromatin modifications.
The same meta-analysis strategy can be adapted for gene pyramiding that has
been successfully deployed for resistance to various plant pathogens (Joshi and
Nayak 2010). In the case of combined biotic and abiotic stresses, the pyramided
genes can be defense genes such as R-genes, pre-invasion defenses (such as callose
deposition), nonhost resistance genes in combination with genes in the hormone
signaling pathways, antioxidant defenses, or ion homeostasis (Fig. 1.6; Kissoudis
et al. 2014).
A second strategy for improving plant tolerance to combined stresses involves
the screening of large collections of germplasm in conjunction with genome-wide
association mapping (Huang and Han 2014). In recent years, genotyping data for
large collections of crop germplasms are becoming available in the public domain
(Hao et al. 2012; Li et al. 2013; Song et al. 2013; Yu and Buckler 2006; Zhang et al.
2014). A reliable phenotypic evaluation of germplasm to various stress combina-
tions of interest can be performed. The genotypic information from public domain
can be exploited to precisely identify genomic regions associated with the traits of
interest. The recent assembly and characterization of association mapping panels
in various crop plants, development of improved statistical methods, user-friendly
tools for association mapping (e.g., GWAPP for Arabidopsis; TASSEL) and suc-
cessful association of candidate genes have begun to realize the power of candidate-
gene association mapping.
1.9 Conclusions/Perspectives
Studies of stress combinations that naturally occur under field conditions must be
a priority for researchers working on abiotic and biotic stresses. Studies of such
combined stresses should exploit the naturally occurring variation in the germplasm
of crop plants to identify novel sources of resistance or tolerance. While impos-
ing stress combinations, it is important to consider the plant developmental stages
that can have the most detrimental agronomic consequences and conduct surveys
of germplasm during these critical stages. Phenomic screening using noninvasive
high-throughput phenotyping platforms will provide a wide spectrum of observa-
tions that span metabolic, physiological, and biochemical parameters. Though the
initial costs are high for these setups, the long-term benefits are beyond compari-
son. Finally, integrating data from multiple omics platforms in conjunction with the
phenotyping data will provide a cogent view of the responses to combined stresses
in different genotypes. This is crucial for identifying the elite germplasm that can
tolerate multiple stresses and provide maximum yields.
Acknowledgments This work was partially supported by the Oklahoma Agricultural Experiment
Station. My sincere thanks to Dr. Yixing Wang for conducting the combined stress experiments in
soybeans reported in this chapter.
References
Agrawal SB, Rathore D, Singh A. Combined effects of enhanced ultraviolet-B radiation and min-
eral nutrients on growth, biomass accumulation and yield characteristics of two cultivars of
Vigna radiata L. J Environ Biol. 2006;27:55–60.
Ahmed IM, Dai H, Zheng W, Cao F, Zhang G, Sun D, et al. Genotypic differences in physiological
characteristics in the tolerance to drought and salinity combined stress between Tibetan wild
and cultivated barley. Plant Physiol Biochem. 2013a;63:49–60.
Ahmed IM, Cao F, Zhang M, Chen X, Zhang G, Wu F. Difference in yield and physiological fea-
tures in response to drought and salinity combined stress during anthesis in Tibetan wild and
cultivated barleys. PLoS ONE. 2013b;8:e77869.
Ahmed IM, Cao F, Han Y, Nadira UA, Zhang G, Wu F. Differential changes in grain ultrastructure,
amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys under
drought and salinity alone and combined stress. Food Chem. 2013c;141:2743–50.
Ainsworth EA, Leakey AD, Ort DR, Long SP. FACE-ing the facts: inconsistencies and interdepen-
dence among field, chamber and modeling studies of elevated [CO2] impacts on crop yield and
food supply. New Phytol. 2008;179:5–9.
Ainsworth EA, Serbin SP, Skoneczka JA, Townsend PA. Using leaf optical properties to detect
ozone effects on foliar biochemistry. Photosynth Res. 2014;119:65–76.
14 R. Mahalingam
Alameda D, Anten NPR, Villar R. Soil compaction effects on growth and root traits of tobacco
depend on light, water regime and mechanical stress. Soil Tillage Res. 2012;120:121–9.
Albertine JM, Manning WJ. Elevated night soil temperatures result in earlier incidence and in-
creased extent of foliar ozone injury to common bean ( Phaseolus vulgaris L.). Environ Pollut.
2009;157:711–3.
Allen LH Jr, Kakani VG, Vu JC, Boote KJ. Elevated CO2 increases water use efficiency by sustain-
ing photosynthesis of water-limited maize and sorghum. J Plant Physiol. 2011;168:1909–18.
Aloni B, Peet M, Pharr M, Karni L. The effect of high temperature and high atmospheric CO2 on
carbohydrate changes in bell pepper ( Capsicum annuum) pollen in relation to its germination.
Physiol Plant. 2001;112:505–12.
Alonso R, Elvira S, Gonzalez-Fernandez I, Calvete H, Garcia-Gomez H, Bermejo V. Drought
stress does not protect Quercus ilex L. from ozone effects: results from a comparative study of
two subspecies differing in ozone sensitivity. Plant Biol (Stuttgart). 2014;16:375–84.
Amtmann A, Troufflard S, Armengaud P. The effect of potassium nutrition on pest and disease
resistance in plants. Physiol Plant. 2008;133:682–91.
Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, Ehlert C, et al. Antago-
nistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates
defense gene expression and disease resistance in Arabidopsis. Plant Cell. 2004;16:3460–79.
Armond PA, Bjorkman O, Staehelin LA. Dissociation of supramolecular complexes in chloroplast
membranes. A manifestation of heat damage to the photosynthetic apparatus. Biochim Biophys
Acta. 1980;601:433–43.
Aroca R, Irigoyen JJ, Sanchez-Diaz M. Drought enhances maize chilling tolerance. II. Photosyn-
thetic traits and protective mechanisms against oxidative stress. Physiol Plant. 2003;117:540–9.
Atkinson NJ, Lilley CJ, Urwin PE. Identification of genes involved in the response of Arabidopsis
to simultaneous biotic and abiotic stresses. Plant Physiol. 2013;162:2028–41.
Bandurska H, Pietrowska-Borek M, Cieslak M. Response of barley seedlings to water deficit and en-
hanced UV-B irradiation acting alone and in combination. Acta Physiol Plant. 2012;34:161–71.
Barnabas B, Jager K, Feher A. The effect of drought and heat stress on reproductive processes in
cereals. Plant Cell Environ. 2008;31:11–38.
Barnaby JY, Fleisher D, Reddy V, Sicher R. Combined effects of CO enrichment, diurnal light lev-
els and water stress on foliar metabolites of potato plants grown in naturally sunlit controlled
environment chambers. Physiol Plant. 2014. doi: 10.1111/ppl.12238.
Behnke K, Ghirardo A, Janz D, Kanawati B, Esperschutz J, Zimmer I, et al. Isoprene function in
two contrasting poplars under salt and sunflecks. Tree Physiol. 2013;33:562–78.
Bengtsson M, Shen Y, Oki T. A SRES-based gridded global population dataset for 1990–2100.
Popul Environ. 2006;28:113–31.
Betzelberger AM, Gillespie KM, McGrath JM, Koester RP, Nelson RL, Ainsworth EA. Effects of
chronic elevated ozone concentration on antioxidant capacity, photosynthesis and seed yield of
10 soybean cultivars. Plant Cell Environ. 2010;33:1569–81.
Bhatia NP, Baker AJ, Walsh KB, Midmore DJ. A role for nickel in osmotic adjustment in
drought-stressed plants of the nickel hyperaccumulator Stackhousia tryonii Bailey. Planta.
2005;223:134–9.
Bilgin DD, Aldea M, O’Neill BF, Benitez M, Li M, Clough SJ, et al. Elevated ozone alters soy-
bean-virus interaction. Mol Plant Microbe Interact. 2008;21:1297–308.
Bilichak A, Ilnystkyy Y, Hollunder J, Kovalchuk I. The progeny of Arabidopsis thaliana plants
exposed to salt exhibit changes in DNA methylation, histone modifications and gene expres-
sion. PLoS ONE. 2012;7:e30515.
Biswas DK, Jiang GM. Differential drought-induced modulation of ozone tolerance in winter
wheat species. J Exp Bot. 2011;62:4153–62.
Biswas DK, Xu H, Li YG, Liu MZ, Chen YH, Sun JZ, et al. Assessing the genetic relatedness of
higher ozone sensitivity of modern wheat to its wild and cultivated progenitors/relatives. J Exp
Bot. 2008;59:951–63.
Bobich EG, Barron-Gafford GA, Rascher KG, Murthy R. Effects of drought and changes in va-
pour pressure deficit on water relations of Populus deltoides growing in ambient and elevated
CO2. Tree Physiol. 2010;30:866–75.
Bohler S, Sergeant K, Jolivet Y, Hoffmann L, Hausman JF, Dizengremel P, et al. A physiological
and proteomic study of poplar leaves during ozone exposure combined with mild drought.
Proteomics. 2013;13:1737–54.
Brosche M, Merilo E, Mayer F, Pechter P, Puzorjova I, Brader G, et al. Natural variation in ozone
sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance.
Plant Cell Environ. 2010;33:914–25.
Brown JE, Lu TY, Stevens C, Khan VA, Lu JY, Wilson CL, et al. The effect of low dose ultraviolet
light-C seed treatment on induced resistance in cabbage to black rot ( Xanthomonas campestris
pv. Canpestris). Crop Prot. 2001;20:873–83.
Burgos A, Szymanski J, Seiwert B, Degenkolbe T, Hannah MA, Giavalisco P, et al. Analysis of
short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and
light. Plant J. 2011;66:656–68.
Caldwell CR. Modification of the cellular heat sensitivity of cucumber by growth under supple-
mental ultraviolet-B radiation. Plant Physiol. 1994;104:395–9.
Campo S, Peris-Peris C, Montesinos L, Penas G, Messeguer J, San Segundo B. Expression of the
maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibil-
ity to pathogen infection. J Exp Bot. 2012;63:983–99.
Casteel CL, O’Neill BF, Zavala JA, Bilgin DD, Berenbaum MR, Delucia EH. Transcriptional
profiling reveals elevated CO2 and elevated O3 alter resistance of soybean ( Glycine max) to
Japanese beetles ( Popillia japonica). Plant Cell Environ. 2008;31:419–34.
Centritto M, Brilli F, Fodale R, Loreto F. Different sensitivity of isoprene emission, respiration
and photosynthesis to high growth temperature coupled with drought stress in black poplar
( Populus nigra) saplings. Tree Physiol. 2011;31:275–86.
Chapuis R, Delluc C, Debeuf R, Tardieu F, Welcker C. Resiliences to water deficit in a phenotyp-
ing platform and in the field: how related are they in maize? Eur J Agron. 2012;42:59–67.
Chen K, Hu G, Keutgen N, Janssesns MJJ, Lenz F. Effects of NaCl salinity and CO2 enrichment
on pepino ( Solanum muricatum Ait.). II. Leaf photosynthetic properties and gas exchange. Sci
Hort. 1999;81:43–56.
Chen LS, Li P, Cheng L. Effects of high temperature coupled with high light on the balance be-
tween photooxidation and photoprotection in the sun-exposed peel of apple. Planta. 2008;228:
745–56.
Comont D, Winters A, Gwynn-Jones D. Acclimation and interaction between drought and elevated
UV-B in A. thaliana: Differences in response over treatment, recovery and reproduction. Ecol
Evol. 2012;2:2695–709.
Conrath U. Molecular aspects of defence priming. Trends Plant Sci. 2011;16:524–31.
Crafts-Brandner SJ, Salvucci ME. Rubisco activase constrains the photosynthetic potential of
leaves at high temperature and CO2. Proc Natl Acad Sci U S A. 2000;97:13430–5.
Crous KY, Zaragoza-Castells J, Ellsworth DS, Duursma RA, Low M, Tissue DT, et al. Light
inhibition of leaf respiration in field-grown Eucalyptus saligna in whole-tree chambers under
elevated atmospheric CO2 and summer drought. Plant Cell Environ. 2012;35:966–81.
Cvikrova M, Gemperlova L, Martincova O, Vankova R. Effect of drought and combined drought
and heat stress on polyamine metabolism in proline-over-producing tobacco plants. Plant
Physiol Biochem. 2013;73:7–15.
de Jong CF, Takken FL, Cai X, de Wit PJ, Joosten MH. Attenuation of Cf-mediated defense re-
sponses at elevated temperatures correlates with a decrease in elicitor-binding sites. Mol Plant
Microbe Interact. 2002;15:1040–9.
de Silva ND, Cholewa E, Ryser P. Effects of combined drought and heavy metal stresses on xylem
structure and hydraulic conductivity in red maple ( Acer rubrum L.). J Exp Bot. 2012;63:5
957–66.
del Amor FM, Cuadra-Crespo P, Walker DJ, Camara JM, Madrid R. Effect of foliar application of
antitranspirant on photosynthesis and water relations of pepper plants under different levels of
CO2 and water stress. J Plant Physiol. 2010;167:1232–8.
16 R. Mahalingam
del Carmen Martinez-Ballesta M, Bastias E, Carvajal M. Combined effect of boron and salinity on
water transport: the role of aquaporins. Plant Signal Behav. 2008;3:739–48.
Delatorre J, Pinto M, Cardemil L. Effects of water stress and high temperature on photosynthetic
rates of two species of Prosopis. J Photochem Photobiol B. 2008;92:67–76.
Demmig B, Winter K, Kruger A, Czygan FC. Zeaxanthin and the heat dissipation of excess light
energy in Nerium oleander exposed to a combination of high light and water stress. Plant
Physiol. 1988;87:17–24.
Diaz M, de Haro V, Munoz R, Quiles MJ. Chlororespiration is involved in the adaptation of Bras-
sica plants to heat and high light intensity. Plant Cell Environ. 2007;30:1578–85.
Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery JR, et al. Widespread dynamic
DNA methylation in response to biotic stress. Proc Natl Acad Sci U S A. 2012;109:E2183–91.
Duursma RA, Barton CV, Eamus D, Medlyn BE, Ellsworth DS, Forster MA, et al. Rooting depth
explains [CO2] x drought interaction in Eucalyptus saligna. Tree Physiol. 2011;31:922–31.
Estavillo GM, Crisp PA, Pornsiriwong W, Wirtz M, Collinge D, Carrie C, et al. Evidence for a
SAL1-PAP chloroplast retrograde pathway that functions in drought and high light signaling in
Arabidopsis. Plant Cell. 2011;23:3992–4012.
Feng H, Li S, Xue L, An L, Wang X. The interactive effects of enhanced UV-B radiation and soil
drought on spring wheat. S Afr J Bot. 2007;73:1334–44.
Fontaine V, Cabane M, Dizengremel P. Regulation of phosphoenolpyruvate carboxylase in Pinus
halepensis needles submitted to ozone and water stress. Physiol Plant. 2003;117:445–52.
Furbank RT, Tester M. Phenomics-technologies to relieve the phenotyping bottleneck. Trends
Plant Sci. 2011;16:635–44.
Garcia-Sanchez F, Syvertsen JP, Martinez V, Melgar JC. Salinity tolerance of “Valencia” orange
trees on rootstocks with contrasting salt tolerance is not improved by moderate shade. J Exp
Bot. 2006;57:3697–706.
Garrett KA, Dendy SP, Frank EE, Rouse MN, Travers SE. Climate change effects on plant disease:
genomes to ecosystems. Annu Rev Phytopathol. 2006;44:489–509.
Geissler N, Hussin S, Koyro HW. Elevated atmospheric CO2 concentration ameliorates ef-
fects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L. J Exp Bot.
2009;60:137–51.
Geissler N, Hussin S, Koyro HW. Elevated atmospheric CO2 concentration enhances salinity tol-
erance in Aster tripolium L. Planta. 2010;231:583–94.
Georgieva K, Sarvari E, Keresztes A. Protection of thylakoids against combined light and
drought by a lumenal substance in the resurrection plant Haberlea rhodopensis. Ann Bot.
2010;105:117–26.
Germ M, Kreft I, Stibilj V, Urbanc-Bercic O. Combined effects of selenium and drought on pho-
tosynthesis and mitochondrial respiration in potato. Plant Physiol Biochem. 2007;45:162–7.
Gillespie KM, Xu F, Richter KT, McGrath JM, Markelz RJ, Ort DR, et al. Greater antioxidant and
respiratory metabolism in field-grown soybean exposed to elevated O3 under both ambient and
elevated CO2. Plant Cell Environ. 2012;35:169–84.
Giraud E, Ho LH, Clifton R, Carroll A, Estavillo G, Tan YF, et al. The absence of ALTERNATIVE
OXIDASE1a in Arabidopsis results in acute sensitivity to combined light and drought stress.
Plant Physiol. 2008;147:595–610.
Grantz DA, Gunn S, Vu HB. O3 impacts on plant development: a meta-analysis of root/shoot al-
location and growth. Plant Cell Environ. 2006;29:1193–209.
Grattan SR, Maas EV. Effect of salinity on leaf P accumulation and injury in soybean. I. Influence
of varying CaCl2/NaCl. Plant Soil. 1988;109:65–71.
Greer DH, Weedon MM. Interactions between light and growing season temperatures on, growth
and development and gas exchange of Semillon ( Vitis vinifera L.) vines grown in an irrigated
vineyard. Plant Physiol Biochem. 2012;54:59–69.
Gunasekera TS, Paul ND, Ayres PG. The effects of ultraviolet-B (UV-B: 290-320 nm) radiation
on blister blight disease of tea ( Camellia sinensis). Plant Pathol (Oxford). 1997;46:179–85.
Haghjou MM, Shariati M, Smirnoff N. The effect of acute high light and low temperature stresses
on the ascorbate-glutathione cycle and superoxide dismutase activity in two Dunaliella salina
strains. Physiol Plant. 2009;135:272–80.
Han Y, Wang L, Zhang X, Korpelainen H, Li C. Sexual differences in photosynthetic activity, ul-

trastructure and phytoremediation potential of Populus cathayana exposed to lead and drought.
Tree Physiol. 2013;33:1043–60.
Hao D, Cheng H, Yin Z, Cui S, Zhang D, Wang H, et al. Identification of single nucleotide poly-
morphisms and haplotypes associated with yield and yield components in soybean ( Glycine
max) landraces across multiple environments. Theor Appl Genet. 2012;124:447–58.
Hartikainen K, Nerg AM, Kivimaenpaa M, Kontunen-Soppela S, Maenpaa M, Oksanen E, et al.
Emissions of volatile organic compounds and leaf structural characteristics of European aspen
( Populus tremula) grown under elevated ozone and temperature. Tree Physiol. 2009;29:1163–
73.
Herbinger K, Tausz M, Wonisch A, Soja G, Sorger A, Grill D. Complex interactive effects of
drought and ozone stress on the antioxidant defence systems of two wheat cultivars. Plant
Physiol Biochem. 2002;40:691–6.
Hewezi T, Leger M, Gentzbittel L. A comprehensive analysis of the combined effects of high light
and high temperature stresses on gene expression in sunflower. Ann Bot. 2008;102:127–40.
Honsdorf N, March TJ, Berger B, Tester M, Pillen K. High-throughput phenotyping to detect
drought tolerance QTL in wild barley introgression lines. PLoS ONE. 2014;9:e97047.
Huang X, Han B. Natural variations and genome-wide association studies in crop plants. Annu
Rev Plant Biol. 2014;65:531–51.
Iyer NJ, Tang Y, Mahalingam R. Physiological, biochemical and molecular responses to a com-
bination of drought and ozone in Medicago truncatula. Plant Cell Environ. 2013;36:706–20.
Jambunathan N, Puckette M, Mahalingam R. Transcriptomic analysis of combined stresses in
plants. In: Jain M, Brar DS, editors. Molecular techniques for crop improvement. New York:
Springer; 2010. pp. 511–24.
Jansen MA, Martret BL, Koornneef M. Variations in constitutive and inducible UV-B toler-
ance; dissecting photosystem II protection in Arabidopsis thaliana accessions. Physiol Plant.
2010;138:22–34.
Johnson SM, Lim FL, Finkler A, Fromm H, Slabas AR, Knight MR. Transcriptomic analysis of Sor-
ghum bicolor responding to combined heat and drought stress. BMC Genomics. 2014;15:456.
Joshi RK, Nayak S. Gene pyramiding—a broad spectrum technique for developing durable stress
resistance in crops. Biotech Mol Biol. 2010;5:51–60.
Kakani VG, Vu JC, Allen LH Jr, Boote KJ. Leaf photosynthesis and carbohydrates of CO(2)-
enriched maize and grain sorghum exposed to a short period of soil water deficit during vegeta-
tive development. J Plant Physiol. 2011;168:2169–76.
Kanani H, Dutta B, Klapa MI. Individual vs. combinatorial effect of elevated CO2 conditions and
salinity stress on Arabidopsis thaliana liquid cultures: comparing the early molecular response
using time-series transcriptomic and metabolomic analyses. BMC Syst Biol. 2010;4:177.
Kandianis CB, Michenfelder AS, Simmons SJ, Grusak MA, Stapleton AE. Abiotic stress growth
conditions induce different responses in kernel iron concentration across genotypically distinct
maize inbred varieties. Front Plant Sci. 2013;4:488.
Karlsson PE, Medin EL, Wallin G, Sellden G, Skarby L. Effects of ozone and drought stress
on the physiology and growth of two clones of Norway spruce ( Picea abies). New Phytol.
1997;136:265–75.
Karni L, Aloni B. Fructokinase and hexokinase from pollen grains of bell pepper ( Capsicum an-
nuum L.): possible role in pollen germination under conditions of high temperature and CO2
enrichment. Ann Bot. 2002;90:607–12.
Kasurinen A, Biasi C, Holopainen T, Rousi M, Maenpaa M, Oksanen E. Interactive effects of el-
evated ozone and temperature on carbon allocation of silver birch ( Betula pendula) genotypes
in an open-air field exposure. Tree Physiol. 2012;32:737–51.
Kaya C, Kirnak H, Higgs D. The effects of high salinity (NaCl) and supplementary phosphorus
and potassium on physiology and nutrition development of Spinach. Bulg J Plant Physiol.
2001;27:47–59.
Keles Y, Oncel I. Response of antioxidative defence system to temperature and water stress com-
binations in wheat seedlings. Plant Sci. 2002;163:783–90.
18 R. Mahalingam
Kets K, Darbah JN, Sober A, Riikonen J, Sober J, Karnosky DF. Diurnal changes in photosynthetic
parameters of Populus tremuloides, modulated by elevated concentrations of CO2 and/or O3
and daily climatic variation. Environ Pollut. 2010;158:1000–7.
Kissoudis C, van de Wiel C, Visser RG, van der Linden G. Enhancing crop resilience to combined
abiotic and biotic stress through the dissection of physiological and molecular crosstalk. Front
Plant Sci. 2014;5:207.
Kivimaenpaa M, Sutinen S, Karlsson PE, Sellden G. Cell structural changes in the needles of Nor-
way spruce exposed to long-term ozone and drought. Ann Bot. 2003;92:779–93.
Kleier C, Farnsworth B, Winner W. Photosynthesis and biomass allocation of radish cv. “Cherry
Belle” in response to root temperature and ozone. Environ Pollut. 2001;111:127–33.
Kornyeyev D, Logan BA, Payton P, Allen RD, Holaday AS. Enhanced photochemical light utiliza-
tion and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing
genes encoding chloroplast-targeted antioxidant enzymes. Physiol Plant. 2001;113:323–31.
Koti S, Reddy KR, Reddy VR, Kakani VG, Zhao D. Interactive effects of carbon dioxide, tempera-
ture, and ultraviolet-B radiation on soybean ( Glycine max L.) flower and pollen morphology,
pollen production, germination, and tube lengths. J Exp Bot. 2005;56:725–36.
Koussevitzky S, Suzuki N, Huntington S, Armijo L, Sha W, Cortes D, et al. Ascorbate peroxidase
1 plays a key role in the response of Arabidopsis thaliana to stress combination. J Biol Chem.
2008;283:34197–203.
Kozlowska-Szerenos B, Bialuk I, Maleszewski S. Enhancement of photosynthetic O2 evolution in
Chlorella vulgaris under high light and increased CO2 concentration as a sign of acclimation
to phosphate deficiency. Plant Physiol Biochem. 2004;42:403–9.
Kunz BA, Cahill DM, Mohr PG, Osmond MJ, Vonarx EJ. Plant responses to UV radiation and
links to pathogen resistance. Int Rev Cytol. 2006;255:1–40.
Labidi N, Ammari M, Snoussi S, Messelini N, Gharbi F, Abdelly C. Stimulated growth rate by
restriction of P availability at moderate salinity but insensitive to P availability at high salinity
in Crithmum maritimum. Acta Biol Hung. 2011;62:302–15.
Lam HM, Xu X, Liu X, Chen W, Yang G, Wong FL, et al. Resequencing of 31 wild and cul-
tivated soybean genomes identifies patterns of genetic diversity and selection. Nat Genet.
2010;42:1053–9.
Lavola A, Nybakken L, Rousi M, Pusenius J, Petrelius M, Kellomaki S, et al. Combination treat-
ment of elevated UVB radiation, CO and temperature has little effect on silver birch ( Betula
pendula) growth and phytochemistry. Physiol Plant. 2013.
Leakey AD, Uribelarrea M, Ainsworth EA, Naidu SL, Rogers A, Ort DR, et al. Photosynthesis,
productivity, and yield of maize are not affected by open-air elevation of CO2 concentration in
the absence of drought. Plant Physiol. 2006;140:779–90.
Lewis JD, Smith RA, Ghannoum O, Logan BA, Phillips NG, Tissue DT. Industrial-age changes in
atmospheric [CO2] and temperature differentially alter responses of faster- and slower-grow-
ing eucalyptus seedlings to short-term drought. Tree Physiol. 2013;33:475–88.
Li P, Bohnert HJ, Grene R. All about FACE-plants in a high-[CO2] world. Trends Plant Sci.
2007;12:87–9.
Li QM, Liu BB, Wu Y, Zou ZR. Interactive effects of drought stresses and elevated CO2 concen-
tration on photochemistry efficiency of cucumber seedlings. J Integr Plant Biol. 2008;50:1307–
17.
Li H, Peng Z, Yang X, Wang W, Fu J, Wang J, et al. Genome-wide association study dissects the
genetic architecture of oil biosynthesis in maize kernels. Nat Genet. 2013;45:43–50.
Li JY, Wang J, Zeigler RS. The 3,000 rice genomes project: new opportunities and challenges for
future rice research. Gigascience. 2014a;3:8.
Li X, Ahammed GJ, Zhang YQ, Zhang GQ, Sun ZH, Zhou J, et al. Carbon dioxide enrichment
alleviates heat stress by improving cellular redox homeostasis through an ABA-independent
process in tomato plants. Plant Biol (Stuttgart). 2014b.
Liu X, Wu H, Ji C, Wei L, Zhao J, Yu J. An integrated proteomic and metabolomic study on the
chronic effects of mercury in Suaeda salsa under an environmentally relevant salinity. PLoS
ONE. 2013;8:e64041.
Lokhande SD, Ogawa K, Tanaka A, Hara T. Effect of temperature on ascorbate peroxidase activ-
ity and flowering of Arabidopsis thaliana ecotypes under different light conditions. J Plant
Physiol. 2003;160:57–64.
Long SP, Ainsworth EA, Leakey AD, Morgan PB. Global food insecurity. treatment of major food
crops with elevated carbon dioxide or ozone under large-scale fully open-air conditions sug-
gests recent models may have overestimated future yields. Philos Trans R Soc Lond B Biol
Sci. 2005;360:2011–20.
Long SP, Ainsworth EA, Leakey AD, Nosberger J, Ort DR. Food for thought: lower-than-expected
crop yield stimulation with rising CO2 concentrations. Science. 2006;312:1918–21.
Loreto F, Barta C, Brilli F, Nogues I. On the induction of volatile organic compound emissions by
plants as consequence of wounding or fluctuations of light and temperature. Plant Cell Envi-
ron. 2006;29:1820–8.
Loveys BR, Egerton JJ, Ball MC. Higher daytime leaf temperatures contribute to lower freeze
tolerance under elevated CO2. Plant Cell Environ. 2006;29:1077–86.
Lu Y. Intraspecific variation in drought response of Populus cathayana growth under ambient and
enhanced UV-B radiation. Ann For Sci. 2009;66:613–24.
Luedemann G, Matyssek R, Fleischmann F, Grams TE. Acclimation to ozone affects host/patho-
gen interaction and competitiveness for nitrogen in juvenile Fagus sylvatica and Picea abies
trees infected with Phytophthora citricola. Plant Biology (Stuttgart). 2005;7:640–9.
Madan P, Jagadish SV, Craufurd PQ, Fitzgerald M, Lafarge T, Wheeler TR. Effect of elevated
CO2 and high temperature on seed-set and grain quality of rice. J Exp Bot. 2012;63:3843–52.
Maenpaa M, Riikonen J, Kontunen-Soppela S, Rousi M, Oksanen E. Vertical profiles reveal im-
pact of ozone and temperature on carbon assimilation of Betula pendula and Populus tremula.
Tree Physiol. 2011;31:808–18.
Maggio A, Chiaranda FQ, Cefariello R, Fagnano M. Responses to ozone pollution of alfalfa ex-
posed to increasing salinity levels. Environ Pollut. 2009;157:1445–52.
Mahoney SR, Ghosh S, Peirson D, Dumbroff EB. Paclobutrazol affects the resistance of black
spruce to high light and thermal stress. Tree Physiol. 1998;18:121–7.
Makumburage GB, Stapleton AE. Phenotype uniformity in combined-stress environments has a
different genetic architecture than in single-stress treatments. Front Plant Sci. 2011;2:12.
Makumburage GB, Richbourg HL, LaTorre KD, Capps A, Chen C, Stapleton AE. Genotype to
phenotype maps: multiple input abiotic signals combine to produce growth effects via attenuat-
ing signaling interactions in maize. G3 (Bethesda). 2013;3:2195–204.
Malthus T. An essay on the principle of population. London: Murray; 1817.
Manes F, Donato E, Vitale M. Physiological response of Pinus halepensis needles under ozone and
water stress conditions. Physiol Plant. 2001;113:249–57.
Masojidek J, Trivedi S, Halshaw L, Alexiou A, Hall DO. The synergistic effect of drought and light
stresses in sorghum and pearl millet. Plant Physiol. 1991;96:198–207.
Mateos-Naranjo E, Redondo-Gomez S, Andrades-Moreno L, Davy AJ. Growth and photosynthetic
responses of the cordgrass Spartina maritima to CO2 enrichment and salinity. Chemosphere.
2010a;81:725–31.
Mateos-Naranjo E, Redondo-Gomez S, Alvarez R, Cambrolle J, Gandullo J, Figueroa ME. Syn-
ergic effect of salinity and CO2 enrichment on growth and photosynthetic responses of the
invasive cordgrass Spartina densiflora. J Exp Bot. 2010b;61:1643–54.
Mavrogianopoulos GN, Spanakin J, Tsikalas P. Effect of carbon dioxide enrichment and salinity
on photosynthesis and yield in melon. Sci Hortic. 1999;79:51–63.
McKee IF, Long SP. Plant growth regulators control ozone damage to wheat yield. New Phytol.
2001;152:41–51.
Medeiros JS, Ward JK. Increasing atmospheric [CO2] from glacial to future concentrations affects
drought tolerance via impacts on leaves, xylem and their integrated function. New Phytol.
2013;199:738–48.
Melgar JC, Syvertsen JP, Garcia-Sanchez F. Can elevated CO(2) improve salt tolerance in olive
trees? J Plant Physiol. 2008;165:631–40.
20 R. Mahalingam
Mirouze M, Paszkowski J. Epigenetic contribution to stress adaptation in plants. Curr Opin Plant
Biol. 2011;14:267–74.
Mishra AK, Rai R, Agrawal SB. Individual and interactive effects of elevated carbon dioxide and
ozone on tropical wheat ( Triticum aestivum L.) cultivars with special emphasis on ROS genera-
tion and activation of antioxidant defence system. Indian J Biochem Biophys. 2013;50:139–49.
Mittler R. Abiotic stress, the field environment and stress combination. Trends Plant Sci.
2006;11:15–9.
Mittler R, Blumwald E. Genetic engineering for modern agriculture: challenges and perspectives.
Annu Rev Plant Biol. 2010;61:443–62.
Monneveux P, Pastenes C, Reynolds MP. Limitations to photosynthesis under light and heat stress
in three high-yielding wheat genotypes. J Plant Physiol. 2003;160:657–66.
Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651–81.
Munoz R, Quiles MJ. Water deficit and heat affect the tolerance to high illumination in hibiscus
plants. Int J Mol Sci. 2013;14:5432–44.
Nanasato Y, Akashi K, Yokota A. Co-expression of cytochrome b561 and ascorbate oxidase
in leaves of wild watermelon under drought and high light conditions. Plant Cell Physiol.
2005;46:1515–24.
Niziolek OK, Berenbaum MR, Delucia EH. Impact of elevated CO2 and increased temperature on
Japanese beetle herbivory. Insect Sci. 2013;20:513–23.
Nogues S, Allen DJ, Morison JI, Baker NR. Ultraviolet-B radiation effects on water relations, leaf
development, and photosynthesis in droughted pea plants. Plant Physiol. 1998;117:173–81.
Nunn AJ, Wieser G, Metzger U, Low M, Wipfler P, Haberle KH, et al. Exemplifying whole-plant
ozone uptake in adult forest trees of contrasting species and site conditions. Environ Pollut.
2007;146:629–39.
Oliveira VF, Silva EA, Zaidan LB, Carvalho MA. Effects of elevated CO2 concentration and
water deficit on fructan metabolism in Viguiera discolor Baker. Plant Biology (Stuttgart).
2013;15:471–82.
Paakkonen E, Vahala J, Pohjola M, Holopainen T, Karenlampi L. Physiological, stomatal and
ultrastructural ozone responses in birch ( Betula pendula Roth) are modified by water stress.
Paoletti E, Grulke NE. Ozone exposure and stomatal sluggishness in different plant physiognomic
classes. Environ Pollut. 2010;158:2664–71.
Paredes M, Quiles MJ. Stimulation of chlororespiration by drought under heat and high illumina-
tion in Rosa meillandina. J Plant Physiol. 2013;170:165–71.
Payton P, Webb R, Kornyeyev D, Allen R, Holaday AS. Protecting cotton photosynthesis during
moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activ-
ity. J Exp Bot. 2001;52:2345–54.
Perez-Lopez U, Robredo A, Lacuesta M, Sgherri C, Munoz-Rueda A, Navari-Izzo F, et al. The
oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2. Physiol
Plant. 2009;135:29–42.
Perez-Lopez U, Robredo A, Lacuesta M, Mena-Petite A, Munoz-Rueda A. Elevated CO2 reduces
stomatal and metabolic limitations on photosynthesis caused by salinity in Hordeum vulgare.
Photosynth Res. 2012;111:269–83.
Peters G, Marland G, Le Quere C, Boden T, Canadell J, Raupach M. Rapid growth of CO2 emis-
sion after the 2008–2009 global financial crisis. Nat Clim Change. 2011;318:113–6.
Picchi V, Iriti M, Quaroni S, Saracchi M, Viola P, Faoro F. Climate variations and phenological
stages modulate ozone damages in field-grown wheat. A three-year study with eight modern
cultivars in Po Valley (Northern Italy). Agric Ecosyst Environ 2010;135 310–7.
Pliura A, Baliuckiene A, Baliuckas V. Phenogenetic response of silver birch populations and
half-sib families to elevated ozone and ultraviolet-B radiation at juvenile age. Environ Pollut.
2008;156:152–61.
Poulson ME, Boeger MR, Donahue RA. Response of photosynthesis to high light and drought
for Arabidopsis thaliana grown under a UV-B enhanced light regime. Photosynth Res.
2006;90:79–90.
Prasch CM, Sonnewald U. Simultaneous application of heat, drought, and virus to Arabidopsis
plants reveals significant shifts in signaling networks. Plant Physiol. 2013;162:1849–66.
Prasad PVV, Pisipati SR, Momcilovic I, Ristic Z. Independent and combined effects of high tem-
perature and drought stress during grain filling on plant yield and chloroplast Ef-Tu expression
in spring wheat. J Agron Crop Sci. 2011;197:430–41.
Puckette MC, Weng H, Mahalingam R. Physiological and biochemical responses to acute ozone-
induced oxidative stress in Medicago truncatula. Plant Physiol Biochem. 2007;45:70–9.
Quiles MJ. Stimulation of chlororespiration by heat and high light intensity in oat plants. Plant Cell
Environ. 2006;29:1463–70.
Rahman MS, Akter S, Al-Amin M. Temperature and saline stress on seedlings of Swietenia mac-
rophylla: a comparative study. Pak J Biol Sci. 2013;16:1765–70.
Ramegowda V, Senthil-Kumar M, Ishiga Y, Kaundal A, Udayakumar M, Mysore KS. Drought
stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in
Nicotiana benthamiana. Int J Mol Sci. 2013;14:9497–513.
Rampino P, Mita G, Fasano P, Borrelli GM, Aprile A, Dalessandro G, et al. Novel durum wheat
genes up-regulated in response to a combination of heat and drought stress. Plant Physiol Bio-
chem. 2012;56:72–8.
Rasmussen S, Barah P, Suarez-Rodriguez MC, Bressendorff S, Friis P, Costantino P, et al. Tran-
scriptome responses to combinations of stresses in Arabidopsis. Plant Physiol. 2013;161:
1783–94.
Riikonen J, Maenpaa M, Alavillamo M, Silfver T, Oksanen E. Interactive effect of elevated tem-
perature and O3 on antioxidant capacity and gas exchange in Betula pendula saplings. Planta.
2009;230:419–27.
Riikonen J, Kontunen-Soppela S, Ossipov V, Tervahauta A, Tuomainen M, Oksanen E, et al. Nee-
dle metabolome, freezing tolerance and gas exchange in Norway spruce seedlings exposed to
elevated temperature and ozone concentration. Tree Physiol. 2012;32:1102–12.
Riikonen J, Kontunen-Soppela S, Vapaavuori E, Tervahauta A, Tuomainen M, Oksanen E. Carbo-
hydrate concentrations and freezing stress resistance of silver birch buds grown under elevated
temperature and ozone. Tree Physiol. 2013;33:311–9.
Rivero RM, Mestre TC, Mittler R, Rubio F, Garcia-Sanchez F, Martinez V. The combined effect
of salinity and heat reveals a specific physiological, biochemical and molecular response in
tomato plants. Plant Cell and Environ. 2014;37:1059–73.
Rizhsky L, Liang HJ, Mittler R. The combined effect of drought stress and heat shock on gene
expression in tobacco. Plant Physiol. 2002;130:1143–51.
Rizhsky L, Liang HJ, Shuman J, Shulaev V, Davletova S, Mittler R. When defense pathways col-
lide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol.
2004;134:1683–96.
Roden JS, Ball MC. The effect of elevated [CO2] on growth and photosynthesis of two Eucalyptus
species exposed to high temperatures and water deficits. Plant Physiol. 1996;111:909–19.
Rohrs-Richey JK, Mulder CP, Winton LM, Stanosz G. Physiological performance of an Alaskan
shrub ( Alnus fruticosa) in response to disease ( Valsa melanodiscus) and water stress. New
Phytol. 2011;189:295–307.
Rollins JA, Habte E, Templer SE, Colby T, Schmidt J, von Korff M. Leaf proteome alterations in
the context of physiological and morphological responses to drought and heat stress in barley
( Hordeum vulgare L.). J Exp Bot. 2013;64:3201–12.
Sainz M, Diaz P, Monza J, Borsani O. Heat stress results in loss of chloroplast Cu/Zn superoxide
dismutase and increased damage to photosystem II in combined drought-heat stressed Lotus
japonicus. Physiol Plant. 2010;140:46–56.
Sakurai G, Iizumi T, Nishimori M, Yokozawa M. How much has the increase in atmospheric CO2
directly affected past soybean production? Sci Rep. 2014;4:4978.
Sales CR, Ribeiro RV, Silveira JA, Machado EC, Martins MO, Lagoa AM. Superoxide dismutase
and ascorbate peroxidase improve the recovery of photosynthesis in sugarcane plants subjected
to water deficit and low substrate temperature. Plant Physiol Biochem. 2013;73:326–36.
22 R. Mahalingam
Sardans J, Penuelas J. Drought changes the dynamics of trace element accumulation in a Mediter-
ranean Quercus ilex forest. Environ Pollut. 2007;147:567–83.
Sawada H, Kohno Y. Differential ozone sensitivity of rice cultivars as indicated by visible injury
and grain yield. Plant Biol (Stuttgart). 2009;11(Suppl 1):70–5.
Schmidt A-M, Ormrod DP, Livingston NJ, Misra S. The interaction of ultraviolet-B radiation and
water deficit in two Arabidopsis thaliana genotypes. Ann Bot. 2000;85:571–5.
Schnurbusch T, Hayes J, Hrmova M, Baumann U, Ramesh SA, Tyerman SD, et al. Boron toxicity
tolerance in barley through reduced expression of the multifunctional aquaporin HvNIP2;1.
Plant Physiol. 2010;153:1706–15.
Schwanz P, Picon C, Vivin P, Dreyer E, Guehl JM, Polle A. Responses of antioxidative systems
to drought stress in pendunculate oak and maritime pine as modulated by elevated CO2. Plant
Physiol. 1996;110:393–402.
Shaik R, Ramakrishna W. Genes and co-expression modules common to drought and bacterial
stress responses in Arabidopsis and rice. PLoS ONE. 2013;8:e77261.
Shaik R, Ramakrishna W. Machine learning approaches distinguish multiple stress conditions us-
ing stress-responsive genes and identify candidate genes for broad resistance in rice. Plant
Physiol. 2014;164:481–95.
Sharma PK, Singhal GS. Effect of water stress on primary photosynthetic process: interaction with
light and temperature. Indian J Biochem Biophys. 1993;30:10–4.
Shukla UC, Joshi PC, Kakkar P. Synergistic action of ultraviolet-B radiation and cadmium on the
growth of wheat seedlings. Ecotoxicol Environ Safety. 2002;51:90–6.
Shukla UC, Murthy RC, Kakkar P. Combined effect of ultraviolet-B radiation and cadmium con-
tamination on nutrient uptake and photosynthetic pigments in Brassica campestris L. seed-
lings. Environ Toxicol. 2008;23:712–9.
Sicher R. Combined effects of CO2 enrichment and elevated growth temperatures on metabo-
lites in soybean leaflets: evidence for dynamic changes of TCA cycle intermediates. Planta.
2013;238:369–80.
Sicher RC, Barnaby JY. Impact of carbon dioxide enrichment on the responses of maize leaf tran-
scripts and metabolites to water stress. Physiol Plant. 2012;144:238–53.
Silberbush M, Ben-Asher J. The effect of NaCl concentration on NO3, K and orthophosphate-P
influx to peanut roots. Sci Hort. 1989;39:279–87.
Silva EN, Ferreira-Silva SL, Fontenele Ade V, Ribeiro RV, Viegas RA, Silveira JA. Photosynthetic
changes and protective mechanisms against oxidative damage subjected to isolated and com-
bined drought and heat stresses in Jatropha curcas plants. J Plant Physiol. 2010;167:1157–64.
Simon-Sarkadi L, Kocsy G, Varhegyi A, Galiba G, de Ronde JA. Genetic manipulation of proline
accumulation influences the concentrations of other amino acids in soybean subjected to simul-
taneous drought and heat stress. J Agric Food Chem. 2005;53:7512–7.
Singh SK, Kakani VG, Surabhi GK, Reddy KR. Cowpea ( Vigna unguiculata [L.] Walp.) geno-
types response to multiple abiotic stresses. J Photochem Photobiol B. 2010;100:135–46.
Sirault XRR, James RA, Furbank RT. A new screening method for osmotic component of salinity
tolerance in cereals using infrared thermography. Funct Plant Biol. 2009;36:970–7.
Slama I, Ghnaya T, Savoure A, Abdelly C. Combined effects of long-term salinity and soil drying
on growth, water relations, nutrient status and proline accumulation of Sesuvium portulacas-
trum. C R Biol. 2008;331:442–51.
Smith AB, Katz BW. U.S. billion-dollar weather and climate disasters: data sources, trends, ac-
curacy and biases. Nat Hazards. 2013;67:387–410.
Song Q, Hyten DL, Jia G, Quigley CV, Fickus EW, Nelson RL, et al. Development and evaluation
of SoySNP50K, a high-density genotyping array for soybean. PLoS ONE. 2013;8:e54985.
Song Y, Yu J, Huang B. Elevated CO2-mitigation of high temperature stress associated with main-
tenance of positive carbon balance and carbohydrate accumulation in Kentucky bluegrass.
PLoS ONE. 2014;9:e89725.
Srivastava G, Kumar S, Dubey G, Mishra V, Prasad SM. Nickel and ultraviolet-B stresses induce
differential growth and photosynthetic responses in Pisum sativum L. seedlings. Biol Trace
Elem Res. 2012;149:86–96.
Sullivan JH, Teramura AH. Field study of the interaction between solar ultraviolet-B radiation and
drought on photosynthesis and growth in soybean. Plant Physiol. 1990;92:141–6.
Sun Z, Huve K, Vislap V, Niinemets U. Elevated [CO2] magnifies isoprene emissions under heat
and improves thermal resistance in hybrid aspen. J Exp Bot. 2013;64:5509–23.
Suzuki N, Rivero RM, Shulaev V, Blumwald E, Mittler R. Abiotic and biotic stress combinations.
New Phytol. 2014;203:32–43.
Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, Lakatos L, et al. Low temperature inhib-
its RNA silencing-mediated defence by the control of siRNA generation. EMBO J. 2003;22:
633–40.
Szucs A, Jager K, Jurca ME, Fabian A, Bottka S, Zvara A, et al. Histological and microarray analy-
sis of the direct effect of water shortage alone or combined with heat on early grain develop-
ment in wheat ( Triticum aestivum). Physiol Plant. 2010;140:174–88.
Takagi M, El-Shemy HA, Sasaki S, Toyama S, Kanai S, Saneoka H, et al. Elevated CO2 concentra-
tion alleviates salinity stress in tomato plant. Acta Agric Scand B Soil Plant Sci. 2009;59:87–96.
Talbi Zribi O, Abdelly C, Debez A. Interactive effects of salinity and phosphorus availability on
growth, water relations, nutritional status and photosynthetic activity of barley ( Hordeum vul-
gare L.). Plant Biol (Stuttgart). 2011;13:872–80.
Talbi Zribi O, Labidi N, Slama I, Debez A, Ksouri R, Rabhi M, et al. Alleviation of phosphorus
deficiency stress by moderate salinity in the halophyte Hrodeum maritimum L. Plant Growth
Regul. 2012;66:75–85.
Tripathi R, Agrawal SB. Evaluation of changes in lipid peroxidation, ROS production, surface
structures, secondary metabolites and yield of linseed ( Linum usitatissimum L.) under individ-
ual and combined stress of ultraviolet-B and ozone using open top chambers. Indian J Biochem
Biophys. 2013a;50:318–25.
Tripathi R, Agrawal SB. Interactive effect of supplemental ultraviolet B and elevated ozone on
seed yield and oil quality of two cultivars of linseed ( Linum usitatissimum L.) carried out in
open top chambers. J Sci Food Agric. 2013b;93:1016–25.
Turtola S. Genotypic variation in drought response of willows grown under ambient and enahnced
UV-B radiation. Environ Exp Bot. 2006;52:80–6.
van de Staaij JW, Tonneijck AE, Rozema J. The effect of reciprocal treatments with ozone and
ultraviolet-B radiation on photosynthesis and growth of perennial grass Elymus athericus. En-
viron Pollut. 1997;97:281–6.
Van Vuuren DP, Meinshausen M, Plattner GK, Joos F, Strassmann KM, Smith SJ, et al. Tem-
perature increase of 21st century mitigation scenarios. Proc Natl Acad Sci U S A. 2008;105:
15258–62.
Vasseur F, Pantin F, Vile D. Changes in light intensity reveal a major role for carbon balance in
Arabidopsis responses to high temperature. Plant Cell Environ. 2011;34:1563–76.
Vile D, Pervent M, Belluau M, Vasseur F, Bresson J, Muller B, et al. Arabidopsis growth under
prolonged high temperature and water deficit: independent or interactive effects? Plant Cell
Environ. 2012;35:702–18.
Vollsnes AV, Eriksen AB, Otterholt E, Kvaal K, Oxaal U, Futsaether CM. Visible foliar injury and
infrared imaging show that daylength affects short-term recovery after ozone stress in Trifo-
lium subterraneum. J Exp Bot. 2009;60:3677–86.
Wagg S, Mills G, Hayes F, Wilkinson S, Cooper D, Davies WJ. Reduced soil water availability
did not protect two competing grassland species from the negative effects of increasing back-
ground ozone. Environ Pollut. 2012;165:91–9.
Wang N, Fang W, Han H, Sui N, Li B, Meng QW. Overexpression of zeaxanthin epoxidase gene
enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress. Physiol
Plant. 2008;132:384–96.
Wei J, Li C, Li Y, Jiang G, Cheng G, Zheng Y. Effects of external potassium (k) supply on drought
tolerances of two contrasting winter wheat cultivars. PLoS ONE. 2013;8:e69737.
Weigel D, Mott R. The 1001 genomes project for Arabidopsis thaliana. Genome Biol. 2009;10:107.
Welfare K, Flowers TJ, Taylor G, Yeo AR. Additive and antagonistic effects of ozone and salinity
on the growth, ion contents and gas exchange of five varieties of rice ( Oryza sativa L.). Envi-
ron Pollut. 1996;92:257–66.
24 R. Mahalingam
Welfare K, Yeo AR, Flowers TJ. Effects of salinity and ozone, individually and in combination,
on the growth and ion contents of two chickpea ( Cicer arietinum L.) varieties. Environ Pollut.
2002;120:397–403.
Wen X, Qiu N, Lu Q, Lu C. Enhanced thermotolerance of photosystem II in salt-adapted plants of
the halophyte Artemisia anethifolia. Planta. 2005;220:486–97.
Wilkinson S, Mills G, Illidge R, Davies WJ. How is ozone pollution reducing our food supply? J
Exp Bot. 2012;63:527–36.
Wolfe MD, Tonsor SJ. Adaptation to spring heat and drought in northeastern Spanish Arabidopsis
thaliana. New Phytol. 2014;201:323–34.
Woo NS, Badger MR, Pogson BJ. A rapid, non-invasive procedure for quantitative assessment of
drought survival using chlorophyll fluorescence. Plant Methods. 2008;4:27.
Xiong L, Yang Y. Disease resistance and abiotic stress tolerance in rice are inversely modulated
by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell. 2003;15:745–59.
Xu P, Chen F, Mannas JP, Feldman T, Sumner LW, Roossinck MJ. Virus infection improves
drought tolerance. New Phytol. 2008;180:911–21.
Yalpani N, Enyedi AJ, Leon J, Raskin I. Ultraviolet-light and ozone stimulate accumula-
tion of salicylic-acid, pathogenesis-related proteins and virus-resistance in tobacco. Planta.
1994;193:372–6.
Yang S, Hua J. A haplotype-specific resistance gene regulated by BONZAI1 mediates tempera-
ture-dependent growth control in Arabidopsis. Plant Cell. 2004;16:1060–71.
Yang H, Shi Y, Liu J, Guo L, Zhang X, Yang S. A mutant CHS3 protein with TIR-NB-LRR-LIM
domains modulates growth, cell death and freezing tolerance in a temperature-dependent man-
ner in Arabidopsis. Plant J. 2010;63:283–96.
Yang F, Jorgensen AD, Li H, Sondergaard I, Finnie C, Svensson B, et al. Implications of high-
temperature events and water deficits on protein profiles in wheat ( Triticum aestivum L. cv.
Vinjett) grain. Proteomics. 2011;11:1684–95.
Yang Y, Chen J, Liu Q, Ben C, Todd CD, Shi J, et al. Comparative proteomic analysis of the ther-
motolerant plant Portulaca oleracea acclimation to combined high temperature and humidity
stress. J Proteome Res. 2012;11:3605–23.
Ye L, Fu X, Ge F. Enhanced sensitivity to higher ozone in a pathogen-resistant tobacco cultivar. J
Exp Bot. 2012;63:1341–7.
York PH, Gruber RK, Hill R, Ralph PJ, Booth DJ, Macreadie PI. Physiological and morphological
responses of the temperate seagrass Zostera muelleri to multiple stressors: investigating the
interactive effects of light and temperature. PLoS ONE. 2013;8:e76377.
Yu J, Buckler ES. Genetic association mapping and genome organization of maize. Curr Opin
Biotechnol. 2006;17:155–60.
Zeppel MJ, Lewis JD, Medlyn B, Barton CV, Duursma RA, Eamus D, et al. Interactive effects
of elevated CO2 and drought on nocturnal water fluxes in Eucalyptus saligna. Tree Physiol.
2011;31:932–44.
Zhang M, Li G, Huang W, Bi T, Chen G, Tang Z, et al. Proteomic study of Carissa spinarum in
response to combined heat and drought stress. Proteomics. 2010;10:3117–29.
Zhang D, Song H, Cheng H, Hao D, Wang H, Kan G, et al. The acid phosphatase-encoding
gene GmACP1 contributes to soybean tolerance to low-phosphorus stress. PLoS Genet.
2014;10:e1004061.
Zhao M, Running SW. Drought-induced reduction in global terrestrial net primary production
from 2000 through 2009. Science. 2010;329:940–3.
Zhao H, Zhao Z, An L, Chen T, Wang X, Feng H. The effects of enhanced ultraviolet-B radi-
ation and soil drought on water use efficiency of spring wheat. J Photochem Photobiol B.
2009;94:54–8.
Zheng YF, Zhang JE, Wu RJ, Liu RN, Zhao Z, Hu CD, et al. Combination effects of enhanced
UV-B radiation and O3 stress on photosynthetic characteristics of winter wheat. Huan Jing Ke
Xue. 2011;32:3023–32.
Zheng YH, Li X, Li YG, Miao BH, Xu H, Simmons M, et al. Contrasting responses of salinity-
stressed salt-tolerant and intolerant winter wheat ( Triticum aestivum L.) cultivars to ozone
pollution. Plant Physiol Biochem. 2012;52:169–78.
Zhou Y, Lam HM, Zhang J. Inhibition of photosynthesis and energy dissipation induced by water
and high light stresses in rice. J Exp Bot. 2007;58:1207–17.
Zhu Y, Qian W, Hua J. Temperature modulates plant defense responses through NB-LRR proteins.
PLoS Pathog. 2010;6:e1000844.
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W. GENEVESTIGATOR. Arabidopsis
microarray database and analysis toolbox. Plant Physiol. 2004;136:2621–32.
Ziska LH. Growth temperature can alter the temperature dependent stimulation of photosynthesis
by elevated carbon dioxide in Albutilon theophrasti. Physiol Plant. 2001;111:322–8.
Chapter 2
The Impact of Enhanced Atmospheric CO2
Concentrations on the Responses of Maize and
Soybean to Elevated Growth Temperatures
Richard C. Sicher and James A. Bunce
2.1 Introduction
The agricultural industry is uniquely dependent upon climate and a changing

climate has the potential to alter crop productivity and affect economic returns to
growers. Atmospheric CO2 levels have risen about 40 % since the advent of the
industrial revolution and this is largely due to fossil-fuel combustion and changes
in land management (IPCC 2007). Because atmospheric CO2 absorbs heat from the
sun, global mean temperatures, over both land and water, increased to an average
of 0.85 °C between 1880 and 2012 (IPCC 2013). Additional increases in the global
mean temperature are likely to occur during the current century and this will have
consequences for both mechanized and subsistence agriculture. The IPCC (2007)
has concluded that global mean temperatures could increase by an additional 4 °C
by the end of the current century, if mitigation measures are not enacted. Moreover,
a report by Hatfield et al. (2008) predicts that agriculture will face a more variable,
future climate with an increased frequency of extreme weather events including,
prolonged drought, intense heat waves, and episodes of drenching rains. Above op-
timal temperatures decrease both the vegetative and reproductive growth of crop
plants but this may be partially offset by greater rates of net photosynthesis due
to CO2 enrichment (Baker and Allen 1989; Boote et al. 2005). Several excellent
reviews exist that discuss the effects of heat and/or abiotic stress (Vierling 1991;
Wahid et al. 2007; Ahuja et al. 2010; Mittler et al. 2011) and of CO2 enrichment
(Kimball et al. 1993; Allen et al. 1996; Sicher and Kim 2011; Barnaby and Ziska
2012) on plants. The current chapter briefly discusses these subjects but principally
focuses on how elevated temperatures and increased atmospheric CO2 concentra-
tions interact to affect the growth and harvestable yields of important crop plants.
R. C. Sicher () · J. A. Bunce
Crop Systems and Global Change Laboratory, Beltsville Agricultural Research Center,
Room 342, Building 001, 10300 Baltimore Avenue, Beltsville, MD 20705, USA
e-mail: Richard.Sicher@ars.usda.gov

28 R. C. Sicher and J. A. Bunce
Our primary focus will be on soybean and maize but, where inadequate data are
available, results for related legumes, tropical grass species, and specific crop plants
also will be cited.
2.2 Positive Effects of CO2 Enrichment on Plants
The carbon for plant growth is derived from CO2 in the atmosphere and the light-
dependent reactions of photosynthesis. The current atmospheric CO2 concentration,
i.e., 396 μmol mol−1, does not saturate rates of photosynthesis for the majority of
terrestrial plants that possess the C3 pathway of photosynthesis (Stitt 1991). Many
important agricultural crops, including rice, cotton, potato, wheat, and soybean,
possess the C3 pathway of photosynthesis. Supra-ambient CO2 concentrations nor-
mally increase rates of photosynthesis, photoassimilate accumulation, and growth
of most terrestrial plants. The conversion of carbon dioxide into organic products
takes place in the chloroplast stroma and is catalyzed by the bifunctional enzyme,
Rubisco. The concentration of CO2 within the chloroplast is estimated to be 10 μM,
which is close to the apparent Michaelis constant ( Km) for the CO2 fixation reaction
of Rubisco. Rubisco also functions as an oxygenase, that competitively inhibits the
carboxylase activity of the enzyme, and the former reaction initiates the first step
in photorespiratory metabolism. Therefore, an increase in atmospheric CO2 concen-
tration is capable of accelerating the rate of CO2 fixation in the chloroplast by si-
multaneously enhancing the carboxylation and inhibiting the oxygenation reactions
of Rubisco (Kobza and Edwards 1987). Stitt (1991) has argued that increasing the
atmospheric CO2 concentration from 396 to 700 μmol mol−1 should accelerate the
net rate of photosynthesis of C3 plants by 25–75 %.
Other plants, including maize, sorghum, and sugar cane, are dependent upon a
second carboxylase enzyme, i.e., phospho(enol) pyruvate carboxylase (PEPCase),
to catalyze the initial reactions of photosynthesis. The immediate products of the
PEPCase reaction are C4 acids, which are subsequently decarboxylated in the vicin-
ity of Rubisco (Sage and Kubien 2003). This raises the intracellular CO2 concen-
tration in a manner that facilitates the carboxylase activity of Rubisco and almost
completely inhibits the oxygenase activity. Unlike C3 plants, photosynthetic rates of
plants possessing the C4 biochemical concentrating mechanism are effectively satu-
rated at ambient atmospheric CO2 levels. Therefore, rates of CO2 fixation, whole
plant growth rates, and harvestable yields of C4 plants are not nearly as responsive
to rising atmospheric CO2 concentrations as that of C3 plants. However, both C3
and C4 plants exhibit stomatal closure in response to elevated CO2 and this has
important consequences for plant–water relations (Bunce 2004). Because high con-
centrations of intracellular CO2 are maintained, partial stomatal closure due to CO2
enrichment normally does not inhibit photosynthetic rates of maize and other C4
plants (Sage 1999). Therefore, growth rates of maize can be positively affected by
CO2 enrichment, in part, because of improved water relations. However, any growth
enhancement of C4 plants due to CO2 enrichment is usually much smaller than that
reported for C3 plants (Kimball et al. 1993; Hatfield et al. 2011).
2 The Impact of Enhanced Atmospheric CO2 Concentrations on the Responses … 29
2.3 Negative Effects of CO2 Enrichment on Plants
CO2 enrichment is broadly beneficial for plant growth, although continuous expo-
sure to elevated CO2 can have a negative impact on plant development. It has been
observed that the C/N ratio is frequently higher in plants grown in elevated than in
ambient CO2 (Baker et al. 1989; Foyer et al. 1994), which suggests that the uptake
and assimilation of N, and possibly other nutrients from the soil, is not commensu-
rate with the C gain due to CO2 enrichment from the atmosphere. In some instances,
plants grown in elevated CO2 can become N deficient, which reduces tissue protein
concentrations and decreases photosynthetic capacity (Stitt 1991). There are exam-
ples where photosynthetic rates of older leaves in the elevated CO2 treatment were
below that of comparable leaves in the ambient CO2 treatment and this occurred
when gas exchange rates were measured at the respective CO2 concentrations used
for plant growth (Sicher and Kremer 1996).
Increased leaf starch levels are almost always observed in leaves of CO2-enriched
plants and this may partly be due to low leaf N concentrations and to accelerated
rates of net CO2 assimilation (Stitt 1991). Some authors (Sasek et al. 1985) argue
that excessive starch levels in the chloroplast can alter the structure of photosyn-
thetic membranes and this physical disruption negatively impacts leaf photosyn-
thetic rates. Leaves of plants grown in CO2-enriched atmospheres can also become
chlorotic, brittle, and malformed (Sasek et al. 1985; Sicher 1998). Low chlorophyll
levels in CO2-enriched tissues have been attributed to nitrogen insufficiency and
to the onset of premature senescence (Sicher and Bunce 1998). Premature senes-
cence as a result of CO2 enrichment has been observed for cereal crops, such as
wheat and barley, but this same treatment delays the onset of senescence in soy-
bean (Rogers et al. 2004). Clearly, alterations in the timing of senescence affect the
overall yield potential of annual crops. In some plant species, the initial stimulation
of photosynthesis in response to CO2 enrichment may be reversed over time as ni-
trogen becomes insufficient and chlorosis develops. This process is known as pho-
tosynthetic acclimation to CO2 enrichment and photosynthetic rates can ultimately
be below that of control plants grown with ambient CO2 concentrations.
2.4 Elevated Temperature Effects on Plant Growth
The relationship between plant growth and temperature is complex. The variation
between day and night temperatures and also mean annual or seasonal temperatures
is an important determinant of plant growth rates. Also, the interaction of tempera-
ture with other environmental variables, such as irradiance, water availability, and
atmospheric CO2 levels, affects plant development. The growth of all plants is char-
acterized by a number of critical temperatures that can be determined empirically.
For example, all plants possess a minimum, maximum, and optimum temperature
for growth (Luo 2011; Table 2.1). The minimum and maximum temperatures are
the lowest and highest temperatures, respectively, that will sustain the growth of
Table 2.1 Responses of reproductive yields of major crop species to temperature. The optimum
and maximum temperatures for reproductive yield ( Topt and Tmax, respectively) are means of day
and night values
Crop Topt,°C Tmax, °C, Yield Yield Yield % decrease
(yield) (yield) ( Topt) t ha−1 (28 °C), t ha−1 (32 °C), t ha−1 (28–32 °C)
Rice 25 36 7.6 6.3 2.9 54
Soybean 26–28 39–40 3.4 3.4 3.1 10
Dry bean 22–24 32 2.9 1.4 0 100
Peanut 23–25 40 3.4 3.2 2.6 20
Sorghum 23–25 35 12.2 11.8 7.0 41
Maize 20–25 35 10.9 – – –
Temperature data are from Hatfield et al. (2011) and Luo (2011). Yield data are from
Dr. V. R. Reddy (personal communication)
a given plant species. Agricultural crops have an optimum temperature for yield
and this is normally below that of the temperature optimum for vegetative growth
(Muchow et al. 1990; Luo 2011). The explanation for this is that lower temperatures
usually extend the growing season, thereby maximizing light interception and
enhancing crop yields. Temperatures above the vegetative and reproductive
growth optima are deleterious, although plants do possess adaptive mechanisms
that facilitate growth and successful reproduction under stress-inducing, elevated
growth temperatures.
2.5 Heat Stress Responses of Plants
Exposing plants to high temperatures for the first time, even for a few hours, can
cause heat stress, which is a dangerous condition that can result in cell damage or
even death (Mittler et al. 2011). Because leaves are thin and have a low heat capac-
ity, cellular injuries can occur within minutes when plants are exposed to acute
heat stress (Sharkey 2005). Cellular damage also occurs at moderately high tem-
peratures but only after longer periods of exposure. The heat stress response of
plants is complex and involves many components including the following: suscep-
tible proteins become inactivated or denaturated (Zhang et al. 2005), membrane
integrity and function is compromised (Howarth 2005); metabolic pathways break
down (Wahid et al. 2007); the assembly and elongation of microtubules is disrupted
(Smertenko et al. 1997); ion fluxes decrease (Schöffl et al. 1999), toxic compounds
and reactive oxygen species (ROS) accumulate and both RNA and protein synthesis
become impaired (Schöffl et al. 1999; Howarth 2005). To cope with heat stress,
plant cells completely reprogram metabolic networks and synthesize stress-related
metabolites, proteins, and lipid constituents (Wahid et al. 2007). Plants that are
pretreated with high temperatures normally have an improved ability to withstand
future heat stress episodes and this occurs by a process known as acquired ther-
motolerance. At the cellular level, acquired heat tolerance requires gene activation
and specific changes to the metabolome and transcriptome. Low molecular weight
metabolites accumulate that function as compatible solutes in the protection of
cellular proteins and membranes (Kaplan et al. 2004). Conversely, processes in-
volved in establishing a basal level of heat tolerance are not upregulated by stress
pretreatments (Qin et al. 2008).
One of the most important and most thoroughly studied aspects of thermotoler-
ance is the accumulation of heat shock proteins (HSP) in response to heat stress
and related environmental stresses (Wang et al. 2004). Families of HSPs vary by
molecular weight, i.e., Hsp60, Hsp70, Hsp90, Hsp100, and small or sHSP, and are
synthesized within a few hours of acute heat stress in plants. These proteins function
as molecular chaperones and are involved in stabilizing and resolubilizing proteins
that have denatured due to heat stress. Specific HSPs can be found in the nucleus,
chloroplast, mitochondria, and in other cellular compartments (Kotak et al. 2007).
This suggests that HSPs are involved in protecting and sustaining numerous, vital
processes throughout the cell.
It is also clear that the oxidative stress is a significant factor in the heat stress re-
sponse of plants and of other species. Heat stress frequently induces the synthesis of
highly reactive molecules including, singlet oxygen, the superoxide radical, hydro-
gen peroxide, and hydroxyl radicals (Wahid et al. 2007). One consequence of ROS
is the peroxidation of membrane lipids, which can lead to membrane leakage and a
loss of membrane integrity. Brief exposures to high temperatures also induce a burst
of hydrogen peroxide in plant cells that may be derived from NADPH oxidase ac-
tivity (Neill et al. 2002). It is believed that this burst of hydrogen peroxide is a signal
for the induction of several heat stress-related genes. Various antioxidant molecules,
including ascorbate and glutathione, can protect against ROS and controlling ROS
is a crucial mechanism in minimizing damage due to heat stress.
2.6 Heat Stress Effects on Photosynthesis
There is broad agreement that photosynthetic reactions within the chloroplast are
among the most highly sensitive to heat stress in higher plants (Berry and Bjorkman
1980; Sharkey 2005). Both light-driven electron transport reactions in the thylakoid
membranes and enzymatic reactions promoting CO2 fixation in the stroma are
thought to be thermolabile (Weis and Berry 1988; Havaux and Gruszecki 1993).
Various lines of evidence suggest that the oxidizing side of photosystem-II was
impaired by heat stress (Havaux and Gruszecki 1993; Heckathorn et al. 1998).
However, the reduction of plastoquinone by photosystem-II is relatively thermotol-
erant and cyclic electron flow involving photosystem-I actually increased with heat
stress (Bukhov et al. 1999; Schrader et al. 2004). The above adjustments decrease
linear electron flow and reduce rates of CO2 fixation.
2.7 Effects of CO2 Enrichment and Heat Stress

on Photosynthesis
Soybean, which possesses C3 photosynthesis, generally has a substantial, long-term

increase in leaf photosynthesis when grown at elevated CO2 (Sicher and Bunce
1998; Bunce 2014; Fig. 2.1a). Under field conditions, soybean exhibits little (Ber-
nacchi et al. 2005) or no downregulation of photosynthesis at elevated CO2 when
measured at high light, except when the plants are under water stress (Sicher and
Bunce 1998). This contrasts with the often substantial downregulation of photosyn-
thesis observed at elevated CO2 in this species when grown in controlled environ-
ment chambers (Sicher et al. 1995; Sims et al. 1998). However, during long-term
growth experiments, single-leaf photosynthetic rates were not increased by CO2 en-
richment when measured at limiting light levels (Rogers et al. 2006; Bunce 2014).
This finding suggested that long-term exposure to elevated CO2 decreased the quan-
tum efficiency of photosynthesis in soybean, similar to that observed for various
other species (Bunce and Ziska 1999; Lewis et al. 1999; Takeuchi et al. 2001).
In plants with C3 photosynthesis, such as soybean, the optimum temperature for
photosynthesis increases with the carbon dioxide concentration, primarily because
Fig. 2.1 Effects of elevated

temperatures and CO2 enrich-
ment on single-leaf photo-
synthetic rates of maize and
soybean. Plants were grown
from seed in naturally sunlit,
temperature-controlled enclo-
sures at Beltsville, MD, and
foliar photosynthetic rates
were determined on sunny
days shortly after canopy
closure. Data are shown for
ambient ( dark fill) or twice
ambient ( no fill) CO2 concen-
trations and are courtesy of
Dr. V. R. Reddy
of the suppression of photorespiration and increased carboxylation rates due to

CO2 enrichment discussed above, i.e., due to changes of the Vc/Vo ratio (Long 1991;
Kirschbaum 1994). This is true whether photosynthesis is light limited or light
saturated. However, elevated temperatures can lower the ratio of the velocity of
carboxylase to the velocity of oxygenase ( Vc/Vo) (Jordan and Ogren 1984). Al-
though a relative increase in photorespiration is a principal effect of elevated tem-
peratures on photosynthesis, it is clear that other factors are also involved. The
temperature at which the optimum rate of photosynthesis occurs largely depends
upon the thermal stability of the RuBP-regeneration system, because the Rubisco
protein itself is stable to at least 45 °C (Bjorkman et al. 1989; Devos et al. 1998).
However, Crafts-Brandner and Salvucci (2000) and Ristic et al. (2009) observed
that Rubisco became deactivated after the prolonged exposure of leaf tissue to
acute heat stress. Briefly, in the inactivate state, the Rubisco enzyme tightly binds
a substrate molecule to the active site, thereby blocking catalytic activity. A sec-
ond protein, Rubisco activase, facilitates removal of the substrate from the active
site and allows Rubisco to become activated and catalytically active. Both in vivo
and in vitro evidence suggests that exposing leaf tissue to elevated temperatures
can inactivate Rubisco activase. Therefore, one of the principal effects of elevated
temperatures on photosynthesis is the conversion of Rubisco from an active to an
inactive state. Lowering the Rubisco activation state decreases the carboxylation
efficiency of photosynthesis and may lead to the production of excess energy that
contributes to photo-oxidative stress (Ort and Baker 2002). However, Wise et al.
(2004) and Kubien and Sage (2008) have argued that decreases in Rubisco activa-
tion state are a secondary effect caused by a reduction in electron transport rates.
According to these authors, the deactivation of Rubisco at elevated temperatures
functions naturally to restore the imbalance between electron transport rates and
rates of CO2 fixation.
The stimulation of photosynthesis by elevated CO2 usually increases strongly
and predictably with temperature (Long 1991). However, at excessively high tem-
peratures, the CO2-dependent stimulation of photosynthesis may be negated by low
rates of Rubp-regeneration. When this situation occurs, the stimulation of photosyn-
thesis by elevated CO2 is highly insensitive to measurement temperatures (Bunce
2007; Ziska 2001; Yamori et al. 2005). Additionally, acclimation of photosynthesis
to seasonal changes in temperature can result in the stimulation of photosynthesis
by elevated CO2 being nearly constant at different times of the year despite sea-
sonal variations in temperature. This phenomenon has been attributed to thermal
acclimation of the photosynthesis system (e.g., Bunce 1998, 2000; Tesky 1997;
Tjoelker et al. 1998).
Above the optimum temperature of photosynthesis, photosynthetic rates may be-
come unstable and decrease continuously with time. There is a critical temperature
below which photosynthesis will completely recover after the plants are returned to
ambient growth temperatures. However, above this critical temperature, irreversible
damage occurs to the photosynthetic machinery of the leaf (Berry and Bjorkman
1980). This makes the assessment of CO2 effects on responses of photosynthesis to
extremely high temperatures difficult. Taub et al. (2000) found that for about 60 %
of the species they examined, cultivating plants in atmospheres containing elevated

CO2 resulted in about a 1 °C increase in the temperature required to damage pho-
tosystem II. This could also be due to decreased stomatal conductance during the
growth at elevated CO2 caused by leaves acclimating to warmer temperatures. A
similar effect on photosynthetic thermal tolerance due to elevated CO2 was report-
ed in wheat (Gutierrez et al. 2009), birch, and aspen trees (Darbah et al. 2010).
However, no effect of elevated CO2 on the thermal tolerance of photosynthesis was
observed with either creosote bush (Naumberg et al. 2004) or Phillyrea angustifo-
lium (Vitale et al. 2008). Soybean photosynthesis has a relatively high temperature
optimum (Harley et al. 1985) and photosynthesis was not damaged by exposures to
temperatures up to 48 °C at either ambient or elevated CO2 when plants were grown
with a daytime temperature of 28 °C (Bunce, unpublished data). Thus, it is unlikely
that soybean photosynthesis suffers from heat damage in any of the locations where
it is currently grown.
As stated above, plants with C4 photosynthetic metabolism, such as maize,
generally exhibit little or no stimulation of leaf photosynthesis when grown at
elevated CO2 (Kim et al. 2007, Fig. 2.1b). However, maize plants in the field
displayed episodic CO2-dependent increases in photosynthetic rates during water
stress events when stomatal conductance was reduced (Leakey et al. 2006). In
maize, photosynthesis can be limited by PEP carboxylase (or C4 cycle) activity,
Rubisco activity, or by Rubp-regeneration capacity. Unlike Rubisco, PEP carbox-
ylase activity is saturated by ambient atmospheric CO2 concentrations. Therefore,
photosynthesis rates of intact maize leaves are only limited by very low sub-
ambient CO2 concentrations. Determining whether Rubisco activity or rates of
Rubp-regeneration are limiting for photosynthesis in C4 species often requires
measuring light response curves, in addition to CO2 response curves (Massad
et al. 2007). Crafts-Brandner and Salvucci (2002) observed that photosynthesis
rates of corn leaves decreased at temperatures above 38 °C. These authors attrib-
uted this to a reduced activation state of Rubisco rather than to either diminished
C4 cycle or electron transport activity (i.e., Rubp-regeneration). Because high in-
tracellular CO2 concentrations are available to Rubisco, C4 species, in general,
tend to have greater optimum temperatures for photosynthesis than do C3 species
(Pearcy and Ehleringer 1984). This is partly because rates of photorespiration are
normally very low in C4 species. Maize evolved at higher elevations in the tropics,
so it is more heat sensitive than many closely related C4 species. Qu et al. (2014)
found that photosynthesis in corn leaves was inhibited by brief exposures to
45 °C and the temperature effect was more acute at elevated than at ambient CO2
(Fig. 2.2). Hamilton et al. (2008) also found that elevated CO2 decreased photo-
synthetic thermal tolerance in maize, as well as in Amarathus retroflexus, another
C4 species, although these earlier treatments were based on air temperature rather
than leaf temperature.

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Fig. 2.2 Percentage reductions in single leaf rates of photosynthesis for Zea mays L. cv. Silver
Queen, after leaf tissue was exposed to 45 °C for 2 h using plants grown in indoor or outdoor
chambers. The “ambient” and “elevated” treatments were with 380 mmol mol−1 ( dark fill) and
560 mmol mol−1 ( gray fill) CO2, respectively. In all cases, stomatal conductance was greater
after heat treatment in comparison to the untreated controls. Data are unpublished results from
Drs. M. Qu and J. Bunce

on Leaf Components and Metabolism
Both CO2 enrichment and supraoptimal temperatures affect a number of metabolic

processes in plants including photosynthesis, photorespiration, and dark respiration.
Consequently, these two environmental factors independently affect concentrations
of primary and secondary metabolites in plant tissues (Kaplan et al. 2004; Prasad
et al. 2004). As mentioned briefly above, CO2 enrichment enhances the accumula-
tion of carbon-containing compounds, such as starch, sucrose and hexoses, and may
decrease levels of many nitrogen-containing metabolites, including soluble amino
acids, photosynthetic proteins, such as Rubisco, and membrane-associated pigment-
protein complexes. These conclusions are true for most C3 plants, although soybean
normally does not exhibit large changes of nitrogen metabolism in response to CO2
enrichment (Campbell 1990; Sicher et al. 1995; Rogers et al. 2006).
As described above, heat stress affects the plant metabolome and leaf metabolites
usually exhibit a greater response to heat stress than those found in other tissues on
the plant (Rizhsky et al. 2004). Summarizing changes of plant metabolites due to
elevated temperatures is complicated by the fact that two fundamentally different
experimental approaches have been used. Some investigators examined metabolite
changes in response to an acute heat shock treatment and other studies involved
modified growth temperatures over longer period of time. These are two related
but different approaches to studying heat stress that can have varying outcomes
(Kaplan et al. 2004). A second problem is that plants are usually adapted to specific
cool or warm environments and this can affect the extent of thermal tolerance ob-
served (Yu et al. 2012). Third, acute heat treatments when applied to plants can
cause leaf tissues to lose water and become desiccated. This is a complication that
can result in indirect treatment effects on foliar metabolite levels.
Although the total dataset is limited, the heat stress metabolome of Arabidopsis
may be smaller than that for cold or drought stress. Kaplan et al. (2004) reported
that 143 and 311 out of 497 real and putative compounds from Arabidopsis rosettes
were affected by a heat and cold shock, respectively. Rizhsky et al. (2004) observed
that 5 of 48 targeted metabolites in Arabidopsis rosettes differed from the controls
after raising the growth temperature from 22 to 35 °C for 6 h. In the latter experi-
ment, it also was observed that 17 of 48 metabolites were altered by water stress. To
our knowledge, similar metabolite analyses from combined stress experiments have
not been performed in other species.
Nonstructural Carbohydrates Elevated growth temperatures decreased par-
titioning to both transitory and storage starch (Geigenberger et al. 1998; Prasad
et al. 2004). However, reports of changes of soluble nonstructural carbohydrates in
response to elevated temperatures in plants have been variable. Sucrose, glucose,
and fructose in leaves of specific crops and forage species frequently remained
unchanged or decreased in response to elevated growth temperatures (Chatterton
et al. 1987; Liu and Huang 2000; Sicher 2013). However, foliar sucrose levels also
increased due to supraoptimal temperatures in reports by other authors (Kaplan
et al. 2004; Yu et al. 2012). Sugar alcohols, or polyols, typically increased in soy-
bean leaflets at elevated growth temperatures. Pinitol, which is a methylated deriva-
tive of inositiol, is particularly abundant in soybean leaves and it accumulates in
response to elevated growth temperatures (Guo and Oosterhuis 1995; Sicher 2013).
This result suggested there was a shift in metabolism from sucrose to pinitol synthe-
sis in response to heat stress. Mannitol, myo-inositol, galactinol and raffinose have
also been observed to accumulate in response to elevated temperatures (Kaplan
et al. 2004; Sicher 2013). The former two compounds are polyols that likely func-
tion as osmolytes or compatible solutes that protect proteins and membranes from
abiotic stress. Galactinol, raffinose, and myo-inositol also are involved in scaveng-
ing ROS (Loewus and Murthy 2000).
Organic Acids Organic acids are normally synthesized from soluble sugars, which
are then converted to amino acids by transamination. In the Arabidopsis literature,
changes of organic acids in response to heat shock were relatively minor. Rizhsky
et al. (2004) reported that hydroxysuccinic acid and lactic acid increased with ris-
ing treatment temperatures. Hydroxysuccinic acid is another name for malic acid,
which, surprisingly, did not respond to heat stress and lactic acid is normally syn-
thesized during anaerobic metabolism. Kaplan et al. (2004) mentioned four organic
acids and all increased with heat stress. These were quinic acid, citramalic acid,
fumarate, and malate. Quinic acid is a cyclic polyol, citramalic or 2-methylmalic
acid is involved in leucine synthesis and the latter two compounds are tricarboxylic
acid (TCA) cycle intermediates with multiple cellular functions.
More recent investigations on crop species have suggested that organic acids may
have a major role in the heat stress responses of plants. Yu et al. (2012) reported that
oxalic, shikimic, malonic, threonic, glyceric and galacturic acids decreased from 55
to 85 % in tall fescue leaves when the growth temperature was maintained at 10 °C
above the optimum for plant growth. These same authors found that pyruvic and
malic acid were unchanged and citric acid increased about twofold in response to
elevated growth temperatures. Sicher (2013) observed that citrate, aconitate, suc-
cinate, fumarate, 2-oxoglutarate and malate decreased from 39 to 94 % in soybean
leaves when the average daytime growth temperature was increased from 28 to
36 °C (Fig. 2.3). All of these organic acids function in the TCA cycle and are impor-
tant in respiratory metabolism, amino acid synthesis, ammonia detoxification, and
nitrogen assimilation. The studies with tall fescue and soybean were longer-term
growth studies using moderate increases in temperature, whereas the Arabidopsis
experiments by Rizhsky et al. (2004), and Kaplan et al. (2004) employed acute heat
shock experiments of 4 and 6 h duration.
Amines Soluble amino acids participate in nitrogen assimilation, protein synthesis
and degradation, and in the manufacture of secondary metabolites. Prior studies
with Arabidopsis and cowpea cells showed that alanine, β-alanine, asparagine,
γ-amino butyric acid (GABA) and putrescine increased in response to heat shock
Fig. 2.3 Effects of heat stress on compounds involved in primary plant metabolism. Values
in parentheses are ratios of metabolite concentrations from leaves of plants grown with 36/28
compared to 28/20 °C (day/night) temperatures. Experiments were performed with ambient
(400 μmol mol−1) CO2 and observed changes in metabolite concentrations werenot observed
when plants were grown with elevated (700 μmol mol−1) CO2. Data are based on results from
Sicher (2013)
(Mayer et al. 1990; Kaplan et al. 2004; Rizhsky et al. 2004). Branched chain amino
acids (BCAA), leucine, isoleucine, and valine, also accumulated in the prior heat
shock studies by Mayer et al. (1990) and Kaplan et al. (2004). Alanine and aspara-
gine can accumulate to very high levels in plant tissues and these two amino acids
function as important storage forms of nitrogen during abiotic stress events. GABA
is a nonprotein amino acid that accumulates, often in combination with alanine, in
affected cells in response to abiotic and biotic stress (Bown and Shelp 1997). Mayer
et al. (1990) argued that GABA accumulation was triggered by low cellular pH, a
condition that is associated with Ca2+ buildup and the activation of glutamate decar-
boxylase, an enzyme involved in the synthesis of GABA from glutamate. Yu et al.
(2012) and Sicher (2013) also observed that GABA increased in plants exposed to
a moderate increase in growth temperature. The BCAAs accumulate in response
to drought stress and these compounds are important precursors in the synthesis of
secondary metabolites (Sicher and Barnaby 2012). Both Yu et al. (2004) and Sicher
(2013) reported that glycine and serine decreased in leaves in response to elevated
growth temperatures. This result was unexpected because elevated temperatures
favor photorespiratory metabolism over CO2 assimilation, and glycine and serine
are important photorespiratory metabolites. However, both serine and glycine may
be involved in other cellular processes that are inhibited by elevated temperatures
(Sicher and Barnaby 2012). Overall, we can conclude that elevated temperatures
cause large changes in amino acid metabolism.
Other Metabolites High temperature stress affects concentrations of phyto-
hormones in various plant tissues and these are likely involved in regulating the
growth and development of plants affected by abiotic stress (Wahid et al. 2007).
Collectively, abscisic acid, ethylene, and salicylic acid have all been associated with
temperature stress and brassinosteroid treatments reportedly improved the thermal
tolerance of certain plant species (Dhaubhadel et al. 1999). Glycine betaine accu-
mulates in many plant species in response to abiotic stress, and may be involved
in the response to heat shock (Sakamoto and Murata 2002). This compound is a
quaternary amine that likely functions as a compatible solute in the protection of
stress-susceptible proteins. Additionally, elevated temperatures also affected prod-
ucts of lipid peroxidation, certain carotenoids, phenolics, and polyamines (Wahid
et al. 2007).
2.9 CO2 Enrichment Mitigates Metabolite Responses

to Elevated Temperatures
The above-described metabolite changes in response to heat stress were measured

using plants exposed to ambient CO2. Again, the data are limited but there are
strong indications that metabolite responses to moderate heat stress were partially
to completely reversed by elevated CO2 treatments. Yu et al. (2012) observed that
the effects of elevated growth temperatures on six amino acids, two sugars, and
three amines were not observed when the CO2 concentrations used for plant growth
were increased from 400 to 800 μmol mol−1. Similarly, Sicher (2013) working with
soybean observed that 28 of 43 metabolites in soybean leaves were altered by in-
creasing the growth temperature to 8 °C under ambient CO2. Conversely, only three
amines in soybean leaflets were affected by the same temperature treatment when
experiments were performed at 700 μmol mol−1 CO2. We are not aware of similar
metabolite studies that have been performed on plants exposed to acute temperature
stress during a heat shock. However, it is likely that CO2 enrichment is capable of
mitigating the effects of elevated temperature stress on plant metabolism.

on Vegetative Growth
Atmospheric CO2 concentrations and air temperatures are important determinants

of plant growth and both of these environmental parameters are likely to be af-
fected by climate change. As discussed above, fertilization with atmospheric CO2
enhances photosynthetic rates and increases biomass formation of C3 plants. There-
fore, significant temperature by CO2 interactions has been observed for many C3
crop plants and observed growth responses to CO2 enrichment are usually enhanced
by moderate increases in air temperature (Boote et al. 2005). One additional reason
that this would occur is that moderately warmer temperatures have the capacity to
extend the length of the growing season (Hatfield et al. 2011). Although elevated
temperatures normally enhance the CO2 fertilization effect, there is a critical point
at which temperature increases become deleterious to growth regardless of CO2
concentrations. Idso et al. (1987) and Kimball et al. (2002) observed that the bio-
mass growth modification ratio increased by 0.08/°C between 12 and 34 °C when
the ambient CO2 concentration was enhanced by 300 μmol mol−1. In contrast to the
above, Allen et al. (1996) observed that for soybean the season-long biomass growth
modification ratio was −0.026 °C and he attributed this to a shortened grain filling
period due to accelerated reproductive development at elevated temperatures. Allen
et al. (1996) also observed that total biomass yields of soybean fell rapidly when
day/night temperatures exceeded 44/34 °C.
The growth of maize normally does not respond to elevated atmospheric CO2
concentrations except during periods of soil moisture deficits (Kim et al. 2006;
Leakey et al. 2006). The latter authors reported that CO2 enrichment increased pho-
tosynthetic rates of maize up to 41 % in the field during periods of water stress.
These authors proposed that CO2 enrichment enhanced intercellular CO2 concen-
trations and that this resulted in increased photosynthetic rates when the stoma-
tal conductance was reduced. Kim et al. (2007) reported that biomass formation,
photosynthesis, and leaf area of maize were unaffected by doubling the ambient
CO2 concentration and that this conclusion was maintained across a wide range of
growth temperatures. These same authors observed that the total above-ground bio-
mass and leaf area were negatively correlated with increasing growth temperatures
between 19/13 and 38.5/32.5 °C when experiments were performed using well wa-
tered plants in naturally sunlit, outdoor environmental chambers. The optimum tem-
perature for maize leaf development was about 31 or 32 °C (Tollenaar et al. 1979;
Kim et al. 2007), when determined with ambient or elevated CO2.

on Flowering/Reproductive Growth and Yield
Considerable research has been performed on predicting the effects of climate

change on crop yields and broad agreement exists on the basic effects of elevated
CO2 and temperature on the yield parameters of various crop species (Table 2.1).
However, there is widespread disagreement regarding the precise magnitude of the
predicted responses of seed yield to carefully defined environmental parameters
(Long et al. 2006). Crop yields are normally determined at numerous locations and
data from each location are based on substantial land areas. It is not affordable to
perform accurate yield determinations on a large scale using elevated CO2 treat-
ments. Therefore, all yield studies using elevated CO2 treatments are based on a
relatively small number of plants at a single location and are potentially subject to
error.
Harvestable yields of soybean are consistently increased by CO2 enrichment and
changes of yield were commensurate with increased rates of net photosynthesis and
total biomass production (Allen et al. 1996; Ainsworth et al. 2002). However, the
harvest index, which is the ratio of seed mass to above-ground biomass, decreased
in response to CO2 enrichment (Baker et al. 1989; Ainsworth et al. 2002). This is
an indication that the soybean plants have a greater capacity to synthesize biomass
in response to elevated CO2 than to utilize it for seed production. Allen and Boote
(2000) reported that soybean yields were increased 34 % in a study based on a sea-
son-long doubling of ambient CO2. Ainsworth et al. (2002) and Ziska et al. (2001)
reported that mean soybean seed yields increased 38 and 40 %, respectively, in re-
sponse to the same doubling of CO2. In addition, Ziska et al. (2001) suggested that
yield increases due to CO2 enrichment varied widely among soybean genotypes,
although genetic differences were not observed for single-leaf photosynthetic rates.
Soybean yields in the USA have increased dramatically since 1924 and the rate of
improvement has accelerated in the last four decades (Specht et al. 1999). Half of
this yield improvement was attributed to genetic and technological advances but
increased atmospheric CO2 concentrations also were identified as a major contribu-
tor to enhanced soybean yields.
The temperature optimum for soybean seed yield is between 23 and 24 °C (Piper
et al. 1998) and rising temperatures are expected to have a negative impact on har-
vestable yields. Diminished yields occur with increasing temperatures up to 40 °C,
which is the point at which crop failure is possible (Allen et al. 1996). It should
be pointed out that soybean is a moderately temperature tolerant species and sig-
nificant yield losses have been observed when air temperatures exceeded 30 °C for
prolonged periods during the growing season. Yield losses due to heat stress can
occur at any point in the growth cycle but temperature effects on yield are usually
greatest during the reproductive growth. Hatfield et al. (2008) and Lobell and Field
(2008) estimated that a 0.8–1.0 °C temperature increase across the Southeastern
USA would result in a 1.3–2.4 % decrease in soybean seed yield. Single-leaf pho-
tosynthetic rates by soybean leaflets are fairly stable between 26 and 36 °C. There-
fore, factors such as shortened grain-filling duration, poor seed set and decreased
seed size are responsible for the yield decreases in soybean that occur at above
optimum temperatures (Boote et al. 2005).
Baker et al. (1989) determined soybean seed yields (g plant−1) using naturally
sunlit controlled environment chambers set to provide 3-day/night temperatures and
ambient or twice ambient CO2 levels. Individual plants grown with 26/19 °C day/
night temperatures and with 330 μmol mol−1 CO2 yielded 9.0 g of seed plant−1. This
increased to 10.1 g seed plant−1 when the temperature was raised to 36/29 °C or to
13.1 g seed plant−1 when the CO2 concentration was doubled to 660 μmol mol−1.
However, the same plants yielded 11.6 g seed plant−1 when grown at the higher tem-
perature with double the ambient CO2 concentration and intermediate results were
observed at intermediate temperatures. The yield enhancement due to CO2 enrich-
ment was 45 and 15 % at the lower and higher growth temperatures, respectively.
Therefore, the beneficial effects of CO2 enrichment on soybean yields diminish at
elevated growth temperatures and disappear at acutely high temperatures.
The effects of elevated temperatures on maize and soybean yields were basi-
cally similar. It is well recognized that elevated temperatures decreased the grain
filling duration of maize and that this negatively affected crop yields (Muchow
et al. 1990). Conversely, Tollenaar and Bruulsema (1988) showed that kernel dry
matter accumulation only varied slightly between 10 and 25 °C. Commuri and Jones
(2001) reported that heat stress decreased overall kernel dry weight and kernel den-
sity. Consequently, the reproductive growth of maize is generally more sensitive
to heat stress than vegetative growth (Allen and Boote 2000; Reddy et al. 2000).
Lobell et al. (2011) and Hawkins et al. (2013) used historical maize yield data to
estimate yield losses due to excessive temperatures. The former paper studied maize
production in southern Africa and determined that each day above 30 °C found re-
duced yields by 1.0–1.7 % depending upon water availability. The latter paper simi-
larly found that maize yields in France decreased in proportion to the number of
days during the growing season with temperatures above 32 °C.
High temperatures decrease maize yields primarily during the reproductive
growth by inducing flower abortion, disrupting fertilization and inhibiting endo-
sperm development. Herrero and Johnson (1980) showed that temperatures above
32.5 °C inhibited maize pollen germination and that this process was affected by the
duration and severity of heat stress. There is also a possibility that maize pollen and
silk become desiccated when exposed to elevated temperatures. Monjardino et al.
(2005) reported that starch and protein synthesis in maize endosperm were inhibited
by 4 days of heat treatment at 35 °C. These authors also observed that kernel sizes
were smaller for the heat-treated samples in comparison with the controls.
Hatfield et al. (2011) summarized the effects of CO2 enrichment on maize and
concluded that seed yields would only increase 3–4 % on average in response to
doubling CO2 levels. The combined effects of CO2 enrichment and elevated temper-
atures on maize yields have not been characterized adequately in field experiments.
However, Prasad et al. (2008) demonstrated that elevated CO2 treatments increased
internal tissue temperatures of grain sorghum and this exacerbated the negative ef-
fects of elevated air temperatures on seed yields. Due to a lack of experimental
data, estimating the combined effects of CO2 and temperature on maize yields has
relied, in part, on crop modeling approaches. Hatfield et al (2011) concluded that
temperatures in the North American Corn Belt would increase to 0.8 °C in the next
30 years when atmospheric CO2 concentrations could reach 440 μmol mol−1. These
authors suggested that these conditions would result in a minimum 2–3 % decrease
in maize grain yields under water-sufficient conditions. Easterling et al. (2007) con-
cluded that a 1–2 °C increase in global mean temperatures would increase maize
yields by a few percent in the mid latitudes, that maize grown in the tropics would
have major yield losses due to temperatures 3–5 °C above today’s values and that
the elevated atmospheric CO2 concentrations would have negligible benefits for
maize production.
2.12 Summary
CO2 enrichment is capable of mitigating the effects of moderate heat stress on

plants, such as soybean, that have the C3 pathway of photosynthesis. Evidence
for this was based on changes of net photosynthetic rate, primary metabolism,
plant growth, and yield. However, the mitigation of heat stress by CO2 enrich-
ment diminishes in soybean and other species as temperatures elevate further and
heat stress becomes more acute. Very high air temperatures, i.e., those that exceed
40.0–42.5 °C, frequently cause irreversible damage to plant tissues and may cause
death or reproductive failure. Unlike soybean, the reversal of moderate heat stress
by CO2 enrichment is almost immeasurable for maize and other plants that pos-
sess the C4 photosynthetic pathway. This is because maize has high internal CO2
concentrations that almost completely saturate rates of photosynthesis in ambient
air. Second, elevated CO2 concentrations induce stomatal closure of many plant
species and this decreases evapotranspiration rates from leaves. The resultant im-
proved water status would certainly benefit maize and soybean in the field during
prolonged exposures to heat stress. Note that acute air temperatures create a de-
mand for lower leaf temperatures and this requires stomatal opening and increased
evapotranspiration rates. Thus, very high temperatures negate the effects of CO2
enrichment on stomatal aperture. Third, plant growth in elevated CO2 is capable of
accelerating or delaying the onset of senescence of several annual crops. Elevated
growth temperatures accelerate plant development and this shortens the growing
season and negatively affects crop production. Therefore, delaying the onset of
senescence via CO2 enrichment should mitigate the effects of a shortened growing
season due to elevated growth temperatures. Conversely, cereals, such as wheat,

exhibit premature senescence in response to CO2 enrichment and the combination
of elevated temperatures and supra-ambient CO2 levels would work synergistically
to decrease yields.
References
Ahuja I, deVos RIC, Bines AM, Hall RD. Plant molecular stress responses face climate change.
Trends Plant Sci. 2010;15:1360–85. doi:10.1016/j.tplants.2010.08.002.
Ainsworth EA, Davey PA, Bernacchi CJ, Dermody OC, Heaton EA, Moore DJ, et al. A meta-
analysis of elevated [CO2] effects on soybean (Glycine max) physiology, growth and yield.
Global Change Biol. 2002;8:695–709. doi:10.1104/pp.106.086256.
Allen LH Jr, Boote KJ. Crop ecosystem responses to climatic change: soybean (Chapter 7). In:
Reddy KR, Hodges HF, editors. Climate change and global crop productivity. New York: CAB
International; 2000. p. 133–60.
Allen LH Jr, Baker JT, Boote KJ. The CO2 fertilization effect: Higher carbohydrate production
and retention as biomass and seed yield. In: Bazzaz FA, Sombroek WG, editors. Global cli-
mate change and agricultural production: direct and indirect effects of changing hydrological,
pedological and plant physiological processes. Rome: Wiley; 1996. p. 65–100.
Barnaby JY, Ziska LH. Plant responses to elevated CO2. In: eLS. Chicester: Wiley; 2012. p. 1–10.
doi:10.1002/9780470015902.a0023718.
Bernacchi CJ, Morgan PB, Ort DR, Long SP. The growth of soybean under free air [CO2]
enrichment (FACE) stimulates photosynthesis while decreasing in vivo Rubisco capacity.
Planta. 2005;220:434–46. doi:10.1007/s00425-004-1320-8.
Berry JA, Bjorkman O. Photosynthetic response and adaptation to temperature in higher plants.
Annu Rev Plant Physiol. 1980;31:491–543. doi:10.1146/annurev.pp.31.060180.002423.
Bjorkman O, Badger MR, Armond PA. Response and adaptation of photosynthesis to high
temperatures. In: Turner NC, and Kramer PJ, editors. Adaptation of plants to water and high
temperature stress. New York: Wiley; 1989. p. 233–49.
Boote KJ, Allen LH, Prasad PVV, Baker JT, Gesch RW, Snyder AM, et al. Elevated temperature
and CO2 impacts on pollination, reproductive growth, and yield of several globally important
crops. J Agric Meteorol. 2005;60:469–74.
Bukhov NG, Weise C, Neimanis S, Heber U. Heat sensitivity of chloroplasts and leaves: leakage
of protons from thylakoids and reversible activation of cyclic electron transport. Photosyn Res.
1999;59:81–93. doi:10.1093/jxb/49.326.1555.
Bunce JA. The temperature dependence of the stimulation of photosynthesis by elevated carbon
dioxide in wheat and barley. J Exp Bot. 1998;49:1555–61. doi:10.1093/jxb/49.326.1555.
Bunce JA. Acclimation to temperature of the response of photosynthesis to increased carbon diox-
ide concentration in Taraxacum officinale. Photosyn Res. 2000;64:89–94.
Bunce JA. Carbon dioxide effects on stomatal responses to the environment and water use by crops
under field conditions. Oecologia. 2004;140:1–10. doi:10.1007/s00442-003-1401-6.
Bunce JA. Effects of elevated carbon dioxide on photosynthesis and productivity of alfalfa in rela-
tion to seasonal changes in temperature. Physiol Molec Biol Plants. 2007;13:243–52.
Bunce JA. Limitations to soybean photosynthesis at elevated carbon dioxide in free-air
enrichment and open top chamber systems. Plant Sci. 2014;226:131–5. doi:10.1016/j.
plantsci.2014.01.002.
Bunce JA, Ziska LH. Impact of measurement irradiance on acclimation of photosynthesis to
elevated CO2 concentration in several plant species. Photosynthetica. 1999;37:509–17.
Chatterton NJ, Harrison PA, Bennett JH, Thornley WR. Fructosan, starch and sucrose concentra-
tions in crested wheatgrass and redtop as affected by temperature. Plant Physiol Biochem.
1987;25:617–23.
Commuri PD, Jones RD. High temperatures during endosperm cell division in maize: A geno-
typic comparison under in vitro and field conditions. Crop Sci. 2001;41:1122–30. doi:10.2135/
cropsci2001.4141122x.
Crafts-Brandner SJ, Salvucci ME. Rubisco activase constrains the photosynthetic potential of
leaves at high temperature and CO2. Proc Natl Acad Sci USA. 2000;97:13430–5. doi:10.1073/
pnas.230451497.
Crafts-Brandner SJ, Salvucci ME. Sensitivity of photosynthesis in a C4 plant, maize, to heat stress.
Plant Physiol. 2002;129:1773–80. doi:101104/pp 002170.
Darbah JNT, Sharkey TD, Calfapietra C, Karnosky DF. Differential response of aspen and birch
trees to heat stress under elevated carbon dioxide. J Environ Pollut. 2010;158:1008–14.
doi:10.1016/J.envpol.2009.10.019.
Devos N, Ingouff M, Loppes R, Matagne RF. Rubisco adaptation to low temperatures: a com-
parative study in psychrophlilic and mesophilic unicellular algae. J Phycol. 1998;34:655–60.
doi:10.1046/j.1529-8817.1998.340655.x.
Dhaubhadel S, Chaudhary S, Dobinson KF, Krishna P. Treatment with 24-epibrassinolide, a brassi-
nosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings. Plant
Mol Biol. 1999;40:333–42.
Easterling WE, Aggarwal PK, Batima P, Brandner KM, Erda L, Howden SM, et al. Food, fibre
and forest products. In: Parry ML, Canziani OF, Palutikof JP, van der Linden PJ, Hansen CE,
editors. Climate change 2007, impacts adaptation and vulnernability. Contribution of working
group II to the Fourth Assassment Report of the Intergovernmental Panel on Climate Change.
2007. Cambridge: Cambridge University Press. p. 273–313.
Foyer CH, Lescure J-C, Lefebvre C, Morot-Gaudry J-F, Vincentz M, Vaucheret H. Adaptations
of photosynthetic electron transport, carbon assimilation, and carbon partitioning in trans-
genic Nicotiana plumbaginfolia plants to changes in nitrate reductase activity. Plant Physiol.
1994;104:171–8. doi:10.1104/pp.104.1.171.
Geigenberger P, Geiger M, Stitt M. High temperature perturbation of starch synthesis is at-
tributable to inhibition of ADP-glucose pyrophosphorylase by decreased levels of glycer-
ate-3-phosphate in growing potato tubers. Plant Physiol. 1998;117:1307–16. doi:10.1104/
pp.117.4.1307.
Gutiérrez D, Gutiérrez E, Pérez P, Morcuende R, Verdejo AL, Martinez-Carrasco R Acclima-
tion to future atmospheric CO2 levels increases photochemical efficiency and mitigates pho-
tochemistry inhibition by warm temperatures in wheat under field chambers. Physiol Plant.
2009;137:86–100. doi:10.1111/j.1399-3054.2009.01256.x.
Guo C, Oosterhuis DM. Pinitol occurrence in soybean plants as affected by temperature and plant
growth regulators. J Exptl Bot. 1995;46:249–53. doi:10.1093/jxb/46.2.249.
Hamilton EW, Heckathorn SA, Joshi P, Wang D, Barua D. Interactive effects of elevated CO2 and
growth temperature on the tolerance of photosynthesis to acute heat stress in C3 and C4 species.
J Integr Plant Biol. 2008;50:1375–87. doi:10.1111/j.1744-7909.2008.00747.x.
Harley PC, Weber JA, Gates DM. Interactive effects of light, leaf temperature, [CO2] and [O2] on
photosynthesis in soybean. Planta. 1985;165:249–63.
Hatfield JL, Boote K, Fay P, Hahn L, Izaurralde C, Kimball BA, et al. Agriculture. In: The effects
of climate change on agriculture, land resources, water resources, and biodiversity. A report by
the U.S. Climate Change Science Program and the Subcommittee on Global Change Research.
Washington, DC: 2008. p. 21–74.
Hatfield JL, Boote KJ, Kimball BA, Ziska LH, Izaurralde RC, Ort D, et al. Climate impacts
on agriculture: implications for crop production. Agron J. 2011;103:351–70. doi:10.2134/
agronj2010.0303.
Havaux M, Gruszecki WI. Heat-and light-induce chlorophyll a fluorescence changes in potato
leaves containing high and low levels of carotenoid zeaxanthin: indications of a regulatory
effect of zewaxanthin on membrane fluidity. Photochem Photobiol. 1993;58:607–17.
Hawkins E, Fricker TE, Challinor AJ, Ferro CAT, Ho CK, Osborne TM. Increasing influence of heat
stress on French maize yields for the 1960s to the 2030s. Global Change Biol. 2013;19:937–47.
doi:10.1111/gcb/12069.
Heckathorn SA, Cowns CA, Sharkey TD, Coleman JS. The small methionine rich chloroplast
heat shock protein protects photosystem-II electron transport during heat stress. Plant Physiol.
1998;116:439–44. doi:10.1104/pp.116.1.439.
Herrero MP, Johnson RR. High temperature stress and pollen viability of maize. Crop Sci.
1980;20:796–800. doi:10.2135/cropsci1980.0011183X002000060030x.
Howarth CJ. Genetic improvements of tolerance to high temperature. In: Ashraf M, Harris PJC,
editors. Abiotic stresses: plant resistance through breeding and molecular approaches. New
York: Haworth Press; 2005. p. 277–300.
Idso SB, Kimball BA, Anderson MG, Mauney JR. Effects of atmospheric CO2 enrichment on
plant growth: the interactive role of air temperature. Agric Ecosys Environ. 1987;20:1–10. doi
:org/10.1010/0167-8809(87)90023-5.
IPCC. 2007. Climate change 2007: the physical science basis. Contribution of Working Group I
to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. [Solo-
mon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL, editors].
2007;Cambridge: Cambridge University Press. p. 996.
IPCC. 2013. Climate change 2013: the physical science basis. Contribution of Working Group I to
the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. [Stocker, T.F.,
D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M.
Midgley, eds.]. 2013; United Kingdom; Cambridge University Press. pp. 1535.
Jordan DB, Ogren WL. The CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygen-
ase. Planta. 1984;161:308–13.
Kaplan F, Kopka J, Haskell DW, Zhao W, Schiller KC, Gatzke N, Sung DY, Guy CL. Exploring the
temperature stress metabolome of Arabidopsis. Plant Physiol. 2004;136:4159–68. doi:10.1104/
pp.104.052142.
Kim S-H, Sicher RC, Bae H, Gitz DC, Baker JT, Timlin DJ, Reddy VR. Canopy photosynthesis,
evapotranspiration, leaf nitrogen, and transcription profiles of maize in response to CO2 enrich-
ment. Global Change Biol. 2006;12:588–600. doi:10.111/j.1365-2486.2006.01110.x.
Kim S-H, Gitz DC, Sicher RC, Baker JT, Reddy VR. Temperature dependence of growth, devel-
opment, and photosynthesis in maize under elevated CO2. Environ Exp Bot. 2007;61:224–36.
doi:10.1016/j.envexpbot.2007.06.005.
Kimball BA, Mauney JR, Nakayama FS, Idso SB. Effects of increasing atmospheric CO2 on veg-
etation. Vegetatio. 1993;104/105:65–75.
Kimball BA, Kobayashi K, Bindi M. Responses of agricultural crops to free-air CO2 enrichment.
Adv Agron. 2002;77:293–368. doi:org/10.1016/S0065-2113(02)77017-X.
Kirschbaum MUF. The sensitivity of C3 photosynthesis to increasing CO2 concentration: a theo-
retical analysis of its dependence on temperature and background CO2 concentration. Plant
Cell Environ. 1994;17:747–54. doi:10.1111/j.1365-3040.1994.tb00167.x.
Kobza J, Edwards GE. Influences of leaf temperature on photosynthetic carbon metabolism. Plant
Physiol. 1987;83:69–74. doi:10.1104/pp.83.1.69.
Kotak S, Larkindale J, Lee U, Koskull-Döring PV, Vierling E, Scharf K-D. Complexity of the
heat stress response in plants. Curr Opin Plant Biol. 2007;10:310–16. doi:org/10.1016/j.
pbi.2007.04.011.
Kubien DS, Sage RF. The temperature response of photosynthesis in tobacco with reduced amounts
of Rubisco. Plant Cell Environ. 2008;31:407–18 doi:10.1111/j.1365-3040.2008.01778.x.
Leakey ADB, Uribelarrea M, Ainsworth EA, Naidu SL, Rogers A, Ort DR, et al. Photosynthesis,
productivity, and yield of maize are not affected by open-air elevation of CO2 concentration in
the absence of drought. Plant Physiol. 2006;140:779–90. doi:10.1104/pp.105.073957.
Lewis JD, Olszyk D, Tingey DT. Seasonal patterns of photosynthetic light response in Douglas-fir
seedlings subjected to elevated atmospheric CO2 and temperature. Tree Physiol. 1999;19:243–
52. doi:10.1093/treephys/19.4-5.243.
Liu X, Huang B. Carbohydrate accumulation in relation to heat stress tolerance in two creeping
bentgrass cultivars. J Amer Soc Hort Sci. 2000;125:442–7.
Lobell DB, Field CB. Estimation of the CO2 fertilization effect using growth rate anomalies in
CO2 and crop yields since 1961. Global Change Biol. 2008;14:39–45. doi:10.111/j.1365-
2486.2007.01476.x.
Lobell DB, Bäenziger M, Magoroskosho C, Vivek B. Nonlinear heat effects on African maize
as evidenced by historical yield trials. Nat Climate Change. 2011;1:42–5. doi:10.10138/ncli-
mate1043.
Loewus FA, Murthy PN. myo-Inositol metabolism in plants. Plant Sci. 2000;150:1–19.
doi:org/10.1016/S0168-9452(99)00150-8.
Long SP. Modification of the response of photosynthetic productivity to rising temperature by
atmospheric CO2 concentrations: has its importance been underestimated. Plant Cell Environ.
1991;14:729–39. doi:10.1111/j.1365-3040.1991.tb01439.x.
Long SP, Ainsworth EA, Leakey ADB, Nösberger J, Ort D. Food for thought: lower than ex-
pected crop yield simulation with rising CO2 concentrations. Science. 2006;312:1918–21.
doi:10.1126/science.1114722.
Luo Q. Temperature thresholds and crop production: a review. Clim Change. 2011;109:583–98.
doi 10.1007/s10584-011-0028-6.
Massad R-S, Tuzet A, Bethenod O. The effect of temperature on C4-type leaf photosynthesis pa-
rameters. Plant Cell Environ. 2007;30:1191–204 doi:10.1111/j.1365-3040.2007.01691.x.
Mayer RR, Cherry JH, Rhodes D. Effects of heat shock on amino acid metabolism of cowpea cells.
Plant Physiol. 1990;94:796–810. doi:10.1104/pp.94.2.796.
Mittler R, Finka A, Goloubinoff P. How do plants feel the heat? Trends Biochem Sci. 2011;37:118–
25. doi:10.1016/j.tibs.2011.11.007.
Monjardino P, Smith AG, Jones RJ. Heat stress effects on protein accumulation of maize endo-
sperm. Crop Sci. 2005;45:1203–10. doi:10.2135/cropsci2003.0122.
Muchow RC, Sinclair TC, Bennett JM. Temperature and solar radiation effects on potential maize
yield across locations. Agron J. 1990;82:338–43.
Naumberg E, Loik ME, Smith SD. Photosynthetic responses of Larrea tridentata to seasonal
temperature extremes under elevated carbon dioxide. New Phytologist. 2004;162:323–330.
doi:10.1111/j.1469-8137.2004.01023.x.
Neill S, Desikan R, Hancock J. Hydrogen peroxide signaling. Curr Opin Plant Biol. 2002;5:388–
95. doi:org/10.1016/S1369-5266(02)00282-0.
Ort DR, Baker NR. A photoprotective role of O2 as an alternative electron sink in photosynthesis.
Curr Opin Plant Biol. 2002;5:193–8. doi:org/10.1016/S1369-5266(02)00259-5.
Pearcy RW, Ehleringer JR. Comparative ecophysiololgy of C3 and C4 plants. Plant Cell Environ.
1984;7:1–13. doi: 10.1111/j.1365-3040.1984.tb01194.x.
Piper EL, Boote KJ, Jones JW. Evaluation and improvement of crop models using regional cultivar
trial data. Appl Eng Agric. 1998;14:435–46.
Prasad PVV, Boote KJ, Vu JCV, Allen LH Jr. The carbohydrate metabolism enzymes sucrose-P
synthase and ADG-pyrophosphorylase in phaseolus bean leaves are up-regulated at elevated
growth carbon dioxide and temperature. Plant Sci. 2004;166:1565–73.
Prasad PVV, Pisipati SR, Mutava RN, Tuinstra MR. Sensitivity of grain sorghum to high tem-
perature stress during reproductive development. Crop Sci. 2008;48:1911–17. doi:10.2135/
cropsci2008.01.0036.
Qin D, Wu H, Peng H, Yao Y, Ni Z, Li Z, et al. Heat stress-responsive transcriptome analysis in
heat susceptible and tolerant wheat ( Triticum aestivum L.) by using Wheat Genome Array.
BMC Genomics. 2008;9:432. doi:10.1186/1471-2164-9-432.
Qu M, Bunce JA, Shi AS. Does elevated CO2 protect damage to photosynthesis by high tempera-
ture via modifying leaf water status in maize seedlings? Photosynthetica 2014;52(2):211–16.
doi:10.1007/s11099-014-0022-0.
Reddy KR, Hodges HF, Kimball BA. Crop ecosystem responses to climatic change: cotton. In:
Reddy KR, Hodges HF, editors. Climate change and global crop productivity. 2000. New York:
CAB Int.; 2000. p. 161–87.
Ristic Z, Momčilović I, Bukovnik U, Prasad PVV, Fu HJ, DeRidder BP, et al. Rubisco activase
and wheat productivity under heat stress conditions. J Exp Bot. 2009;60:4003–14. doi:10.1093/
jxb/erp241.
Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R. When defense pathways col-
2004;134:1683–96. doi:10.1104/pp.103.033431.
Rogers A, Allen DJ, Davey PA, Morgan PB, Ainsworth EA, Bernacchi CJ, Cornic G, Dermody O,
Dohleman FG, Heaton EA, Mahoney J, Zhu X-G, Delucia EH, Ort DH, Long SP. Leaf pho-
tosynthesis and carbohydrate dynamics of soybeans growth throughout their life-cycle under
free-air carbon dioxide enrichment. Plant Cell Environ. 2004;27:449–58. doi 10.1111/j.1365-
3040.2004.01163.x.
Rogers A, Gibon Y, Stitt M, Morgan P, Bernacchi CJ, Ort DR, et al. Increased C availability at el-
evated carbon dioxide concentration improves N assimilation in a legume. Plant Cell Environ.
2006;29:1651–58. doi:10.1111/j.1365-3040.2006.01549.x.
Sage RF, Kubien DS. Quo Vadis? An ecological perspective on global change and the future of C4
plants. Photosyn Res. 2003;77:209–25.
Sakamoto A, Murata N. The role of glycine betaine in the protection of plants from stress:
clues from transgenic plants. Plant Cell Environ. 2002;25:163–71. doi:10.1046/j.0016-
8025.2001.00790.x.
Sasek TEW, DeLucia EH, Strain BR. Reversibility of photosynthetic inhibition in cotton after long-
term exposure to elevated CO2 concentrations. Plant Physiol. 1985;78:619–22. doi:10.1104/
pp.78.3.619.
Schöffl F, Prandl R, Reindl A. Molecular responses to heat stress. In: Shinozaki K, Yamaguchi-
Shinozaki K, editors. Molecular responses to cold, drought, heat and salt stress in higher plants.
Austin: RG Landes Co.; 1999. p. 81–98.
Schrader SM, Wise RR, Wacholtz WF, Ort DR, Sharkey TD. Thylakoid membrane responses
to moderately high leaf temperature in Pima cotton. Plant Cell Environ. 2004;27:725–35.
doi:10.1111/j.1365-3040.2004.01172.x.
Sharkey TD. Effects of moderate heat stress on photosynthesis: importance of thylakoid reactions,
rubisco deactivation, reactive oxygen species, and thermotolerance provided by isoprene. Plant
Cell Environ. 2005;28:269–77. doi:10.1111/j.1365-3040.2005.01324.x\
Sicher RC. Yellowing and photosynthetic decline of barley primary leaves in response to
atmospheric CO2 enrichment. Physiol Plant. 1998;103:193–200. doi:10.1034/j.1399-
3054.1998.1030206.x.
Sicher R Combined effects of CO2 enrichment and elevated growth temperatures on metabolites in
soybean leaflets. Planta. 2013;238:369–80. doi: 10.1007/s00425-013-1899-8.
Sicher R, Barnaby JY. Impact of carbon dioxide enrichment on responses of maize leaf tran-
scripts and metabolites to water stress. Physiol Plant. 2012;144:238–52. doi:10.111/j.1399-
3054.2011.01555.x.
Sicher RC, Bunce JA. Evidence that premature senescence affects photosynthetic decline of wheat
flag leaves during growth in elevated carbon dioxide. Int J Plant Sci. 1998;159:798–804. http://
www.jstor.org/stable/2475150.
Sicher RC, Kim S-H Photosynthesis, growth and maize yields in the context of global change. In:
Prioul J-L, Thévenot C, Molnar T, editors. Advances in maize, essential reviews in experimen-
tal biology, Vol 3. London: Society for Experimental Biology; 2011. p. 373–92.
Sicher RC, Kremer DF. Rubisco activity is altered in a starchless mutant of Nicotiana sylvestris
grown in elevated carbon dioxide. Env Exptl Bot. 1996;36:385–91. doi:org/10.1016/S0098-
8472(98)01031-3.
Sicher RC, Kremer DF, Bunce JA. Photosynthetic acclimation and photosynthate partitioning in
soybean leaves in response to carbon dioxide enrichment. Photosyn Res. 1995;46:409–17.
Sims DA, Luo Y, Seemann JR. Comparison of photosynthetic acclimation to elevated CO2 and
limited nitrogen supply in soybean. Plant Cell Environ. 1998;21:945–52. doi:10.1046/j.1365-
3040.1998.00334.x.
Smertenko A, Draber P, Viklicky V, Opatrny Z. Heat stress affects the organization of micro-
tubules and cell division in Nicotiana tabacum cells. Plant Cell Environ. 1997;20:1534–42.
doi:10.1046/j.1365-3040.1997.d01-44.x.
Specht JE, Hume DJ, Kumundi SV. Soybean yield potential- a genetic and physiological perspec-
tive. Crop Sci. 1999;39:1560–70. doi:10.2135/cropsci1999.3961560x.
Stitt M. Rising CO2 levels and their significance for carbon flow in photosynthetic cells. Plant Cell
Environ. 1991;14:741–62. doi:10.1111/j.1365-3040.1991.tb01440.x.
Takeuchi Y, Kubiske ME, Isebrands JG, Pregtizer KS, Hendrey G, Karnosky DF. Photosynthesis,
light and nitrogen relationships in a young deciduous forest canopy under open-air CO2 enrich-
ment. Plant Cell Environ. 2001;24:1257–68. doi:10.1046/j.0016-8025.2001.00787.x.
Taub DR, Seemann JR, Coleman JS. Growth in elevated CO2 protects photosynthesis against
high-temperature damage. Plant Cell Environ. 2000;23:649–56. doi:10.1046/j.1365-
3040.2000.00565.x.
Tesky RO. Combined effects of elevated CO2 and air temperature on carbon assimilation of Pinus
taeda trees. Plant Cell Environ. 1997;20:373–80. doi:10.1046/j.1365.3040.1997.d012-75.x.
Tjoelker MG, Oleksyn J, Reich PB. Seedlings of five boreal tree species differ in acclimation of net
photosynthesis to elevated CO2 and temperature. Tree Physiol. 1998;18:715–26. doi:10.1093/
treephys/18.11.715.
Tollenaar M, Bruulsema TW. Effects of temperature on rate and duration of kernel dry matter ac-
cumulation of maize. Can J Plant Sci. 1988;68:935–40. doi:10.4141/cjps88-113.
Tollenaar M, Daynard TB, Hunter RB. Effect of temperature on rate of leaf appearance and flow-
ering date in maize. Crop Sci. 1979;19:363–366. doi:10.2135/cropsci1979.0011183X001900
030022x.
Vierling E. The roles of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol Biol.
1991;42:579–620. doi:10.1146/annurev.pp.42.060191.003051.
Vitale L, Arena C, De Santo AV, D’Ambrosio N. Effects of heat stress on gas exchange and photo-
system II (PSII) photochemical activity of Phillyrea angustifolia exposed to elevated CO2 and
substaturating irradiance. Botany. 2008;86:435–41. doi:10.1139.b07-132.
Wahid A, Gelani S, Ashraf M, Foolad MR. Heat tolerance in plants: an overview. Environ Exptl
Bot. 2007;61:199–223. doi:org/10.1016/j.envexpbot.2007.05.011.
Wang W, Vincour B, Shoseyov O, Altman A. Role of heat shock proteins and molecular chaperones
in abiotic stress. Trends Plant Sci. 2004;9:244–52. doi:10.1016/j.tplants.2004.03.006.
Weis E, Berry JAB. Plants and high temperature stress. In: Long SP, Woodward FI, editors. Plants
and high temperature stress. Cambridge: Company of Scientists; 1988. p. 329–46.
Wise RR, Olson AJ, Schrader SM, Sharkey TD. Electron transport is the functional limitation
of photosynthesis in field-grown Pima cotton plants at high temperature. Plant Cell Environ.
2004;27:717–24. doi:10.1111/j.1365-3040.2004.01171.x.
Yamori W, Noguchi K, Tersahima I. Temperature acclimation of photosynthesis in spinach leaves:
analyses of photosynthetic components and temperature dependencies of photosynthetic par-
tial reactions. Plant Cell Environ. 2005;28:536–47. doi:10.1111/j.2005.01299.x.
Yu J, Du H, Xu M, Huang B. Metabolic responses of heat stress under elevated atmospheric CO2
concentration in a cool-season grass species. J Amer Soc Hort Sci. 2012;137:221–8.
Zhang J-H, Huang WD, Liu Y-P, Pan QH. Effects of temperature acclimation pretreatment on the
ultrastructure of mesophyll cells in young grape plants ( Vitis vinifera L. cv. Jingxiu) under
cross-temperature stresses. J Integr Plant Biol. 2005;47:959–70.
Ziska LH. Growth temperature can alter the temperature dependent stimulation of photosyn-
thesis by elevated carbon dioxide in Abultion theophrasti. Physiol. Plant. 2001;111:322–8.
doi:10.1034/j.1399-3054.2001.1110309.x.
Ziska LH, Bunce JA, Caulfield FA. Rising atmospheric carbon dioxide and seed yields of soybean
genotypes. Crop Sci. 2001;41:385–91. doi:10.2135/cropsci2001.412385x.
Chapter 3
Investigating the Effect of Elevated CO2
in the Growth Environment of Salt-Stressed
Plants Using Integrated Omic Analyses
Matthaios-Emmanouil P. Papadimitropoulos and Maria I. Klapa
3.1 Introduction
In an era, when the need for food of consistent and high quality throughout the
globe is of great interest, while the changes in the environment, including the green-
house effect, are altering the conditions of plant growth, furthering our understand-
ing of how the plants respond to various stresses at the molecular level becomes a
major objective for molecular plant physiologists, agricultural engineers, and the
food industry. Major abiotic stress factors for plants that are under investigation
individually but mainly in combination are the extreme cold or heat, the drought or
flooding, the soil or water salinity, chemicals and pollutants like heavy metals and
pesticides, the oxidative stress (i.e., the reactive oxygen species (ROS), the ozone),
the nutrient deprivation in soil, and changes in the composition of the atmosphere,
mainly the increase in the carbon dioxide (CO2) concentration.
Among these, the investigation of the salinity effect on plant growth has intensified
in the recent years, because high soil or water salinity is a major environmental stress
and a substantial constraint to crop production. Increased salinization of arable land
is expected to have devastating global effects, estimated to result in 50 % land loss
by the middle of the twenty-first century (Wang et al. 2003). Hot and dry climates
favor water evaporation, leading thus to an increase in the salt concentration. Heavy
or low quality irrigation may also contribute to an increase in salinity. The problem
M. I. Klapa () · M.-E. P. Papadimitropoulos
Metabolic Engineering and Systems Biology Laboratory, Institute of Chemical
Engineering Sciences, Foundation for Research & Technology-Hellas (FORTH/ICE-HT),
Stadiou St., Platani, GR-265 04, Patras, Greece
e-mail: mklapa@iceht.forth.gr
M.-E. P. Papadimitropoulos
Division of Genetics, Cell Biology & Developmental Biology, Department of Biology,
University of Patras, Patras, Greece
M. I. Klapa
Departments of Chemical & Biomolecular Engineering & Bioengineering,
University of Maryland, College Park, MD 20742, USA

50 M.-E. P. Papadimitropoulos and M. I. Klapa
is even greater in the coastal areas, where the seawater enters the aquifer, increas-
ing thus the soil salinity in intensively cultivated areas (Mahajan and Tuteja 2005).
As the growing of hydroponic cultures in greenhouses gains momentum as a means
for consistent plant and product quality independently of the place of plant growth
around the globe (Jones 2005), this trend has also contributed in the past decade to
an increase in the studies about the effect of varying water salinity on plant growth.
On the other hand, considering the elevation of the CO2 concentration in the envi-
ronment due to the greenhouse effect, which can drastically change the physiology
of the plants and the quality of crop production in the future (Solomon et al. 2007),
the particular stress has been the subject of molecular plant physiology studies for
many years. This is also due to the fact that CO2 is the major carbon source for the
plants and its increase at moderate levels and for moderate durations has been shown
to be beneficial for the plant growth, especially when the plants are also under the
influence of other stresses, including salinity (Takagi et al. 2009; Geissler et al. 2010;
Kanani et al. 2010; Perez-Lopez et al. 2012; Ratnakumar et al. 2013). Therefore, the
combined effect of high soil, but mainly water, salinity, and elevated CO2 on plants
has been under investigation by agricultural engineers and plant physiologists not
only in the context of the greenhouse effect but also for the development of plant
growth optimization strategies in the presence of salinity stress.
In the system biology era, the investigation of the molecular mechanisms underly-
ing plant growth and response under various stresses has been enhanced by the high-
throughput biomolecular (i.e., omic) analyses. The latter enable the simultaneous
quantification of the concentration of tens to hundreds to thousands of molecular
quantities from the RNA to protein to small molecule (i.e., metabolic) level. How-
ever, these are new technologies, most at the stage of standardization, and the current
number of omic analyses in plants is not extensive, especially in the case of inte-
grated analyses at various molecular levels of cellular function. Moreover, the inves-
tigation of intact plants using omic analyses presents unique challenges over similar
investigations in cell cultures or other biological systems, among which are the cur-
rent lack of full genome sequence information for most plants, long life cycles, and
poorly controlled conditions in field experiments. In this chapter, we present the tran-
scriptomic and metabolomic studies of salinity and elevated CO2 stresses in plants,
applied individually or in combination, emphasizing on the integrated analyses of
both levels of cellular function. The specifications of the experimental design for the
plant growth and the omic analyses, the challenges of such experiments, the acquired
results, and future directions for research and practice are also discussed.
3.2 Physiological Characteristics of the Plant Response

to High Salinity and/or Elevated CO2
3.2.1 High Soil and/or Water Salinity
High soil salinity can affect plants in multiple ways. High salt depositions in the
soil generate low water potential in the root zone, making it difficult for the plants
3 Investigating the Effect of Elevated CO2 in the Growth Environment … 51
to pump water. Thus, the physiology of high-salinity-stressed plants resembles the

physiology of drought-stressed plants (Mahajan and Tuteja 2005). To avoid loss of
water through osmosis, plants have to increase the osmotic pressure of their cells.
In light of this need, under salinity stress plant cells tend to accumulate metabolites
that act as osmolytes, e.g., proline (Delauney and Verma 1993; Ford 1984). The
net effect of this metabolic “deviation” is that the plants have to use part of their
resources towards the production of osmolytes, thus decreasing carbon flux towards
their growth (Kanani et al. 2010). Moreover, under high-salinity stress plants tend
to close their stomata to reduce water loss by transpiration. Carbon dioxide fixa-
tion through the Calvin cycle is then reduced and the photosynthesis rate declines
(Chaves et al. 2009). Furthermore, reduced CO2 in the chloroplasts combined with
intense light enhances the photoproduction of ROS (Asada 2006). High levels of
sodium may also have deleterious effect on the functioning of some of the enzymes
(Niu et al. 1995). Lower photosynthesis and transpiration levels in combination
with a lower flux towards the plant growth cause a decrease in the development and
productivity of the salinity-stressed plants (Cuartero and Fernandez-Munoz 1999;
Shannon and Grieve 1999). It is thus apparent that the salinity stress is a substantial
constraint to crop production especially in the arid and semiarid climates (Wang
et al. 2003). In greenhouses, the problem of high-salinity stress may be even more
intense, especially in the case of poor quality water in combination with high tem-
peratures (Ayers and Westcot 1985). In addition, in hydroponics, the salinity of the
small volume nutrient solution can increase rapidly, especially in closed systems
with nutrient solution recycling (Magan et al. 2008).
3.2.2 Elevated CO2 in the Growth Environment of the Plants
Short-term enrichment of the CO2 in the growth environment of the plants up to

three times the current ambient (375 ppm) level has a positive impact in the plants
as it stimulates photosynthesis and reduces stomatal conductance (Ainsworth and
Rogers 2007). However, the long-term exposure of plants to elevated CO2 leads to
photosynthetic acclimation and reduced CO2 uptake (Rogers and Ellsworth 2002).
Positive responses to elevated CO2 are mainly attributed to the competitive inhibi-
tion of the photorespiration by the carbon dioxide. Increase in the CO2 levels in
the growth environment of the plants increases carbon fixation. The elevated CO2
conditions can also enhance growth through improved plant water relations, since
the increased CO2 slows down the transpiration by inducing the partial closure of
stomatal guard cells of the leaves (Prior et al. 2011).
3.2.3 Combined Application of High Salinity and Elevated CO2
Based on the observed physiological characteristics of the plants under short-term

elevated CO2 and high-salinity treatment, the former perturbation can be beneficial
for the plant growth, while the latter initiates a series of negative physiological con-
sequences on plants upon its application. Thus, it is of interest to investigate how
this physiological “divergence” between the effect of these stresses on the plants
when they are applied separately, is finally manifested when these perturbations are
combined on plants. Interestingly, physiological (Geissler et al. 2010; Perez-Lopez
et al. 2009, 2012; Ratnakumar et al. 2013; Takagi et al. 2009) and high-through-
put omic studies (Kanani et al. 2010) have shown that short-term application of
elevated CO2 in the growth environment of salt-stressed plants can alleviate the
negative effect of high salinity on the plant growth. Different justifications have
been provided for this observation, with more prevalent the one supporting that
the additional CO2 contributes to the maintenance of the redox homeostasis of the
plants (Perez-Lopez et al. 2009). According to the presently single integrated tran-
scriptomic and metabolomic analysis of the combined high-salinity and short-term
elevated CO2 perturbations on Arabidopsis thaliana plant liquid cultures presented
below (Kanani et al. 2010), a major reason for the positive effect of the elevated
CO2 on the salt-stressed plants is the availability of additional carbon resources. The
latter enable the plants to produce the required osmoprotectant metabolites while at
the same time maintaining their normal growth rate.
3.3 Integrated High-Throughput Biomolecular Analyses

in Plant Systems Biology
The technologies for high-throughput biomolecular analysis (omics) have revolu-

tionized the way in which questions are approached in life sciences. Rather than
examining a small number of genes and/or reactions at any one time, we can now
begin to look at gene expression and protein activity in the context of networks and
systems of interacting genes and gene products (Sussman et al. 2009). Because our
knowledge of this domain is still not extensive, investigations are now routinely
moving from being purely “hypothesis driven” to being largely “data driven” with
analysis based on a search for biologically relevant patterns from which network
structures could be inferred. Recent developments have shown that educated use of
the existing biological knowledge in the application of data mining methods can in-
deed lead to the reconstruction of the active biomolecular networks at each level of
molecular function that characterize a particular physiology (“knowledge”-driven
approach). These technological advances have created enormous opportunities for
accelerating the pace of science. One can now envision the possibility of obtain-
ing a comprehensive picture of the mechanisms underlying the cellular function,
its regulation, and the interactions of an organism with its environment. While the
greatest attention to date has been paid to gene sequence and transcriptional expres-
sion analysis using mainly microarrays, it is becoming increasingly clear that these
alone cannot be used to accurately determine cellular function and system physiol-
ogy. Rather, a comprehensive analysis of biological systems requires the integration
of all fingerprints of cellular function (Vidal 2009), i.e., genome sequence, tran-
scriptional, proteomic, and metabolic profiles, and flux distributions. While each of
these fingerprints has significant value on its own, the picture that emerges from any
single approach is quite limited in nature. Gene transcription is a necessary but not
Fig. 3.1 Omic profiling analyses are multistep procedures with potential sources of systematic
biases at any stage
a sufficient condition for high in vivo protein production. Regulation of translation,

RNA and protein stability, and posttranslational modifications can alter the linear
relationship between the message and the corresponding protein. Additionally, a
protein could be present in high concentration, but it may lack the requisite con-
ditions (substrate concentration, cofactors, etc.) for activity in the actual cellular
environment. Moreover, in the context of the regulatory networks, a modification in
the expression levels of a gene is not expected to alter only the concentration of the
corresponding protein and the activity of the corresponding biochemical reaction,
but it would also affect other parts of the cellular networks depending on the role of
this gene in cellular function. Therefore, it is the integration of all of these molecu-
lar profiles for a systematically perturbed cellular system that can provide insight
about the function of unknown genes, the relationship between gene and metabolic
regulation, and even the reconstruction of the gene regulation network (Klapa and
Quackenbush 2003; Vidal 2009). To succeed in the challenge of quantitative sys-
tems biology, however, major issues concerning the quantification capabilities and
sources of biases of these multistep molecular analyses (Fig. 3.1) need to be thor-
oughly resolved for each level of molecular function and for the specific needs of
each investigated biological system. They range from limitations in the available
experimental protocols, to lack of data analysis and visualization techniques for
upgrading the information content of the acquired measurements.
In comparison with the profiling analyses of other molecular levels of cellu-

lar function, the main asset of transcriptomics derives from the relative simplicity
of its subject: mRNA is a polymer of only four different subunits, unlike proteins
that are composed of 20 different amino acids and have various 3D structures and
metabolites that have great chemical diversity. Thus, a single method of extraction
and detection can theoretically identify and quantify every transcript in a tissue
sample. As a result, transcriptomic studies tend to identify at least one order of
magnitude more gene products than proteome studies (Baginsky 2009; Deyholos
2010). Moreover, the protein and metabolic levels are highly dynamic and environ-
ment sensitive. Currently, microarrays (Pease et al. 1994; Schena et al. 1995) have
been the main platform used for transcriptomic studies in plants and in general in
most biological systems. Microarrays have proven to be a reliable technological
platform for the study of gene expression patterns, because of their relatively high
sensitivity, specificity, accuracy, throughput, and cost-efficiency. However, array-
based technologies are limited to the analysis of known transcripts. This limitation
can be bypassed with transcriptome analysis based on the next-generation (“deep”)
sequencing platforms (Wang et al. 2009), which have not yet gained adequate mo-
mentum in plant physiology studies. Recently, Mizuno et al. (2010) conducted a
study on the transcriptional effects of salinity stress on rice using both RNA deep se-
quencing and microarrays. RNA sequencing predicted the expression of more than
3000 transcripts not previously annotated by the Rice Annotation Project. Some of
the unannotated genes were differentially expressed in response to salinity stress
(Mizuno et al. 2010).
Metabolomic analyses provide the link between gene expression and the meta-
bolic phenotype, the latter being very sensitive to the physiological responses caused
by environmental perturbations on the plants. It has been estimated that about tens
of thousand primary and secondary metabolism intermediates (metabolites) occur
in the plant kingdom (Fiehn 2002). The metabolite concentration profile is affected
and also affects the metabolic reaction rates, being thus a fingerprint of the meta-
bolic state of the cells and tissues. Most metabolites act as regulatory molecules of
protein functions and interactions, their accurate quantification being of additional
importance for deciphering the molecular mechanisms that impose the physiology
of the plants under specific conditions. Because of the chemical diversity of me-
tabolites, metabolomic analysis is subject to analytical constraints that limit the
number of metabolites that can be identified and quantified in a single sample. Cur-
rently, there is no extraction protocol and technological platform that can detect and
quantify the total metabolome. Most often, extraction protocols of polar and semi-
polar compounds are used in the metabolomics studies, as they capture a larger
chemical diversity range. The most common technological platforms used for me-
tabolome analysis are liquid or gas chromatography coupled with mass spectrome-
try (LC–MS or GC–MS), capillary electrophoresis coupled with mass spectrometry
(CE–MS), and nuclear magnetic resonance (NMR) spectroscopy. Each platform
has certain analytical limitations and a single platform can detect only a fraction of
the total metabolome. The combined use of multiple analytical techniques, if avail-
able, can increase the fraction of the observable metabolome. Depending on the
tissue, such a protocol and analytical technique will extract the components of the
primary metabolism, like sugars (monosaccharides and oligosaccharides), organic

acids, amino acids, phosphate compounds, and amines, providing thus an extensive
perspective of the primary (central carbon) metabolism. The primary metabolism is
indicative of the energy, redox homeostasis, and growth demands of the plant cells,
while it produces all the precursors of the cellular macromolecules and secondary
metabolites. The changes in the primary metabolism reflect core perturbations in
the metabolic physiology of the plants. Conserved among species metabolic re-
sponses to environmental stress acclimation should be observable within the prima-
ry metabolism. On the other hand, the secondary metabolism is more diverse among
species and presumably reflects the successful adaptation of a species to particular
environmental stresses through the acquisition of novel biosynthetic capacities of
its primary metabolism (Sanchez et al. 2008a). Therefore, even if there are differ-
ences in the secondary metabolism, they can be inferred from the changes in the
concentration profile of precursor molecules in the primary metabolism.
A major advantage of the high-throughput biomolecular analyses is that by ob-
serving a large number of molecular quantities at the same time, correlations be-
tween the activity of various molecular pathways can be determined, new knowl-
edge can be extracted, and the biomolecular networks at different levels of cellular
function (e.g., gene regulation, protein interaction, or metabolic networks) can be
reconstructed. To this end, we are in search of multi-compound biomarker profiles
and patterns of expression, rather than single molecules that can be sensitive sensors
of changes in the physiology of the plants. Thus, the acquired datasets have to be
analyzed with multivariate statistical methods, attempting to identify either clusters
of genes or gene products that have similar expression or concentration, respec-
tively, profiles among various physiological conditions, or physiological states that
are of similar omic profiles. For this purpose, clustering, e.g., hierarchical clustering
(HCL), analysis, and dataset dimension reduction and visualization, e.g., principal
component analysis (PCA), methods are used. Customized multivariate significance
analysis methods for omic data, like significance analysis for microarrays (SAM)
(Tusher et al. 2001), have been developed enabling the identification of the genes or
gene products, the change in the expression or concentration, respectively, of which
is characteristic of the difference between two sets of physiological conditions. In
the case of time-series experiments, particular modifications of PCA (Scholz et al.
2005) and SAM analyses (Dutta et al. 2007) have been proposed to take into con-
sideration that the physiological states of the plants at the different time points are
not independent, but rather part of the same physiological history.
3.4 Omic Analyses of Salinity Stress on Plants
3.4.1 Metabolomic Analyses
The effect of salt stress on plant metabolic physiology using metabolomic analyti-
cal platforms has been investigated in the context of maize (Gavaghan et al. 2011),
barley (Widodo et al. 2009), and grapevine (Cramer et al. 2007) in addition to the
studies using the model organism A. thaliana (Kanani et al. 2010; Kim et al. 2007;
Gong et al. 2005). Major aspects of each study that need to be considered when at-
tempting to unify their results are the selected level of the salt stress and the duration
of the treatment. Treatment durations can be categorized into: (a) short term, i.e., up
to 24–30 h, (b) mid-term, i.e., from few days up to one week, and (c) long term, i.e.,
longer than one week up to few months.
Gavaghan et al. (2011) studied the mid-term responses of maize to high-salinity
(i.e., 50 and 150 mM NaCl) stress using NMR spectroscopy. They observed a
significantly increased concentration of sucrose, γ-aminobutyric acid (GABA),
glycine-betaine, and free amino acids, including alanine, in the roots of the salt-
stressed plants. The changes correlated with the salt concentration, suggesting thus
a response mechanism for the plants to maintain osmotic balance. The concentra-
tions of citrate, malate, succinate, and α-ketoglutarate declined in the shoot extracts
in response to the salinization. The depletion of these tricarboxylic acid (TCA)
cycle intermediates implies that the TCA cycle flux is reduced in the shoots as a
result of the salt stress, hence the plant growth and energy metabolism is slowed
down or arrested. Differences between the responses of salt tolerant and salt sensi-
tive cultivars to salinity stress were observed in rice plants after long-term treatment
with 100 mM NaCl (Zuther et al. 2007). Even the tolerant cultivars did not have
common responses to salinity stress, but formed physiological response subgroups.
One common response to salinity stress for most cultivars was the depletion of TCA
cycle intermediates, in agreement with the results of the previously described maize
study. Hence, both studies suggest that the acclimation to high salt concentrations
has a high demand for energy, competing thus with the plant growth.
Lu et al. studied the mid-term response to the salinity (i.e., 100 mM NaCl) stress
of two varieties of soybean using GC–MS and LC–MS metabolomics (Lu et al.
2013). In leaf samples from salt-stressed plants of both varieties, they observed a
significant reduction in the concentration of alanine, sucrose, and TCA cycle in-
termediates and a significant increase in the concentration of abscisic acid (ABA),
glycine, serine, and sugar alcohols, such as lactitol and maltitol, compared to the
control conditions. ABA is a plant hormone that accumulates under drought stress
and causes stomata closure. The ABA-induced stomata closure reduces transpira-
tion, thus preventing further water loss from the leaves in times of low water avail-
ability (Steuer et al. 1988). Sugar alcohols and amino acids can act as osmolytes and
their increase under salt stress is a response mechanism for the plants to maintain
osmotic balance, balancing the decreased water potential associated with the sodi-
um ion accumulation in the vacuoles and the extracellular volume, as stated above.
The reduction in the concentration of sucrose and TCA cycle intermediates suggests
the high energy cost for the acclimation to salinity stress that was observed in all rel-
evant studies discussed so far. The accumulation of osmolytes under salinity stress
has also been observed in grapevines after mid- and long-term treatment (Cramer
et al. 2007). The shoot concentrations of fructose, glucose, proline, glycine, and
malate increased in the salinized compared to the control plants. The observed in-
crease in the malate concentration was consistent with the significant increase in the
transcripts of the glyoxysomal and chloroplastic malate dehydrogenases and the de-
creased abundance of transcripts of the cytoplasmic and mitochondrial malate dehy-
drogenases. Moreover, the accumulation of proline was consistent with an increase
in the transcript abundance for delta 1-pyrroline-5-carboxylate synthetase (P5CS),
the enzyme that catalyzes the first two steps in the proline biosynthetic pathway.
Kanani et al. (2010) observed that after a short-term (i.e., 30 h) continuous ex-
posure to high salinity, A. thaliana plant liquid cultures accumulated fatty acids and
sterols including tocopherol, a known antioxidant. A significant increase was also
observed in the levels of homo-serine, β-alanine, methionine, glycine, N-acetyl-
glutamate, allantoin, and the TCA cycle intermediates from citrate to fumarate
throughout the treatment period. Homoserine and methionine are precursors of the
S-adenosyl-methionine, which is required along with glycine for the biosynthesis of
glycine-betaine, the main osmoprotectant in A. thaliana, and along with β-alanine
for the production of β-alanine-betaine. Polyamines and betaines are known osmo-
lytes in plants. As it was the case with the previously discussed studies, these ob-
servations are in accordance with the need of the plants to produce osmoprotectants
and antioxidants to counteract the stress conditions. At the same time, the increased
production of amino acids/amine group containing metabolites that are precursors
of osmoprotectants and antioxidants was accompanied by a significant decrease
in the concentration of metabolic intermediates that are required for plant growth.
Moreover, based on their time-series analysis, Kanani et al. were able to observe
a change in the metabolic physiology of the plants even from the first hour of the
salinity treatment.
Sanchez et al. (2008a) studied comparatively the metabolic responses of A. thali-
ana, Lotus japonicus, and rice after long exposure and potential acclimation of the
plants to salinity stress (i.e., 75, 150, and 100 mM NaCl for each plant species,
respectively). They reported a salinity dose-dependent increase in the concentra-
tion of sucrose and amino acids like proline, glycine, serine, threonine, leucine,
and valine, in all the three species. The TCA cycle intermediates, citrate, succinate,
malate, and other organic acids, such as oxalic and maleic acids, which are direactly
related with the TCA cycle flux, exhibited conserved reduction in their pool sizes in
response to long-term salinity stress. Reduction was also observed in the concentra-
tions of the glycolysis intermediates glucose, fructose, glucose-6-phosphate, and
fructose-6-phosphate. The authors suggest that a reason for the reduced acid levels
under salt stress may be their involvement in the compensation of the ionic imbal-
ance. At physiological pH levels, organic acids exist as carboxylic anions and coun-
terbalance inorganic anions, so a depletion of organic acids may actually reflect
preferential uptake of anions compared to cations. Moreover, the increased amino
acid biosynthesis may also serve the plants to absorb excess ammonium while pro-
ducing osmolytes. Excess organic acids could be recruited from the TCA cycle and
sequestered into the biosynthesis pathways of amino acids and amines. Thus, the
maintenance of the charge balance, the ammonium detoxification, and the compat-
ible solute accumulation could all be met by a common mechanism.
Gong et al. (2005) compared the short-term responses to salinity (i.e., 150 mM
NaCl) stress of A. thaliana and Thellungiella halophila, a species related to
A. thaliana with extreme tolerance to a variety of abiotic stresses, including low

humidity, freezing, and high salinity. As expected, metabolites that act as osmolytes,
i.e., proline, galatinol, and glycine, increased under salt stress in both species. It has
to be noted that, at the control conditions, the concentration of several compounds
that have protective functions was much higher in T. halophila than in the A. thali-
ana plants. Maybe, this concentration profile could partly justify the T. halophila
surviving mechanisms under extreme salt concentrations. Widodo et al. (2009)
studied the long-term responses of two barley cultivars to salinity (i.e., 100 mM
NaCl) stress using GC–MS. After three weeks of high-salinity treatment, the more
sensitive cultivar ceased growing, while the tolerant resumed similar growth to the
control plants. At the metabolic level, the sensitive cultivar exhibited an increase
in the levels of proline, GABA, and the polyamine putrescine, most in accordance
with the previous salinity studies. They suggested, however, that the observed in-
crease in these metabolites is not an adaptive response to salinity but an indication
of slower growth or tissue necrosis. On the other hand, in the tolerant plants, the
levels of TCA cycle intermediates and hexose phosphates increased in response to
salt. However, the response of each cultivar to salinity stress depended heavily on
the duration of its exposure to high salinity.
3.4.2 Transcriptomic Analyses
Many DNA microarray transcriptomic studies of the plant response to high salinity
have been reported in the literature (Sanchez et al. 2008b, 2011; Beritognolo et al.
2011; Bazakos et al. 2012; Kanani et al. 2010; Legay et al. 2009; Jankangram et al.
2011; Gong et al. 2005; Chao et al. 2005; Evers et al. 2012; Wang et al. 2013; Cra-
mer et al. 2007). Main common observations of these studies are: (a) the significant
decrease in the transcripts related to photosynthesis, i.e., the photosystem I and
II subunits, Calvin cycle enzymes, RuBisCO subunits and the RuBisCO activase,
protein synthesis and energy metabolism pathways (Beritognolo et al. 2011; Kanani
et al. 2010; Legay et al. 2009; Gong et al. 2005; Chao et al. 2005; Evers et al. 2012;
Wang et al. 2013) and (b) the simultaneous significant increase in the abundance of
transcripts related to signaling, membrane transporters, and the synthesis of osmo-
protectants and antioxidants (Deyholos 2010). These observations are in agreement
with the known decrease in the photosynthesis rate of the salinized plants based on
physiological studies (Chaves et al. 2009) while providing molecular insights about
this decrease. Interestingly, however, Cramer et al. report an increase in the tran-
script levels of the photosystem I and II subunits and the RuBisCO activase after
long exposure of grapevines to progressive salinity stress (Cramer et al. 2007). This
could be a secondary response of the specific species after long exposure to salin-
ity stress that ensures the survival of the plant. It also underlines the significance
of considering all parameters of the experimental design, including the treatment
duration and strength, when trying to integrate the results among different studies.
In the salt-stressed plants, the abundance of transcripts encoding proteins related
to cellular growth like histones (Kanani et al. 2010; Gong et al. 2005) and the as
primary metabolism (Beritognolo et al. 2011; Legay et al. 2009; Evers et al. 2012)
was significantly decreased, suggesting that the salinity stress affects in a negative
way the plant-growth-related pathways at the transcriptional level.
The levels of transcripts encoding late embryogenesis abundant (LEA) (Sanchez
et al. 2011; Legay et al. 2009; Chao et al. 2005; Wang et al. 2013; Cramer et al.
2007) and heat shock proteins (HSP) (Beritognolo et al. 2011; Legay et al. 2009)
tend to increase after exposure of plants to high salinity. LEA proteins are small hy-
drophilic, largely unstructured, and thermostable proteins that are synthesized in the
seeds during maturation. It is believed that they play a protective role against desic-
cation through multiple functions, including ion binding, hydration buffering, and
membrane and protein stabilization (Battaglia et al. 2008). Most HSPs have been
shown to act as molecular chaperones, which are responsible for protein synthesis,
targeting, maturation, stabilization, refolding under stress conditions, and degrada-
tion in a broad array of normal cellular processes. Moreover, the HSPs participate in
the membrane stabilization under stress conditions (Wang et al. 2003).
Consistent with the findings from metabolomics, most transcriptomic studies
of the salinity effect on plants record increased the abundance of gene transcripts
involved in the biosynthesis of osmolytes (Sanchez et al. 2011; Legay et al. 2009;
Gong et al. 2005; Chao et al. 2005; Evers et al. 2012). The abundance of transcripts
related to the ROS scavenging and detoxification has in some studies been reported
as increasing (Beritognolo et al. 2011; Gong et al. 2005; Chao et al. 2005; Cramer
et al. 2007) and in some others as decreasing (Legay et al. 2009; Evers et al. 2012;
Wang et al. 2013), after the plants are exposed to salinity stress. This discrepancy
could be an indication that in some cases ROS act as signaling molecules for the
salinity stress and have thus to attain high concentrations to trigger other reactions,
or it could just be a consequence of different durations of plant exposure to stress.
Transcripts that encode ion and amino acid transporters also accumulate in the
plants after exposure to the salinity stress (Beritognolo et al. 2011; Kanani et al.
2010; Legay et al. 2009; Gong et al. 2005).
The activity of the salt overly sensitive (SOS) signaling pathway is of particular
interest regarding the response of the plants to the salt stress. This pathway is re-
sponsible for the extracellular and vacuolar sequestration of the Na+ ions with H+/
Na+ antiporters, a process of high significance for the ion homeostasis of the plants
(Zhu 2002). The Na+ ion increase caused by the salt stress could be detrimental to
the plants, causing membrane disorganization, impaired nutrient and water acquisi-
tion, metabolic toxicity, inhibition of photosynthesis, and the production of ROS
(Niu et al. 1995). In a transcriptomic analysis of A. thaliana plant liquid cultures
under salt stress, the activity of the SOS pathway was indeed observed as signifi-
cantly increased at the transcriptional level (Kanani et al. 2010).
3.4.3 Results of Integrated Metabolomic and Transcriptomic

Analyses
Some of the above-mentioned studies carried out both transcriptomic and

metabolomic analyses on the same set of plants, in an effort to comprehensively
investigate the changes in the physiology of the plants due to the high-salinity stress
at the two molecular levels of cellular function. Some observations were consistent
between the transcriptional and metabolic level, indicating regulation of the relevant
response mechanisms at the transcriptional level, which are then by consequence re-
flected at the metabolic level too. For example, Cramer et al. (2007) and Gong et al.
(Gong et al. 2005) reported that the proline accumulation after salinity treatment
was consistent with the observed increase in the abundance of transcripts encod-
ing enzymes in the proline biosynthesis pathway (Cramer et al. 2007; Gong et al.
2005). However, other results at the metabolic level would not have been directly
predictable if only the transcriptomic information had been available, indicating thus
regulatory mechanisms that are active at the metabolic level. There are also pro-
cesses that are not directly involved in metabolism and cannot thus be directly ob-
servable through the metabolic profiles, but only through the transcriptomic p rofiles,
like photosynthesis, ethylene signaling, and others. Integrated omic analyses at mul-
tiple molecular levels are thus required for the comprehensive understanding of all
physiological changes due to a particular stress.
3.5 Omic Analyses of Elevated CO2 Stress on Plants
The effect of the elevated CO2 concentration in the growth environment of the
plants has been extensively studied with both physiological and high-throughput
biomolecular analysis studies at the transcriptional, protein, and metabolic levels,
mainly in the context of long-term (i.e., 1–2 weeklong) adaptation to high CO2 en-
vironments. The main reason for these studies has been to investigate how the plants
will change their physiology in response to the greenhouse effect. Li et al. (2008)
conducted a free-air CO2 enrichment (FACE) experiment to study the metabolic and
transcriptional effects of elevated CO2 (i.e., 550 ppm) in the growth environment of
two A. thaliana ecotypes. At the metabolic level, they observed an increase in the
concentration of sugars, like maltose, glucose, fructose, and galactose, and of TCA
cycle organic acid intermediates, along with a decrease in the levels of most amino
acids, with the exception of the aromatic amino acids tryptophan and phenylalanine,
the concentration of which increased under elevated CO2. In accordance with the
metabolomic results, transcriptomic analysis indicated an increase in the concen-
trations of transcripts related to the cell wall formation and metabolic processes
like the glycolysis, the TCA cycle, and the anthocyanin and flavonoid biosynthesis.
Moreover, transcripts related to the amino acid biosynthesis were downregulated or
did not change, with the exception of those involved in the tryptophan and phenyl-
alanine biosynthesis. The abundance of transcripts related to photosynthesis, like
the photosystem I and II subunits, as well as Calvin cycle enzymes, was reduced
in plants treated with elevated CO2 for long durations. The amount of transcripts
encoding chloroplast-localized proteins unrelated to light capture and fixation func-
tions also declined significantly. The authors suggested that these changes reflect ni-
trogen deprivation. Increased photosynthetic CO2 fixation altered the apparent C:N
balance. The findings of Miyagi et al. (2011) were consistent with this h ypothesis.
After four weeks of growth in 1000 ppm of CO2, Rumex obtusifolius plants had
increased levels of TCA cycle intermediates, especially citrate and fumarate, while
the amino acid levels, apart from phenylalanine and tryptophan, decreased. On the
other hand, plants that were grown under elevated CO2 in a medium rich in nitrogen
exhibited increased levels of TCA cycle intermediates and amino acids compared to
the control or just nitrogen-rich conditions. In contrast with the above findings, Ka-
plan et al. (2012) reported decreased levels of TCA cycle intermediates and glycine
and increased levels of sugars in A. thaliana plants after long-time exposure to 1200
and 4000 ppm of CO2. In the same plants, the concentration of transcripts related
to starch synthesis and catabolism increased, with a simultaneous decrease in tran-
scripts related to photosynthesis, like the photosystem and RuBisCO subunits. The
amount of transcripts for genes that are inducible by ABA and jasmonic acid was
also increased by elevated CO2. The authors suggested that the elevated CO2 condi-
tions reduce respiration and act as a stressor for plants. All these discrepancies in the
findings from the discussed studies underline the importance of carefully examining
the physiological conditions to which each study refers with respect to the duration
and severity of treatment, the plant species that is investigated, the tissue or cell type
analyzed, and the type of plant culture (hydroponic or other), to accurately interpret
and potentially generalize the observed results.
Dutta et al. (2009) examined the responses of A. thaliana plant liquid cultures to
elevated CO2 (i.e., 10,000 ppm) over a short period of 30 h in a time-series experi-
ment using integrated metabolomic and transcriptomic analyses. It was observed
that the plants which were grown in the elevated CO2 environment had decreased
pools of all the three organic acids (glycerate, glyoxylate, glycolate) and serine
in the photorespiration pathway and decreased expression of the photorespirato-
ry pathway genes at most of the examined time points. Interestingly, differences
were observed between the responses of the plants at the earlier compared to the
later time points of the experiment. Specifically, during the first six hours of the
experiment, the levels of most amino acids (i.e., glutamine, asparagine, aspartate,
arginine, valine, isoleucine, glycine, methionine, lysine, and GABA) increased.
An increase was also observed in the levels of the TCA cycle intermediates citrate
and isocitrate. However, beyond twelve hours of continuous exposure to elevated
CO2 conditions, the levels of almost all amino acids decreased. The transcriptomic
analysis showed that at the early time points, the abundance of transcripts associ-
ated with the ribosomes decreased, whereas at the later time points many of the
transcripts related with photosynthesis had a reduced abundance in response to the
elevated CO2, implying thus potential closure of stomata after a twelve hour expo-
sure to elevated CO2.
The above-mentioned data suggest that after a particular duration of growth under
elevated CO2 conditions, the plants seem to acclimate to the particular environment
and the expression of genes related to photosynthesis declines. However, carbon
fixation remains higher than in the ambient CO2 conditions. Thus, after a certain
duration of exposure to elevated CO2, the carbon to nitrogen ratio increases and the
nitrogen becomes the limiting factor for the plant growth. Therefore, the levels of
amino acids are expected to decrease after a long-time exposure to elevated CO2.
3.6 Omic Analysis of Plant Response to Combined

High-Salinity and Elevated CO2 Perturbations
Despite the fact that physiological measurements in different plants and trees have
indicated that the elevated CO2 conditions can alleviate the negative effect of salinity
stress in plants at least for short-term treatments (Geissler et al. 2010; Perez-Lopez
et al. 2012; Perez-Lopez et al. 2009; Ratnakumar et al. 2013; Takagi et al. 2009), to
the best of our knowledge, there has currently been only one study, which has moni-
tored the molecular response of the plants to combined salinity stress and elevated
CO2, using the high-throughput biomolecular (omic) analyses. Kanani et al. (2010)
integrated GC-MS metabolomics and DNA microarray transcriptomics to study the
growth of A. thaliana plant liquid cultures in a high-salinity (i.e., 50 mM NaCl) me-
dium and elevated CO2 (10,000 ppm) environment, for the first 30 h of continuous
treatment in a time-series experiment. The plants had grown under constant light,
temperature, and humidity and the same conditions were maintained throughout the
treatment period. The authors support this setup, as it minimizes any contributions
to the observed physiological changes from any other parameter but the two inves-
tigated factors. The authors report that the effect of the salinity stress was stronger
than that of the elevated CO2 conditions at both the transcriptional and metabolic
levels. Interestingly, there was a strong similarity over time between the transcrip-
tomic responses of the plants exposed to high salinity and those exposed to the
combined stress. This similarity suggests that the early transcriptional response of
the plant cultures to the salinity stress is robustly active independently of the co-oc-
currence of the elevated CO2 conditions. For example, the SOS signaling pathway
is upregulated at the transcriptional level under both high-salinity and the combined
perturbation conditions. The major finding of this analysis, however, was that the
observed physiological consequences of the combined stress at the metabolic level
was different from what would have been expected based only on the transcriptomic
profiles. Specifically, the combinatorial effect of the elevated CO2 conditions and
the salinity stress on the metabolic physiology of the plants was milder than that of
the salinity stress alone, implying that the elevated CO2 conditions are an alleviat-
ing factor for the salt-stressed samples. The analysis of the metabolomic profiles
indicated that this beneficiary role of the elevated CO2 can be primarily attributed
to the provision of additional resources to the salt-stressed plants. Using these ad-
ditional resources the plants can activate their response machinery against high sa-
linity and produce osmoprotectants and antioxidants, without having, however, to
sacrifice substrates needed for plant growth. This conclusion was based on the fact
that, under the combined stress, the concentrations of the TCA cycle intermediates
citrate, aconitate, and isocitrate, and the amino acids alanine, valine, lysine, and
asparagine, which contribute to protein synthesis, were observed at similar values
as in the control metabolic state. At the same time, metabolic precursors of osmo-
protectants that exhibited increased concentration in the salt-stressed plants (i.e.,
S-adenosyl-methionine and glycine, which are precursors of glycine–betaine and
β-alanine, which is a precursor of β-alanine-betaine) retained their concentrations
in the plants subjected to the c ombined high-salinity and elevated CO2 perturbation.
The alleviating role of the elevated CO2 in the growth environment of salt-stressed
plants was also supported by the downregulation of transcripts related to the ethyl-
ene signaling, a pathway that is characteristically upregulated at the transcriptional
level in plants exposed to salt stress. These observations suggest that the controlled
use of the CO2 in greenhouses could offer a pragmatic solution for counteracting
the negative effect of high soil or water salinity and lead to plant crops of consistent
quality and yield.
3.7 Experimental Design Specifications of Integrated

Omic Analyses of Combined Salinity and Elevated
CO2 Stresses
A controlled study of the combined effect of high salinity and elevated CO2 on the
plants should include at least four plant groups: plants grown in a control medium
or soil and the ambient CO2 concentration (i.e., the control group), plants grown in
a high-salinity medium or soil and the ambient CO2 concentration (i.e., the high-sa-
linity group), plants grown in a control medium or soil and an elevated CO2 concen-
tration (i.e., the elevated CO2 group), and plants grown in a high-salinity medium
or soil and an elevated CO2 concentration (i.e., the combined perturbation group).
In this type of studies, hydroponic cultures provide a more controlled system over
the soil-grown plants, as the effect of any perturbations in other growth parameters,
e.g., the nutrient composition of the soil, are minimized. Comparison between the
four measured physiological states can provide information about changes in the
physiology of the plants due to stresses that are not directly measured. For example,
the comparison between the omic profile of the combined perturbation and the high-
salinity groups can provide information about the effect of the elevated CO2 on the
salt-stressed plants, even when this experiment has not been carried out. This is how
the alleviating role of the elevated CO2 on salt-stressed plants was identified in Ka-
nani et al. (2010). Depending on the investigated species (or cultivars or ecotypes),
the imposed salinity should be high enough to act as stressor for the plants, but
not too high to cause tissue necrosis. In the high-salinity experiments, the utilized
salt concentration usually ranges from 50 to 150 mM, reaching 250 mM in some
studies of halophile species. In most reported studies, elevated CO2 conditions are
characterized by concentrations between 500 and 1500 ppm to simulate the plant
responses to the predicted increase in the ambient CO2 due to the greenhouse effect
(Kaplan et al. 2012; Li et al. 2008; Miyagi et al. 2011). However, in some studies, a
much greater CO2 concentration has been used to ensure changes in the physiology
of the plants due to this perturbation (Kaplan et al. 2012; Dutta et al. 2009). Apart
from growth chambers, FACE facilities have also been used to study the effect of
the elevated CO2 on the plants (Li et al. 2008). At these facilities, horizontal or verti-
cal pipes are placed in a 1 m to 30 m diameter circle around the experimental plot,
and emit CO2-enriched air around the plants (Ainsworth and Long 2005).
As discussed above, the responses of the plants to various perturbations depend

also on the duration of the treatment. Time-series experiments are preferable to
gain deeper insight into the physiology of the plants as a function of the exposure
duration to stress. The first sampling should be carried out just before the perturba-
tion/treatment is applied to monitor the physiological state of the plants at the time
zero of the experiment. The rest of the samplings should be scheduled in such a
way to allow for the determination of the short-, mid-, and long-term responses
of the particular plant species (or cultivar or ecotype) to the applied perturbation/
treatment. When scheduling the sampling points, the circadian rhythm of the plants
and the difference in the timescale of the response between the transcriptional and
the metabolic processes should also be taken into consideration. Cramer et al. con-
ducted an interesting time-series experiment to study the metabolic responses of the
grapevine to high salinity and drought (Cramer et al. 2007). Instead of using one
fixed concentration of NaCl for the high-salinity “perturbed” plants, they started the
experiment with zero salt concentration in the irrigating solution and increased it
gradually over time. This experimental design enabled them to make the separation
between the plant responses due to the water-deficit effects and those arising from
ionic effects within the plants.
Three to six plants per group and per time point usually provide an adequate num-
ber of biological replicates for the extraction of accurate results from omic analyses.
In most of the studies cited above, leaves were sampled. Leaves are the main photo-
synthetic organs of plants and their reaction to elevated CO2 and the salt stress is of
great interest. However, the first tissues that experience the salinity of the medium
or the soil are the roots, so it would be of value for the roots to be sampled and their
response to the applied perturbation(s) to be studied in comparison with that of the
leaves. Immediately after sampling, the collected samples should be frozen in liquid
nitrogen and kept at − 80 °C until further processing. Freezing with liquid nitrogen
is essential to stop all the enzymatic processes in the samples and to protect thermo-
sensitive molecules like sugar phosphates and mRNA molecules from degradation.
If the collected amount permits, the same sample should be divided and used for the
extraction of mRNAs, proteins, and metabolites for integrated omic analyses.
Great care should be paid to eliminate or correct for various systematic biases
introduced at various stages of the multistep omic analyses (see Fig. 3.1). For
example, in a typical metabolomic analysis, injection errors or unequal division
of a sample into replicates could affect the metabolic profiles. These types of er-
rors affect equally all metabolites detected in a profile. To account for these biases,
internal standard normalization is required. The selected internal standard should
not be produced by the plant species of interest. Ribitol or isotopes of known me-
tabolites are commonly used as internal standards in plant MS metabolomics (Fiehn
et al. 2001; Roessner et al. 2000). Errors that affect specific metabolites may also
occur. In the case of GC–MS metabolomics, the metabolites have to be deriva-
tized so that they are converted to their volatile, nonpolar, and thermostable deriva-
tives. The most common method for derivatization involves the conversion of the
original metabolites to their methoxime (MEOX) and trimethylsilyl (TMS) deriva-
tives (Roessner et al. 2000). However, some metabolites produce more than one
d erivative with relative concentrations depending on the derivatization conditions

and time. Thus, the derivatization step itself can introduce biases affecting each
metabolite in a different way depending on its structure, concentration, and relative
affinity for the derivatizing agent. If these biases are not identified and properly ac-
counted for, they will skew the measured metabolite concentration profile providing
a faulty perspective of the metabolic state of the samples. Kanani and Klapa (2007)
proposed a normalization method for this type of biases. Proper normalization of
the omic profiles at each molecular level before data analysis is quite crucial to es-
tablish comparability between the samples and avoid assigning biological meaning
to experimental biases.
In addition, in integrated transcriptomic and metabolomic analysis of (combined)
stresses in plants, the final interpretation of the results should take into consideration
the different specifics of the two analyses. Transcriptomic analyses are based on the
comparison of the concentration profile of mRNA transcripts in an equal amount
of total mRNA between for all samples. It is considered that the cells of the same
species produce equal amount of total mRNA independently of their physiologi-
cal state. Thus, the transcriptomic analysis of a biological sample among different
physiological states provide a measure of the change in the composition of the total
mRNA of this sample among different physiological conditions. On the other hand,
the amount of the acquired metabolite extract of a biological sample can change
between states. The internal standard is added to allow for sample normalization
per unit of mass of the investigated biological system. In this way, it is mainly the
change in the amounts and to a lesser extent the change in the relative concentra-
tions of the metabolites in the extract that governs the observed differences among
the metabolic profiles of the various samples (Kanani et al. 2010).
3.8 Conclusions
In the post-genomic era, high-throughput biomolecular (omic) analyses have been

used to gain insight into the molecular response of the plants to abiotic stresses.
Among the mostly investigated abiotic stresses are the high soil or water salinity
and the elevated CO2 in the growth environment of the plants. The most popular
omic analyses for this type of stresses have been the MS metabolomics and the
transcriptomics based on DNA microarrays. While physiological studies have in-
dicated that the elevated CO2 can alleviate the negative effect of the salinity stress
in plants, only one time-series omic analysis study has been reported so far for
the combined implementation of elevated CO2 and salinity stresses on plants. The
particular study on A. thaliana plant liquid cultures indicated that the elevated CO2
provides additional resources to the plants allowing them to produce the required
osmoprotectants to counteract the salinity stress without having to sacrifice their
growth (Kanani et al. 2010). However, for the insights garnered from this model
system study to be directly applicable in crop improvement and production, species
and cultivars of commercial value for the food industry and/or agro-biotechnology
should be studied. Plants such as tomato or pepper are of great interest because they
are mainly cultivated in greenhouses where the levels of CO2 could be adjusted. The
identification of molecular biomarkers for the salinity stress in plants could help
monitoring the progress of its negative effect on the plant growth and yield of salt-
stressed plants. The latter can further our understanding of the underlying molecular
mechanisms and assist us in devising methods for the educated use of elevated CO2
conditions to alleviate the salinity impact, supporting plant cultivation processes of
consistent quality and yield.
Acknowledgments We gratefully acknowledge the “PHYTOALATOTITA” research Grant

No. 09ΣΥΝ-22–797 of the “Cooperation I” Action/Sub-Action I “Small-Scale Cooperative Proj-
ects” of the Greek General Secretariat for Research and Technology (GSRT-GR), funded by the
European Social Fund (ESF), and National Resources of Greece under the Operational Programs
“COMPETITIVENESS and ENTREPRENEURSHIP” of the National Strategic Reference Frame-
work (NSRF) 2007–2013, for funding the PhD fellowship of Mr. M.-E. P. Papadimitropoulos.
References
Ainsworth EA, Long SP. What have we learned from 15 years of free-air CO2 enrichment (FACE)?
A meta-analytic review of the responses of photosynthesis, canopy properties and plant produc-
tion to rising CO2. New Phytol. 2005;165(2):351–71. doi:10.1111/j.1469-8137.2004.01224.x.
Ainsworth EA, Rogers A. The response of photosynthesis and stomatal conductance to rising
[CO2]: mechanisms and environmental interactions. Plant Cell Environ. 2007;30(3):258–70.
doi:10.1111/j.1365-3040.2007.01641.x.
Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their func-
tions. Plant Physiol. 2006;141(2):391–6. doi:10.1104/pp.106.082040.
Ayers RS, Westcot DW. Water quality for agriculture. Rome: Food And Agriculture Organization
of the United Nations; 1985.
Baginsky S. Plant proteomics: concepts, applications, and novel strategies for data interpretation.
Mass Spectrom Rev. 2009;28(1):93–120. doi:10.1002/Mas.20183.
Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA. The enigmatic LEA
proteins and other hydrophilins. Plant Physiol. 2008;148(1):6–24. doi:10.1104/pp.108.120725.
Bazakos C, Manioudaki ME, Therios I, Voyiatzis D, Kafetzopoulos D, Awada T, et al. Compar-
ative transcriptome analysis of two olive cultivars in response to NaCl-stress. PLoS ONE.
2012;7(8):e42931. doi:10.1371/journal.pone.0042931.
Beritognolo I, Harfouche A, Brilli F, Prosperini G, Gaudet M, Brosche M, et al. Comparative
study of transcriptional and physiological responses to salinity stress in two contrasting
Populus alba L. genotypes. Tree Physiol. 2011;31(12):1335–55. doi:10.1093/treephys/tpr083.
Chao DY, Luo YH, Shi M, Luo D, Lin HX. Salt-responsive genes in rice revealed by cDNA micro-
array analysis. Cell Res. 2005;15(10):796–810. doi:10.1038/sj.cr.7290349.
Chaves MM, Flexas J, Pinheiro C. Photosynthesis under drought and salt stress: regulation mecha-
nisms from whole plant to cell. Ann Bot. 2009;103(4):551–60. doi:10.1093/aob/mcn125.
Cramer GR, Ergul A, Grimplet J, Tillett RL, Tattersall EA, Bohlman MC et al. Water and salinity
stress in grapevines: early and late changes in transcript and metabolite profiles. Funct Integr
Genomics. 2007;7(2):111–34. doi:10.1007/s10142-006-0039-y.
Cuartero J, Fernandez-Munoz R. Tomato and salinity. Sci Hortic. 1999;78(1–4):83–125.
doi:10.1016/S0304-4238(98)00191-5.
Delauney AJ, Verma DPS. Proline biosynthesis and osmoregulation in plants. Plant J. 1993;
4(2):215–23. doi:10.1046/j.1365-313X.1993.04020215.x.
Deyholos MK. Making the most of drought and salinity transcriptomics. Plant Cell Environ.
2010;33(4):648–54. doi:10.1111/j.1365-3040.2009.02092.x.
Dutta B, Snyder R, Klapa MI. Significance analysis of time-series transcriptomic data: a meth-
odology that enables the identification and further exploration of the differentially expressed
genes at each time-point. Biotechnol Bioeng. 2007;98(3):668–78. doi:10.1002/bit.21432.
Dutta B, Kanani H, Quackenbush J, Klapa MI. Time-series integrated “omic” analyses to elu-
cidate short-term stress-induced responses in plant liquid cultures. Biotechnol Bioeng.
2009;102(1):264–79. doi:10.1002/Bit.22036.
Evers D, Legay S, Lamoureux D, Hausman JF, Hoffmann L, Renaut J. Towards a synthetic view of
potato cold and salt stress response by transcriptomic and proteomic analyses. Plant Mol Biol.
2012;78(4–5):503–14. doi:10.1007/s11103-012-9879-0.
Fiehn O. Metabolomics-the link between genotypes and phenotypes. Plant Mol Biol. 2002;48(1–2):
155–71. doi:10.1007/978-94-010-0448-0_11.
Fiehn O, Kloska S, Altmann T. Integrated studies on plant biology using multiparallel techniques.
Curr Opin Biotechnol. 2001;12(1):82–6. doi:10.1016/S0958-1669(00)00165-8.
Ford CW. Accumulation of low-molecular weight solutes in water-stressed tropical legumes. Phy-
tochemistry. 1984;23(5):1007–15. doi:10.1016/S0031-9422(00)82601-1.
Gavaghan CL, Li JV, Hadfield ST, Hole S, Nicholson JK, Wilson ID, et al. Application of NMR-
based metabolomics to the investigation of salt stress in maize ( Zea mays). Phytochem Anal.
2011;22(3):214–24. doi:10.1002/pca.1268.
Geissler N, Hussin S, Koyro HW. Elevated atmospheric CO2 concentration enhances salinity tol-
erance in Aster tripolium L. Planta. 2010;231(3):583–94. doi:10.1007/s00425-009-1064-6.
Gong Q, Li P, Ma S, Indu Rupassara S, Bohnert HJ. Salinity stress adaptation competence in the
extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana.
Plant J. 2005;44(5):826–39. doi:10.1111/j.1365-313X.2005.02587.x.
Jankangram W, Thammasirirak S, Jones MG, Hartwell J, Theerakulpisut P. Proteomic and tran-
scriptomic analysis reveals evidence for the basis of salt sensitivity in Thai jasmine rice ( Oryza
sativa L. cv. KDML 105). Afr J Biotechnol. 2011;10(72):16157–66. doi:10.5897/Ajb11.1559.
Jones JB. Hydroponics: a practical guide for the soilless grower. Florida: CRC Press; 2005.
Kanani HH, Klapa MI. Data correction strategy for metabolomics analysis using gas chroma-
tography-mass spectrometry. Metab Eng. 2007;9(1):39–51. doi:10.1016/j.ymben.2006.08.001.
Kanani H, Dutta B, Klapa MI. Individual vs. combinatorial effect of elevated CO2 conditions and
salinity stress on Arabidopsis thaliana liquid cultures: comparing the early molecular response
using time-series transcriptomic and metabolomic analyses. BMC Syst Biol. 2010;4:177.
doi:10.1186/1752-0509-4-177.
Kaplan F, Zhao W, Richards JT, Wheeler RM, Guy CL, Levine LH. Transcriptional and metabolic
insights into the differential physiological responses of Arabidopsis to optimal and supraopti-
mal atmospheric CO2. PLoS ONE. 2012;7(8). doi:10.1371/journal.pone.0043583.
Kim JK, Bamba T, Harada K, Fukusaki E, Kobayashi A. Time-course metabolic profiling in
Arabidopsis thaliana cell cultures after salt stress treatment. J Exp Bot. 2007;58(3):415–24.
doi:10.1093/jxb/erl216.
Klapa MI, Quackenbush J. The quest for the mechanisms of life. Biotechnol Bioeng. 2003;
84(7):739–42. doi:10.1002/bit.10858.
Legay S, Lamoureux D, Hausman JF, Hoffmann L, Evers D. Monitoring gene expression of potato
under salinity using cDNA microarrays. Plant Cell Rep. 2009;28(12):1799–816. doi:10.1007/
s00299-009-0780-5.
Li PH, Ainsworth EA, Leakey ADB, Ulanov A, Lozovaya V, Ort DR, et al. Arabidopsis transcript
and metabolite profiles: ecotype-specific responses to open-air elevated [CO(2)]. Plant Cell
Environ. 2008;31(11):1673–87. doi:10.1111/j.1365-3040.2008.01874.x.
Lu Y, Lam H, Pi E, Zhan Q, Tsai S, Wang C, et al. Comparative metabolomics in Glycine max and
Glycine soja under salt stress to reveal the phenotypes of their offspring. J Agric Food Chem.
2013;61(36):8711–21. doi:10.1021/jf402043m.
Magan JJ, Gallardo M, Thompson RB, Lorenzo P. Effects of salinity on fruit yield and quality if
tomato grown in soil-less culture greenhouses in Mediterranean climatic conditions. Agr Water
Manage. 2008;95:1041–55. doi:10.1016/j.agwat.2008.03.011.
Mahajan S, Tuteja N. Cold, salinity and drought stresses: an overview. Arch Biochem Biophys.
2005;444(2):139–58. doi:10.1016/j.abb.2005.10.018.
Miyagi A, Takahara K, Kasajima I, Takahashi H, Kawai-Yamada M, Uchimiya H. Fate of 13C in

metabolic pathways and effects of high CO2 on the alteration of metabolites in Rumex obtusi-
folius L. Metabolomics. 2011;7(4):524–35. doi:10.1007/s11306-010-0272-1.
Mizuno H, Kawahara Y, Sakai H, Kanamori H, Wakimoto H, Yamagata H, et al. Massive parallel
sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in
rice ( Oryza sativa L. ). BMC Genomics. 2010;11:683. doi:10.1186/1471-2164-11-683.
Niu XM, Bressan RA, Hasegawa PM, Pardo JM. Ion homeostasis in NaCl stress environments.
Plant Physiol. 1995;109(3):735–42. doi:10.1104/pp.109.3.735.
Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, Fodor SPA. Light-generated oligonucle-
otide arrays for rapid DNA-sequence analysis. Proc Natl Acad Sci U S A. 1994;91(11):5022–6.
doi:10.1073/pnas.91.11.5022.
Perez-Lopez U, Robredo A, Lacuesta M, Sgherri C, Munoz-Rueda A, Navari-Izzo F, et al. The
oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2. Physiol
Plant. 2009;135(1):29–42. doi:10.1111/j.1399-3054.2008.01174.x.
Perez-Lopez U, Robredo A, Lacuesta M, Mena-Petite A, Munoz-Rueda A. Elevated CO2 reduces
Photosynth Res. 2012;111(3):269–83. doi:10.1007/s11120-012-9721-1.
Prior SA, Runion GB, Marble SC, Rogers HH, Gilliam CH, Torbert HA. A review of elevated
atmospheric CO2 effects on plant growth and water relations: implications for horticulture.
Hortscience. 2011;46(2):158–62.
Ratnakumar P, Rajendrudu G, Swamy PM. Photosynthesis and growth responses of peanut
( Arachis hypogaea L.) to salinity at elevated CO2. Plant Soil Environ. 2013;59(9):410–6.
Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L. Simultaneous analysis of metabo-
lites in potato tuber by gas chromatography-mass spectrometry. Plant J. 2000;23(1):131–42.
doi:10.1046/j.1365-313x.2000.00774.x.
Rogers A, Ellsworth DS. Photosynthetic acclimation of Pinus taeda (loblolly pine) to long-term
growth in elevated pCO(2) (FACE). Plant Cell Environ. 2002;25(7):851–8. doi:10.1046/
j.1365-3040.2002.00868.x.
Sanchez DH, Siahpoosh MR, Roessner U, Udvardi M, Kopka J. Plant metabolomics reveals con-
served and divergent metabolic responses to salinity. Physiol Plant. 2008a;132(2):209–19.
doi:10.1111/j.1399-3054.2007.00993.x.
Sanchez DH, Lippold F, Redestig H, Hannah MA, Erban A, Kramer U, et al. Integrative func-
tional genomics of salt acclimatization in the model legume Lotus japonicus. Plant J.
2008b;53(6):973–87. doi:10.1111/j.1365-313X.2007.03381.x.
Sanchez DH, Pieckenstain FL, Szymanski J, Erban A, Bromke M, Hannah MA, et al. Compara-
tive functional genomics of salt stress in related model and cultivated plants identifies and
overcomes limitations to translational genomics. PLoS ONE. 2011;6(2):e17094. doi:10.1371/
journal.pone.0017094.
Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns
with a complementary DNA microarray. Science. 1995;270(5235):467–70. doi:10.1126/sci-
ence.270.5235.467.
Scholz M, Kaplan F, Guy CL, Kopka J, Selbig J. Non-linear PCA: a missing data approach. Bio-
informatics. 2005;21(20):3887–95. doi:10.1093/bioinformatics/bti634.
Shannon MC, Grieve CM. Tolerance of vegetable crops to salinity. Sci Hortic. 1999;78(1–4):5–38.
doi:10.1016/S0304-4238(98)00189-7.
Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, et al. Climate change 2007:
the physical science basis. Contribution of working group I. Fourth Assessment Report of the
Intergovernmental Panel on Climate Change. Cambridge, Cambridge University Press; 2007.
Steuer B, Stuhlfauth T, Fock HP. The efficiency of water use in water stressed plants is increased
due to ABA induced stomatal closure. Photosynth Res. 1988;18(3):327–36. doi:10.1007/
BF00034837.
Sussman MR, Huttlin EL, Wohlbach DJ. Democratization and integration of genomic profiling
tools. Methods Mol Biol. 2009;553:373–93. doi:10.1007/978-1-60327-563-7_20.
Takagi M, El-Shemy H, Sasaki S, Toyama S, Kanai S, Saneoka H, et al. Elevated CO2 concentration
alleviates salinity stress in tomato plant. Acta Agr Scand B Soil Plant Sci. 2009;59(1):87–96.
doi:10.1080/09064710801932425.
Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radi-
ation response. Proc Natl Acad Sci U S A. 2001;98(9):5116–21. doi:10.1073/pnas.091062498.
Vidal M. A unifying view of 21st century systems biology. FEBS Lett. 2009;583(24):3891–4.
doi:10.1016/j.febslet.2009.11.024.
Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures: to-
wards genetic engineering for stress tolerance. Planta. 2003;218(1):1–14. doi:10.1007/s00425-
003-1105-5.
Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev
Genet. 2009;10(1):57–63. doi:10.1038/Nrg2484.
Wang YP, Yang L, Zheng ZM, Grumet R, Loescher W, Zhu JK, et al. Transcriptomic and physi-
ological variations of three Arabidopsis ecotypes in response to salt stress. PLoS ONE.
2013;8(7). doi:10.1371/journal.pone.0069036.
Widodo, Patterson JH, Newbigin E, Tester M, Bacic A, Roessner U. Metabolic responses to salt
stress of barley ( Hordeum vulgare L. ) cultivars, Sahara and Clipper, which differ in salinity
tolerance. J Exp Bot. 2009;60(14):4089–103. doi:10.1093/jxb/erp243.
Zhu JK. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol. 2002;53:247–73.
doi:10.1146/annurev.arplant.53.091401.143329.
Zuther E, Koehl K, Kopka J. Comparative metabolome analysis of the salt response in breed-
ing cultivars of rice. Advances in molecular breeding toward drought and salt tolerant crops.
Berlin: Springer; 2007.
Chapter 4
Combination of Elevated CO2 Levels and Soil
Contaminants’ Stress in Wheat and Rice
Hongyan Guo, Hui Zhou, Yaodan Zhang, Wenchao Du, Yuanyuan Sun,
Ying Yin, Daping Pei, Rong Ji, Jichun Wu, Xiaorong Wang and Jianguo Zhu
4.1 Introduction
With increasing global industrialization, the atmospheric CO2 concentration has

risen from approximately 280 mmol mol−1 in preindustrial times to approximately
380 mmol mol−1 now, and it is expected to continue increasing in the future (IPCC
2007). Elevated atmospheric CO2 levels can stimulate photosynthesis (Zhang et al.
2008), enhance carbon deposition in soil (Hill et al. 2007), and change the rhizo-
sphere conditions of the plant, leading to increases in biomass and yields of crops
(Delucia et al. 1997; Lieffering et al. 2004; Liu et al. 2008).
Some industrial, mining, and agricultural activities have contaminated soils
with heavy metals, and such pollution is increasingly becoming a serious environ-
mental problem. In China, more than 2.0 × 107 ha of agricultural land is reportedly
Reprinted (adapted) with permission from elevated CO2 levels affects the concentrations of
copper and cadmium in crops grown in soil contaminated with heavy metals under fully open-air
field conditions. Copyright (2011) American Chemical Society.
H. Guo () · H. Zhou · Y. Zhang · W. Du · Y. Yin · D. Pei · R. Ji · X. Wang

State Key Laboratory of Pollution Control and Resource Reuse, School of Environment,
Nanjing University, Nanjing 210093, China
e-mail: hyguo@nju.edu.cn
J. Zhu
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,
Chinese Academy of Science, Nanjing 210008, Jiangsu, China
e-mail: jgzhu@issas.ac.cn
Y. Sun · J. Wu
State Key Laboratory of Pollution Control and Resource Reuse, School of Earth Sciences
and Engineering, Hydrosciences Department, Nanjing University, Nanjing 210093, China
H. Guo
Institute for Climate and Global Change Research (ICGCR), Nanjing University,
Nanjing 210093, China

72 H. Guo et al.
contaminated with heavy metals (Huang et al. 2009). Heavy metals are toxic to
plants, animals, and humans at different concentrations, and are known to cause
significant environmental damage and human health problems (Huang et al. 2009;
Mulligan et al. 2001; Nahmani et al. 2005; Maksymiec 2007). Of the heavy metals,
copper (Cu) is an essential micronutrient for plants and animals. However, when in
excess, Cu can interfere with numerous physiological processes, resulting in cell
toxicity. Cadmium (Cd) is a toxic trace element in the environment that can be eas-
ily taken up by plants and subsequently transferred to humans through food chains.
In the future, further increases in global CO2 levels and contamination with
heavy metals are likely. More research is needed to investigate the response of
crops grown in soils contaminated with metals under elevated CO2 levels. In the
few reports available, Duval et al. (Duval et al. 2011) indicated that CO2 alters the
distribution of contaminant elements in ecosystems; Wu et al. (2009) showed that
elevated CO2 level increases cesium (Cs) concentrations in rice shoots and roots;
Li et al. (2010) reported that elevated CO2 levels decrease or barely affect Cu con-
centrations in six rice varieties grown in contaminated soils, but increase Cd levels
in three rice varieties. These studies highlight the need for a better understanding of
the mechanisms by which CO2 and heavy metals jointly affect crop growth and up-
take of metals, especially from the viewpoint of food safety. One also must consider
that these studies were conducted in open-top chambers (OTCs). Long et al. (2006)
reported that the effects on plants grown in OTCs are often greater than on plants
grown under open air. Therefore, the results of such studies cannot be extrapolated
to address the effect of long-term, more realistic CO2 fumigation on plants. One
way to approach such a study is to use free-air CO2 enrichment (FACE). FACE
experiments are conducted in open fields, allow the best simulation of elevated CO2
environments (Long et al. 2006), and have been carried out in many countries (Lief-
fering et al. 2004; Andrews and Schlesinger 2001; Hoosbeek et al. 2007).
With the aim of predicting future food safety and the combined stress of CO2 and
soil pollution, we used a full-size (14 m diameter) FACE system in farm fields in
Jiangsu Province, China, to investigate the effect of elevated atmospheric CO2 on
Cd and Cu levels in two important crops worldwide, rice and wheat.
4.2 Materials and Methods
4.2.1 Experimental Site
The FACE system was established in the town of Xiaoji, Jiangdu County, Jiangsu
Province, China (119°42’E, 32°35’N). Here, rice–wheat rotation system is prac-
ticed. This region lies within the northern subtropical monsoon climate. The annual
mean temperature is 14–16 °C, and the mean annual precipitation is 980 mm. The
annual length of the nonfrost period is approximately 220 days. The soil is Shaji-
ang-Aquic Cambosols with a sandy–loamy texture.
4 Combination of Elevated CO2 Levels and Soil Contaminants’ Stress … 73
4.2.2 FACE System
The FACE system has been described in detail by Liu et al. (2002) and Okada et al.
(2001). In brief, the FACE system consists of octagonal plots located in different
paddies having similar soils and agronomic histories. The plots have either elevat-
ed CO2 levels (hereafter called FACE plots) or ambient CO2 conditions (hereafter
called ambient plots). Each plot is ca. 80 m2. In the FACE plots, plants were exposed
to elevated CO2 levels within rings 12.5 m in diameter that emitted pure CO2 from
the periphery toward the center through emission tubes located about 50–60 cm
above the canopy. In the ambient plots, plants were grown under ambient CO2 con-
ditions. To minimize the CO2 contamination, ambient plots were at least 90 m away
from the nearest FACE ring. The season-long average CO2 concentration of the
ambient plots was about 370 μmol mol−1. The CO2 concentration in the FACE plots
was constantly controlled at about 200 μmol mol−1 higher than in the ambient plots.
4.2.3 Crop Cultivation and Sample Preparation
Rice ( Oryza sativa L. cv. Wu Xiang jing 14) and wheat ( Triticum aestivum L. cv.
Yangmai 14) plants were grown in plastic pots in soils collected from a local farm.
The properties of the soils are shown in Table 4.1. Fresh soil was sieved through
a 3-mm sieve and kept in the dark until used. Soils were spiked with either Cu
or Cd; control soil was not spiked. Specified amounts of Cd in the form of a dis-
solved solution of CdCl2·2H2O were added and thoroughly mixed into the soil as
0, 0.5, and 2.0 mg kg−1. Specified amounts of Cu in the form of a dissolved solu-
tion of CuSO4·5H2O were added and thoroughly mixed into the soil as 0, 50, and
400 mg kg−1. The spiked and unspiked soils were then watered to field water capac-
ity and kept in the dark for 6 months. Prepared soil was placed in plastic pots (5 kg
soil per pot; 20 cm in diameter, 35 cm in height). Two FACE plots and two ambient
plots were used in this experiment; plants treated with Cu were grown on one FACE
plot and one ambient plot, and plants treated with Cd were grown on another FACE
plot and another ambient plot. Each treatment consisted of three replicate pots.
The experiments were conducted from June 2006 to October 2008. Rice–wheat
rotation was used. The first rice seeds were planted in June 2006, and the plants
were harvested in October 2006. The first wheat sowing was in November 2006,
with harvest in May 2007. The second rice sowing was in June 2007, with harvest
in October 2007. The second wheat sowing was in November 2007, with harvest in
May 2008. The third rice sowing was in June 2008.
Table 4.1 Physical and chemical characteristics of soils used in this study
Soil Organic Sand (%) Silt (%) Clay (%) pH Total Cu Total Cd
spiked matter (mg kg−1) (mg kg−1)
with (g kg−1)
Cu 17.3 57.8 28.5 13.7 6.92 21.7 0.15
Cd 18.4 56.1 29.6 14.3 7.21 18.9 0.11
74 H. Guo et al.
The rice and wheat plants from the first sowing were sampled at the mid-tiller-
ing, panicle-initiation, and grain-maturity growth stages, and those from the second
sowing were only sampled at grain maturity. Leaves for enzyme assays were frozen
in liquid nitrogen when sampling and stored at −80 °C. Soils were sampled at the
rice grain-maturity growth stage of the second sowing (October 2007) for pH analy-
sis and metal fractionations. Rice roots were sampled at the panicle-initiation stage
of the third sowing and stored at − 40 °C for microscopy (August 2008).
4.2.4 Analysis of Antioxidant Enzyme Activities in Leaves
The preparation method for crude enzyme referred to the method proposed by Cho
and Seo (2005). The activities of superoxide dismutase (SOD) were measured by
nitroblue tetrazolium (NBT) photoreduction method (Dhindsa et al. 1981). The
methods for determining the activities of catalase (CAT), peroxidase (POD), and
ascorbate peroxidase (APX) were as described earlier (Cakmak and Horst 1991;
Cakmak et al. 1993; Asada 1984).
4.2.5 Determination of Cd and Cu
The sampled plants were thoroughly washed with tap water and then with deionized
water, and were then oven-dried to a constant weight at 70 °C. The dried samples
were ground, weighed, and digested with concentrated HNO3/HClO4 (4:1 v/v; Li
et al. 2001). The Cd and Cu concentrations in the digested solution were analyzed
by atomic absorption spectroscopy (AAS; Thermo Solaar M6, USA).
4.2.6 Determination of Soil pH and Sequential Extraction of Soil
The pH of the soil was measured in a 0.01 M CaCl2 solution at a 1:2.5 ratio of soil
to solution (w/v) using a pH meter. Cu and Cd fractionation in soil was determined
by sequential extraction using the method of the Commission of the European Com-
munities (Community Bureau of Reference; BCR). The method is described in de-
tail by Quevauviller et al. (1997).
4.2.7 Scanning Electron Microscopy
Fresh rice roots were thoroughly washed with deionized water. The first 1 cm of
each root tip was cut and coated with gold (ca. 1 nm thickness) for 60 s using a
sputter coater (HITACHI E-1010, Japan). The samples were viewed with a scanning
electron microscope (SEM; S-3400N II, Hitachi, Japan).
4.2.8 Statistics
Data were expressed as means ± standard deviation ( n = 3; n represents three repli-

cate pots) and analyzed statistically using the SPSS software program (SPSS Inc.,
USA, version 16.0). The data were analyzed with a two-way analysis of variance
(ANOVA) approach, i.e., Cd or Cu treatment (three levels) and CO2 treatment (two
levels). The mean values from the ambient plots were compared to the mean values
from the FACE plots on each day of measurement using Tukey’s test. The unilateral
t test was also performed to distinguish among enzyme activities. The difference
between the means was considered significant at p < 0.05.
4.3 Results and Discussion
4.3.1 Changes in Enzyme Activities
The changes in the activities of enzymes in the leaves of rice at mid-tillering growth
stage are shown in Figs. 4.1 and 4.2. In Cu treatment group, the activity of CAT in
leaves of rice grown on FACE plots with 50 and 400 mg kg−1 Cu in the soil was
41.9 and 40.0 % lower, respectively, than that in leaves of rice grown on ambient
plots (Fig. 4.1a). The activity of APX in leaves of rice grown on FACE plots with
400 mg kg−1 Cu was 18.9 % lower than that in leaves of rice grown on ambient
plots (Fig. 4.1b). The activity of POD in leaves of rice grown on FACE plots with 0
(control) mg kg−1 Cu added was 39.3 % higher than that in leaves of rice grown on
ambient plots, while no significant differences were found between the FACE and
ambient plots either 50 or 400 mg kg−1 Cu treatment group (Fig. 4.1c). The activity
of SOD in leaves of rice grown on FACE plots with 0 (control) mg kg−1 Cu was
30.9 % higher than that in leaves of rice grown on ambient plots. No significant dif-
ferences were found between the FACE and ambient plots either 50 or 400 mg kg−1
Cu treatment group (Fig. 4.1d). In Cd treatment group, the activity of CAT in leaves
of rice grown on FACE plots with 0.5 mg kg−1 Cd was 34.0 % lower than that in
leaves of rice grown on ambient plots (Fig. 4.2a). The activity of APX in leaves of
rice grown on FACE plots with 0.5 mg kg−1 Cd was 18.9 % lower than that in leaves
of rice grown on ambient plots (Fig. 4.2b). No significant differences on the activ-
ity of POD were found between the FACE and ambient plots in each Cd treatment
group (Fig. 4.2c). The activity of SOD in leaves of rice grown on FACE plots with
0 (control) mg kg−1 Cd added was 46.3 % higher than that in leaves of rice grown
on ambient plots, while that was 50.4 % lower in 0.5 mg kg−1 Cd treatment group
(Fig. 4.2d).
In Cu treatment groups, the changes in the activities of enzymes in the leaves of
wheat at mid-tillering and panicle-initiation growth stages are shown in Fig. 4.3.
No significant differences in CAT activities were found between the FACE and
ambient plots in each Cd treatment group at mid-tillering growth stage (Fig. 4.3a).
76 H. Guo et al.
Fig. 4.1 Changes in the activities of CAT (a), APX (b), POD (c), SOD (d) in the leaves of rice at
mid-tillering growth stage. Rice plants grown in soil with 0, 50, or 400 mg kg−1 Cu, under either
ambient CO2 levels or elevated CO2 levels ( FACE). Values represent means ± SD. An asterisk
indicates a significant difference between FACE and ambient conditions ( p < 0.05). CAT cata-
lase, APX ascorbate peroxidase, POD peroxidase, SOD superoxide dismutase, FACE free-air CO2
enrichment
At p anicle-initiation growth stage, the activity of CAT in leaves of wheat grown

on FACE plots with 50 mg kg−1 Cu was 24.8 % higher than that in leaves of wheat
grown on ambient plots, while that was 35.3 % lower in 400 mg kg−1 Cu treatment
group (Fig. 4.3b). No significant changes in the activity of APX in the leaves of
wheat at mid-tillering and panicle-initiation growth stages were observed (Fig. 4.3c,
d). The activity of POD in leaves of wheat grown on FACE plots with 400 mg kg−1
Cu added was lower than that in leaves of wheat grown on ambient plots at mid-
tillering growth stage, as well as panicle-initiation growth stage (Fig. 4.3e, f). In all
Cu treatment groups, the activities of SOD in leaves of wheat grown on FACE plots
were lower than that in leaves of wheat grown on ambient plots (Fig. 4.3g, h). The
changes in the activities of enzymes in the leaves of wheat at mid-tillering and pan-
icle-initiation growth stages in response to Cd treatment are shown in Fig. 4.4. At
mid-tillering growth stage, the activity of CAT in leaves of wheat grown on FACE
plots with added Cd was higher than that in leaves of wheat grown on a mbient plots.
Fig. 4.2 Changes in the activities of CAT (a), APX (b), POD (c), SOD (d) in the leaves of rice
at mid-tillering growth stage. Rice plants grown in soil with 0, 0.5, or 2 mg kg−1 Cd, under either
ambient CO2 levels or elevated CO2 levels ( FACE). Values represent means ± SD. An asterisk
indicates a significant difference between FACE and ambient conditions ( p < 0.05). CAT cata-
lase, APX ascorbate peroxidase, POD peroxidase, SOD superoxide dismutase, FACE free-air CO2
enrichment
Interestingly, CAT activity was lower in 2 mg kg−1 Cd treatment group at panicle-

initiation growth stage (Fig. 4.4a, b). In all Cd treatment groups, the activities of
APX, POD, and SOD in leaves of wheat grown on FACE plots were higher than that
in leaves of wheat grown on ambient plots (Fig. 4.4c, h).
Studies have shown that metals such as Cu and Cd exhibit the ability to produce
reactive oxygen species (ROS) such as superoxide ion, hydrogen peroxide, and hy-
droxyl radical, resulting in lipid peroxidation (Stohs and Bagchi 1995; Skorzyn-
ska-Polit et al. 2006; Schraudner et al. 1997). The excessive production of ROS
in chloroplasts of plants has proven to be the main cause of oxidative damage in
leaves (Foyer and Noctor 2003). Plants have evolved an antioxidant defense system
including enzyme system and nonenzymatic system to avoid damage caused by
ROS. The antioxidant defense system plays an important role in the ROS removal
and protective defense reaction (Hernandez et al. 2001). SOD is a major scaven-
ger of superoxide ion, its enzymatic action results in the formation of hydrogen
78 H. Guo et al.
Fig. 4.3 Changes in the activities of CAT (a), APX (c), POD (e), SOD (g) in the leaves of wheat
at mid-tillering growth stage and changes in the activities of CAT (b), APX (d), POD (f), SOD (h)
in the leaves of wheat at panicle-initiation growth stage. Wheat plants grown in soil with 0, 50, or
400 mg kg−1 Cu, under either ambient CO2 levels or elevated CO2 levels ( FACE). Values represent
means ± SD. An asterisk indicates a significant difference between FACE and ambient conditions
( p < 0.05). CAT catalase, APX ascorbate peroxidase, POD peroxidase, SOD superoxide dismutase,
FACE free-air CO2 enrichment
Fig. 4.4 Changes in the activities of CAT (a), APX (c), POD (e), SOD (g) in the leaves of wheat
at mid-tillering growth stage and changes in the activities of CAT (b), APX (d), POD (f), SOD (h)
in the leaves of wheat at panicle-initiation growth stage. Wheat plants grown in soil with 0, 0.5, or
2 mg kg−1 Cd, under either ambient CO2 levels or elevated CO2 levels ( FACE). Values represent
means ± SD. An asterisk indicates a significant difference between FACE and ambient conditions
( p < 0.05). CAT catalase, APX ascorbate peroxidase, POD peroxidase, SOD superoxide dismutase,
80 H. Guo et al.
p eroxide and molecular oxygen, while CAT decomposes hydrogen peroxide into
water (Dionisio-Sese and Tobita 1998). For higher plants, the induction of POD ac-
tivity is a comprehensive reaction to harmful stress, which may be associated with
oxidation reactions of membrane (Lin and Kao 2002). The most important reducing
substrate for hydrogen peroxide detoxification is ascorbate, APX uses two mol-
ecules of ascorbate to reduce hydrogen peroxide to water (Noctor and Foyer 1998).
In this study, at mid-tillering growth stage, the activities of enzymes (CAT, APX,
POD, and SOD) in leaves of rice grown on FACE plots with Cu and Cd added
groups were lower than that in leaves of rice grown on ambient plots. We speculate
that elevated CO2 levels might alleviate oxidative stress in leaves of rice polluted
by Cu and Cd. Some research suggested that elevated concentrations of CO2 caused
a significant reduction in the activities of SOD and CAT in leaves of plant, and
the oxidative stress of plant was alleviated to a certain extent (Polle et al. 1993;
Schwanz et al. 1996). It was suggested that the activity of SOD in leaves of beech
( Fagus sylvatica L.) was inhibited with elevating CO2 levels as a result of increase
of NADPH which was the intermediate of photosynthesis and the activity of CAT
decreased with elevating CO2 levels because the respiration rate of plant slowed
down and the concentration of hydrogen peroxide which was the product of respira-
tion decreased (Polle et al. 1997).
In Cu treatment groups, the activities of enzymes in the leaves of wheat grown
on FACE plots at mid-tillering and panicle-initiation growth stages were lower than
that in leaves of wheat grown on ambient plots, while the trend was opposite in Cd
treatment groups. We surmise that the absorption of Cu and Cd was different in
wheat under different atmospheric conditions. Elevated CO2 levels increased the
absorption of Cd, resulted in the increase of oxidative stress. Increase in ROS prob-
ably served as an inciting factor that increased the activities of antioxidant enzymes.
4.3.2 Copper Concentration in Plants
In this 2-year study, elevated CO2 levels significantly led to lower Cu concentration
in both rice and wheat (Figs. 4.5 and 4.6). At the mid-tillering stage of the first rice
season, the Cu concentrations in shoots of rice grown on FACE plots with 50 and
400 mg kg−1 Cu in the soil were 23.0 and 22.9 % lower, respectively, than in shoots
of rice grown on ambient plots (Fig. 4.5a, p < 0.05). At the panicle-initiation stage,
the Cu concentration in shoots of rice grown on FACE plots with 50 mg kg−1 Cu was
22.2 % lower than that in shoots of rice grown on ambient plots (Fig. 4.5b, p < 0.05).
At grain maturity during the first rice season, the Cu concentration in the shoots of
rice grown on FACE plots was 34.1, 16.1, and 19.7 % lower (Fig. 4.5c, p < 0.05) than
their counterparts grown on ambient plots with 0 (control), 50, and 400 mg kg−1 Cu,
respectively, and the Cu concentration in the grains of rice grown on FACE plots
with 400 mg kg−1 Cu (Fig. 4.5d, p < 0.05) was 8.8 % lower than in grains of rice
grown on ambient plots. A similar trend was detected in samples from the second
year. At grain maturity during the second rice season, the Cu concentration in the
35 Ambient
18 Ambient 12 Ambient
30 FACE
15 FACE 10 FACE
25
12 8
20
9 6
15
10 6 4
Cu concentration
Cu concentration
Cu concentration
rioe shoots (mg kg-1)

5 3 2
a 0 b 0 c 0
10 Ambient 10 Ambient 10 Ambient

FACE FACE
8 8 8 FACE
6 6 6
4 4 4
Cu concentration
Cu concentration
Cu concentration

2 2 2
d 0 e 0 f 0
0 50 400
4 Combination of Elevated CO2 Levels and Soil Contaminants’ Stress …
Cu added to soil (mg kg-1)
Fig. 4.5 Copper concentrations in tissues of rice plants grown in soil with 0, 50, or 400 mg kg−1 Cu, under either ambient CO2 levels or elevated CO2 levels
( FACE). a Rice shoots at the mid-tillering growth stage of the first rice sowing, b rice shoots at the panicle-initiation growth stage of the first rice sowing, c
rice shoots at grain maturity of the first sowing, d rice seeds at grain maturity of the first season, e rice shoots at grain maturity of the second season, and f rice
seeds at grain maturity of the second season. Values represent means ± SD. An asterisk indicates a significant difference between FACE and ambient conditions
( p < 0.05). FACE free-air CO2 enrichment
81
82 H. Guo et al.
shoots of rice grown on FACE plots with 0 (control) and 400 mg kg−1 Cu was 18.6
and 12.6 % (Fig. 4.5e, p < 0.05) lower than shoots of rice grown on ambient plots,
and the Cu concentration in the grains of rice grown on FACE plots with 0 (con-
trol), 50, and 400 mg kg−1 Cu was 25.5, 20.3, and 14.2 % lower than in grains of
rice grown on ambient plots (Fig. 4.5f, p < 0.05). Similar results were observed for
wheat (Fig. 4.6).
In the previous studies of uncontaminated soils, Manderscheid et al. (1995) found
that elevated CO2 levels led to lower concentrations of Ca, S, Mg, Fe, and Zn in the
wheat grain. Fangmeier et al. (1999) reported that elevated CO2 levels resulted in
lower Ca, S, and Fe concentrations in spring wheat. Yang et al. (2007) showed that
the Cu content of milled rice grain under elevated CO2 levels was 20 % lower than
that of ambient atmosphere. In an OTC experiment with contaminated soils and el-
evated CO2 levels, Li et al. (2010) reported that the Cu concentration in rice grains
was significantly lower than that of ambient atmosphere. In the short term, lower
Cu concentrations in crops probably alleviate the Cu toxicity and have important
positive implications for the food quality of crops harvested from soils contami-
nated with Cu. In this study, the SEM images of rice roots showed that exposure to
elevated CO2 levels alleviated Cu stress and increased the root hair density. When
the plants were grown with 2 mg kg−1 Cd on either FACE or ambient plots, the root
hair density of rice was low (Fig. 4.7). However, when the plants were grown with
400 mg kg−1 Cu on FACE plots, the root hair density was markedly higher than that
of plants grown on ambient plots with 400 mg kg−1 Cu (Fig. 4.7). In the long term,
depending on the magnitude of the effect, Cu deficiency in crops has the potential
to contribute to health problems.
4.3.3 Cadmium Concentration in Plants
Elevated CO2 levels resulted in higher Cd concentrations in the tissues of both

wheat and rice, especially in those plants grown in soils contaminated with high
levels of Cd (Figs. 4.8 and 4.9). At the mid-tillering stage of the first rice season
(Fig. 4.8a), the Cd concentration in shoots of rice grown on the FACE and ambi-
ent plots did not differ significantly. At the panicle-initiation stage, the Cd concen-
trations in shoots of rice grown on FACE plots with 0.5 and 2 mg kg−1 Cd were
55.7 and 7.8 % higher, respectively, than in shoots of rice grown on ambient plots
(Fig. 4.8b, p < 0.05). At grain maturity of both the first and second rice season, the
Cd concentration in shoots of rice grown on FACE plots with 2 mg kg−1 Cd was 11.3
and 21.5 % higher ( p < 0.05), respectively, than in shoots of rice grown on ambient
plots. But the Cd concentration in shoots of rice grown on FACE and ambient plots
with 0 and 0.5 mg kg−1 Cd did not significantly differ (Fig. 4.8c, e). The Cd concen-
tration in seeds was not significantly affected by elevated CO2 levels in the first rice
season (Fig. 4.8d). In the second rice season, the Cd concentration in seeds of plants
grown on FACE plots with 2 mg kg−1 Cd was 38.8 % higher than in seeds of plants
grown on ambient plots ( p < 0.05), but the Cd concentration in seeds of rice grown
Ambient 18 Ambient Ambient
18 24
FACE 15 FACE FACE
15 20
12 12 16
9 9 12
6 6 8
Cu concentration in
Cu concentration in
Cu concentration in
3 4
wheat shoots (mg kg-1)

3
a 0 b 0 c 0
10 Ambient
24 Ambient 12
FACE Ambient
8 20 FACE 10
FACE
8
6 16
12 6
4
8 4
2
Cu concentration in
Cu concentration in
Cu concentration in
4 2

wheats shoots (mg kg-1)

d 0 e 0 f 0
0 50 400
Cu added to soil (mg kg-1)
−1
Fig. 4.6 Copper concentrations in tissues of wheat plants grown in soil with 0, 50, or 400 mg kg Cu added, under either ambient CO2 levels or elevated CO2
levels ( FACE). a Wheat shoots at the mid-tillering growth stage of the first season, b wheat shoots at the panicle-initiation growth stage of the first season, c
wheat shoots at grain maturity of the first season, d wheat seeds at grain maturity of the first season, e wheat shoots at grain maturity of the second season, and
f wheat seeds at grain maturity of the second season. Values represent means ± SD. An asterisk indicates a significant difference between FACE and ambient
conditions ( p < 0.05). FACE free-air CO2 enrichment
83
84 H. Guo et al.
Fig. 4.7 Scanning electron microscopy images of rice root tips at the panicle-initiation growth
stage. Plants were grown in soil with (a) and (c) 400 mg kg−1 Cu on ambient plots, (b) and (d)
400 mg kg−1 Cu on FACE plots, (e) and (g) 2 mg kg−1 Cd on ambient plots, and (f) and (h) 2 mg
kg−1 Cd on FACE plots. (a), (b), (e), and (f) cross section; (c), (d), (g), and (h) longitudinal section
2.5 1 Ambient
3 Ambient Ambient
FACE
2.5 2 FACE 0.8
FACE
2 0.6
1.5
1.5
1 0.4
1
shoots (mg kg-1)

shoots (mg kg-1)
shoots (mg kg-1)
0.5 0.2
Cd concentration in rice
0.5
a 0 b 0 c 0
Ambient 0.18
0.12 Ambient 1 Ambient
FACE FACE 0.15
0.8 FACE
0.09
0.12
0.6
0.06 0.09
0.4
0.06
shoots (mg kg-1)

shoots (mg kg-1)
shoots (mg kg-1)
0.03 0.2
0.03
d 0 e 0 f 0
0 0.5 2
Cd added to soil (mg kg-1)
Fig. 4.8 Cadmium concentrations in tissues of rice plants grown in soil with 0, 0.5, or 2.0 mg kg−1 Cd, under either ambient CO2 levels or elevated CO2 levels
( FACE). a Rice shoots at the mid-tillering growth stage of the first season, b rice shoots at the panicle-initiation growth stage of the first season, c rice shoots at
grain maturity of the first season, d rice seeds at grain maturity of the first season, e rice shoots at grain maturity of the second season, and f rice seeds at grain
maturity of the second season. Values represent means ± SD. An asterisk indicates a significant difference between FACE and ambient conditions ( p < 0.05).
85
86
6 1.6 Ambient 2
Ambient Ambient
FACE FACE
5 FACE 1.6
1.2
4
1.2
3 0.8
0.8
2
0.4
cd concentration in
cd concentration in
cd concentration in
0.4

1

a 0 b 0 c 0
1.5 1.5 Ambient
Ambient 2 Ambient
FACE FACE
1.2 1.6 FACE 1.2
0.9 1.2 0.9
0.6 0.8 0.6
cd concentration in
0.3
cd concentration in
0.4 0.3
wheat grain (mg kg-1)

wheat grain (mg kg-1)
cd concentration in
d 0 e 0 f 0
0 0.5 2
cd added to soil (mg kg-1)
Fig. 4.9 Cadmium concentrations in tissues of wheat plants grown in soil with 0, 0.5, or 2.0 mg kg−1 Cd, under either ambient CO2 levels or elevated CO2 levels
( FACE). a Rice shoots at the mid-tillering growth stage of the first season, b rice shoots at the panicle-initiation growth stage of the first season, c rice shoots at
grain maturity of the first season, d rice seeds at grain maturity of the first season, e rice shoots at grain maturity of the second season, and f rice seeds at grain
maturity of the second season. Values represent means ± SD. An asterisk indicates a significant difference between FACE and ambient conditions ( p < 0.05).
H. Guo et al.
on FACE and ambient plots with 0 and 0.5 mg kg−1 Cd did not significantly differ
(Fig. 4.8f). Similar results were observed for wheat (Fig. 4.9).
In this study, the Cd concentration in wheat grains of all samples far exceeded
the legal limits (wheat flour: 0.1 mg kg−1; Ministry of Health 2005). The Cd con-
centration in rice seeds of the first and second seasons from plants grown on either
FACE or ambient plots was below the legal limits (rice: 0.2 mg kg−1; Ministry of
Health 2005). But after exposure to elevated CO2 level, the Cd concentration in
rice seeds of second season is more close to the legal limits than that of the first
season. Such increasing trends of Cd concentrations in rice seeds under elevated
CO2 suggest that the levels of these toxic metals could exceed the legal limit in the
future. Cadmium can accumulate in the human body and damage kidneys, bones,
and reproductive system (Jarup and Akesson 2009). To keep the Cd levels in creati-
nine in urine below 1 μg Cd g−1 in 95 % of the population by age 50, the European
Food Safety Authority (EFSA 2009) has suggested that the average daily dietary Cd
intake should not exceed 0.36 μgCd/kg body weight, which corresponds to a weekly
dietary intake of 2.52 μg Cd/kg body weight (EFSA 2009). For an average adult of
60 kg with a daily intake of 261.1 g rice or wheat (Pan et al. 2007), this estimated
weekly dietary intake the levels of Cd far exceeds the levels suggested by EFSA in
all of the wheat samples from this study grown in control and contaminated soils
and in rice samples grown in highly contaminated soil and elevated CO2 levels. Li
et al. (2010)also found significantly higher Cd concentrations in three rice variet-
ies grown on contaminated soils under elevated CO2 levels. In China, farmland
polluted by Cd has reached 20 × 104 ha and produces 14.6 × 108 kg of agricultural
products annually (Li et al. 2003). Since almost the entire population in China cur-
rently depends on rice and wheat as staple foods, the high, toxic concentrations of
Cd accumulated in crops threaten food quality and safety. This threat will increase
as the CO2 levels increase in the future.
4.3.4 Variations in Soil pH, and Cu and Cd Fractionation in Soil
After the second rice harvest (October 2007), the pH of the soil was slightly but
significantly lower in the FACE plots than in the ambient plots (Table 4.2). Elevated
CO2 levels also led to the changes in the available Cu and Cd in the soil. Compared
to soil from ambient plots, the acid-extractable fraction of Cu in soil from FACE
plots with 50 and 400 mg kg−1 Cu was 10.5 and 16.4 % higher ( p < 0.05), and the
reducible fraction of Cu in soil from FACE plots was 3.9 and 7.9 % lower ( p < 0.05),
respectively. Compared to the soil from ambient plots, the acid-extractable and re-
ducible fractions of Cd in soil from FACE plots with 2 mg kg−1 Cd were 4.7 and
6.9 % higher ( p < 0.05), and the oxidizable and residual fractions of Cd were 45.9
and 7.1 % lower ( p < 0.05), respectively.
Several studies have indicated that elevated CO2 levels lower the pH of rhizo-
sphere soils, favor the release of elements into soil solution, and as a result, help
the plant to take up more elements. DeLucia et al. (1997) reported that elevated
88 H. Guo et al.
Table 4.2 pH of soil after the second harvest of rice (November 2007) from FACE and ambient
plots to which Cu or Cd was added
Heavy metal pH
Ambient plots FACE plots
Cu (0 mg kg−1) (control) 7.06 ± 0.02 6.85 ± 0.03*
Cu (50 mg kg−1) 7.04 ± 0.03 7.11 ± 0.03
Cu (200 mg kg ) −1
6.93 ± 0.06 6.80 ± 0.01*
Cd (0 mg kg ) (control)
−1
7.48 ± 0.11 7.38 ± 0.03*
Cd (0.5 mg kg ) −1
7.36 ± 0.01 7.31 ± 0.02*
Cd (2 mg kg−1) 7.35 ± 0.01 7.06 ± 0.07*
Values represent means ± SD. An asterisk indicates a significant difference in pH between FACE
and ambient conditions ( p < 0.05)
CO2 levels increased the concentration of oxalate in the soil, and that this low mo-
lecular weight organic acid solubilized inorganic phosphorus, making it available
for uptake by the plant. Andrews and Schlesinger (2001) observed an increase in
cation concentration in the deep soil (200 cm) in the third year of CO2 fumigation,
and proposed that the observed increase in cation availability was caused by the
increased organic acid content. Wu et al. (2009) showed that elevated CO2 levels
lowered the pH by 0.2–0.4 units compared to ambient CO2 levels, which implies
that the lower pH in the rhizosphere zone could help the plants take up more Cs. Li
et al. (2010) reported that the decrease in pH of 0.04–0.15 in the rhizosphere soil
of rice was due to elevated CO2 levels, and considered that this slightly decreas-
ing trend might be linked to higher Cd concentrations in rice. Cheng et al. (2010)
reported that elevated CO2 levels significantly increased the concentration of Ca2+
and Mg2+ in soil solution and reduced the solution pH, and total cations in plant
biomass were also significantly higher under elevated CO2 levels. In this study
after the second rice harvest, especially for heavy-metal-contaminated soils, the
pH of the soil also exhibited a decreasing trend and the acid-extractable fraction
of metals in soils exhibited an increasing trend at elevated CO2 levels. It is known
that the mobility and bioavailability of heavy metals in the acid-extractable form
are greater than that of other fractions (Mulligan et al. 2001). These changes can
link elevated CO2 levels to the increasing phytoavailability of heavy metals and
are probably sufficient to explain the higher Cd concentrations in rice and wheat
in this study. Thus, we propose that at elevated CO2 levels, the exudation of low
molecular weight organic compounds by the roots of plants lowers the pH of rhi-
zosphere soils, facilitates metal solubility and bioavailability, and increases the up-
take of metal by plants. But if the soils are contaminated with little or no Cd under
elevated CO2 conditions, the slight decrease in the pH of the soil will not lead to a
significant uptake of Cd by crops.
Since in this study the bioavailability of both Cu and Cd increased under el-
evated CO2 levels, we were surprised that Cd concentrations in the crops increased,
but Cu concentrations decreased. There are a few possible explanations for these
contrasting results (IPCC 2007). Many studies have shown that elevated CO2 levels
increase plant growth and yields (Liu et al. 2008; Ziska et al. 1996; Moya et al.
1998; Kim et al. 2003), including a study using a same FACE system that reported
that elevated CO2 levels enhance hybrid rice grain yield by 34 % (Liu et al. 2008).
Recently, Duval et al. (2011) indicated that CO2 alters the distribution of contami-
nant elements in ecosystems, with contaminant elements accumulating in plants
and declining in soil, both likely explained by the CO2 stimulation of plant biomass.
Li et al. (2010) reported that although higher Cd concentrations and lower Cu con-
centration in rice grown on contaminated soils under elevated CO2 were detected,
elevated CO2 still significantly increased the total uptake of Cu and Cd owing to the
change in biomass. Similarly, the higher concentrations of Cd and lower concen-
trations of Cu in crops observed in this study were probably due to the change in
biomass under elevated CO2 conditions (Zhang et al. 2008). As reported, elevated
CO2 levels increased the exudation of low molecular weight organic compounds
by the roots of plants (Delucia et al. 1997; Andrews and Schlesinger 2001), but the
binding strength of Cd and Cu to organic compounds differs (Groenenberg et al.
2010). Cu has a relatively high binding affinity to organic matter, whereas Cd has
a relatively weak affinity. This could have an influence on the uptake of Cd and Cu
under elevated CO2 levels, leading to differences (Hill et al. 2007). Cations, such as
Ca2+, Mg2+, Na+, and K+, interfere with the heavy metal bioavailability and allevi-
ate metal toxicity owing to cation competition (Voigt et al. 2006; Luo et al. 2008;
Wang et al. 2008; Li et al. 2009). Kinraide et al. (2004) reported that the addition
of Ca2+ and Mg2+ alleviates metal toxicity, but the relative ameliorative effective-
ness of Ca2+ and Mg2+ depends upon the metal. Cheng et al. (2010), who used
a similar FACE system, reported that elevated CO2 levels significantly stimulate
Ca2+ and Mg2+ release from soil. In this study, increased Ca2+ and Mg2+ in solution
in soil could have decreased both Cu and Cd uptake owing to cation competition,
but the relative effectiveness for Cu and Cd could differ based on the plant species.
Experimental evidences supporting the above explanations are lacking. Additional
research is needed to investigate the relationship between elevated CO2 levels and
the increased phytoavailability of heavy metals and to elucidate the different mech-
anisms of the uptake of these two metals. The data presented here were obtained
from crops grown in artificially contaminated soils in pots. More data need to be
collected from crops grown under a wide range of soil conditions and realistic field
conditions to make better predictions on the combined effects of elevated CO2 lev-
els and multimetal-contaminated soils on the metal uptake by crops and thereby on
their contribution to food quality and safety.
Acknowledgments This work was supported by Program for New Century Excellent Talents
in University (NECT-12-0266) and the National Natural Science Foundation of China (Grant
No. 20777034 and 40110817), the Knowledge Innovation Program of Chinese Academy of Sci-
ences (Grant No. KZCX3-SW−440), the China Postdoctoral Science Foundation, and the Jiangsu
Planned Projects for Postdoctoral Research Funds, and the Fundamental Research Funds for the
Central Universities (Grant No.1085021108). The main instruments and apparatus of the FACE
system were supplied by the Japan National Institute for Agro-Environmental Sciences (NIAES)
and the Japan Agricultural Research Centre for Tohoku Region (NARCT).
90 H. Guo et al.
References
Andrews JA, Schlesinger WH. Soil CO2 dynamics, acidification, and chemical weathering in a tem-
perate forest with experimental CO2 enrichment. Glob Biogeochem Cycles. 2001;15:149–62.
Asada K. Chloroplasts-formation of active oxygen and its scavenging. Methods Enzymol.
1984;105:422–9.
Cakmak I, Horst WJ. Effect of aluminum on lipid-peroxidation, superoxide-dismutase, catalase,
and peroxidase-activities in root-tips of soybean (Glycine-Max). Physiol Plant. 1991;83:463–8.
Cakmak I, Strbac D, Marschner H. Activities of hydrogen peroxide-scavenging enzymes in germi-
nating wheat seeds. J Exp Bot. 1993;44:127–32.
Cheng L, Zhu J, Chen G, Zheng X, Oh NH, Rufty TW, et al. Atmospheric CO2 enrichment facili-
tates cation release from soil. Ecol Lett. 2010;13:284–91.
Cho UH, Seo NH. Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydro-
gen peroxide accumulation. Plant Sci. 2005;168:113–20.
Delucia EH, Callaway RM, Thomas EM, Schlesinger WH. Mechanisms of phosphorus acquisi-
tion for ponderosa pine seedlings under high CO2 and temperature. Ann Bot. 1997;79:111–20.
Dhindsa RS, Plumbdhindsa P, Thorpe TA. Leaf senescence-correlated with increased levels of
membrane-permeability and lipid-peroxidation, and decreased levels of superoxide-dismutase
and catalase. J Exp Bot. 1981;32:93–101.
Dionisio-Sese ML, Tobita S. Antioxidant responses of rice seedlings to salinity stress. Plant Sci.
1998;135:1–9.
Duval BD, Dijkstra P, Natali SM, Megonigal JP, Ketterer ME, Drake BG, et al. Plant-soil dis-
tribution of potentially toxic elements in response to elevated atmospheric CO2. Environ Sci
Technol. 2011;45:2570–4.
EFSA. Scientific panel on contaminants in the food chain. Cadmium in food. EFSA J.
2009;980:1–139.
Fangmeier A, De Temmerman L, Mortensen L, Kemp K, Burke J, Mitchell R, et al. Effects on
nutrients and on grain quality in spring wheat crops grown under elevated CO2 concentrations
and stress conditions in the European, multiple-site experiment ‘ESPACE-wheat’. Eur J Agron.
1999;10:215–29.
Foyer CH, Noctor G. Redox sensing and signalling associated with reactive oxygen in chloro-
plasts, peroxisomes and mitochondria. Physiol Plant. 2003;119:355–64.
Groenenberg JE, Koopmans GF, Comans RNJ. Uncertainty analysis of the nonideal competitive
adsorption-donnan model: effects of dissolved organic matter variability on predicted metal
speciation in soil solution. Environ Sci Technol. 2010;44:1340–6.
Hernandez JA, Ferrer MA, Jimenez A, Barcelo AR, Sevilla F. Antioxidant systems and O2-/H2O2
production in the apoplast of pea leaves. Its relation with salt-induced necrotic lesions in minor
veins. Plant Physiol. 2001;127:817–31.
Hill PW, Marshall C, Williams GG, Blum H, Harmens H, Jones DL, et al. The fate of photosyn-
thetically-fixed carbon in Lolium perenne grassland as modified by elevated CO2 and sward
management. New Phytol. 2007;173:766–77.
Hoosbeek MR, Vos JM, Meinders MBJ, Velthorst EJ, Scarascia-Mugnozza GE. Free atmospheric
CO2 enrichment (FACE) increased respiration and humification in the mineral soil of a poplar
plantation. Geoderma. 2007;138:204–12.
Huang YZ, Hu Y, Liu YX. Combined toxicity of copper and cadmium to six rice genotypes ( Oryza
sativa L. ). J Environ Sci China. 2009;21:647–53.
IPCC. In: Climate change 2007; the physical science basis. Contribution of working group I to
the fourth assessment report of the intergovernmental panel on climate change (ed. Solomon
S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL). Cambridge:
Cambridge University Press; 2007.
Jarup L, Akesson A. Current status of cadmium as an environmental health problem. Toxicol Appl
Pharmacol. 2009;238:201–8.
Kim HY, Lieffering M, Kobayashi K, Okada M, Mitchell MW, Gumpertz M. Effects of free-air
CO2 enrichment and nitrogen supply on the yield of temperate paddy rice crops. Field Crops
Res. 2003;83:261–70.
Kinraide TB, Pedler JF, Parker DR. Relative effectiveness of calcium and magnesium in the al-
leviation of rhizotoxicity in wheat induced by copper, zinc, aluminum, sodium, and low pH.
Plant and Soil. 2004;259:201–8.
Li XD, Poon CS, Liu PS. Heavy metal contamination of urban soils and street dusts in Hong Kong.
Appl Geochem. 2001;16:1361–8.
Li KQ, Liu ZG, Lu XL, Yang JC, Zhang ZJ, Zhu QS. Uptake and distribution of cadmium in dif-
ferent rice cultivars (in Chinese). J Argo-Environ Sci. 2003;22:529–32.
Li LZ, Zhou DM, Wang P, Jin SY, Peijnenburg WJGM, Reinecke AJ, et al. Effect of cation com-
petition on cadmium uptake from solution by the earthworm Eisenia Fetida. Environ Toxicol
Chem. 2009;28:1732–8.
Li ZY, Tang SR, Deng XF, Wang RG, Song ZG. Contrasting effects of elevated CO2 on Cu and
Cd uptake by different rice varieties grown on contaminated soils with two levels of metals:
Implication for phytoextraction and food safety. J Hazard Mater. 2010;177:352–61.
Lieffering M, Kim HY, Kobayashi K, Okada M. The impact of elevated CO2 on the elemental
concentrations of field-grown rice grains. Field Crops Res. 2004;88:279–86.
Lin CC, Kao CH. Osmotic stress-induced changes in cell wall peroxidase activity and hydrogen
peroxide level in roots of rice seedlings. Plant Growth Regul. 2002;37:177–83.
Liu G, Han Y, Zhu JG, Okada M, Nakamura H, Yoshimoto M. Rice-wheat rotational FACE plat-
form: I. System structure and control (in Chinese). Chinese J Appl Ecol. 2002;13:1253–8.
Liu HJ, Yang LX, Wang YL, Huang JY, Zhu JG, Wang YX, et al. Yield formation of CO2-en-
riched hybrid rice cultivar Shanyou 63 under fully open-air field conditions. Field Crops Res.
2008;108:93–100.
Long SP, Ainsworth EA, Leakey ADB, Nosberger J, Ort DR. Food for thought: lower-than-expect-
ed crop yield stimulation with rising CO2 concentrations. Science. 2006;312:1918–21.
Luo XS, Li LZ, Zhou DM. Effect of cations on copper toxicity to wheat root: implications for the
biotic ligand model. Chemosphere. 2008;73:401–6.
Maksymiec W. Signaling responses in plants to heavy metal stress. Acta Physiol Plant.
2007;29:177–87.
Manderscheid R, Bender J, Jager HJ, Weigel HJ. Effects of Season Long CO2 Enrichment on Ce-
reals.II. Nutrient Concentrations and Grain Quality. Agric Ecosyst Environ. 1995;54:175–85.
Ministry of Health PR China M. Maximum levels of contaminants in foods. China National Stan-
dard GB2762 (China). 2005. p. 55–63.
Moya TB, Ziska LH, Namuco OS, Olszyk D. Growth dynamics and genotypic variation in tropi-
cal, field-grown paddy rice ( Oryza sativa L.) in response to increasing carbon dioxide and
temperature. Glob Change Biol. 1998;4:645–56.
Mulligan CN, Yong RN, Gibbs BF. Remediation technologies for metal-contaminated soils and
groundwater: an evaluation. Eng Geol. 2001;60:193–207.
Nahmani J, Capowiez Y, Lavelle P. Effects of metal pollution on soil macroinvertebrate burrow
systems. Biol Fertil Soils. 2005;42:31–9.
Noctor G, Foyer CH. Ascorbate and glutathione: keeping active oxygen under control. Ann Rev
Plant Physiol Plant Mol Biol. 1998;49:249–79.
Okada M, Lieffering M, Nakamura H, Yoshimoto M, Kim HY, Kobayashi K. Free-air CO2 enrich-
ment (FACE) using pure CO2 injection: system description. New Phytol. 2001;150:251–60.
Pan XQ, Yan BJ, Shi ZM, Dai Y. Investigation on dietary status among rural and urban residents
in Jiangsu (in Chinese). Jiangsu J Prev Med. 2007;18:6–9.
Polle A, Pfirrmann T, Chakrabarti S, Rennenberg H. The effects of enhanced ozone and enhanced
carbon-dioxide concentrations on biomass, pigments and antioxidative enzymes in spruce nee-
dles ( Picea-Abies L). Plant Cell Environ. 1993;16:311–6.
Polle A, Eiblmeier M, Sheppard L, Murray M. Responses of antioxidative enzymes to elevated
CO2 in leaves of beech ( Fagus sylvatica L.) seedlings grown under a range of nutrient regimes.
92 H. Guo et al.
Quevauviller P, Rauret G, LopezSanchez JF, Rubio R, Ure A, Muntau H. Certification of trace

metal extractable contents in a sediment reference material (CRM 601) following a three-step
sequential extraction procedure. Sci Tot Environ. 1997;205:223–34.
Schraudner M, Langebartels C, Sandermann H. Changes in the biochemical status of plant cells
induced by the environmental pollutant ozone. Physiol Plant. 1997;100:274–80.
Schwanz P, Haberle KH, Polle A. Interactive effects of elevated CO2, ozone and drought stress
on the activities of antioxidative enzymes in needles of Norway spruce trees ( Picea abies, [L]
Karsten) grown with luxurious N-supply. J Plant Physiol. 1996;148:351–5.
Skorzynska-Polit E, Pawlikowska-Pawlega B, Szczuka E, Drazkiewicz M, Krupa Z. The activity
and localization of lipoxygenases in Arabidopsis thaliana under cadmium and copper stresses.
Plant Growth Regul. 2006;48:29–39.
Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal-ions. Free Radic Biolo Med.
1995;18:321–36.
Voigt A, Hendershot WH, Sunahara GI. Rhizotoxicity of cadmium and copper in soil extracts.
Environ Toxicol Chem. 2006;25:692–701.
Wang P, Zhou DM, Kinraide TB, Luo XS, Li LZ, Li DD, et al. Cell membrane surface poten-
tial ( ψ0) plays a dominant role in the phytotoxicity of copper and arsenate. Plant Physiol.
2008;148:2134–43.
Wu HB, Tang SR, Zhang XM, Guo JK, Song ZG, Tian SA, et al. Using elevated CO2 to increase
the biomass of a Sorghum vulgare x Sorghum vulgare var. sudanense hybrid and Trifolium
pratense L. and to trigger hyperaccumulation of cesium. J Hazard Mater. 2009;170:861–70.
Yang LX, Wang YL, Dong GC, Gu H, Huang JY, Zhu JG, et al. The impact of free-air CO2 enrich-
ment (FACE) and nitrogen supply on grain quality of rice. Field Crops Res. 2007;102:128–40.
Zhang YB, Duan BL, Qiao YZ, Wang KY, Korpelainen H, Li CY. Leaf photosynthesis of Betula
albosinensis seedlings as affected by elevated CO2 and planting density. For Ecol Manage.
2008;255:1937–44.
Ziska LH, Manalo PA, Ordonez RA. Intraspecific variation in the response of rice ( Oryza
sativa L.) to increased CO2 and temperature: growth and yield response of 17 cultivars. J Exp
Bot. 1996;47:1353–9.
Chapter 5
Tolerance to Combined Stress of Drought
and Salinity in Barley
Imrul Mosaddek Ahmed, Umme Aktari Nadira, Noreen Bibi, Guoping Zhang
and Feibo Wu
5.1 Introduction
Drought and salinity stresses occur naturally (Dai 2011), and have been expanding
worldwide due to human activities such as deforestations, salt mining (Ghassemi
et al. 1995), poor irrigation water (Marcum and Pessarakli 2006), and escalating
emissions of greenhouse gases (IPCC 2000). Currently, more than 800 million hect-
ares (ha) of land are affected by salinity (Munns 2005), and about one third of the
world’s arable land has experienced yield reduction due to cyclical or unpredict-
able drought (Chaves and Oliveira 2004), which are causing a great threat to crop
production. For example, China, India, and the USA, the world’s three major grain
producers and exporters, have been suffering serious water shortages in many major
agricultural regions. In China, according to the survey by the Ministry of Water Re-
sources, over 25.67 million ha of farmland was annually affected by drought stress
during the 15th 5-year plan, which caused production reduction of 3.5 × 1010 kg and
economic losses of more than 230 billion Chinese Yuan (http://mt.china-papers.
com/1/?p=185213).
Generally, the co-occurrence of several abiotic stresses, rather than an individual
stress condition, is even worse for crop production (Mittler 2006). For example,
the combined effects of salinity and drought on yield are more detrimental than the
effects of each stress alone, as observed in potato (Levy et al. 2013), wheat (Yousfi
et al. 2012), and barley (Yousfi et al. 2010). However, most studies to date have ad-
dressed the effects of single stresses on plant (Zhao et al. 2010; Wu et al. 2013), and
little is known about the physiological and molecular mechanisms underlying the
acclimation of plants to a combination of salinity and drought (Mittler 2006). Recent
studies have revealed that the response of plants to a combination of different abiot-
ic stresses is unique and cannot be directly extrapolated from the response of plants
F. B. Wu () · I. M. Ahmed · N. Bibi · U. A. Nadira · G. P. Zhang

Department of Agronomy, College of Agriculture and Biotechnology, Zijingang Campus,
Zhejiang University, Hangzhou 310058, China
e-mail: wufeibo@zju.edu.cn

94 I. M. Ahmed et al.
to each of the different stresses individually (Rollins et al. 2013; Iyer et al. 2013).
Breeding of stress-tolerant crops is the most efficient strategy to maintain yield in
stress-prone marginal land. It is thus important to identify genetic resources with
high tolerance to abiotic stresses, especially those co-occurring in the field, such as
salinity and drought, and to understand its mechanisms.
Barley ( H. vulgare L.) is the fourth most important cereal crop in the world in
terms of production. For its versatile properties, it has been used for animal feed,
human food, and beverage (Koornneef et al. 1997). Barley as a staple food is at-
tracting renewed attention, especially in Asia and northern Africa, because of its
nutritional value (Baik and Ullrich 2008). In addition to its agricultural importance,
barley is a genetic model for other crops. However, much of the genetic variation
for improving abiotic stress tolerance has been lost during the process of domesti-
cation, selection, and modern breeding (Zhao et al. 2010). Even more, barley has
a wider ecological range than any other cereals and is widespread in temperate,
subtropical, and arctic areas, from sea level to heights of more than 4500 m in the
Andes and Himalayas (Bothmer et al. 1995). Barley can be grown on soils unsuit-
able for wheat, and at altitudes unsuitable for wheat or oats. Because of its salt and
drought tolerance, barley thrives in nearly every corner of the earth, including ex-
tremely dry areas near deserts. Barley is a short-season, early-maturing, diploid, and
self-pollinating crop, thus it is also an ideal model plant for genetic study of drought
and salinity tolerance (Li et al. 2007). Several papers have summarized research on
barley abiotic stress tolerance including drought and salinity tolerance (Zhao et al.
2010; Wu et al. 2013). In this chapter, we review the impact of salinity and drought
stress applied singly and in combination in barley through morphological, physi-
ological, biochemical, molecular, cellular, and ultrastructural approaches.
5.2 Drought Stress and Tolerance
Drought is a meteorological term and is commonly defined as a period without

significant rainfall or a deficiency of water supply. Generally, drought stress occurs
when the available water in the soil is reduced and atmospheric conditions cause
continuous loss of water by transpiration or evaporation. Hence, a continuous short-
fall in precipitation (meteorological drought) coupled with higher evapotranspira-
tion demand leads to agricultural drought (Mishra and Cherkauer 2010). Agricul-
tural drought is the lack of ample moisture required for normal plant growth and de-
velopment to complete the life cycle (Manivannan et al. 2008). Although droughts
can persist for several years, even a short, intense drought can cause significant
damage and harm the local economy. Drought is a worldwide problem, constraining
global crop production and quality seriously and recent global climate change has
made this situation more serious (Apel and Hirt 2004; Forster et al. 2004; Zhao et al.
2010; Budak et al. 2013).
Drought stress is also considered to be a moderate loss of water, which leads to
stomatal closure and limitation of gas exchange. Desiccation is a much more exten-
5 Tolerance to Combined Stress of Drought and Salinity in Barley 95
sive loss of water that can potentially lead to gross disruption of metabolism and cell
structure and eventually to the cessation of enzyme-catalyzing reactions. Drought
is characterized by the reduction of water content, turgor, total water potential, wilt-
ing, closure of stomata, and decrease in cell enlargement and growth. Barley is one
of the most important cereal crops grown in many developing countries, where it is
often subject to extreme drought stress that significantly affects production (Cec-
carelli et al. 2007). Investigating the drought-tolerance mechanisms in barley could
facilitate a better understanding of the genetic bases of drought tolerance, and fa-
cilitate the effective use of genetic and genomic approaches for crop improvement.
5.3 Salinity Stress and Tolerance
Salinity-affected soils are classified into two types: saline and sodic soils. Some-
times, a third type can be categorized as saline-sodic soils. Salt’s negative effects
on plant growth have initially been associated with the osmotic stress component
caused by decreases in soil water potential and, consequently, restriction of water
uptake by roots.
In agriculture, salt stress severely affects the growth and economic yield of many
important crops (Maas and Hoffman 1977). Compared with other cereal crops, in-
cluding wheat, rice, rye, and oat, barley is highly tolerant to salinity, thus offering
a means for efficient utilization of saline soil and improvement of productivity in
these environments. However, barley still suffers from salt toxicity in many areas of
the world. On the other hand, dramatic differences can be found among and within
the barley species, providing the potential for developing cultivars with improved
salt tolerance. It is predicted that the genetic improvement of salt tolerance will be
an important aspect of barley breeding in the future.
5.4 Overlap Between Salinity and Drought Stresses
Salinity and drought stress show a high degree of similarity with respect to physi-
ological, biochemical, molecular, and genetic effects (Sairam and Tyagi 2004).
Physiological drought occurs when soluble salt levels in the soil solution are high
enough to limit water uptake due to low water potential, thereby inducing drought
stress (Lee et al. 2004). The major difference between the low-water-potential en-
vironments caused by salinity versus drought is the total amount of water available.
During drought, a finite amount of water can be obtained from the soil profile by
the plant, causing ever-decreasing soil water potential. In most saline environments,
a large amount of water is at a constant, but under low water potential. Plants have
a chance to adjust their osmotic potential, which prevent loss of turgor and gener-
ate a lower water potential that allows plants to access water in the soil solution for
growth (Taiz and Zeiger 2006).
Both stresses lead to cellular dehydration, which causes osmotic stress and re-
moval of water from the cytoplasm into the intracellular space resulting in a reduc-
tion of the cytosolic and vacuolar volumes. Early responses to water and salt stress
are largely identical except for the ionic component in the cells of plants under salt
stress. These similarities include metabolic processes, e.g., a decrease of photosyn-
thesis or increase in the levels of the plant hormonal processes, such as abscisic
acid (ABA). High intracellular concentrations of sodium and chloride ions are an
additional problem of salinity stress (Bartels and Sunkar 2005). Plants use com-
mon pathways and components in response to stresses, a concept known as cross-
tolerance, which allows plants to acclimate to a range of different stresses after
exposure to one specific stress (Pastori and Foyer 2002; Tuteja et al. 2007). Thus, a
salinity-tolerant species could also be drought tolerant or vice versa, and has similar
mechanisms to cope with those stresses (Ashraf and O’Leary 1996).
5.5 Mechanisms of Acclimation or Adaptation to Drought

and Salinity Stress
Drought and soil salinity are among the most damaging abiotic stresses affecting to-
day’s agriculture. It is understandable that plants are under periodic water stress be-
cause of the unpredictable nature of rainfall. Salt stress is often observed in irrigated
areas, hydraulic lifting of salty underground water, or spread of seawater in coastal
areas. Plants have evolved mechanisms to perceive the incoming stresses and to
cope with them by rapid regulation of their physiology and metabolism. Very often,
such regulations and responses include feed-forward mechanisms for stress reduc-
tion that are in addition to the responses that are seen after stresses have caused irre-
versible damage to physiological functions. A good example of such a feed-forward
mechanism is the ability of plants to regulate their water loss through partial closure
of stomata and/or reduced leaf development, long before there is a substantial loss
of their leaf turgor or some irreversible damage to inner membrane systems (Zhang
et al. 2006a). The physiological responses of plants to survive under water stress
include leaf wilting, a reduction in leaf area, leaf abscission, and the stimulation of
root growth by directing nutrients to the underground parts of the plants. Besides,
the effects of water deficit become more detrimental during reproductive stages of
the plant (flowering and seed development), as the translocation of photosynthetic
assimilates from leaf to root is reduced which cannot grow more deep in search of
water and nutrients. In addition, ABA, the plant stress hormone, induces the closure
of leaf stomata, thereby reducing water loss through transpiration, and decreasing
the rate of photosynthesis. These responses improve the water-use efficiency of the
plant on the short term (Muhammad and Asghar 2012).
5.6 Effects of Drought and Salinity Stress on Plant

Morphology and Yield
5.6.1 Growth and Development
Plant responses to drought and salinity are complex and involve adaptive changes
and/or deleterious effects. The decrease in the water potential occurring in both
abiotic stresses results in reduced cell growth, root growth, and shoot growth and
also causes inhibition of cell expansion and reduction in cell wall synthesis (Chai-
tanya et al. 2003). According to these authors, drought (likely to salinity) affects
the regular metabolism of the cell such as carbon-reduction cycle, light reactions,
energy charge, and proton pumping and leads to the production of toxic molecules.
Literature has affirmed that plant responses to salt and water stress have much in
common. For example, according to Munns (2002), salinity brings a decrease in
water uptake by plants as the osmotic potential in the root vicinity will become high
and a kind of exosmosis may occur. This will slow down the growth rate, along
with a suite of metabolic changes identical to those caused by water stress. Ahmed
et al. (2013a) observed that barley plants treated with single or combined stress of
salinity (S) and drought (D) showed a significant decrease in plant height, shoot,
and root dry/fresh weights, with the largest reduction in the combined stress (D + S).
Therefore, most mechanisms to tolerate abiotic stresses like drought and salinity are
detrimental to plant development (Fig. 5.1).
Drought Salinity
Disruption of membrane Excessive toxicity of Na+and

integrity, dehydration, solute _
Cl ions to cells, physiological
leakage and metabolic drought to plant
dysfunction
Stress induced
injury
Activation Activation
Detoxification
of stress of stress
signaling
genes genes
Regulation of cell
division and
expansion Damage Ionic and osmotic
Restructuring of control and
cell membrane homeostasis via
repair SOS pathway or
and synthesis of Growth
osmolytes related pathways
inhibition
Stress
tolerance
Fig. 5.1 Possible drought and salt stress tolerance mechanisms in barley plants
5.6.2 Yield
Many yield-determining physiological processes in plants respond to water stress.

Yield is a quantitative trait and many physiological processes are involved. For
water stress, severity, duration and timing of stress, as well as responses of plants
after stress removal, and interaction between stress and other factors are extreme-
ly important (Plaut 2003). For instance, water stress applied at preanthesis re-
duced time to anthesis, while at postanthesis it shortened the grain-filling period
in triticale genotypes (Estrada-Campuzano et al. 2008). In barley ( H. vulgare),
drought stress reduces grain yield by decreasing the number of tillers, spikes,
and grains per plant and individual grain weight. Postanthesis drought stress was
detrimental to grain yield regardless of the stress severity (Samarah 2005). In
maize, water stress reduced yield by delaying silking, thus increasing the anthe-
sis-to-silking interval. This trait was highly correlated with grain yield, specifi-
cally ear and kernel number per plant (Cattivelli et al. 2008). Following heading,
drought had little effect on the rate of kernel filling in wheat, but its duration (time
from fertilization to maturity) was shortened and dry weight reduced at maturity
(Wardlaw and Willenbrink 2000).
Crop growth in saline medium is severely affected at different stages of the
plant life cycle. It was suggested by Shannon et al. (1994), that overall plant
response depends upon the concentration of salts in the tissue, composition of
salts, the exposure time, and climatic conditions as well. The commonly ob-
served adverse effects of salinity on Brassica species include the reduction in
plant height, yield, as well as deterioration of the quality of the product (Kumar
1995). In barley and wheat, salinity stress lowered grain yield by reducing grain
number and individual grain size (Harris et al. 2010). The plasticity of grain num-
ber and stability of grain size was found in another study in response to salinity
(Sadras 2007). Ahmed et al (2013b) observed that the reduction in spike length
was noticeably less in Tibetan wild barley than cultivated barley treated with sin-
gle or combined stress of salinity and drought. Moreover, the 1000-grain yield
and the filled grains per spike measurements were correlated, which may explain
the yield loss in cultivated barley compared to Tibetan wild barley under com-
bined drought and salinity during the anthesis stage. The decline in yield decline
was possibly associated with the reduction in spikelet fertility and grain filling
(Ahmed et al. 2013b).
In summary, prevailing drought and salinity reduce the plant growth and
development, increase flower abscission, reduce grain size due to poor grain filling
which arises due to the reduction in the partitioning of photosynthetic assimilate,
and decrease carbohydrate metabolism.
5.7 Physiological and Biochemical Bases for Drought

and Salinity Tolerance in Barley
5.7.1 Plant Water Relations
Leaf water potential, relative water content (RWC), stomatal movements,

transpiration, leaf and canopy temperatures are the important characteristics that
influence plant water relations. RWC represents plant water status including water
uptake by the roots as well as water loss by transpiration, thus reflect the metabolic
activity in plant tissue, and hence used as a most meaningful index for water stress
tolerance. A decrease in the RWC in response to drought stress has been noted in
a wide variety of plants (Nayyar and Gupta 2006). Furthermore, an exposure of
plants to drought stress substantially decreased the leaf water potential, RWC, and
transpiration rate, with a concomitant increase in leaf temperature as documented in
the previous study (Siddique et al. 2001). Although the components of plant water
relations are affected by reduced availability of water, stomatal opening and clos-
ing are more strongly affected. In barley, the application of the different watering
regimes decreased the RWC, midday leaf water potential ( ψw), and leaf osmotic
potential ( ψo) (Robredo et al. 2010).
Osmotic effects of salt on plants are due to the lowered soil water potential in the
root zone and thus resemble drought stress by affecting the ability of plants to extract
water from the soil and to maintain turgor (Sohan et al. 1999). However, at low or
moderate salt concentrations (higher soil water potential), plants accumulate solutes
and maintain a potential gradient for the influx of water. Under such conditions,
Shannon et al. (1984) reported that growth may be moderated, but unlike drought
stress, the plant is not water deficient. Several authors found that water potential and
osmotic potential of plants became more negative with an increase in salinity, where-
as the turgor pressure increased (Meloni et al. 2001; Gulzar et al. 2003). Vysotskaya
et al. (2010) reported a similar decrease in leaf water potential with increasing salt
concentration in wild barley species (“20–45” and T-1). At 75 mM NaCl, “20–45”
plants were characterized by less inhibition of leaf area, root fresh weight, leaf water
content, and leaf water potentials than T-1 species and were, therefore, considered
more tolerant to salt stress. According to Vysotskaya et al. (2010), these investiga-
tors, it was concluded that, under high salt concentration, plants (1) sequester more
NaCl in the leaf that lower the osmotic potential and (2) reduce the root hydraulic
conductance causing water stress in the leaf tissue. The combined stress of drought
and salinity depressed water potential, RWC in cultivated barley, but was unchanged
in Tibetan wild barley relative to control (Ahmed et al. 2013a).
5.7.2 Photosynthesis
Photosynthesis, together with cell growth, is among the primary processes to be af-
fected by drought (Chaves 1991) or by salinity (Munns et al. 2006). The effects can
be direct, as the decreased CO2 availability caused by diffusion limitations through
the stomata and the mesophyll (Flexas et al. 2007) or the alterations of photosyn-
thetic metabolism (Lawlor and Cornic 2002) or they can arise as secondary effects,
namely oxidative stress. Anjum et al. (2011) indicated that drought stress in maize
led to considerable decline in net photosynthesis, transpiration rate, stomatal con-
ductance, water-use efficiency, intrinsic water-use efficiency, and intercellular CO2
as compared to well-watered control.
Suppression of the photosynthetic capacity by salinity stress has been reported
in a number of plant species (Robinson et al. 1983; Ball and Farquhar 1984; Perez-
Lopez et al. 2012) and might be due to lower stomatal conductance, depression in
specific metabolic processes in carbon uptake, inhibition in photochemical capaci-
ty, or a combination of these (Dubey 1997). Tavakkoli et al. (2011) reported specific
ion toxicities of Na+ and Cl− reducing the growth of four barley genotypes grown in
varying salinity treatments. High Na+, Cl−, and NaCl separately reduced the growth
of barley; however, the reductions in growth and photosynthesis were greatest un-
der NaCl stress and were mainly additive of the effects of Na+ and Cl− stress. High
concentrations of Na+ reduced photosynthesis mainly by reducing stomatal con-
ductance. Salt-tolerant species, Barque73, had significantly greater photosynthetic
rate and water-use efficiency than those of Sahara, Clipper, and Tadmor. It was
concluded that high salt tolerance of the Barque73 was associated with a high CO2
assimilation rate, and water-use efficiency.
5.7.3 Chlorophyll Contents
Chlorophyll is one of the major components of photosynthesis, and decrease in

chlorophyll content under drought stress has been considered as a peculiar symptom
of oxidative stress and may be the result of pigment photooxidation and chloro-
phyll degradation. Drought stress caused a large decline in chlorophyll a content,
chlorophyll b content, and total chlorophyll content in different sunflower varieties
(Manivannan et al. 2007). Barley plants grown under drought showed inhibition of
chlorophyll synthesis as demonstrated by reduced SPAD (soil-plant analyses devel-
opment analyses, based on chlorophyll meter readings) values (Zhao et al. 2010).
Guo et al. (2009) reported that, after 13 days of drought stress, Martin and HS41-1
(drought tolerant) had much higher chlorophyll contents than Moroc9-75 (drought
sensitive).
The chlorophyll contents of leaves decrease in general under salt stress. The
oldest leaves start to develop chlorosis and drop-off with prolonged period of salt
stress (Hernandez et al. 1995; Gadallah 1999; Agastian et al. 2000). However, Wang
and Nil (2000) have reported that chlorophyll content increases under conditions
of salinity in Amaranthus. Salinity causes significant decreases in Chl-a, Chl-b,
and carotenoid in leaves of barley (Vysotskaya et al. 2010). Ahmed et al. (2013b)
reported that barley plants grown under combined drought and salinity treatment
showed a marked reduction in chlorophyll content (Chl-a, Chl-b, and carotenoids),
accompanied by a sharp decrease in net photosynthesis (Pn), stomatal conductance
(gs), and transpiration rate (Tr). These results indicate that photosynthetic inhibition
was caused by stomatal factors and by chlorophyll synthesis inhibition.
5.7.4 Chlorophyll Fluorescence
Chlorophyll fluorescence analysis has proven to be a sensitive method for the de-
tection and quantification of changes induced in the photosynthetic apparatus. The
chlorophyll fluorescence is based on the measurement of fluorescence signal of
dark-adapted plants exposed to continuous light (Govindjee 1995). The dark-adapt-
ed samples show characteristic changes in the intensity of chlorophyll fluorescence
during the illumination by continuous lights and this effect is called fluorescence
induction of Kautsky’s effect. When barley plants are exposed to drought, the val-
ues of maximal quantum yield of PSII ( Fv/Fm) decrease, which is a reliable sign of
photoinhibition (Guo et al. 2009).
Salt stress leads to a decrease in the efficiency of photosynthesis and is known
to influence the chlorophyll content and chlorophyll a fluorescence of barley leaves
(Fedina et al. 2003). Chlorophyll a fluorescence parameters have been used to
study high salt-induced damage to PSII. By measuring 77 K fluorescence emis-
sion spectra in dark grown wheat leaves under high salt conditions, it was shown
that salt stress inhibits the chlorophyll accumulation by restraining several steps in
porphyrin formation (Abdelkader et al. 2008). Delayed fluorescence measurements
in Arabidopsis thaliana seedlings have also proved to be useful as a marker for
detecting damage caused by salt stress (Zhang et al. 2008). A significant decrease in
Fv/Fm by combined drought and salinity (D + S) suggested a possible inhibition of
PSII photochemistry, which could be due to insufficient energy transfer from light
harvesting chlorophyll complex to the reaction center. Compared with Tibetan wild
barley (XZ5), greater decrease in Fv/Fm in cultivated barley (CM72) indicated that
PSII of the latter was more sensitive to D + S, suggesting that a higher protective
capacity for PSII could be an important tolerance mechanism for barley genotypes
(Ahmed et al., 2013a).
5.7.5 Plant Nutrition
Decreasing water availability under drought generally results in limited total nutri-
ent uptake and their diminished tissue concentrations in crop plants. An important
effect of water deficit is on the acquisition of nutrients by the root and their transport
to shoots (Farooq et al. 2009). In general, moisture stress induces an increase in N,
a definitive decline in P and no definitive effects on K (Garg 2003). Influence of
drought on plant nutrition may also be related to limited availability of energy for
the assimilation of NO3− / NH +4 , PO34− , and SO 24 − : they must be converted in energy-
dependent processes before these ions can be used for growth and development
of plants (Grossman and Takahashi 2001). As nutrient and water requirements are
closely related, fertilizer application is likely to increase the efficiency of crops
in utilizing available water. This indicates a significant interaction between soil
moisture deficits and nutrient acquisition. It was shown that N and K uptake was
hampered under drought stress in cotton (McWilliams 2003). Likewise, P and PO3− 4
contents in the plant tissues diminished under drought, possibly because of low-
ered PO3−4 mobility as a result of low moisture availability (Peuke and Rennenberg
2004). In general, drought stress reduces the availability, uptake, translocation, and
metabolism of nutrients. A reduced transpiration rate due to water deficit reduces
the nutrient absorption and efficiency of their utilization (Farooq et al. 2009).
Salinity hampers the uptake of macro- and micronutrients and the concentrations
of sodium (Na+) and chloride (Cl−) in the plant increase, and the concentrations of
potassium (K+) and calcium (Ca+) are reduced (Mansour et al. 2005). This together
result in inhibition of plant growth due to limitation in the absorption of other ions
and nutrients required for growth. It has also been reported that the accumulation
of Na+ and Cl− in both cellular and extracellular compartments competes with K+,
Ca+, magnesium (Mg2+), and manganese (Mn2+), whereas Cl− restricts the absorp-
tion of nitrate, phosphate, and sulfate ions (Termaat and Munns 1986; Romero and
Maranon 1994) and ultimately limits plant growth. Further, high levels of salinity
may also affect the transport of Cl− and Na+ by inhibiting the specific transport
systems of these ions (Maathuis 2006). Ahmed et al. (2013) reported that combined
stress (D + S) resulted in higher increase in Ca, Mn, and Fe concentrations in shoots
of wild barley (XZ5) than that of cultivated barley (CM72). Concerning root min-
eral concentrations, drought or salinity stress alone and in combination significantly
increased Ca concentrations in both genotypes, while no significant effect on Zn
and Cu concentrations was observed. Drought alone and D + S markedly increased
Mn concentration in XZ5, but had no effect on CM72 under salinity and D + S treat-
ments. Maintaining higher translocation of Ca, Mn, and Fe maybe an important
way to reduce D + S stress or beneficial to improve plant tolerance to drought and
salinity stress (Ahmed et al. 2013a).
5.7.6 Oxidative Stress and Enzymatic Regulation
The generation of reactive oxygen species (ROS) is one of the earliest biochemical
responses of eukaryotic cells to biotic and abiotic stresses (Apel and Hirt 2004). The
production of ROS in plants acts as a secondary messenger to trigger subsequent
defense reactions in plants. The most common ROS are hydrogen peroxide (H2O2),
superoxide, the hydroxyl radical, and singlet oxygen that formed as a natural by-
product of the normal metabolism of oxygen and is crucial in cell signaling. The
overproduction of ROS leads to oxidative stress and can cause damage to cellular
components.
To minimize the impact of oxidative stress, plants have evolved a complex
system of enzymatic antioxidants, superoxide dismutase (SOD), catalase (CAT),
peroxidase (POD), glutathione reductase (GR), and ascorbate peroxidase (APX),
and nonenzymatic antioxidants, ascorbic acid, α-tocopherol, reduced glutathione,
β-carotene, Polyamines (PAs), salicylates, compatible solutes such as proline (Pro),
glycine betaine (GB), and zeaxanthin that accumulate in higher plants under drought
and salinity stress (Ozkur et al. 2009).
Plants enhance the production of antioxidants in order to minimize the detrimental
effects of oxidative stress to normalize their metabolic activities under drought- and
salinity-induced oxidative stress (Fig. 5.2). Different antioxidants have roles in pro-

tecting cells in specific compartments and in particular conditions. It is generally
accepted that O 2− might be converted to H2O2 and then metabolized to water by APX
and GR in plants to maintain membrane structures (Foyer and Fletcher 2001). Like-
wise, several other antioxidant enzyme molecules are responsible to counteract the
deleterious effects of ROS. Initially, SOD catalyzes the conversion of O 2− to H2O2
that is further reduced to water by APX by using ascorbate as an electron donor
(Scandalios 2005). Elevated accumulation of antioxidant enzymes such as SOD,
CAT, GR, APX, and POD is involved in lowering oxidative injury in caper bush
seedlings under drought stress (Ozkur et al. 2009). Yang et al. (2009) reported an
increase in the activity of CAT, SOD, POD, APX, and GR at 25 % field capacity as
compared with 100 % field capacity. Seckin et al. (2010) observed the opposite pat-
terns in the activities of SOD, CAT, POD, APX, and GR enzymes in response to NaCl
stress in H. marinum and H. vulgare. Thus, the antioxidant system of H. marinum
Drought and salinity

stress
Excess ROS production

(1O2, H2O., OH., O22-, H2O2)
Antioxidant defense
AsA,
GABA, α-toc, GSH POD
Carotenoids, Proteins, lipids, GR. CAT, SOD,
Alkaloids, enzymes, DNA GST, APX
Phenols DHAR, MDHAR
Reduction of oxidative damage
Stress tolerance
Fig. 5.2 Role of antioxidant enzymes in the ROS scavenging mechanism. Exposure to drought
1
and salinity leads to generation of ROS, including singlet oxygen ( O2 ), perhydroxyl radical
2−
( H2O), superoxide hydroxyl radicals (O2 ), hydroxyl radicals ( OH), and hydrogen peroxide
( H2O2). ROS reactive oxygen species, SOD superoxide dismutase, CAT catalase, POD peroxidase,
GR glutathione reductase, APX ascorbate peroxidase, GABA γ-aminobutyric acid, GSH reduced
glutathione, MDHAR monodehydroascorbate reductase, DHAR dehydroascorbate reductase, GST
glutathione S-transferase
functioned at higher rates to suppress an increased ROS formation under salt stress.
The significant increase in the activities of SOD, POD, APX, and GR in the NaCl-
stressed leaves of H. marinum was highly correlated with the temporal regulation
of the constitutive isoenzymes as well as the induction of new isoenzymes. Lower
level of lipid peroxidation also revealed a higher free radical-scavenging capacity
and protection mechanism of H. marinum against high salinity (300 mM NaCl) than
H. Vulgare. Our previous reports (Ahmed et al. 2013b) indicated that CM72 had a
higher malondialdehyde (MDA) content than XZ5 not only under D + S treatments
but also under drought alone, suggesting less oxidative damage in Tibetan wild
barley than cv. CM72. The essential role of antioxidative systems for maintaining a
balance between the overproduction of ROS and their scavenging to keep them at
appropriate levels for signaling and reinstatement of metabolic homeostasis is well
established.
5.7.7 Compatible Solutes
Compatible solutes are low molecular weight and highly water-soluble compounds
that are usually nontoxic even at high cytosolic concentrations. Plants accumulate
compatible solutes, such as Pro and GB, sugars in response to drought and salinity
to facilitate water uptake (Hare et al. 1998; Ashraf and Foolad 2007). In addition to
osmotic adjustments, these osmolytes were suggested to be important for protecting
cells against increased levels of ROS accumulation under stress conditions. Major
contributors to osmotic adjustment were revealed to be K+ in the early stages of stress
and molecules including GB, Pro, and glucose, in the late stress (Nio et al. 2011).
Pro accumulates in the cytosol and the vacuole during stress (McNeil et al. 1999)
and was shown to protect plant cells against damages caused by 1 O 2 or HO (Matysik
et al. 2002). By quenching 1 O 2 and directly scavenging HO, Pro might be able to
protect proteins, DNA, and membranes (Smirnoff and Cumbes 1989; Matysik et al.
2002). In the recent study, drought stress alone and D + S combined stress caused a
marked increase in GB content in XZ5 and XZ16, more so than in CM72 (Ahmed
et al. 2013b). Enhanced GB levels in Tibetan wild barley may exert protection on
enzyme activity, including enzymes associated with sugar and amino acid metabo-
lism (Chen et al. 2007), leading to greater increases in soluble sugars and Pro in Ti-
betan wild barley than control. Thus, it is proposed that the two Tibetan wild barley
genotypes may acquire more protection than cv. CM72 under stressed environment
due to the elevated levels of GB and the greater osmotic protection from higher
levels of soluble sugars and Pro.
5.7.8 Plant Secondary Metabolism
Plant produces a large variety of secondary metabolites through several metabolic

pathways in normal condition. But different stresses either biotic or abiotic trigger
the plant secondary metabolism that results in enhanced production of plant
secondary products. Generally, precursors of secondary metabolic pathways are the

products of the primary metabolism. To a large extent, secondary metabolites derive
from three biosynthetic routes, namely the phenyl propanoid, isoprenoid, and alka-
loid pathways. The major source of aromatic secondary metabolites in plants is the
phenylpropanoid pathway (Irti and Faoro 2009).
Elevated phenol and flavonoid content were observed under single and combined
stresses in the two Tibetan wild genotypes (Ahmed et al. 2014). In salt stressed
H. vulgare, significantly higher concentration of flavonoids was observed (Ali and
Abbas 2003). The content of protochatechuic acid, caffeic, and chlorogenic acids
was increased following drought stress in Matricaria chamomilla (Kováčik et al.
2009). Ahmed et al. (2013c) also observed that the increase of phenolic compounds
in the tissue prevented the formation of ROS in Tibetan wild and cultivated bar-
ley under combined drought and salinity stresses. In addition, the induced expres-
sion of genes related to secondary metabolism ( GST, PPO, SKDH, PAL, CAD, and
chi2) was demonstrated under all stress conditions in wild barley and accompanied
an increase in the activities of the respective enzymes, with the greatest increase
observed in XZ5. During rehydration and recovery, the activities of all enzymes
increased except for phenylalanine ammonialyase (PAL) and cinnamyl alcohol
dehydrogenase (CAD), which increased only in XZ5 (Ahmed et al. 2014).
5.7.9 Ultra-Morphology of Plants
Drought and salt stress leads to disintegration of fine structure of chloroplast, insta-
bility of the pigment protein complexes, destruction of chlorophylls, and changes in
the quantity and composition of carotenoids (Dubey 1997). A wide array of varia-
tion has been observed in many studies regarding the effects of salinity stress on
chloroplast ultrastructure like swelling of thylakoid membranes of chloroplast in
the mesophyll cells of sweet potato leaves (Mitsuya et al. 2000) and also reduced
numbers and depth of the grana stacks, and enlargement of starch grains in the
chloroplasts of potato (Bruns and Hecht-Buchholz 1990). Hernández et al (1995)
observed disorganized thylakoid structure of the chloroplasts, increased number
and size of plastoglobuli, and decreased starch content in chloroplasts of plants ex-
posed to drought and salinity stress. Whereas, chloroplasts aggregation, distortion
of cell membranes with no signs of grana or thylakoid in chloroplasts were observed
in tomato plants exposed to salt stress (Khavari-Nejad and Mostofi 1998). Eleva-
tion in the level of NaCl increased swelling of thylakoids and reduced chlorophyll
fluorescence in barley seedlings (Zahra et al. 2014). Chloroplasts and mitochondria
were affected in a variety-specific manner under all adverse treatments. The organ-
elles of the drought-tolerant wheat cultivar Katya were better preserved than those
in the sensitive variety Sadovo. Leaf ultrastructure can be considered as one of the
important characteristics in the evaluation of the drought susceptibility of different
wheat varieties (Grigorova et al. 2012). The effect of drought and salinity alone and
in combination on endosperm starch and protein composition varied with genotypes
and treatments. Under drought stress, the endosperm of CM72 grains had smaller
starch granules, especially B-type granules, which were located adjacent to crushed
cell layer (CCL), while many A-type starch granules in this region were either pit-
ted or showed surface erosion. The appearance of pitting can be associated with
degradation of the proteinaceous layer, exposing the starch granule to severe stress.
However, XZ5 and XZ16 showed more protein deposited on the surface of starch
granules under drought stress (Ahmed et al. 2013c).
5.8 Identification of QTLs Controlling Drought

and Salinity Tolerance in Barley
Quantitative trait locus (QTL) mapping is a powerful approach for locating genomic
regions controlling complex traits (Gyenis et al. 2007). By linking phenotypic and
genotypic data, QTL mapping enables the identification of the action, interaction,
numbers and chromosomal locations of loci affecting particular traits (Miles and
Wayne 2008). Large numbers of barley mapping populations have been developed
to map genes and QTLs controlling agronomic and quality traits (Table 5.1) and
have been reviewed by Fox et al. (2003). Several barley populations have been
developed to map the QTLs for drought tolerance in both controlled environments
and Mediterranean field trials. These included Tadmor x (ER/Apm) RIL population
(Teulat et al. 1998), Derkado x B83-12 DH population (Forster et al. 2004), Apex
x ISR101-23 (Pillen et al. 2003), and Barke x Hor11508 populations (Talame et al.
2004).
Kalladan et al. (2013) used advanced backcross quantitative trait locus (AB-
QTL) analysis of a BC3-doubled haploid population developed between the culti-
vated parent Brenda ( H. vulgare ssp. vulgare) and the wild accession HS584 ( H.
vulgare ssp. spontaneum) to study the contribution of wild barley in improving
various agronomic and seed quality traits under postanthesis drought. QTL analysis
indicated that wild barley contributed favorably to most of the traits studied under
both control and drought conditions. A total of seven hotspot QTL regions with
colocalizing QTL for various traits harbored more than 80 % of the stable QTL de-
tected in their study. For yield and 1000-grain weight and their respective drought-
tolerance indices, most of the QTLs were derived from Brenda. On the other hand,
for traits like seed length and seed nitrogen content, all the QTLs were contributed
by HS584, the parent with higher trait value.
Many QTL studies carried out using wild barley as a donor parent for various
traits indicated that it is a potential source for trait improvement (Nevo 1992; Volis
et al. 2000; Pillen et al. 2004; Li et al. 2005, 2006; Rostoks et al. 2005; Schmalen-
bach et al. 2009; Schnaithmann and Pillen 2013). In addition, H. vulgare ssp.
spontaneum was also found to possess positive alleles for abiotic stresses such as
drought and salt (Talame et al. 2004; Suprunova et al. 2007; Ceccarelli et al. 2007;
Lakew et al. 2011, 2013). Major hindrances to the utilization of wild species in crop
improvement using conventional breeding are the quantitative nature of most of the
agronomic traits and the linkage drag of undesirable genes present in wild species
Table 5.1 Enhancing drought and salinity tolerance in barley lines/varieties using marker-assisted selection
Stress QTL used QTL donor line/cultivar Line/cultivar developed Trait improved References
Drought AB-QTL Apex/ISR101–23 BC2F2 population For various agronomic and Pillen et al. (2003)
malting quality traits
Drought AB-QTL Brenda/HS584 BC3-doubled haploid Improved various agronomic Kalladan et al. (2013)
population developed and seed quality traits
Drought 81 QTLs were used, out Hordeum spontaneum Backcross population Improved grain yield, and Baum et al. (2003),
of which six (1H-3, 2H-1, reduced negative impact of Talame et al. (2004),
3H-2, 4H-3, 1H-5, 3H-1 drought on grain filling Tuberosa and Salvi (2006)
and 3H-4) were for grain
yield
Salinity Identified QTLs were Steptoe x Morex and Two doubled haploid Those of QTLs controlling Mano and Takeda (1997)
located on chromosomes Harrington x TR306 (DH) populations salt tolerance at germination
2H, 1H, 4H, 6H, and 5H and at the seedling stage
Salinity A spring barley collection From a wide This study can be used Improved biomass Long et al. (2013)
of 192 genotypes were geographical range for targeting candidate production, chlorophyll
used to identify QTLs on gene(s) for salt tolerance content, plant height, tiller
chromosome 6H and 4H and uptake/transportation number, leaf senescence and
by a 1000 SNP marker set of both Na+ and Cl− which shoot Na+, shoot Cl− and
are important factors for shoot, root Na+/K+ contents
salt-tolerance improve-
5 Tolerance to Combined Stress of Drought and Salinity in Barley
ment of barley
Drought and MQTL were located Hordeum vulgare MWG2262BE37M33-160 In this study, 26 genes (some Li et al. (2013)
salinity on chromosomes 2H and E36M50-81- genes with two functions)
(drought) and 5H (salinity) E38M61-302, could be associated with antioxidation
better for developing (7), electron/ion transporta-
makers for salinity and tion (11), Ca+2 ion binding
drought stresses tolerance (3), ATP binding/ATPase
activity (7), or phosphoryla-
tion (7) were colocated with
the five-type MQTL
107
QTL quantitative trait locus, MQTL meta-QTL, AB-QTL advanced backcross QTL, SNP single nucleotide polymorphism
(Wang and Chee 2010). One of the breeding strategies to overcome the problem of
linkage drag associated with wild genotypes during breeding programs is AB-QTL
analysis, which combines QTL detection with the introduction of favorable alleles
into the targeted variety (Tanksley and Nelson 1996). In barley, AB-QTL analysis
was first reported by Pillen et al. (2003) using a BC2F2 population developed be-
tween the cultivar Apex and the wild accession ISR101-23 for various agronomic
and malting quality traits. Some of the other studies for improving drought toler-
ance in barley include Baum et al. (2003), Ceccarelli et al. (2004), Forster et al.
(1997), Grando et al. (2001), and Ivandic et al. (2003).
Wild barley H. spontaneum has been recognized as an important source for
drought tolerance. A QTL identified on chromosome 4H from H. spontaneum con-
sistently increased grain yield across six test environments with an average yield
increase of 7.7 % (Pillen et al. 2003). Talame et al. (2004) identified two QTLs
on chromosomes 2H and 5H with relative yield increase ranging from 12 to 22 %
under dry conditions. These QTLs could be used as target chromosome regions for
the integration of wild barley genes for yield improvement under drought. Lu et al.
(1999) suggested that drought tolerance in wild barley is related to their differ-
ing genetic abilities of osmotic adjustment under drought conditions. Thus, further
genetic mapping and marker-assisted transfer of the osmotic-adjustment genes har-
bored in the wild progenitor could improve resistance of cultivated barley grown in
water-limited environments.
Traditional QTL mapping or biparental QTL mapping based on a single segre-
gating population derived from two homozygous parental genotypes has been the
commonly used approach for genetic dissection of salt tolerance in barley and to
identify candidate genes (Mano and Takeda 1997; Xue et al. 2009; Ellis et al. 2002;
Witzel et al. 2009). This approach provides valuable information on genomic re-
gions that control quantitative traits but it also has limitations due to poor sampling
of the allelic variation present in the barley gene pool for each of the loci affect-
ing salt tolerance, lack of segregation, and poor resolution of this type of mapping
QTLs. Mano and Takeda (1997) identified QTLs controlling salt tolerance at ger-
mination and the seedling stage in barley by interval mapping analysis using marker
information from two doubled haploid (DH) populations derived from the crosses,
Steptoe × Morex, and Harrington × TR306. The results revealed that the QTLs
for salt tolerance at germination in the DH lines of Steptoe x Morex were located
on chromosomes 4H, 6H, and 5H, and in the DH lines of Harrington/TR306 on
chromosomes 1H and 5H. In both DH populations, the most effective QTLs were
found at different loci on chromosome 5H. Genetic linkage between salt tolerance
at germination and ABA response was found from QTL mapping. The QTLs for
the most effective ABA response at germination were located very close to those
for salt tolerance on chromosome 5H in both crosses. The QTLs for salt tolerance
at the seedling stage were located on chromosomes 2H, 1H, 6H, and 5H in the DH
lines of Steptoe x Morex, and on chromosome 5H in the DH lines of Harrington x
TR306. Their positions were different from those of QTLs controlling salt tolerance
at germination, indicating that salt tolerance at germination and at the seedling stage
was controlled by different loci.
Long et al. (2013) demonstrated that a spring barley collection of 192 genotypes
from a wide geographical range was used to identify QTLs for salt-tolerance traits
by means of an association mapping approach using a 1000 single nucleotide poly-
morphism (SNP) marker set. Linkage disequilibrium (LD) decay was found with
marker distances spanning 2–8 cM depending on the methods used to account for
population structure and genetic relatedness between genotypes. The association
panel showed large variation for traits that were highly heritable under salt stress,
including biomass production, chlorophyll content, plant height, tiller number, leaf
senescence, shoot Na+, shoot Cl−, and shoot, root Na+/K+ contents. The significant
correlations between these traits and salt tolerance (defined as the biomass pro-
duced under salt stress relative to the biomass produced under control conditions)
indicate that these traits contribute to (components of) salt tolerance. Association
mapping was performed using several methods to account for population structure
and minimize false-positive associations. This resulted in the identification of a
number of genomic regions that strongly influenced salt tolerance and ion homeo-
stasis, with a major QTL controlling salt tolerance on chromosome 6H, and a strong
QTL for ion contents on chromosome 4H (Long et al. 2013).
Recently, Li et al. (2013) confirmed that the distribution of meta-QTL (MQTL)
was similar to that of the initial QTL. Many of these MQTL were located on chro-
mosomes 2H (drought) and 5H (salinity). It inferred that chromosomes 2H and
5H were important for barley abiotic stress tolerance. As expected from trait cor-
relations, 22.8 % of these MQTL displayed overlapping confidence intervals (CIs).
These overlapping regions were mainly on chromosomes 1H, 2H, and 4H. The
results indicated that the tolerance to diverse abiotic stresses were associated with
each other in barley (Li et al. 2013).
5.9 Molecular Approaches for Improvement

of Modern Barley
The high-throughput omics analysis, including transcriptomics, proteomics, and me-

tabolomics, will improve comprehensive understanding of drought and salt stress-
induced changes in gene-protein‑metabolite (Urano et al. 2010; Sicher et al. 2012).
Transcriptomics and proteomics analysis have been widely used in salt-tolerance stud-
ies (Du et al. 2008; Zhang et al. 2012). Currently, metabolomics are developed and
applied in understanding multiple physiological processes in plants, in combination
with other platforms such as transcript profiling and proteomics. Major approaches
currently used in plant metabolomics are metabolic fingerprinting, metabolite profil-
ing, and targeted analysis. Main analysis methods include gas chromatography-mass
spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capil-
lary electrophoresis-mass spectrometry (CE-MS), Fourier transformation cyclotron
resonance-mass spectrometry (FT-ICR-MS), and nuclear magnetic resonance (NMR;
Nicholson et al. 1999; Shulaev et al. 2008). In recent years, metabolomics analysis is
being widely used to investigate abiotic stress tolerance of plants (Shulaev et al. 2008;
Oliver et al. 2011). In barley root, the metabolite profiling was analyzed in response
to drought (Sicher et al. 2012), and combined stress of high temperature and drought
(Rizhsky et al. 2004). Metabolome changes were also reported in cultivated barleys
in response to salt stress (Widodo et al. 2009, Wu et al. 2013). In these context, sev-
eral categories of genes which respond to the stress could be differentiated (Fig. 5.3):
genes that encode protective but metabolically inactive polypeptides, such as dehy-
drins, chaperones (including proteases), genes for metabolic pathways leading to the
synthesis of low-molecular osmolytes which increase stress tolerance, radical scaven-
gers, or compounds with both functions, and regulatory proteins such as transcription
factors, protein kinases, phospholipase C, or 14-3-3 proteins.
Most of the drought- and salt-tolerance genes belong to large gene families with
high-sequence similarity distribute in a genome, which brings difficulty in identify-
ing the specific locus for a specific function. More recently, genomic technologies
have provided high-throughput integrated approaches (Bartels and Sunkar 2005) to
investigate global gene expression responses not only to drought but also to other
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Fig. 5.3 Stress tolerance factors produced in adaptive responses of a barley plant to drought and
salinity stress. CBF C-repeat binding factor, MYB myeloblastosis oncogenes, LEA late embryo-
genesis abundant, INA ice nucleation-active protein, MYC v-myc avian myelocytomatosis viral
oncogene homolog, bZIP basic leucine zipper, MAPK mitogen-activated protein kinase, MAPKK
mitogen-activated protein kinase kinase, HVA1 ABA-inducible protein PHV A1, WRKY c-terminal
wrky domain, NAC nascent polypeptide-associated complex protein
abiotic stresses (Chaves et al. 2003). Microarray profiling under drought stress has
been carried out in different plant species such as Arabidopsis (Oono et al. 2003),
rice (Rabbani et al. 2003), barley (Ozturk et al. 2002; Talame’ et al. 2007), and
wheat (Mohammadi et al. 2007). These studies identified differentially expressed
transcripts of genes involved in photosynthesis, ABA synthesis and signaling, bio-
synthesis of osmoprotectants, protein stability and protection, reactive oxygen de-
toxification, water uptake, and a myriad of transcription factors including several
members of the zinc finger, WRKY (c-terminal wrky domain), and bZIP (basic
leucine zipper) families. Du et al. (2011) showed that two dehydrin genes might
contribute to improved drought and salt tolerance of Tibetan and wild barley. Hv-
WRKY38 is a barley gene coding for a WRKY protein, whose expression is in-
volved in cold and drought stress response which was mapped close to the QTL
region (Mare et al. 2004). Hv-WRKY38 was early and transiently expressed during
exposure to low nonfreezing temperature, in ABA-independent manner. Further-
more, it showed a continuous induction during dehydration and freezing treatments.
The aquaporin, dehydrin, C-repeat binding factor (CBF) genes, and Hv-WRKY38
may be putative candidate genes that underlie the QTL effect on salt tolerance.
Differentially regulated proteins predominantly had functions not only in photo-
synthesis but also in detoxification, energy metabolism, and protein biosynthesis.
The analysis indicated that de novo protein biosynthesis, protein quality control
mediated by chaperones and proteases, and the use of alternative energy resources,
i.e., glycolysis, play important roles in adaptation to drought and heat stress (Rollins
et al. 2013).
Transcriptional factors (TFs) play important roles in the regulation of gene ex-
pression in response to abiotic stresses such as drought and salinity. TFs are power-
ful targets for genetic engineering of stress tolerance, because overexpression of a
single TF can lead to the up-regulation or down-regulation of a wide array of stress
response genes. Until now, transcription factors have been the most appealing tar-
gets for transgenic barley improvement, due to their role in multiple stress-related
pathways. Dehydration-responsive element-binding protein 1 (DREB1)/CBF and
DREB2 gene function in ABA-independent gene expression while ABA-responsive
element (ABRE)-binding protein (AREB)/ABRE binding factor (ABF) functions in
ABA-dependent gene expression. NAC (nascent polypeptide-associated complex
protein) and MYB (myeloblastosis oncogenes)/MYC (v-myc avian myelocytomato-
sis viral oncogene homolog) are involved in abiotic stress-responsive gene expression
(Uauy et al. 2006). In another study, a barley LEA protein, HVA1 (ABA-inducible
protein PHV A1), was also overexpressed in wheat, and the overexpressors were
observed to have better drought tolerance (Bahieldin et al. 2005). Transgenic wheat
obtained with Arabidopsis DREB and HVA1 protein overexpression was also shown
to produce higher yield in the field under drought conditions, but further studies are
required to confirm their performance under different environments (Bahieldin et al.
2005). The transformation of oat and rice with the barley HVA1 gene also improved
drought and salt tolerance (Xu et al. 1996; Oraby et al. 2005). It is not unreasonable
to predict in the following decades: genetically modified (GM) wheat will be trans-
ferred to the fields as a common commercial crop. However, to pace this process,
new transgenics methodologies should be developed since the current methods are
laborious and time-consuming. In a recent study, drought enhancement of bread
wheat was established with the overexpression of barley HVA1, using a novel tech-
nique, which combines doubled haploid technology and Agrobacterium-mediated
genetic transformation (Chauhan and Khurana 2011). Most of the transformed genes
are from model plants such as Arabidopsis and rice or from wheat and barley cul-
tivars. These approaches could be applied to wild relatives whose genes may have
stronger effects. This hypothesis awaits experimental confirmation and field testing.
Plant miRNAs are approximately 20–24-nucleotide noncoding RNAs that spe-
cifically base pair to and induce the cleavage of target mRNAs or cause transla-
tional inhibition (Zhang et al. 2006b; Shukla et al. 2008). They have diverse roles
in plant development, such as phase transition, leaf morphogenesis, floral organ
identity, developmental timing, and other aspects of plant development (Lu and
Huang 2008; Rubio-Somoza and Weigel 2011). To date, numerous miRNAs from
diverse plant species have been identified and functionally characterized in plant
development as well as stress response to biotic and abiotic environmental factors
(Eldem et al. 2013). More than 40 miRNA families in plants have been associated
with response to abiotic stress such as salt and drought (Sunkar 2010; Covarrubias
and Reyes 2010). For instance, miR167, miR168, miR171, and miR396 were found
to be drought-responsive miRNAs in Arabidopsis (Liu et al. 2008). In search of
potential miRNAs involved in drought response in barley, some of the miRNAs,
such as miR156, miR171, miR166, and miR408, were observed as differentially
expressed upon dehydration (Kantar et al. 2011). miR166 is an example of many
drought-responsive miRNAs that were previously characterized as crucial for cell
development. It posttranscriptionally regulates class-III homeodomain-leucine zip-
per ( HD-Zip III) transcription factors, which were demonstrated to be important
for lateral root development, axillary meristem initiation, and leaf polarity (Hawker
and Bowman 2004; Boualem et al. 2008). It is likely that differential regulation
of miRNAs in different tissues is important for adaptation to stress in plants. For
example, four miRNAs displayed tissue-specific regulation during dehydration in
barley: miR166 was up-regulated in leaves, but down-regulated in roots; and mi-
R156a, miR171, and miR408 were induced in leaves, but unaltered in roots (Kantar
et al. 2011). Studying drought-responsive miRNAs and their target gene expression
in individual cell types will provide greater insights into miRNA target networks
that operate in a cell- or tissue-specific manner under drought stress. Zhou et al.
(2013) reported that the overexpression of miR319 impacts plant development and
enhances plant drought and salt tolerance. The miR319-mediated down-regulation
of target genes in transgenic plants may have caused changes in various biologi-
cal processes, including those associated with water retention capacity, leaf wax
synthesis, and salt uptake beneficial to plants responding to salinity and water defi-
ciency. The manipulation of miR319 target genes provides novel molecular strate-
gies to genetically engineer crop species for enhanced resistance to environmental
stress. An increasing understanding of the role of miRNAs in drought and salinity
tolerance will enable the use of miRNA-mediated gene regulation to enhance plant
drought and salinity tolerance.
Although tremendous efforts have been applied to breed drought- and s alt-tolerant
barley by conventional and molecular approaches, truly drought and salt-tolerant
barley cultivars have not been produced that can go to farmer’s field. The promising
drought- and/or salt-tolerant genotypes are still in the laboratory and experimental
fields. To overcome this bottleneck from the laboratory to the farmer’s field, breed-
ing programs should target specific environments and pyramid tolerance genes be-
cause drought and salt stresses are complex and variable in different environments
and in different years.
5.10 Conclusions and Future Perspectives
Crop production under field conditions can be decreased by several abiotic stresses
and the studies on multifactor interactions are of greater importance than analyses
of only one stress. A combination of drought and salinity stress affects the plants to
a larger degree and plant reaction cannot be directly extrapolated from the response
of plants to individual effect of these two stresses. In the case of drought toler-
ance, plants potentiate to maintain the metabolic activities even at lower level of
tissue water potential by accumulating intracellular osmoprotectants such as Pro,
GB, amino acids, and soluble sugars. Besides, scavenging of ROS by enzymatic
and nonenzymatic antioxidants, cell membrane stability, expression of aquaporin,
and stress-related proteins such as LEA (late embryogenesis abundant) are also the
vital mechanisms of drought and salinity stress tolerance.
Marker-trait associations are being identified by the development of a high den-
sity SNP assay platform that provides sufficient marker density for genome-wide
scans and LD-led gene identification (Waugh et al. 2009). Projects are aiming to
exploit the discriminatory LD observed in landrace and wild barley populations for
fine mapping and gene identification (e.g., ExBarDiv: http://pgrc.ipk-gatersleben.
de/barleynet/projects_exbardiv.php). Highly significant associations can be identi-
fied between genome-wide SNPs and drought and salt tolerances in wild progeni-
tors, landraces, and varieties. These approaches offer the possibility of identifying
novel allelic variation that may be of considerable value to future crop improvement
(Waugh et al. 2009).
Advances are still needed to efficiently explore the extensive reservoir of drought
and salt-tolerant alleles within wild germplasm deciphering: (1) the molecular net-
works those lost during domestication and modern breeding (Fu and Somers 2009);
(2) the high-throughput screening of wild germplasm for drought/salt tolerance and
their regulation of fitness components; (3) the molecular basis of chromosomal re-
combination; and (4) the potential regulatory relationship between coding and non-
coding regions. This will increase the availability of sequence information and will
encourage new breeding strategies by transferring single and multiple interacting
networked loci/QTLs from wild relatives to commercial varieties via marker-assisted
selection. The International Triticeae Mapping Initiative and the Barley Genome Se-
quencing Consortia are serving as platforms for international collaborative projects
that will ensure the use of extensive drought- and salt-tolerance gene pools for ce-
real crop improvement.
References
Abdelkader AF, Aronsson H, Solymosi K, Böddi B, Sundqvist C. Chlorophyll accumulation, pro-

tochlorophyllide formation and prolamellar body conversion are held back in wheat leaves
exposed to high salt stress, photosynthesis. Energy from the Sun. Berlin: Springer; 2008.
pp. 1133–6.
Agastian P, Kingsley S, Vivekanandan M. Effect of salinity on photosynthesis and biochemical
characteristics in mulberry genotypes. Photosynthetica. 2000;38:287–90.
Ahmed IM, Dai HX, Zheng W, Cao FB, Zhang GP, Sun DF, Wu FB. Genotypic differences in
physiological characteristics in the tolerance to drought and salinity combined stress between
Tibetan wild and cultivated barley. Plant Physiol Biochem. 2013a;63:49–60.
Ahmed IM, Cao F, Zhang M, Chen X, Zhang G, Wu F. Difference in yield and physiological fea-
tures in response to drought and salinity combined stress during anthesis in Tibetan wild and
cultivated barleys. PloS ONE. 2013b;8(10):e77869.
Ahmed IM, Cao FB, Han Y, Nadira UA, Zhang GP, Wu FB. Differential changes in grain ultra-
structure, amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys
under drought and salinity alone and combined stress. Food Chem. 2013c;141:2743–50.
Ahmed IM, Nadira UA, Bibi N, Cao FB, Zhang GP, Wu FB. Differential changes in leaf physiol-
ogy and secondary metabolism between Tibetan wild and cultivated barleys under drought
and salinity alone and combined stress and subsequent recovery. Environ Expt Bot. 2014 (Ac-
cepted).
Ali R, Abbas H. Response of salt stressed barley seedlings to Phenylurea. Plant Soil Environ.
2003;49:158–62.
Anjum SA, Xie X, Wang L, Saleem MF, Man C, Lei W. Morphological, physiological and bio-
chemical responses of plants to drought stress. Afr J Agric Res. 2011;6:2026–32.
Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction.
Ann Rev Plant Biol. 2004;55:373–99.
Ashraf M, Foolad M. Roles of glycine betaine and proline in improving plant abiotic stress resis-
tance. Environ Exp Bot. 2007;59:206–16.
Ashraf M, O’leary J. Responses of some newly developed salt-tolerant genotypes of spring wheat
to salt stress: 1. yield components and ion distribution. J Agron Crop Sci. 1996;176:91–101.
Bahieldin A, Mahfouz HT, Eissa HF, Saleh OM, Ramadan AM, Ahmed IA, Dyer WE, El-Itriby HA
Madkour MA. Field evaluation of transgenic wheat plants stably expressing the HVA1 gene for
drought tolerance. Physiol Planta. 2005;123:421–7.
Ball MC, Farquhar GD. Photosynthetic and stomatal responses of two mangrove species, Ae-
giceras corniculatum and Avicennia marina, to long term salinity and humidity conditions.
Plant Physiol. 1984;74:1–6.
Bartels D, Sunkar R. Drought and salt tolerance in plants. Crit Rev Plant Sci. 2005;24:23–58.
Baik B-K, Ullrich SE. Barley for food: characteristics, improvement, and renewed interest. J Ce-
real Sci. 2008;48(2):233–42.
Baum M, Grando S, Backes G, Jahoor A, Sabbagh A, Ceccarelli S. QTLs for agronomic traits in
the Mediterranean environment identified in recombinant inbred lines of the cross _Arta_ _ H.
spontaneum 41-1. Theor Appl Genet. 2003;107:1215–225.
Bothmer R von, Jacobsen N, Baden C, Jorgensen RB, Linde-Laursen I. An ecogeographical study
of the genus Hordeum. Systematic and ecogeographic studies on crop genepools 7, 2nd ed.
Rome: IPGRI; 1995. p. 129.
Boualem A, Fergany M, Fernandez R, Troadec C, Martin A, Morin H, Sari M-A, Collin F, Flowers
JM, Pitrat M. A conserved mutation in an ethylene biosynthesis enzyme leads to an dromon-
oecy in melons. Science. 2008;321(5890):836–8.
Bruns S, Hecht-Buchholz C. Light and electron microscope studies on the leaves of several potato
cultivars after application of salt at various development stages. Potato Res. 1990;33:33–41.
Budak H, Kantar M, Kurtoglu KY. Drought tolerance in modern and wild wheat. The Scientific
World J. 2013. p. 16.
Cattivelli L, Rizza F, Badeck F-W, Mazzucotelli E, Mastrangelo AM, Francia E, Mare C, Tondelli
A, Stanca AM. Drought tolerance improvement in crop plants: an integrated view from breed-
ing to genomics. Field Crops Res. 2008;105:1–14.
Ceccarelli S, Grando S, Baum M, Udupa SM. Breeding for drought resistance in a changing cli-
mate. Challenges and strategies for dryland agriculture. Madison: Crop Science Society of
America Inc. and American Society of Agronomy Inc.; 2004. pp 167–90.
Ceccarelli S, Grando S, Baum M. Participatory plant breeding in water-limited environments. Exp
Agric. 2007;43(04):411–35.
Chaitanya K, Jutur P, Sundar D, Reddy AR. Water stress effects on photosynthesis in different
mulberry cultivars. Plant Growth Regul. 2003;40:75–80.
Chauhan H, Khurana P. Use of doubled haploid technology for development of stable drought
tolerant bread wheat ( Triticum aestivum L.) transgenics. Plant Biotech J. 2011;9(3):408–17.
Chaves MM. Effects of water deficits on carbon assimilation. J Exp Bot. 1991;42:1–16.
Chaves MM, Oliveira M. Mechanisms underlying plant resilience to water deficits: prospects for
water-saving agriculture. J Exp Bot. 2004;55:2365–84.
Chaves MM, Maroco JP, Pereira JS. Understanding plant responses to drought-from genes to the
whole plant. Func Plant Biol. 2003;30:239–64.
Chen ZH, Cuin TA, Zhou M, Twomey A, Naidu BP, Shabala S. Compatible solute accumulation
and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. J Exp Bot.
2007;58:4245–55.
Covarrubias AA, Reyes JL. Post-transcriptional gene regulation of salinity and drought responses
by plant microRNAs. Plant Cell Environ. 2010;33(4):481–9.
Dai A. Drought under global warming: a review. wiley interdisciplinary reviews. Climate Change.
2011;2:45–65.
Dubey R. Photosynthesis in plants under stressful conditions. Handb Photosynth. 1997;2:717–37.
Du J, Huang YP, Xi J, Cao MJ, Ni WS. Functional gene-mining for salt-tolerance genes with the
power of Arabidopsis. Plant J. 2008;56:653–64.
Du JB, Yuan S, Chen YE, Sun X, Zhang ZW, Xu F, Yuan M, Shang J, Lin HH. Comparative ex-
pression analysis of dehydrins between two barleyvarieties, wild barley and Tibetan hulless
barley associated with different stress resistance. Acta Physiol Planta. 2011;33:567–74.
Eldem V, Okay S, Unver T. Plant microRNAs: new players in functional genomics. Turk J Agric
For. 2013;37:1–21.
Ellis RP, Forster BP, Gordon DC, Handley LL, Keith RP, Lawrence P, Meyer R, Powell W, Rob-
inson D, Scrimgeour CM, Young G, Thomas WT. Phenotype/genotype associations for yield
and salt tolerance in a barley mapping population segregating for two dwarfing genes. J Exp
Bot. 2002;53:1163–76.
Estrada-Campuzano G, Miralles DJ, Slafer GA. Genotypic variability and response to water stress
of pre-and post-anthesis phases in triticale. Eur J Agron. 2008;28:171–7.
Farooq M, Wahid A, Kobayashi N, Fujita D, Basra S. Plant drought stress: effects, mechanisms
and management, sustainable agriculture. Berlin: Springer; 2009. pp. 153–88.
Fedina IS, Grigorova ID, Georgieva KM. Response of barley seedlings to UV-B radiation as af-
fected by NaCl. J Plant Physiol. 2003;160:205–8.
Flexas J, Diaz-Espejo A, Galm ESJ, Kaldenhoff R, Medrano HOL, Ribas-Carbo M. Rapid varia-
tions of mesophyll conductance in response to changes in CO2 concentration around leaves.
Forster BP, Rzussell JR, Ellis RP, Handley LL, Robinson D, Hackett CA, Nevo E, Waugh R,
Gordon DC, Keith R, Powell W. Locating genotypes and genes for abiotic stress tolerance in
barley: a strategy using maps, markers and the wild species. New Phytol. 1997;137:141–7.
Forster BP, Ellis RP, Moir J, Talame V, Sanguinet MC, Tuberosa R, This D, Teulat-Merah B,
Ahmed I, Mariy S, Bahri H, Ouahabi ME, Zoumarou-Wallis N, El-Fellah M, Salem MB. Geno-
type and phenotype associations with drought tolerance in barley tested in North Africa. Ann
Appl Biol. 2004;144:157–68.
Fox GP, Panozzo J, Li C, Lance R, Inkerman PA, Henry RJ. Molecular basis of barley quality.
Crop Pasture Sci. 2003;54(12):1081–101.
Foyer C, Fletcher J. Plant antioxidants: colour me healthy. Biologist (London). 2001;48:115–20.
Fu YB, Somers D. Genome-wide reduction of genetic diversity in wheat breeding. Crop Sci.
2009;49:161–8.
Gadallah M. Effects of proline and glycinebetaine on Vicia faba responses to salt stress. Biol
Planta. 1999;42:249–57.
Garg B. Nutrient uptake and management under drought: nutrient-moisture interaction. Curr Ag-
ric. 2003;27:1–8.
Ghassemi F, Jakeman AJ, Nix HA Salinisation of land and water resources: human causes, extent,
management and case studies. Sydney: UNSW; 1995. (CAB, Wallingford).
Govindjee. Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust J Plant Physiol.
1995;22:131–60.
Grando S, Von Bothmer R, Ceccarelli S. Genetic diversity of barley: use of locally adapted germ-
plasm to enhance yield and yield stability of barley in dry areas. In: Cooper HD, Spillane C
and Hodgkin T (eds.). Broadening the genetics base of crop production. Rome: FAO; 2001.
pp. 351–71.
Grigorova B, Vassileva V, Klimchuk D, Vaseva I, Demirevska K, Feller U. Drought, high tem-
perature, and their combination affect ultrastructure of chloroplasts and mitochondria in wheat
( Triticum aestivum L.) leaves. J Plant Interact. 2012;7(3):2012.
Grossman A, Takahashi H. Macronutrient utilization by photosynthetic eukaryotes and the fabric
of interactions. Ann Rev Plant Biol. 2001;52:163–210.
Gulzar S, Khan MA, Ungar IA. Salt tolerance of a coastal salt marsh grass. Commu Soil Sci Plant
Anal. 2003;34:2595–605.
Guo P, Baum M, Grando S, Ceccarelli S, Bai G, Li R, Von Korff M, Varshney RK, Graner A,
Valkoun J. Differentially expressed genes between drought-tolerant and drought-sensitive
barley genotypes in response to drought stress during the reproductive stage. J Exp Bot.
2009;60:3531–44.
Gyenis L, Yun JS, Smith KP, Steffenson BJ, Bossolini E, Sanguineti MC, Muehlbauer GJ. Genetic
architecture of quantitative trait loci associated with morphological and agronomic trait differ-
ences in a wild by cultivated barley cross. Genome. 2007;50:714–23.
Hare P, Cress W, Van Staden J. Dissecting the roles of osmolyte accumulation during stress. Plant
Cell Environ. 1998;21:535–53.
Harris BN, Sadras VO, Tester M. A water-centred framework to assess the effects of salinity on the
growth and yield of wheat and barley. Plant Soil. 2010;336:377–89.
Hawker NP, Bowman JL. Roles for Class III HD-Zip and KANADI genes in Arabidopsis root
development. Plant Physiol. 2004;135(4):2261–70.
Hernandez J, Olmos E, Corpas F, Sevilla F, Del Rio L. Salt-induced oxidative stress in chloroplasts
of pea plants. Plant Sci. 1995;105:151–67.
IPCC. Emissions scenarios. In: Nakicenovic N, Swart R, editors. Special report of the intergov-
ernmental panel on climate change. Cambridge: Cambridge University Press; 2000. p. 570.
Iriti M, Faoro F. Ozone-induced changes in plant secondary metabolism, climate change and crops.
Berlin: Springer; 2009. pp 245–68.
Ivandic V, Thomas W, Nevo E, Zhang Z, Forster B. Associations of simple sequence repeats with
quantitative trait variation including biotic and abiotic stress tolerance in Hordeum sponta-
neum. Plant Breed. 2003;122:300–4.
Iyer NJ, Tang Y, Mahalingam R. Physiological, biochemical and molecular responses to combina-
tion of drought and ozone in Medicago truncatula. Plant Cell Environ. 2013;36:706–20.
Kantar M, Lucas SJ, Budak H. miRNA expression patterns of Triticum dicoccoides in response to
shock drought stress. Planta. 2011;233(3):471–84.
Kalladan R, Worch S, Rolletschek H, Harshavardhan VT, Kuntze L, Seiler C, Sreenivasulu N, Röder
MS. Identification of quantitative trait loci contributing to yield and seed quality parameters
under terminal drought in barley advanced backcross lines. Mol Breeding. 2013;32(1):71–90.
Khavari-Nejad R, Mostofi Y. Effects of NaCl on photosynthetic pigments, saccharides, and chloro-
plast ultrastructure in leaves of tomato cultivars. Photosynthetica. 1998;35:151–4.
Koornneef M, Alonso-Blanco C, Peeters A. Genetic approaches in plant physiology. New Phytol.
1997;137:1–8.
Kováčik J, Klejdus B, Hedbavny J, Bačkor M. Salicylic acid alleviates NaCl-induced changes in
the metabolism of Matricaria chamomilla plants. Ecotoxicol. 2009;18:544–54.
Kumar D. Salt tolerance in oilseed Brassicas-present status and future prospects. Plant Breed Ab-
str. 1995;65:1438–47.
Lakew B, Eglinton J, Henry RJ, Baum M, Grando S, Ceccarelli S. The potential contribution of
wild barley ( Hordeum vulgare ssp. spontaneum) germplasm to drought tolerance of cultivated
barley ( H. vulgare ssp. vulgare). Field Crops Res. 2011;120:161–8.
Lakew B, Henry R, Ceccarelli S, Grando S, Eglinton J, Baum M. Genetic analysis and phenotypic
associations for drought tolerance in Hordeum spontaneum introgression lines using SSR and
SNP markers. Euphytica. 2013;189:9–29.
Lawlor D, Cornic G. Photosynthetic carbon assimilation and associated metabolism in relation to
water deficits in higher plants. Plant Cell Environ. 2002;25:275–94.
Lee G, Duncan RR, Carrow RN. Salinity tolerance of seashore paspalum ecotypes: shoot growth
responses and criteria. Hort Sci. 2004;39:1138–42.
Levy D, Coleman WK, Veilleux RE. Adaptation of potato to water shortage: irrigation manage-
ment and enhancement of tolerance to drought and salinity. Am J Potato Res. 2013;90:1–21.
Li J, Huang XQ, Heinrichs F, Ganal MW, Ro¨der MS. Analysis of QTLs for yield, yield com-
ponents, and malting quality in a BC3-DH population of spring barley. Theor Appl Genet.
2005;110:356–63.
Li JZ, Huang XQ, Heinrichs F, Ganal MW, Ro¨der MS. Analysis of QTLs for yield components,
agronomic traits, and disease resistance in an advanced backcross population of spring barley.
Genome. 2006;49:454–66.
Li C, Zhang GP, Lance R. Recent advances in breeding barley for drought and saline stress tol-
erance. In: Advances in molecular breeding toward drought and salt tolerant crops. Berlin:
Springer; 2007. pp. 603–26.
Li W-T, Liu C, Liu Y-X, Pu Z-E, Dai S-F, Wang J-R, Lan X-J, Zheng Y-L, Wei Y-M. Meta-analysis
of QTL associated with tolerance to abiotic stresses in barley. Euphytica. 2013;189(1):31–49.
Liu H-H, Tian X, Li Y-J, Wu C-A, Zheng C-C. Microarray-based analysis of stress-regulated mi-
croRNAs in Arabidopsis thaliana. RNA. 2008;14(5):836–43.
Long NV, Dolstra O, Malosetti M, Kilian B, Graner A, Visser RG, van der Linden CG. As-
sociation mapping of salt tolerance in barley ( Hordeum vulgare L.). Theor Appl Genet.
2013;126(9):2335–51.
Lu Z, Tamar K, Neumann PM, Nevo E. Physiological characterization of drought tolerance in wild
barley ( Hordeum spontaneum)from the Judean Desert. Barley Genet Newsl. 1999;29:36–9.
Lu X-Y, Huang X-L. Plant miRNAs and abiotic stress responses. Biochem Biophys Res Commun.
2008;368:458–62.
Maas E, Hoffman G. Crop Salt Tolerance\-Current Assessment. J Irri Drain Div. 1977;103(2):115–
34.
Maathuis FJ. The role of monovalent cation transporters in plant responses to salinity. J Exp Bot.
2006;57:1137–47.
Manivannan P, Jaleel CA, Sankar B, Kishorekumar A, Somasundaram R, Lakshmanan GA, Pan-
neerselvam R. Growth, biochemical modifications and proline metabolism in Helianthus an-
nuus L. as induced by drought stress. Colloids Surf B Biointerfaces. 2007;59:141–9.
Manivannan P, Jaleel CA, Somasundaram R, Panneerselvam R. Osmoregulation and antioxidant

metabolism in drought-stressed Helianthus annuus under triadimefon drenching. Comptes
Rend Biol. 2008;331:418–25.
Mano Y, Takeda K. Mapping quantitative trait loci for salt tolerance at germination and the seed-
ling stage in barley ( Hordeum vulgare L.). Euphytica. 1997;94:263–72.
Mansour M, Salama K, Ali F, Abou Hadid A. Cell and plant responses to NaCl in Zea mays L.
cultivars differing in salt tolerance. Gen Appl Plant Physiol. 2005;31:29–41.
Marcum K, Pessarakli M. Salinity tolerance and salt gland excretion efficiency of bermuda grass
turf cultivars. Crop Sci. 2006;46:2571–4.
Mare C, Mazzucotelli E, Crosatti C, Francia E, Stanca AM, Cattivelli L. Hv-WRKY38: a
new transcription factor involved in cold- and drought-response in barley. Plant Mol Biol.
2004;55(3):399–416. doi:10.1007/s11103-004-0906-7.
Matysik J, Bhalu B, Mohanty P. Molecular mechanisms of quenching of reactive oxygen species
by proline under stress in plants. Curr Sci. 2002;82:525–32.
McNeil SD, Nuccio ML, Hanson AD. Betaines and related osmoprotectants. Targets for metabolic
engineering of stress resistance. Plant Physiol. 1999;120:945–9.
McWilliams D. Drought strategies for cotton. Cooperative Extension Service, Circular 582 Col-
lege of Agriculture and Home Economics. New Mexico State University, USA; 2003.
Meloni DA, Oliva MA, Ruiz HA, Martinez CA. Contribution of proline and inorganic solutes to
osmotic adjustment in cotton under salt stress. J Plant Nutr. 2001;24:599–612.
Miles CM, Wayne M. Quantitative trait locus (QTL) analysis. Nat Educ. 2008;1 (1).
http://www.nature.com/scitable/topicpage/Quantitative-Trait-Locus-QTL-Analysis-53904.
Accessed 29 June 2012.
Mishra V, Cherkauer KA. Retrospective droughts in the crop growing season: implications to corn
and soybean yield in the Midwestern United States. Agric For Meteorol. 2010;150:1030–45.
Mitsuya S, Takeoka Y, Miyake H. Effects of sodium chloride on foliar ultrastructure of sweet po-
tato ( Ipomoea batatas Lam.) plantlets grown under light and dark conditions in vitro. J Plant
Physiol. 2000;157:661–7.
2006;11:15–9.
Mohammadi M, Kav NNV, Deyholos MK. Transcriptional profiling of hexaploid wheat ( Triti-
cum aestivum L.) roots identifies novel, dehydration-responsive genes. Plant Cell Environ.
2007;30:630–45.
Muhammad W, Asghar A. Mechanism of drought tolerance in plant and its management through
different methods. Cont J Agric Sci. 2012;5:10–25.
Munns R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002;25:239–50.
Munns R. Genes and salt tolerance: bringing them together. New Phytol. 2005;167:645–63.
Munns R, James RA, Läuchli A. Approaches to increasing the salt tolerance of wheat and other
cereals. J Exp Bot. 2006;57:1025.
Nayyar H, Gupta D. Differential sensitivity of C3 and C4 plants to water deficit stress: Association
with oxidative stress and antioxidants. Environ Exp Bot. 2006;58:106–13.
Nevo E, editors. Origin, evolution, population genetics and resources for breeding of wild barley,
Hordeum spontaneum in the Fertile Crescent. Barley: genetics, biochemistry, molecular biol-
ogy and biotechnology. Oxford: CAB; 1992. (The Alden Press)
Nicholson JK, Lindon JC, Holmes E. ‘Metabonomics’: understanding the metabolic responses of
living systems to pathophysiological stimuli via multivariate statistical analysis of biological
NMR spectroscopic data. Xenobiotica. 1999;29:1181–9.
Nio S, Cawthray G, Wade L, Colmer T. Pattern of solutes accumulated during leaf osmotic adjust-
ment as related to duration of water deficit for wheat at the reproductive stage. Plant Physiol
Biochem. 2011;49:1126–37.
Oliver MJ, Guo LN, Alexander DC, Ryals JA, Wone BW. A sister group contrast using untargeted
global metabolomic analysis delineates the biochemical regulation underlying desiccation tol-
erance in Sporobolus stapfianus. Plant Cell. 2011;23:1231–48.
Oono Y, Seki M, Nanjo T, Narusaka M, Fujita M. Monitoring expression profiles of Arabidopsis

gene expression during rehydration process after dehydration using ca. 7000 full-length cDNA
microarray. Plant J. 2003;34:868–87.
Oraby HF, Ransom CB, Kravchenko AN, Sticklen MB. Barley HVA1 gene confers salt tolerance
in R3 transgenic oat. Crop Sci. 2005;45:2218–27.
Ozkur O, Ozdemir F, Bor M, Turkan I. Physiochemical and antioxidant responses of the perennial
xerophyte Capparis ovata Desf. to drought. Environ Exp Bot. 2009;66:487–92.
Ozturk ZN, Talame V, Deyholos M, Michalowski CB, Galbraith DW. Monitoring large-
scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol Biol.
2002;48:551–73.
Pastori GM, Foyer CH. Common components, networks, and pathways of cross-tolerance to stress.
The central role of “redox” and abscisic acid-mediated controls. Plant Physiol. 2002;129:460–8.
Pérez-López U, Robredo A, Lacuesta M, Mena-Petite A, Muñoz-Rueda A. Elevated CO2 reduces
Photosynth Res. 2012;111:269–83.
Peuke A, Rennenberg H. Carbon, nitrogen, phosphorus, and sulphur concentration and parti-
tioning in beech ecotypes ( Fagus sylvatica L.): phosphorus most affected by drought. Trees.
2004;18:639–48.
Pillen K, Zacharias A, Lèon J. Advanced backcross QTL analysis in barley ( Hordeum vulgare L.).
Theor Appl Genet. 2003;107:340–52.
Pillen K, Zacharias A, Lèon J. Comparative AB-QTL analysis in barley using a single exotic donor
of Hordeum vulgare ssp.spontaneum. Theor Appl Genet. 2004;108:1591–601.
Plaut Z. Plant exposure to water stress during specific growth stages. In: Trimble WS (ed.). Ency-
clopedia of water science. London: Taylor & Francis; 2003.
Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K. Monitoring expression profiles of rice
genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA
microarray and RNA gel-blot analyses. Plant Physiol. 2003;133:1755–67.
Rizhsky L, Liang HJ, Shuman J, Shulaev V, Davletova S. When defense pathways collide
the response of Arabidopsis to a combination of drought and heat stress. Plant Physiol.
2004;134:1683–96.
Robinson SP, Downton WJS, Millhouse JA. Photosynthesis and ion content of leaves and isolated
chloroplasts of salt-stressed spinach. Plant Physiol. 1983;73:238–42.
Robredo A, Pérez-López U, Lacuesta M, Mena-Petite A, Muñoz-Rueda A. Influence of water
stress on photosynthetic characteristics in barley plants under ambient and elevated CO2 con-
centrations. Biol Planta. 2010;54:285–92.
Rollins J, Habte E, Templer S, Colby T, Schmidt J, von Korff M. Leaf proteome alterations in
the context of physiological and morphological responses to drought and heat stress in barley
( Hordeum vulgare L.). J Exp Bot. 2013;64:3201–12.
Romero J, Marañón T. Long-term responses of Melilotus segetalis to salinity. I. Growth and parti-
tioning. Plant Cell Environ. 1994;17:1243–8.
Rostoks N, Mudie S, Cardle L, Russell J, Ramsay L, Booth A, Svensson JT, Wanamaker SI, Walia
H, Rodriguez EM. Genome-wide SNP discovery and linkage analysis in barley based on genes
responsive to abiotic stress. Mol Genet Genomics. 2005;274:515–27.
Rubio-Somoza I, Weigel D. MicroRNA networks and developmental plasticity in plants. Trends
Plant Sci. 2011;16:258–64.
Sadras VO. Evolutionary aspects of the trade-off between seed size and number in crops. Field
Crops Res. 2007;100:125–38.
Sairam R, Tyagi A. Physiology and molecular biology of salinity stress tolerance in plants. Curr
Sci Bangalore. 2004;86:407–21.
Samarah NH. Effects of drought stress on growth and yield of barley. Agron Sustain Dev.
2005;25:145–9.
Scandalios J. Oxidative stress: molecular perception and transduction of signals triggering antioxi-
dant gene defenses. Braz J Medical Biol Res. 2005;38:995–1014.
Schmalenbach I, Le’on J, Pillen K. Identification and verification of QTLs for agronomic traits
using wild barley introgression lines. Theor Appl Genet. 2009;118:483–97.
Schnaithmann F, Pillen K. Detection of exotic QTLs controlling nitrogen stress tolerance among
wild barley introgression lines. Euphytica. 2013;189:1–22.
Seckin B, Turkan I, Sekmen AH, Ozfidan C. The role of antioxidant defense systems at differential
salt tolerance of Hordeum marinum Huds. (sea barleygrass) and Hordeum vulgare L. (culti-
vated barley). Environ Exp Bot. 2010;69:76–85.
Shannon M. Breeding, selection, and the genetics of salt tolerance. In: Staples RC, Toeniessen GA,
editors. Salinity tolerance in plants. New York: Wiley; 1984. pp. 232–53.
Shannon MC, Grieve CM, Francois LE. Whole-plant response to salinity. Plant-environment inter-
actions. New York: Marcel Dekker; 1994. pp. 199–244.
Shukla LI, Chinnusamy V, Sunkar R. The role of microRNAs and other endogenous small RNAs
in plant stress responses. Biochim Biophys Acta. 2008;1779:743–8.
Shulaev V, Cortes D, Miller G, Mittler R. Metabolomics for plant stress response. Physiol Planta.
2008;132:199–208.
Sicher RC, Timlin D, Bailey B. Responses of growth and primary metabolism of water-stressed
barley roots to rehydration. J Plant Physiol. 2012;169(7):686–95.
Siddique M, Hamid A, Islam M. Drought stress effects on water relations of wheat. Bot Bull Acad
Sinica. 2001;41:35–9.
Smirnoff N, Cumbes QJ. Hydroxyl radical scavenging activity of compatible solutes. Phytochem.
1989;28:1057–60.
Sohan D, Jasoni R, Zajicek J. Plant-water relations of NaCl and calcium-treated sunflower plants.
Environ Exp Bot. 1999;42(2):105–11.
Sunkar R. Micrornas with macro-effects on plant stress responses. In: Davey J (ed.). Seminars in
cell & developmental biology. Amsterdam: Elsevier; 2010. pp. 805–11.
Suprunova T, Krugman T, Distelfeld A, Fahima T, Nevo E, Korol A. Identification of a novel gene
(Hsdr4) involved in water-stress tolerance in wild barley. Plant Mol Biol. 2007;64:17–34.
Taiz L, Zeiger E. Stress physiology. In: Taiz L, Zeiger E, editors. Plant physiology. Sunderland:
Sinauer Associates; 2006. pp. 671–81.
Talame’ V, Ozturk ZN, Bohnert HJ, Tuberosa R. Barley transcript profiles under dehydration
shock and drought stress treatments: a comparative analysis. J Exp Bot. 2007;58:229–40.
Talame V, Sanguineti MC, Chiapparino E, Bahri H, Salem MB, Forster BP, Ellis RP, Rhouma
S, Zoumarou W, Waugh R, Tuberosa R. Identification ofHordeum spontaneumQTL alleles
improving field performance of barley grown under rainfed conditions. Ann Appl Biol.
2004;144:309–19.
Tanksley SD, Nelson JC. Advanced backcross QTL analysis: a method for the simultaneous dis-
covery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines.
Theor Appl Genet. 1996;92:191–203.
Tavakkoli E, Fatehi F, Coventry S, Rengasamy P, McDonald GK. Additive effects of Na + and Cl−
ions on barley growth under salinity stress. J Exp Bot. 2011;62:2189–203.
Termaat A, Munns R. Use of concentrated macronutrient solutions to separate osmotic from NaCl-
specific effects on plant growth. Func Plant Biol. 1986;13:509–22.
Teulat B, This D, Khairallah M, Borries C, Ragot C, Sourdille P, Leroy P, Monneveux P, Charrier
A. Several QTLs involved in osmotic-adjustment trait variation in barley ( Hordeum vulgare
L.). Theor Appl Genet. 1998;96:688–98.
Tuberosa R, Salvi S. Genomics approaches to improve drought tolerance in crops. Trends Plant
Sci. 2006;11:405–12.
Tuteja A, Choi W, Ma M, Mabry JM, Mazzella SA, Rutledge GC, McKinley GH, Cohen RE. De-
signing superoleophobic surfaces. Science. 2007;318:1618–22.
Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J. A NAC gene regulating senescence im-
proves grain protein, zinc, and iron content in wheat. Science. 2006;314:1298–301.
Urano K, Kurihara Y, Seki M, Shinozaki K. ‘Omics’ analyses of regulatory networks in plant abi-
otic stress responses. Curr Opin Plant Biol. 2010;13:132–8.
Volis S, Mendlinger S, Orlovsky N. Variability in phenotypic traits in core and peripheral popula-
tions of wild barley Hordeum spontaneum Koch. Hereditas. 2000;133:235–47.
Vysotskaya L, Hedley PE, Sharipova G, Veselov D, Kudoyarova G, Morris J, Jones HG. Effect of
salinity on water relations of wild barley plants differing in salt tolerance. AoB Plants. 2010.
doi:10.1093/aobpla/plq006.
Wang Y, Nil N. Changes in chlorophyll, ribulose biphosphate carboxylase-oxygenase, glycine be-
taine content, photosynthesis and transpiration in Amaranthus tricolor leaves during salt stress.
J Hortic Sci Biotechnol. 2000;75:623–27.
Wang B, Chee PW. Application of advanced backcross quantitative trait locus (QTL) analysis in
crop improvement. J Plant Breed Crop Sci. 2010;2:221–32.
Wardlaw I, Willenbrink J. Mobilization of fructan reserves and changes in enzyme activities in
wheat stems correlate with water stress during kernel filling. New Phytol. 2000;148:413–22.
Waugh R, Jannink JL, Muehlbauer GJ, Ramsay L. The emergence of whole genome association
scans in barley. Currt Opin. Plant Biol. 2009;12:218–22.
Widodo, Patterson JH, Newbigin E, Tester M, Baci A. Metabolic responses to salt stress of barley
( Hordeum vulgare L.) cultivars, Sahara and Clipper, which differ in salinity tolerance. J Exp
Bot. 2009;60:4089–103.
Witzel K, Weidner A, Surabhi GK, Varshney RK, Kunze G, BuckSorlin GH, Borner A, Mock HP.
Comparative analysis of the grain proteome fraction in barley genotypes with contrasting salin-
ity tolerance during germination. Plant Cell Environ. 2009;33:211–22.
Wu D, Cai S, Chen M, Ye L, Chen Z, Zhang H, Dai F, Wu FB, Zhang GP. Tissue metabolic re-
sponses to salt stress in wild and cultivated barley. PloS ONE. 2013;8:e55431.
Xue DW, Huang YZ, Zhang XQ, Wei K, Westcott S, Li CD, Chen MC, Zhang GP, Lance R. Iden-
tification of QTLs associated with salinity tolerance at late growth stage in barley. Euphytica.
2009;169:187–96.
Xu D, Duan X, Wang B, Hong B, Ho THD, Wu R. Expression of a late embryogenesis related
protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic
rice. Plant Physiol. 1996;110:249–57.
Yang F, Xu X, Xiao X, Li C. Responses to drought stress in two poplar species originating from
Water & nitrogen use efficiency in plants and crops. Marston, Lincolnshire. UK: 15–16 De-
cember 2010; 2009.
Yousfi S, Serret MD, Araus JL, Draye X, Foulkes J, Hawkesford M, Murchie E. A comparative
effect of salinity and drought on growth, ion concentration and δ13C and δ15N in barley. As-
socia Appl Biol. 2010;105:73–81.
Yousfi S, Serret MD, Márquez AJ, Voltas J, Araus JL. Combined use of δ13C, δ18O and δ15N
tracks nitrogen metabolism and genotypic adaptation of durum wheat to salinity and water
deficit. New Phytol. 2012;194:230–44.
Zahra J, Nazim H, Cai S, Han Y, Wu D, Zhang B, Haider SI, Zhang G. The influence of salinity
on cell ultrastructures and photosynthetic apparatus of barley genotypes differing in salt stress
tolerance. Acta Physiol Planta. 2014;36(5):1261–9.
Zhang J, Jia W, Yang J, Ismail AM. Role of ABA in integrating plant responses to drought and salt
stresses. Field Crops Res. 2006a;97:111–9.
Zhang B, Pan X, Cobb GP, Anderson TA. Plant microRNA: a small regulatory molecule with big
impact. Dev Biol. 2006b;289:3–16.
Zhang X, Liu S, Takano T. Overexpression of a mitochondrial ATP synthase small subunit gene
( AtMtATP6) confers tolerance to several abiotic stresses in Saccharomyces cerevisiae and Ara-
bidopsis thaliana. Biotech Lett. 2008;30:1289–94.
Zhang H, Han B, Wang T, Chen SX, Li HY. Mechanisms of plant salt response, insights from
proteomics. J Proteome Res. 2012;11:49–67.
Zhao J, Sun H, Dai H, Zhang G, Wu F. Difference in response to drought stress among Tibet wild
barley genotypes. Euphytica. 2010;172:395–403.
Zhou M, Li D, Li Z, Hu Q, Yang C, Zhu L, Luo H. Constitutive expression of a miR319 gene alters
plant development and enhances salt and drought tolerance in transgenic creeping bentgrass.
Plant Physiol. 2013;161:1375–91.
Chapter 6
Combined Abiotic Stress in Legumes
Santiago Signorelli, Esteban Casaretto, Jorge Monza and Omar Borsani
6.1 Legume Family: Agronomic Relevance
A major source of protein in the human diet is of animal origin. The production of
beef and mutton is based on natural pastures or supplementation based on grains
(feedlot). Sown pastures can be monospecific or may be ultrasimple, simple or
complex of different species of the same botanical family or a family of different
botanical blends. Within the latter group, are mixtures of grasses and legumes.
From the point of view of human and animal consumption, legumes belonging
to the subfamily Papilonideae are relevant. This includes seeds and forage legumes
such as peanut, beans, chickpea, broad beans, lentils, soybean, among others. Some
species of the genus Medicago, Lotus and Adesmia can be used as forage or green
manure, thus enhancing the contents of nitrogen in the soils.
Forage legumes have been widely spread in the world due to the great agro-
nomic importance that they possess. The species of this plant family are an invalu-
able component of pastures, mainly due to their ability to fix atmospheric nitrogen
through symbiotic association with several bacteria collectively called rhizobia.
Second, legumes have a high nutritional value, especially proteins and minerals
(Ca+2 and Mg+2), which makes them essential for the production of forage. Legume
crops also play a critical role as main protein sources in vegetarian diets. Tolerance
O. Borsani () · E. Casaretto · J. Monza · S. Signorelli

Laboratorio de Bioquímica, Departamento de Biología Vegetal, Facultad de Agronomía,
Universidad de la República, Av. E. Garzón 780, 12900 Montevideo, Uruguay
e-mail: oborsani@fagro.edu.uy
E. Casaretto
e-mail: ecasaretto@fagro.edu.uy
J. Monza
e-mail: jmonza@fagro.edu.uy
S. Signorelli
e-mail: ssignorelli@fagro.edu.uy

124 S. Signorelli et al.
to environmental abiotic stress is one of the ways to improve the productivity of

legumes and aid in harnessing their potential nutritional value. Identification of
biochemical and physiological characters which contribute to improve the yield in
legumes under limiting conditions is a main objective of plant breeders for agri-
cultural and cattle-rearing regions. Thus, this chapter intends to provide an under-
standing of the mechanisms involved in the combined stress-tolerance responses in
legumes.
6.2 Environmental Stresses Induce Varied Plant

Responses
Plants are frequently subjected to stress—environmental condition that adversely

affects the growth, development and productivity thereof. Biotic stress can be im-
posed by organisms such as viruses, bacteria and fungi, while abiotic stress can be
due to an excess or deficit in some environmental factor. Among the environmental
conditions that cause damage are excess water, water deficit, soil salinity, extreme
temperatures, insufficient mineral nutrients in the soil and high- or low-light radia-
tion (Bohnert and Sheveleva 1998; Bray et al. 2000).
Resistance or susceptibility to stress depends on the species, genotype and stage
of development of the plant. Resistance mechanisms can be grouped into two cat-
egories—those that prevent exposure to stress and the other that results in tolerance.
Certain morphological features such as sunken stomata and deep roots are examples
of resistance mechanism that can prevent stress. However, other mechanisms of
resistance are achieved by acclimation, i.e. the maintenance of internal homeostasis
of the various organelles in response to changing environmental factors
(Bray et al. 2000).
Plants acclimate to manage the different types of stress triggering a wide range of
responses from the perception of stress at the cellular level, leading to the activation
of a very large number of genes. Key components of the stress response are the stim-
ulus itself, transducers, signal molecules, transcription regulators, responsive genes
that trigger morphological, biochemical and physiological adaptation involved in
this situation. In turn, the duration and severity with which stress is imposed deter-
mine how the plant will respond (Pastori and Foyer 2002; Bray et al. 2000).
Unlike resistance to biotic factors, resistance to water stress and other abiotic
factors, despite being clearly genetic, is not a result of the action of a specific gene
(Zhu et al. 1997). The ability of plants to withstand water stress is a multigenic trait
and biochemical pathways responsible for products or processes that improve the
overall strength can act additively, and also synergistically (Bohnert et al. 1995).
It is reported that several genes responsive to water stress not only perform their
functions protecting cells by producing metabolically important proteins under wa-
ter deficit but also in the regulation of genes involved in signal transduction in
response to stress. Thus, these gene products are classified into two groups: The
first group includes proteins that are involved in stress tolerance such as channel
6 Combined Abiotic Stress in Legumes 125
proteins involved in the movement of water across membranes, enzymes necessary

for the biosynthesis of osmolytes, proteases and macromolecules that can protect
membranes, among others. The second group includes factors involved in the regu-
lation of signal transduction and gene expression, such as protein kinases, transcrip-
tion factors and 14-3-3 proteins, among others (Bray 1997; Shinozaki and Yamagu-
chi-Shinozaki 1997).
Higher temperatures primarily affect photosynthesis, in particular CO2 as-
similation because Rubisco activation is inhibited. Plants exposed to excessive
temperatures have specific metabolic cellular response characterized by low
protein synthesis, and induction of the synthesis of heat shock proteins (HSPs).
In addition to altering the pattern of gene expression, the high temperature can
damage cellular structures such as organelles and cytoskeleton (Bray et al. 2000;
Tang et al. 2007).
Water stress and high temperatures interact strongly with each other and have
opposite effects on photosynthesis. For example, in response to high temperature,
plants open their stomata to cool their leaves by transpiration, but if there is also
water deficit condition, plants would not be able to open the stomata and hence leaf
temperature will increase (Rizhsky et al. 2002). While both types of stress have
been extensively studied individually, few studies (Lu and Zhang 1999; Rizhsky
et al. 2002; Rizhsky et al. 2004) focused on impacts of combined heat and water
stress—a common situation prevailing under field conditions. It is possible that
combination of these stress factors can alter the metabolism of the plant differently,
compared to when a single stress is imposed (Xu and Zhou 2006).
6.2.1 Plants Response to Water Stress
Water deficit is one of the most widespread environmental factor stresses that occurs
when the transpiration rate exceeds the absorption of water from the root system.
Water deficit at the cellular level may result in an increase of solute concentration,
changes in cell volume, disruption of water potential gradient, turgor loss, loss of
membrane integrity and protein denaturation. The ability of the plant to respond to
water deficit and survive depends on mechanisms that involve the integration of
cellular responses throughout the plant (Bray et al. 2000).
Water deficit is a common plant environmental stress that dramatically limits
growth and development. Water stress can trigger a significant decrease in crop
productivity and quality, especially evident in grain and forage legumes. Lotus
japonicus is a well-established model legume closely related to forage legumes
such as Lotus corniculatus, Lotus tenuis and Lotus uliginosus (Choi et al. 2004;
Díaz et al. 2005a). Alfalfa is a legume species with great plasticity that can suc-
ceed in semiarid, subhumid and humid regions and for that reason is called the
“queen of forage legumes”. However, it requires well-aerated and deep soils and
is morphologically and physiologically adapted to withstand prolonged water defi-
ciencies. In marked contrast to their drought-tolerant nature, these plants are very
sensitive to a lack of oxygen that is common in flooding soils.
Legumes are typically subjected to a variety of different environmental stresses

such as water stress. At the cellular level, this stress induces overproduction of reac-
tive oxygen species (ROS; Fig. 6.1), such as hydrogen peroxide (H2O2), superoxide
radical (O2●−) and hydroxyl radical (●OH), which are responsible for oxidative dam-
age associated with stress (Dat et al. 2000). Plants respond to stress using differ-
ent enzymatic and non-enzymatic antioxidant systems. Oxidative stress responses
may involve increased activity of superoxide dismutase (SOD), catalase (CAT)
and ascorbate–glutathione cycle activities such as glutathione reductase (GR) or
ascorbate peroxidase (APX), which can confer greater tolerance against a specific
environmental stress (Sade et al. 2011). Increased levels of non-enzymatic soluble
antioxidants including glutathione (GSH), ascorbic acid and tocopherols are also
produced in response to water stress-induced oxidative stress (Feng et al. 2004).
Plant antioxidant defence systems normally provide adequate protection against
ROS damage under optimal growth conditions. The generation of higher levels of
ROS may overcome the defence provided by these systems and result in oxida-
tive stress (Mittler 2002; Noctor and Foyer 1998; Valderrama et al. 2006). Cellular
damage caused by oxidative stress includes lipid peroxidation, which increases in
various tissues during water stress and is also a common marker of oxidative stress
(Sade et al. 2011).
In response to water deficit, plant cells also accumulate low-molecular-mass
compounds termed compatible solutes, mainly proline, glycine betaine, sugars
and polyols, in the cytoplasm to control the ionic balance in the vacuoles (Parida
and Das 2005). Among these solutes, proline has been associated with different
Fig. 6.1 ROS production in the chloroplast. Chl chlorophyll, Chl* excited chlorophyll. PSI pho-
tosystem I, PSII photosystem II. Cyt cytochrome, PQ plastoquinone, PC plastocyanin. Superoxide
(O2●−) can be produced by electron transfer to oxygen. Hydrogen peroxide (H2O2) is produced
from superoxide by spontaneous dismutation or SOD activity. Hydroxyl radicals (●OH) are pro-
duced from hydrogen peroxide by homolysis or Fenton reaction in the presence of Fe3+. Singlet
oxygen is generated from oxygen by energy transfer from excited chlorophylls
functions, such as being a free radical scavenger, a cell redox balancer, a cytosolic
pH buffer and a stabilizer for subcellular structures, especially during osmotic and
salt stresses (Szabados and Savouré 2010).
During drought establishment, plants exhibit a decrease in stomatal conductance
with the consequent decrease in CO2 assimilation. Stomatal closure has been con-
sidered as the main reason for the inhibition of photosynthesis under drought. How-
ever, it was demonstrated that limiting stomatal water losses is not so important
to maintain photosynthetic activity. For example, it has been observed in leaves
of various species, reductions in photosynthesis occur without apparent effects on
stomatal conductance (Teskey et al. 1986; Hutmacher and Krieg 1983), suggest-
ing that factors independent of stomatal behaviour impact photosynthesis in plants
subjected to drought.
The use of split root system has helped in gaining knowledge about the impact
of drought on the process of nodulation in legumes (Larrianzar et al. 2014). Nod-
ule number is mainly regulated at the systemic level through a signal which is
produced by nodule/root tissue, translocated to the shoot and transmitted back
to the root system. This process involves shoot Leu-rich repeat receptor-like
kinases. In contrast, local and systemic mechanisms regulate nitrogenase activ-
ity in nodules (Esfahani et al. 2014). Under drought and heavy metal stress, the
regulation is mostly local, whereas the application of exogenous nitrogen seems
to exert a regulation of nitrogen fixation both at the local and systemic levels
(Marino et al. 2007).
6.2.2 Response of Plants to Heat Stress
High temperature at early sowing resulted in poor crop establishment due to fail-
ure of seed germination, emergence and reduced vigour (Khalaffalla 1985; Weaich
et al. 1996). In such situations, avoidance mechanisms, such as transpiration, leaf
rolling, hairiness or wax layers, may play a role in dissipating the heat load. How-
ever, in general, transpiration is the most important heat-dissipating system through
latent heat loss (Kramer 1983).
Plants exposed to high temperatures, at least 5 °C above their optimal growing
conditions, exhibit cellular and metabolic responses required for the plants to
survive under this condition (Guy 1999). These effects include changes in the
organization of organelles, cytoskeletal reorganization and membrane functions,
accompanied by a decrease in the synthesis of some proteins and overexpression
of HSPs, the production of phytohormones such as abscisic acid (ABA) and
antioxidants and other protective molecules (Bita and Gerats 2013; Maestri et al.
2002; Bray et al. 2000). Under heat stress, about 5 % of plant transcripts (∼ 1500
genes) are up regulated, twofold or more (Rizhsky et al. 2004; Larkindale and
Vierling 2008; Finka et al. 2011). A significant fraction of these transcripts encode
heat-induced chaperones. For example, 88 out of 1780 in Arabidopsis thaliana, and
117 out of 1509 in wheat, are associated with HSP-based protection mechanism
(Liu et al. 2008; Ginzberg et al. 2009; Bokszczanin and Fragkostefanakis 2013).
There are many transcripts-encoding proteins involved in calcium signalling;

protein phosphorylation; phytohormone signalling; sugar and lipid signalling and
metabolism; RNA metabolism; translation, primary and secondary metabolisms;
transcription regulation and responses to different biotic and abiotic stresses
(Mittler et al. 2012; Huve et al. 2011). Changes in ambient temperature are sensed
by plant sensors positioned in various cellular compartments. The increased fluidity
of the membrane leads to activation of lipid-based signalling cascades and to an in-
creased Ca2+ influx. Signalling by these routes leads to the production of osmolytes
and antioxidants as a response to heat stress. This stress also brings about changes
in respiration and photosynthesis and thus leads to a shortened life cycle and dimin-
ished plant productivity (Barnabás et al. 2008).
The early effects of heat stress comprise of structural alterations in chloroplast–
protein complexes and reduced activity of enzymes (Ahmad et al. 2010). The photo-
chemical modifications in the carbon flux of the chloroplast stroma and those of the
thylakoid membrane system are considered the primary sites of heat injury (Wise
et al. 2004), as photosynthesis and the enzymes of the Calvin–Benson cycle, in-
cluding ribulose 1,5-bisphosphate carboxylase (Rubisco) and Rubisco activase are
very sensitive to low increases of temperature, and it is suggested to be one of the
primary determinants of heat-dependent reduction in photosynthesis (Maestri et al.
2002; Morales et al. 2003). Heat inactivation of Rubisco is reversible (Salvucci and
Crafts-Brandner 2004; Kim and Portis 2005). However, moderate heat stress has
been shown to alter the thylakoid permeability and electron transport (Schrader
et al. 2007; Zhang and Sharkey 2009), and this inhibition of electron transport is
associated with enhanced membrane permeability, disorganization of photosystem
II (PSII) and antenna tertiary structure, and disruption of the water splitting and
oxygen evolving system (Huve et al. 2011). Other specific responses of heat stress
on photosynthetic membranes include the swelling of grana stacks and an aberrant
stacking. Such structural changes are accompanied by ion leakage from leaf cells
exposed to heat and changes in energy allocation to the photosystems (Wahid and
Shabbir 2005; Allakhverdiev et al. 2008). The maintenance of cellular membrane
function under heat stress is thus essential for sustained photosynthetic and respira-
tory performance (Chen et al. 2010). The detrimental effects of heat on chlorophyll
and the photosynthetic apparatus are also associated with the production of ROS
(Guo et al. 2007). By increasing chlorophyllase activity and decreasing the amount
of photosynthetic pigments, heat stress ultimately reduces the plant photosynthetic
and respiratory activity (Sharkey and Zhang 2010).
Homeostasis, in general, including biosynthesis and compartmentalization of
metabolites, is disturbed in high-temperature-challenged plant tissues (Maestri et al.
2002). Among the primary metabolites, accumulating in response to heat stress are
proline, glycine betaine or soluble sugars (Wahid 2007).
Heat stress results in the misfolding of newly synthesized proteins and the dena-
turation of existing proteins. Protein thermostability is provided in part by chaper-
ones (Ellis 1990). In this sense, the exacerbation of combined heat and other stress
could be due to the loss of function of some enzymes that are overexpressed in
response to other stress.
6.3 Effect of Water Stress–Heat Stress Combination

on Different Plant Processes
L. corniculatus and Trifolium pratense are legumes used in agriculture as a forage

source. These species are both perennial herbaceous plants used in temperate grass-
land and can be nodulated by rhizobia. Nevertheless, lotus is better suited to soils
with water restriction and has a superior tolerance to water stress (Peterson et al.
1992). In the field, mainly during summer, these plants are commonly exposed to
environmental stresses such as water stress and high temperatures, which in fact are
considered to be the most important environmental factors limiting plant growth
and development (Berry and Bjorkman 1980; Yordanov et al. 1986; Sinsawat et al.
2004).
6.3.1 Proline Accumulation
The accumulation of proline is known to be a good indicator of water stress in

L. corniculatus (Díaz et al. 2005b). However, the responses to combination of
stresses are not a mere additive effect of the single stresses. For example, some
plants that tend to accumulate proline in water stress conditions replace it with
sucrose as the major osmoprotectant when subjected to a combination of water
stress and heat stress (Rizhsky et al. 2004). In L. corniculatus water stress and
heat individually produce proline accumulation, but concomitant imposition of
both stresses produced a higher accumulation of proline. In contrast, Trifolium
Pratense-accumulated proline in water stress conditions but not under heat stress
and the imposition of the combined stress produced only a slight increase in pro-
line concentration compared to unstressed plants (Signorelli et al. 2013b). There-
by, for L. corniculatus, proline accumulation is a parameter that can be used as
a stress marker to assess water stress and heat stress conditions, as well as the
combination of both. However, proline accumulation in legumes cannot always
be considered a good indicator of stress condition when two or more stresses are
present. It is also known that proline accumulation under heat stress decreases the
thermotolerance of the plant, probably because of an enhancement in the produc-
tion of ROS via the Pro/P5C cycle (Lv et al. 2011). In T. pratense, it was suggested
that blocking proline accumulation might be a strategy to avoid self-toxicity during
heat stress (Signorelli et al. 2013b). This hypothesis correlated with the lipid per-
oxidation estimated by thiobarbituric reactive substances (TBARS), as T. pratense
did not show an increase in lipid peroxidation under heat conditions. Moreover, T.
pratense has a lower lipid peroxidation content than L. corniculatus when water
stress and heat stress are combined—a treatment in which L. corniculatus accumu-
lates the highest levels of proline.
On the other hand, it has been demonstrated that proline can act as an osmo-
lyte under severe dehydration (Verslues and Sharp 1999). The non-accumulation
of proline and the greater leaf area of T. pratense are important disadvantages of
this species compared to L. corniculatus when water loss must be prevented. In a

comparative analysis of L. corniculatus and T. pratense subjected to water stress
and heat, it was observed that T. pratense did not survive 5 days of combined stress,
while lotus was still alive (Signorelli et al. 2013b). In concordance, higher dry-
matter yield was observed in L. corniculatus compared to T. pratense under field
conditions subjected to summer water stress (Peterson et al. 1992).
6.3.2 Oxidative Stress
Most stresses induce ROS and alter the antioxidant–enzymatic response (Mahalin-
gam and Fedoroff 2003). However, little is known about how two or more stresses
affect the ROS production and the antioxidant response. Alterations induced by
water and heat stress on antioxidant response and oxidative damage in the model
legume L. japonicus (Sainz et al. 2010), in the forage legumes L. corniculatus and
T. pretense has been reported (Signorelli et al. 2013b).
SOD is the main enzymatic system responsible for cell detoxification and is well
documented in several plant species to increase in response to water deficit and heat
stress (Alscher et al. 2002). In L. corniculatus, the activity of Mn-SOD and Fe-SOD
increased as a consequence of water stress and combined stress (Fig. 6.2), but it did
not change under heat stress (Fig. 6.2). In the related model specie L. japonicus,
Cu/Zn-SOD immunodetection and the isoenzyme-specific activity assays con-
firmed that high-temperature treatment provoked a reduction in the Cu/Zn-SOD
protein content and activity. This is consistent with a failure to convert O2●− to H2O2
in the combined heat–drought condition. Additionally, in spite of the decreased Cu/
ZnSOD in the high-temperature treatment, the accumulation of O2●− remains low,
Fig 6.2 SOD activity under drought and combined heat and drought stress. a SOD isoforms pro-
file. C control; D drought; H heat at 42 °C; D + H drought + heat at 42 °C. 40 and 200 mg of protein
were loaded in L. corniculatus and T. pratense, respectively. The gel is the most representative of
three replicates of native gels. b Total in vitro SOD activity. C control; D drought; H heat at 42 °C;
D + H drought + heat at 42 °C. One unit of SOD was defined as the amount of enzyme that inhibits
the rate of cytochrome c reduction by 50 %. Bars indicate the relative standard deviation. (Figure
modified from Signorelli et al. 2013b)
and this is likely because high temperature does not induce accumulation of this
ROS (Sainz et al. 2010).
In T. pratense, however, no changes were observed in the activities of any SOD
isoforms. The results of the quantitative enzyme activity assay demonstrated that
total SOD activity is 2.6-fold greater in L. corniculatus than in T. pratense, and it
is affected by the stress treatments. Heat did not modify the SOD activity in L. cor-
niculatus, but the combination with water stress led to same level activity observed
under water stress (Fig. 6.2). T. pratense showed a slight increase in the SOD activ-
ity by heat stress and combined stress (Fig. 6.2). In this case, for both legumes the
response of SOD activity in the combined stress was the addition of responses in the
individuals’ stresses. It could be concluded that if one of the stresses that produce
the induction of SOD activity is present, the induction of SOD activity will be war-
ranted in the combined stress. In L. japonicus, heat stress led to a decrease on Cu/
Zn-SOD contents, which also was observed under a combination of heat and water
deficit (Sainz et al. 2010).
In L. corniculatus, CAT activity only increases during the combination of wa-
ter stress and heat. However, in T. pratense, CAT enzyme activity increased with
reference to control in response to water deficit, heat stress and combined stress,
although no differences were observed among these stresses. In T. pratense, it was
observed that any stress was able to induce CAT activity and the combination of
both stresses did not lead to an additive effect on the enzyme activity. For L. cor-
niculatus, it seems that any individual stress is not sufficient to induce CAT activ-
ity; however, the combination of stresses led to the induction of CAT, suggesting
that more than one signal is required to induce this enzyme. In L. japonicus, the
combination of heat and water deficit led to an increase in CAT activity, that was
much higher than the activity observed when the stressors were imposed individu-
ally (Sainz et al. 2010).
Interestingly, the APX activity in L. corniculatus was inhibited by water stress
condition, while in T. pratense, this activity was inhibited only in the combined
stress treatment. This enzyme is inactivated by nitration (Begara-Morales et al.
2014), which is reported to occur under several abiotic stresses (Corpas et al. 2013).
For example, for L. japonicus, a closely related species, it was observed that water
deficit induces a nitro-oxidative stress that was also reducing APX activity (Si-
gnorelli et al. 2013c). We speculate that the different stressful situations are also
inducing nitro-oxidative stress in these plants, and this could explain the decay in
enzyme activity.
Both L. corniculatus and T. pratense leaves showed O2●− accumulation only in
the water deficit–heat stress combination, as was previously observed in the model
legume L. japonicus (Sainz et al. 2010). The higher SOD activity in water stress
conditions with respect to controls, would allow this species to deal with the O2●−
induced mainly by water stress. However, the increase of Mn-SOD and Fe-SOD
isoform activity by water stress was lost under high-temperature conditions, result-
ing in an increase of O2●− in the combined treatment. In L. japonicus, similar results
were obtained with Cu/Zn-SOD, showing that deleterious effects of heat stress on
SOD activity might be a general response for this legume genus (Sainz et al. 2010).
The differences detected between both species are mainly explained by changes
in the Cu/Zn- SOD isoforms. In T. pratense, H2O2 accumulation showed the same
pattern; however, in L. corniculatus, the highest accumulation of ROS was observed
under water deficit. These results clearly demonstrate that combination of stress
situations cannot be always considered the additive responses of individual stresses.
L. corniculatus showed an increase in TBARS content as a consequence of wa-
ter deficit, heat stress and a combination of these. But T. pratense did not produce
any increase in TBARS content under heat stress. As proline antioxidant protection
function under stress conditions is now in discussion (Signorelli et al. 2013a), the
absence of proline accumulation in T. pratense may be an advantage under heat
stress by avoiding the Pro/P5C cycle which, as previosuly mentioned, could result
in higher ROS production via the Pro/P5CS cycle (Lv et al. 2011). However, proline
accumulation might be critical under combined stress because the osmolyte func-
tion seems to be important when water stress is established.
6.3.3 Photosynthesis
Water stress and heat combination affects the rate of photosynthesis due to an
increase in photoinhibition, a process that can be enhanced when more types of
abiotic stress coexist (Takahashi and Murata 2008). Under stress conditions, the
possibility of overexcitation of PSII increases. This can cause a decline in the
photosynthetic rate as the process of photoinhibition increases due to the neces-
sity to dissipate, through nonradiative processes, the excess of absorbed energy
(Takahashi and Murata 2008; Baker 2008). Because the capacity of photopro-
tection is limited, certain conditions can lead to damage and loss of active PSII
reaction centres. Under severely high temperatures, in combination with water
stress, the photosynthetic apparatus is the primary site of damage. On the con-
trary, photosystem I is more resistant to heat than PSII (Sayed et al. 1989; Hu
et al. 2004; Havaux 1993). Once photoinhibition is established, the PSII reaction
centre is simultaneously repaired via removal, synthesis and replacement of de-
graded D1 protein (Ohad et al. 1984; Kyle and Ohad 1986), a protein of reaction
centre of PSII (Fig. 6.1). The observed photoinhibitory damage is the net result of
a balance between photodamage and the repair process (Samuelsson et al. 1985;
Lidholm et al. 1987; Shyam and Sane 1989). Several studies have reported a good
correlation between changes in chlorophyll fluorescence parameters in response
to environmental stresses, such as heat, chilling, freezing and salinity (Bonnecar-
rére et al. 2011; Smillie and Hetherington 1983; Yamada et al. 1996; Hakam et al.
2000). Others authors have linked the decrease in the maximum quantum yield
of PSII ( FV/FM) to the physical dissociation of the PSII reaction centres that lead
to photoinhibition, and this assay was used to identify tolerant wheat cultivars
(Abdullah et al. 2011).
In L. corniculatus, no changes of the maximum quantum efficiency, evaluated
as FV/FM, were observed in any treatment until the 5th day, when the combined
treatment showed a significant decrease in the FV/FM parameter. In contrast, in T.

pratense, this fluorescence parameter slightly decreased from the 1st day in the
heat and combined treatment, but no changes were observed under water stress
conditions.
L. corniculatus showed a slight decrease in the amount of D1 protein after water
stress treatment. However, there was no decrease in the protein content when the
control and heat conditions were compared. The D1-complex profile of T. pratense
was also analysed, and the western blot showed a very different result when com-
pared with the L. corniculatus profile. The total D1 protein content in T. pratense
did not change in any treatment, but a difference was found in the ratio between the
free protein and the complex form. In the treatments where heat was involved, an
increase in free D1 protein together with a decrease in the D1-complex form was
evident, but it should be considered that this result might be a consequence of the
high hydrophobicity of these complexes, which makes their isolation difficult. Re-
garding D2, in L. corniculatus, the results were similar to those observed with D1;
namely, when water stress was present in the treatments, a reduction in the amount
of D2 protein was observed. Surprisingly, in the combined treatment, the D2 protein
was not detected. In contrast, in T. pratense, no significant changes were observed
in D2 protein levels.
The chlorophyll fluorescence parameter that was evaluated showed that L. cor-
niculatus had a significant decrease in the maximum quantum efficiency at the end
of the combined treatment. The low FV to FM ratio indicated photoinhibition, a
process that can act as determinant of plant performance during a stress condition
(Abdullah et al. 2011). In T. pratense, only a small decrease in the FV/FM values was
observed from the 1st day and in the treatments involving heat stress.
Analysis of PSII proteins in the two legumes with contrasting water stress
responses shows an effect that is stress- and species-specific. The D1 and D2
subunit content is decreased in L. corniculatus in both treatments involving water
stress, showing certain adaptability in response to water stress. Interestingly, the
decrease in the D2 levels was pronounced in the combined treatment, and this is
well correlated with the decrease in the maximum quantum efficiency, suggesting
the presence of a disassembling process. The D2 subunit is of particular interest
because it represents the initial point for the assembly of the PSII as a whole (de
Vitry et al. 1989; Komenda et al. 2004; Minai et al. 2006). The expression of the
gene that encodes the D2 subunit of the PSII reaction centre is regulated post-
transcriptionally by an RNA-binding protein (Schwarz et al. 2007). Modifications
induced by the stress in this post-transcriptional regulation could be a possible
explanation for the absence of D2 in L. corniculatus subjected to the combined
stress treatment.
In T. pratense, the total content of D1 and D2 did not change, but we observed
an increase in the free form of D1 in treatments involving heat. One possible ex-
planation is that the turnover of D1 is taking place in the heat treatments, and this
is evident based on the increase of free D1 together with a reduction of the D1–D2
complex, as well as a decrease in the FV/FM values.
6.4 Waterlogging and Salinity: A Combined Stress

in Legumes
Salt stress is certainly one of the most serious environmental factors limiting the
productivity of crop plants (Ashraf and O’Leary1999). Salinity reduces the ability
of plants to take up water, causing rapid reductions in growth rate, along with an
array of metabolic changes identical to those caused by water stress (Munns 2002).
High salt concentration in the external solution of plant cells produces several
deleterious consequences. First, salt stress causes an ionic imbalance (Niu et al.
1995). The homeostasis of not only Na+ and Cl− but also K+ and Ca+2 ions is dis-
turbed (Rodriguez-Navarro 2000; Hasegawa et al. 2000; Serrano et al. 1999). As a
result, plant survival and growth will depend on adaptations that re-establish ionic
homeostasis, thereby reducing the duration of cellular exposure to ionic imbalance.
Second, high concentrations of salt impose a hyperosmotic shock by decreasing
water and causing loss of cell turgor. This negative effect in the plant cell is thought
to be similar to the effects caused by drought. Third, reduction of chloroplast stro-
mal volume and generation of ROS, in salt-induced water stress, are also thought to
play important roles in inhibiting photosynthesis (Price and Hendry 1991). On the
molecular level, these responses are manifested as changes in the pattern of gene
expression (Maggio et al. 2002).
The process of salinization results from the interaction between climate, geo-
morphology, hydrology, land use and surface water properties and dynamics of the
salts. Regions with salinity are frequently associated with geographical localization
with inundation events; thus it is not infrequent that salt and flood stress occurs
simultaneously.
Salinity and waterlogging interact adversely to reduce production of crops and
pastures, as very few species used in agriculture can tolerate the combination of
both stresses (Barrett-Lennard 2003). Moreover, annual pasture legumes are par-
ticularly sensitive to combined salinity and waterlogging (Bennett et al. 2009).
One of the most important consequences of energy limitation under anoxia is
altered redox state of the cell. Under low oxygen pressure conditions, the interme-
diate electron carriers in electron transport chain become reduced, affecting redox-
active metabolic reactions. Therefore, for maintaining redox homeostasis cells need
to regulate NADH to NAD ratio under flooding (Chirkova et al. 1992). Saturated
electron transport components, the highly reduced intracellular environment and
low-energy supply are the factors favourable for ROS generation. The consequenc-
es of ROS formation depend on the intensity of the stress as well as on the physico-
chemical conditions in the cell (i.e. antioxidant status, redox state and pH). As was
mentioned for other stresses, ROS accumulation may cause damage to different cell
structures and biomolecules. H2O2 production during O2 deprivation was observed
in the plant cells (Blokhina et al. 2001), and its degradation was found to play an
important role in waterlogging tolerance in non-legume plants (Lin et al. 2004).
A trait that is essential for root survival during water logging or flooding is the
development of aerenchyma (Armstrong 1979). Aerenchymas are cortical airspaces
that provide a low-resistance internal pathway for the movement of O2 from the
shoots to the roots, where it is consumed in respiration and may also reoxidize
the rhizosphere (Armstrong 1970; Armstrong 1971, 1979). In legumes, aerenchyma
may also be important for supplying O2 and N2 to root nodules (Walker et al. 1983;
James et al. 1992; Zook et al. 1986; Pugh et al. 1995). Tolerance of Melilotus siculus
to waterlogging is associated with the production of a highly porous phellem, a type
of secondary aerenchyma, on taproots and upper lateral roots (Verboven et al. 2011).
Studies with plant species sensitive or tolerant to flooding–salt stress combina-
tion have shown that the rate of transport of Na+ and Cl− to the shoot is critical to
define the response. The ions transport rate increases significantly under combined
stress in comparison with salinity alone (Barrett-Lennard 2003). For more tolerant
species, there is only small or even no increase in shoot Na+ and Cl− in response to
combined salinity and waterlogging (Colmer and Flowers 2008), presumably due
to better root aeration. Moreover, in perennial legumes such as Trifolium repens L.
(Rogers and West 1993) and Liolaemus tenuis (Teakle et al. 2007), high root po-
rosity was associated with better shoot ion regulation under combined salinity and
waterlogging. Comparisons of annual pasture legumes in growth, ion regulation and
root porosity demonstrate that M. siculus has exceptional tolerance to combinations
of salinity and waterlogging (Teakle et al. 2012). Enhanced root aeration would
avoid energy deficits that could impair ion transport processes in roots, which de-
termines delivery of Na+ and Cl− to shoots via the xylem (Barrett-Lennard 2003;
Teakle et al. 2007; Colmer and Flowers 2008). Thus, traits of importance for toler-
ance to combined salinity and waterlogging are likely to include high root porosity,
leading to decreased shoot Na+ and Cl− concentrations.
6.5 Metabolic Changes in Responses to Stress

Combination
It is well known that the effect of a combination of different stresses on plants

can be quite different from those generated when plants are subjected to individual
types of stress (Rizhsky et al. 2002). Table 6.1 represents a summary of how the
combination of different stresses affects some parameters in legumes.
With reference to antioxidant responses, different patterns are observed when
more than one stress is imposed. However, it seems that in most cases the addition
of other stress did not alter the response. It implies that the signal molecules that
induce the expression of antioxidant enzymes probably are the same in different
stresses and so the imposition of both stresses is redundant. In other cases, the effect
of simultaneous stresses produces deleterious effects. For example, for APX and
CAT, one stress produces the induction of the activity (or at least a normal level of
activity), but the imposition of two stresses could produce a more nitrosative condi-
tion in the cell leading to the nitration of the enzyme, which is known to decrease
the activity of these enzymes.
Table 6.1 Effects of stress combination on main parameters studied in legumes

Response
Evaluated Negative Unchanged Additive Synergistic
parameter correlation response effect
APX
SOD
Antioxidant enzymes CAT
GR
POX
H2O2
Oxidative stress TBARS
Electrolyte leakage
O2○-
Photosynthetic activity FV/FM
CP47
D1
D2
Metabolites Proline
Ascorbic acid
Darker shading indicates that the particular response is supported by more evidence. Data obtained
from following legume species under various combined stresses: L. corniculatus, T. pratense
subjected to combined drought and heat (Signorelli et al. 2013b), L. japonicus subjected to
combined drought and heat (Sainz et al. 2010), Vigna unguiculata subjected to combined CO2,
UV-B radiation and temperature stress (Singh et al. 2010), Vigna radiata (Siddiqui 2013)
and Phaseolus vulgaris subjected to combined zinc and high irradiance stress (Michael and
Krishnaswamy 2011).
APX ascorbate peroxidase, SOD superoxide dismutase, CAT catalase, GR glutathione reductase,
H2O2 hydrogen peroxide, TBARS thiobarbituric reactive substances, O2●− superoxide radical,
Fv/FM photosystem II, POX peroxidase
Among the oxidative stress markers, synergistic effect was the most
commonly observed response. Most stresses are accompanied by an increment
of ROS production, and the source of ROS is different for different stresses
(Mahalingam and Fedoroff 2003; Wrzaczek et al. 2013). Thus, when more than
one stress is present, it induces ROS from different organelles, and hence the
total ROS tends to be higher in combined stress scenarios. Less commonly, a
negative correlation or an unchanged response is observed. In one case of negative
correlation observed for H2O2, it was suggested that the reduction in SOD activity
in combined stress as opposed to in single stress was responsible for the lower H2O2
in the former. In the other case, induction of CAT activity only in the combination
of stress was suggested to be the cause of lower H2O2 levels.
Photosynthetic activity does not show a defined pattern, maybe due to lack
of information. Even with the limited data, it can be seen that in all the cases
examined, D1 was unchanged by the imposition of combined stresses. D2 protein
had a synergistic effect in combined stress. It is important to point out that in
drought and heat stress were considered in these studies, and some of these respons-
es were observed in T. pratense and in two related species such as L. japonicus
and L. corniculatus. Other species should be evaluated to see the conservation
in the response of D2, which is suggested to disassemble to induce inhibition of
photosystem activity, and protect cells from oxidative damage caused by its own
activity.
6.6 Forage Legumes Field Productivity and Combined

Environmental Stress
Legumes have a high level of productive diversification and flexible utilization. The
same species can be usefully exploited for different purposes such as soil protection
from erosion; green manure crop; mulching; cover crop in vineyards, orchards and
firebreak lines; high quality honey production; landscape enhancement and medici-
nal use. Consequently, forage legumes were adapted to a wide range of soil types,
climatic conditions and management systems (Sánchez-Díaz 2001).
Legumes, as many other crops, have been bred to maximize productivity (forage
or grain). But this productivity is always affected by adverse environmental factors.
Perennial forage legumes are a good model to analyse the responses of adaptability
of plants under field conditions. This is because during the whole plant growth and
development cycle, plants are subjected to various types of abiotic stresses, both
singly and in combinations.
Low temperatures and periods of water saturation in soils are common during
the winters in many regions and in the other side periods of low water regime
combined with high temperatures are common during summers. To these we must
add other combinations of stresses such as periods of high radiation or toxic ions
(Na+ or heavy metals) produced by changes in the physicochemical conditions of
the soils.
Further, abiotic stress can affect the legume plants at different developmental
stages. So legumes growing under field conditions must have adaptation process
triggered by stress in seedling, vegetative or reproductive stages. For example, for
seedling emergence, the optimal conditions in the field are established at the end of
winter (Fig. 6.3).
Legumes are adapted to different environmental conditions by setting the de-
velopmental stages, such as reseedling capacity that is an important characteristic
for the perpetuation of L. corniculatus. Yield of L. corniculatus during 3 years with
seed set and without seed set, reveal the importance in reseedling (Fig. 6.4, Ayala
and Carámbula 2009).
Fig. 6.3 Seedlings

emergence of L. tenuis
during a typical of temperate
zones from south hemisphere
(Ayala and Carámbula 2009)
Fig. 6.4 Seasonal dry matter production of L. corniculatus under two-seed set management. With
seed set ( solid line) and without seed set ( dashed line). (Ayala and Carámbula 2009)
This suggests that the tolerance in reproductive stages should be accompanied

by physiological responses to deal with water restriction and high temperatures.
Another key physiological mechanism in the survival of legumes is their ability to
mobilize carbohydrates to storage tissues that can be located on the crown, root or
rhizome (Castillo et al. 2012). However, it is critical that the photosynthetic activ-
ity remains active during the stress period to achieve significant accumulation of
sugars allowing regrowth of the shoot after stress.
6.7 Breeding Approaches for Improving Tolerance

to Combined Abiotic Stresses
Selection for one abiotic stress tolerance in the field is very challenging due to in-
teractions among the different stresses. Thus, the only strategy to identify the traits
to be applied in field for breeding tolerant genotypes is by performing experiments
under controlled environment conditions. Regardless of the screening method, a

key objective for plant breeders is to develop an effective set of stress combination
markers that can be used to improve legume crop species. Controlled environmental
conditions allow the dissection of each one of different stress effect and the identi-
fication of principal targets affecting plant tolerance. Breeding for stress tolerance
requires efficient screening procedures, identification of key traits in diverse donor
or tolerant lines and understanding their inheritance and molecular genetics. Statis-
tical package applied to plant breeding will facilitate the identification of markers
in a multi-trait multi-environment way (Malosetti et al. 2004).
Several quantitative trait locus (QTL) studies relating to various abiotic stress
tolerances have already been reported showing it is possible to improve and ac-
celerate the breeding process in plant species without sequenced genomes (Chan-
dra et al. 2004). In order to transfer these traits, classical breeding requires the
establishment of rapid and cost-effective screening procedures and implementing
these using breeding approaches such as association mapping or genomic selection
procedures.
For the complete sequencing of the different important legumes, genome
opens the possibility of fine mapping of the QTLs. In this perspective, gene
identification for combined stress tolerance in legumes using genetic map
information and genome data is an achievable goal (Heffner et al. 2009; Hirayama
and Shinozaki 2010).
Phenotypic and physiological characterization along with RNA sequencing anal-
ysis of plants subjected to drought, heat, salt, flooding stress or their combination
would confirm that the simultaneous imposition of different types of stress pres-
ents unique but varied aspects that includes alteration of respiration rate, decreased
photosynthesis, stomatal closure, high leaf temperature and redox homeostasis.
Thus, deep phenotyping methodologies, genome-based selection and massive RNA
sequencing technologies emerge as a promising avenue for the development of mul-
tiple abiotic stress-tolerant crops.
References
Abdullah F, Hareri F, Naaesan M, Ammar MA, ZuherKanbar O. Effect of drought on different

physiological characters and yield component in different varieties of Syrian durum wheat. J
Agric Sci. 2011;3(3):127–33.
Ahmad A, Diwan H, Abrol YP Global climate change, stress and plant productivity. In: Pareek A,
Sopory KS, Bohnert HJ, Govindjee, editors. Abiotic stress adaptation in plants: physiological,
molecular and genome foundation. Dordrecht: Springer; 2010. p. 503–21.
Allakhverdiev SI, Kreslavski VD, Klimov VV, Los DA, Carpentier R, Mohanty P. Heat stress: an
overview of molecular responses in photosynthesis. Photosynth Res. 2008;98(1–3):541–50.
Alscher RG, Erturk N, Heath LS. Role of superoxide dismutases (SODs) in controlling oxidative
stress in plants. J Exp Bot. 2002;53(372):1331–41.
Armstrong W. Rhizosphere oxidation in rice and other species: a mathematical model based on the
oxygen flux component. Physiol Plant. 1970;23:623–30.
Armstrong W. Radial oxygen loss from intact rice roots as affected by distance from the apex,
respiration and waterlogging. Physiol Plant. 1971;25:192–7.
Armstrong W. Aeration in higher plants. Adv Bot Res. 1979;7:225–332.

Ashraf M, O’Leary JW. Changes in soluble proteins in spring wheat stressed with sodium chloride.
Biol Plant. 1999;42:113–7.
Ayala W, Carámbula M El valor agronómico del género Lotus. Unidad de Comunicación y Trans-
ferencia de Tecnología de INIA, Montevideo, Uruguay; 2009.
Baker NR. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Annu Rev Plant Biol.
2008;59:89–113.
Barnabás B, Jager K, Feher A. The effect of drought and heat stress on reproductive processes in
cereals. Plant Cell Environ. 2008;31(1):11–38.
Barrett-Lennard EGT. The interaction between waterlogging and salinity in higher plants: causes,
consequences and implications. Plant Soil. 2003;253:35–54.
Begara-Morales JC, Sanchez-Calvo B, Chaki M, Valderrama R, Mata-Perez C, Lopez-Jaramillo
J, Padilla MN, Carreras A, Corpas FJ, Barroso JB. Dual regulation of cytosolic ascorbate
peroxidase (APX) by tyrosine nitration and S-nitrosylation. J Exp Bot. 2014;65(2):527–38.
Bennett SJ, Barrett-Lennard EG, Colmer TD. Salinity and waterlogging as constraints to saltland
pasture production: a review. Agric Ecosyst Environ. 2009;129:349–60.
Berry JA, Bjorkman O. Photosynthetic response and adaptation to temperature in higher plants.
Ann Rev Plant Phys Plant Mol Biol. 1980;31:491–543.
Bita CE, Gerats T. Plant tolerance to high temperature in a changing environment: scientific funda-
mentals and production of heat stress-tolerant crops. Front Plant Sci. 2013;4:273.
Blokhina OB, Chirkova TV, Fagerstedt KV. Anoxic stress leads to hydrogen peroxide formation in
plant cells. J Exp Bot. 2001;52(359):1179–90.
Bohnert HJ, Sheveleva E. Plant stress adaptations-making metabolism move. Curr Opin Plant
Biol. 1998;1(3):267–74.
Bohnert HJ, Nelson DE, Jensen RG. Adaptations to environmental stresses. Plant Cell.
1995;7(7):1099–111.
Bokszczanin KL, Fragkostefanakis S. Perspectives on deciphering mechanisms underlying plant
heat stress response and thermotolerance. Front Plant Sci. 2013;4:315.
Bonnecarrére V, Borsani O, Díaz P, Capdevielle F, Blanco P, Monza J. Response to photoxidative
stress induced by cold in japonica rice is genotype dependent. Plant Sci. 2011;180(5):726–32.
Bray EA. Plant responses to water deficit. Trends Plant Sci. 1997;2:48–54.
Bray EA, Bailey-Serres J, Weretilnyk E Responses to abiotic stresses. In: Buchanan BB, Gruis-
sem W, Jones RL, editors. Biochemistry and molecular biology of plants. Rockville: American
society of plant physiologists; 2000. P. 1158–1203.
Castillo A, Rebuffo M, Dalla Rizza M, Folle G, Santiñaque F, Borsani O, Monza J. Generation
and characterization of interspecific hybrids of Lotus uliginosus x Lotus corniculatus. Crop
Sci. 2012;52:1572–82.
Chandra S, Buhariwalla HK, Kashiwagi J, Harikrishna S, Rupa Sridevi K, Krishnamurthy L, Ser-
raj R, Crouch JH Identifying QTL-linked markers in markers-deficient crops. In: Fisher T,
Turner N, Angust J et al., editors. Proceeding of the fourth international crop science congress.
The Regional Institute Ltd., Gosford, Australia; 2004.
Chen J, Wang P, Mi HL, Chen GY, Xu DQ. Reversible association of ribulose-1, 5-bisphosphate
carboxylase/oxygenase activase with the thylakoid membrane depends upon the ATP level and
pH in rice without heat stress. J Exp Bot. 2010;61(11):2939–50.
Chirkova TV, Zhukova TM, Bugrova MP. Redox reactions of plant cells in response to short-term
anaerobiosis. Vestnik SPBGU. 1992;3:82–6.
Choi HK, Mun JH, Kim DJ, Zhu H, Baek JM, Mudge J, Roe B, Ellis N, Doyle J, Kiss GB, Young
ND, Cook DR. Estimating genome conservation between crop and model legume species. Proc
Natl Acad Sci U S A. 2004;101(43):15289–94.
Colmer TD, Flowers TJ. Flooding tolerance in halophytes. New Phytol. 2008;179(4):964–74.
Corpas FJ, Palma JM, Del Rio LA, Barroso JB. Protein tyrosine nitration in higher plants grown
under natural and stress conditions. Front Plant Sci. 2013;4:29.
Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F. Dual action of the
active oxygen species during plant stress responses. Cell Mol Life Sci. 2000;57(5):779–95.
de Vitry C, Olive J, Drapier D, Recouvreur M, Wollman FA. Posttranslational events leading to

the assembly of photosystem II protein complex: a study using photosynthesis mutants from
Chlamydomonas reinhardtii. J Cell Biol. 1989;109(3):991–1006.
Díaz P, Borsani O, Monza J Lotus-related species and their agronomic importance. In: Márquez
AJ, editor. Lotus japonicus handbook. Heidelberg: Springer; 2005a. p. 25–37.
Díaz P, Borsani O, Márquez M, Monza J. Osmotically induced proline accumulation in
Lotus corniculatus leaves affected by light and nitrogen source. Plant Growth Regul.
2005b;46:223–32.
Ellis RJ. Molecular chaperones: the plant connection. Science. 1990;250(4983):954–9.
Esfahani MN, Sulieman S, Schulze J, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS (2014)
Mechanisms of physiological adjustment of N2 fixation in chickpea ( Cicer arientum L.)
during early stages of water deficit: Single or multi-factor controls? Plant J doi:1111/
tpj.12599.
Feng Z, Jin-Kui G, Ying-Li Y, W en-LiangH, Li-Xin Z. Changes in pattern of antiozidant enzyma
in wheat wxposed to water deficit and rewatering. Acta Physiol Plant. 2004;26:345–52.
Finka A, Mattoo RU, Goloubinoff P. Meta-analysis of heat- and chemically upregulated chaperone
genes in plant and human cells. Cell Stress Chaperones. 2011;16(1):15–31.
Ginzberg I, Barel G, Ophir R, Tzin E, Tanami Z, Muddarangappa T, de Jong W, Fogelman E. Tran-
scriptomic profiling of heat-stress response in potato periderm. J Exp Bot. 2009;60(15):4411–
21.
Guo TR, Zhang GP, Zhang YH. Physiological changes in barley plants under combined toxicity of
aluminum, copper and cadmium. Colloids Surf B Biointerfaces. 2007;57(2):182–8.
Guy C. Molecular responses of plants to cold shock and cold acclimation. J Mol Microbiol Bio-
technol. 1999;1(2):231–42.
Hakam P, Khanizadeh S, DeEll JR, Richer C. Assessing chilling tolerance in roses using chloro-
phyll fluorescence. Hortscience. 2000;35(2):184–6.
Hasegawa M, Bressan R, Zhu J-K, Bhonert H. Plant cellular and molecular responses to high salin-
ity. Annu Rev Plant Physiol. 2000;51:493–9.
Havaux M. Rapid photosynthetic adaptation to heat stress triggered in potato leaves by moderately
elevated temperatures. Plant Cell Environ. 1993;16:461–7.
Heffner EL, Sorrells ME, Jannink JL. Genomic selection for crop improvement. Crop Sci.
2009;49:1–12.
Hirayama T, Shinozaki K. Research on plant abiotic stress responses in the post-genome era: past,
present and future. Plant J. 2010;61:1041–52.
Hu ZH, Xu YN, Jiang GZ, Luang TY. Degradation and inactivation of photosystem I complexes
during linera heating. Plant Sci. 2004;166:1177–83.
Hutmacher RB, Krieg DR. Photosynthetic rate control in cotton: stomatal and nonstomatal factors.
Plant Physiol. 1983;73(3):658–61.
Huve K, Bichele I, Rasulov B, Niinemets U. When it is too hot for photosynthesis: heat-induced
instability of photosynthesis in relation to respiratory burst, cell permeability changes and H(2)
O(2) formation. Plant Cell Environ. 2011;34(1):113–26.
James EK, Minchin FR, Sprent JI. The physiology and nitrogen-fixing capability of aquatically
and terrestrially grown Neptunia plena: the importance of nodule oxygen supply. Ann Bot
1992;69:181–7.
Khalaffalla AM. Effect of sowing date, ridge direction, plant orientation and population on faba
grain yield. FABIS Newslett. 1985;12:11–2.
Kim K, Portis AR Jr. Temperature dependence of photosynthesis in Arabidopsis plants with modi-
fications in Rubisco activase and membrane fluidity. Plant Cell Physiol. 2005;46(3):522–30.
Komenda J, Reisinger V, Muller BC, Dobakova M, Granvogl B, Eichacker LA. Accumulation
of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center
complex in Synechocystis PCC 6803. J Biol Chem. 2004;279(47):48620–9.
Kramer PJ. Water relations of plants. Orlando:Academic; 1983.
Kyle DJ, Ohad I. The mechanism of photoinhibition in higher plants and green algae. In: Stae-
helin AL, Arntzen CJ, editors. Encyclopedia of plant physiology. Berlin:Springer; 1986;Vol.
19:pp. 468–75.
Larkindale J, Vierling E. Core genome responses involved in acclimation to high temperature.

Plant Physiol. 2008;146(2):748–61.
Larrainzar E, Gil-Quintana E, Arrese-Igor C, Gonzalez EM, Marino D Split-root systems applied
to the study of the legume-rhizobial symbiosis: what have we learned? J Integr Plant Biol.
2014. doi:10.1111/jipb.12231.
Lidholm J, Gustafsson P, Oquist G. Photoinhibition of photosynthesis and its recovery in a green
alga Chlamydomonas reinhardtii. Plant Cell Physiol. 1987;28:1133–40.
Lin KHR, Weng CC, Lo HF, Chen JT. Study of the root antioxidative system of tomatoes and
eggplants under waterlogged conditions. Plant Sci. 2004;167:355–65.
Liu HT, Gao F, Li GL, Han JL, Liu DL, Sun DY, Zhou RG. The calmodulin-binding protein
kinase 3 is part of heat-shock signal transduction in Arabidopsis thaliana. Plant J.
2008;55(5):760–73.
Lu C, Zhang J. Effects of water stress on photosystem II photochemistry and its thermostability in
wheat plants. J Exp Bot. 1999;50:1199–206.
Lv WT, Lin B, Zhang M, Hua XJ. Proline accumulation is inhibitory to Arabidopsis seedlings dur-
ing heat stress. Plant Physiol. 2011;156(4):1921–33.
Maestri E, Klueva N, Perrotta C, Gulli M, Nguyen HT, Marmiroli N. Molecular genetics of heat
tolerance and heat shock proteins in cereals. Plant Mol Biol. 2002;48(5-6):667–81.
Maggio A, Miyazaki S, Veronese P, Fujita T, Ibeas JI, Damsz B, Narasimhan ML, Hasegawa PM,
Joly RJ, Bressan RA. Does proline accumulation play an active role in stress-induced growth
reduction? Plant J. 2002;31(6):699–712.
Mahalingam R, Fedoroff NV Stress response, cell death and signalling: the many faces of ROS.
Physiol Plant. 2003;119:56–68.
Malosetti M, Voltas J, Romagosa I, Ullrich SE, van Eeuwijk FA. Mixed models including
environmental covariables for studying QTL by environment interaction. Euphytica.
2004;137:139–45.
Marino D, Frendo P, Ladera R, Zabalza A, Puppo A, Arrese-Igor C, Gonzalez EM. Nitrogen
fixation control under drought stress. Localized or systemic. Plant Physiol. 2007;143:1968–74.
Michael PI, Krishnaswamy M. The effect of zinc stress combined with high irradiance stress
on membrane damage and antioxidative response in bean seedlings. Environ Exp Bot.
2011;74:171–7.
Minai L, Wostrikoff K, Wollman FA, Choquet Y. Chloroplast biogenesis of photosystem II
cores involves a series of assembly-controlled steps that regulate translation. Plant Cell.
2006;18(1):159–75.
Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002;7(9):405–10.
Mittler R, Finka A, Goloubinoff P. How do plants feel the heat? Trends Biochem Sci.
2012;37(3):118–25.
Morales D, Rodríguez P, Dell’Amico J, Nicolas E, Torrecillas A, Sánchez-Blanco MJ. High tem-
perature preconditioning and thermal shock imposition affects water relations, gas exchange
and root hydraulic conductivity in tomato. Biol Plant. 2003;47:203–8.
Munns R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002;25(2):239–
50.
Niu X, Bressan RA, Hasegawa PM, Pardo JM. Ion homeostasis in NaCl stress environments. Plant
Physiol. 1995;109(3):735–42.
Noctor G, Foyer CH. Ascorbate and glutathione: keeping active oxygen under control. Annu Rev
Plant Phys. 1998;49:249–79.
Ohad I, Kyle DJ, Arntzen CJ. Membrane protein damage and repair: removal and replace-
ment of inactivated 32-kilodalton polypeptides in chloroplast membranes. J Cell Biol.
1984;99(2):481–5.
Parida AK, Das AB. Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ
Safe. 2005;60(3):324–49.
Pastori GM, Foyer CH. Common components, networks, and pathways of cross-tolerance
to stress. The central role of “redox” and abscisic acid-mediated controls. Plant Physiol.
2002;129(2):460–8.
Peterson PR, Sheaffer CC, Hall MH. Drought effects on perennial forage legume yield and quality.
Agron J. 1992;84(5):774–9.
Price AH, Hendry GAF. Iron-catalyzed oxygen radical formation and its possible contribution to
drought damage in nine native grasses and three cereals. Plant Cell Environ. 1991;14:477–84.
Pugh R, Witty JF, Mytton LR, F.R. M. The effect of waterlogging on nitrogen fixation and nodule
morphology in soil grown white clover ( Trifolium repens L.). J Exp Bot. 1995;46:285–90.
Rizhsky L, Liang H, Mittler R. The combined effect of drought stress and heat shock on gene
expression in tobacco. Plant Physiol. 2002;130(3):1143–51.
Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R. When defence pathways
collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiol.
2004;134(4):1683–96.
Rodriguez-Navarro A. Potassium transport in fungi and plants. Biochim Biophys Acta.
2000;1469(1):1–30.
Rogers ME, West DW. The effects of rootzone salinity and hypoxia on shoot and root growth in
Trifolium species. Ann Bot. 1993;72:503–9.
Sade B, Soylu S, Yetim E. Drought and oxidative stress. Afr J Biotechnol. 2011;10(54):11102–9.
Sainz M, Diaz P, Monza J, Borsani O. Heat stress results in loss of chloroplast Cu/Zn superoxide
dismutase and increased damage to photosystem II in combined drought-heat stressed Lotus
japonicus. Physiol Plant. 2010;140(1):46–56.
Salvucci ME, Crafts-Brandner SJ. Inhibition of photosynthesis by heat stress: the activation state
of Rubisco as a limiting factor in photosynthesis. Physiol Plant. 2004;120(2):179–86.
Samuelsson G, Lonneborg A, Rosenquist E, Gustafsson P, Oquist G. Photoinhibition and reac-
tivation of photosynthesis in the cyanobacterium Anacystis nidulans. Plant Cell Physiol.
1985;83:992–5.
Sánchez-Díaz M Adaptation of legumes to multiple stresses in Mediterranean-type environments.
In: Delgado I, Lloveras J, editors. Quality in lucerne and medics for animal production. vol
Options Méditerranéennes: Série A. Séminaires Méditerranéens. CHIEAM, Zaragoza, 2001.
p. 145–151.
Sayed OH, Earnshaw MJ, Emes MJ. Photosynthetic response of different varieties of wheat to
high temperature. II. Effect of heat stress on photosynthetic electron transport. J Exp Bot.
1989;40:633–8.
Schrader SM, Kleinbeck KR, Sharkey TD. Rapid heating of intact leaves reveals initial effects of
stromal oxidation on photosynthesis. Plant Cell Environ. 2007;30(6):671–8.
Schwarz C, Elles I, Kortmann J, Piotrowski M, Nickelsen J. Synthesis of the D2 protein of
photosystem II in Chlamydomonas is controlled by a high molecular mass complex con-
taining the RNA stabilization factor Nac2 and the translational activator RBP40. Plant Cell.
2007;19(11):3627–39.
Serrano R, Mulet JM, Ríos G, Márquez JA, de Larriona IF, Leube MP, Mendizabal I, Pascual-
Ahuir A, Proft MRR, Montesinos C. A glimpse of the mechanism of ion homeostasis during
salt stress. J Exp Bot. 1999;50:1023–36.
Sharkey TD, Zhang R. High temperature effects on electron and proton circuits of photosynthesis.
J Integr Plant Biol. 2010;52(8):712–22.
Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in water-stress
response. Plant Physiol. 1997;115(2):327–34.
Shyam R, Sane PV. Photoinhibition of photosynthesis and its recovery in low and high light ac-
climatized blue-green alga (Cyanobacterium) Spirulina platensis. Biochem Physiol Pflanz.
1989;185:211–9.
Siddiqui ZS. Effects of double stress on antioxidant enzyme activity in Vigna radiata (L.) Wilczek.
Acta Bot Croat. 2013;72(1):145–56.
Signorelli S, Arellano JB, Melo TB, Borsani O, Monza J. Proline does not quench singlet oxygen:
evidence to reconsider its protective role in plants. Plant Physiol Biochem. 2013a;64:80–3.
Signorelli S, Casaretto E, Sainz M, Diaz P, Monza J, Borsani O. Antioxidant and photosystem II
responses contribute to explain the drought-heat contrasting tolerance of two forage legumes.
Plant Physiol Biochem. 2013b;70C:195–203.
Signorelli S, Corpas FJ, Borsani O, Barroso JB, Monza J. Water stress induces a differential and
spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus.
Plant Sci. 2013c;201–202:137–46.
Singh SK, Kakani VG, Surabhi G-K, Reddy KR () Cowpea ( Vigna unguiculata [L.] Walp.) geno-
types response to multiple abiotic stresses. J Photochem Photobiol B: Biol. 2010;100:135–46.
Sinsawat V, Pandy J, Leipner P, Stamp P, Fracheboud Y. Effect of heat stress on the photosynthetic
apparatus in maize ( Zea maize L.) grown at control or high temperature. Environ Exp Bot.
2004;52:123–9.
Smillie RM, Hetherington SE. Stress tolerance and stress-induced injury in crop plants measured
by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light. Plant
Physiol. 1983;72(4):1043–50.
Szabados L, Savouré A. Proline: a multifunctional amino acid. Trends Plant Sci. 2010;15(2):89–97.
Takahashi S, Murata N. How do environmental stresses accelerate photoinhibition? Trends Plant
Sci. 2008;13(4):178–82.
Tang Y, Wen X, Lu Q, Yang Z, Cheng Z, Lu C. Heat stress induces an aggregation of the
light-harvesting complex of photosystem II in spinach plants. Plant Physiol. 2007;143(2):629–38.
Teakle N, Flowers T, Real D, Colmer T. Lotus tenuis tolerates the interactive effects of salinity
and waterlogging by ‘excluding’ Na+ and Cl- from the xylem. J Exp Bot. 2007;58(8):2169–80.
Teakle NL, Bowman S, Barrett-Lennard EG, Real D, Colmer TD. Comparisons of annual pasture
legumes in growth, ion regulation and root porosity demonstrate that Melilotus siculus has
exceptional tolerance to combinations of salinity and waterlogging. Environ Exp Bot.
2012;77:175–84.
Teskey RO, Fites JA, Samuelson LJ, Bongarten BC Stomatal and nonstomatal limitations to
net photosynthesis in Pinus taeda L. under different environmental conditions. Tree Physiol.
1986;2(1_2_3):131–142.
Valderrama R, Corpas FJ, Carreras A, Gomez-Rodriguez MV, Chaki M, Pedrajas JR, Fernandez-
Ocana A, Del Rio LA, Barroso JB. The dehydrogenase-mediated recycling of NADPH is a key
antioxidant system against salt-induced oxidative stress in olive plants. Plant Cell Environ.
2006;29(7):1449–59.
Verboven P, Pedersen O, Herremans E, Ho QT, Nicolai BM, Colmer TD, Teakle N. Root aeration
via aerenchymatous phellem: three-dimensional micro-imaging and radial O2 profiles in
Melilotus siculus. New Phytol. 2011;193(2):420–31.
Verslues P, Sharp R. Proline accumulation in maize ( Zea mays L) Primary roots at low water
potentials. II Metabolic source of increased proline deposition in the elongation zone. Plant
Physiol. 1999;119:1349–60.
Wahid A. Physiological implications of metabolite biosynthesis for net assimilation and heat-stress
tolerance of sugarcane ( Saccharum officinarum) sprouts. J Plant Res. 2007;120(2):219–28.
Wahid A, Shabbir A. Induction of heat stress tolerance in barley seedlings by pre-sowing seed
treatment with glycinebetaine. Plant Growth Regul. 2005;46:133–41.
Walker BA, Pate JS, Kuo J. Nitrogen fixation by nodulated roots of Viminaria juncea (Schrad. &
Wendl.) Hoffmans. (Fabaceae) when submerged in water. Aust J Plant Physiol. 1983;10:409–21.
Weaich K, Briston KL, Cass A. Modeling preemergent maize shoot growth. II. High temperature
stress conditions. Agron J. 1996;88:391–7.
Wise R, Olson A, Schrader S, Sharkey T. Electron transport is the functional limitation of pho-
tosynthesis in field-grown pima cotton plants at high temperature. Plant Cell Environ.
2004;27:717–24.
Wrzaczek M, Brosche M, Kangasjarvi J. ROS signalling loops—production, perception, regula-
tion. Curr Opin Plant Biol. 2013;16:575–582.
Xu ZZ, Zhou GS. Combined effects of water stress and high temperature on photosynthesis,
nitrogen metabolism and lipid peroxidation of a perennial grass Leymus chinensis. Planta.
2006;224(5):1080–90.
Yamada M, Hidaka T, Fukamachi H. Heat tolerance in leaves of tropical fruit crops as measured
by chlorophyll fluorescence. Sci Hortic. 1996;37:39–48.
Yordanov I, Dilova R, Petkova T, Pangelova V, Goltsev K-H. Mechanisms of the temperatures

damage and acclimation of the photosynthetic apparatus. Photobiochem Photobiophys.
1986;12:147–55.
Zhang R, Sharkey TD. Photosynthetic electron transport and proton flux under moderate heat
stress. Photosynth Res. 2009;100(1):29–43.
Zhu JK, Hasagawa PM, Bressan RA. Molecular aspects of osmotic stress in plants. Crit Rev Plant
Sci. 1997;163:253–77.
Zook DM, Erwin DC, Stolzy LH. Anatomical, morphological, and physiological responses of
alfalfa to flooding. Plant Soil. 1986;96:293–6.
Chapter 7
Interactive Effects Between Ozone
and Drought: Sorrow or Joy?
Sacha Bohler, Ann Cuypers and Jaco Vangronsveld
7.1 Introduction
The industrial revolutions of the eighteenth and nineteenth centuries marked the
beginning of industry and technology, as we know it today (Ashton 1997; Hull
1999). In the eighteenth century, the first commercially available steam engine was
one of many breakthroughs that improved transport and industrial processes. Unfor-
tunately, it was also the first step towards the extensive use of fossil fuels, initially
in the form of coal. The nineteenth century brought forward the invention of the
combustion engine using fuel derived from petrol. The increasing use of fossil fuels
also marked the dawn of anthropogenic pollution, which has increased ever since
and reached its preliminary peak in the twenty-first century.
In the 1970s, acid rain was the major concern of environmentalists (van Breemen
et al. 1984; Shortle and Bondietti 1992), and damaged vast areas of vegetation. Lat-
er, in the 1980s, depletion of the ozone layer had everybody worried (Solomon et al.
1986). Today, climate change is on the mind of the general population, including
policy makers and researchers. Even though still largely rejected by climate change
opponents, the reports of the Intergovernmental Panel on Climate Change (IPCC)
presents ample proof that the earth’s average temperature is increasing, polar ice
caps are melting, ocean levels are rising and extreme weather conditions are becom-
ing more and more frequent (Solomon et al. 2007; Stocker et al. 2014). This is for a
S. Bohler () · A. Cuypers · J. Vangronsveld

Centre for Environmental Sciences, Environmental Biology, Hasselt University, Agoralaan
Building D, 3590 Diepenbeek, Belgium
e-mail: sacha.bohler@uhasselt.be
A. Cuypers
e-mail: ann.cuypers@uhasselt.be
J. Vangronsveld
e-mail: jaco.vangronsveld@uhasselt.be

148 S. Bohler et al.
larger part due to anthropogenic atmospheric pollution, primarily brought on by the

combustion of fossil fuels. Molecules like carbon dioxide (CO2), methane (CH4),
nitric and sulphur oxides (NOx, SOx) are directly emitted into the atmosphere, while
other molecules like ozone (O3) are formed from reactions between pollutants and
atmospheric constituents. CO2 and ozone are two of the main greenhouse gases that
cause the retention of heat in the atmosphere and lead to the observed increase in
temperatures.
7.2 Ozone
Ozone is a secondary pollutant, i.e. it is not directly emitted, but produced as a

consequence of primary pollutants. Nitric dioxide (NO2), mostly emitted by car
and industrial exhausts, reacts with solar UV radiation and fragments to form nitric
monoxide (NO) and an activated oxygen atom (O•−). The reaction between O•− and
atmospheric oxygen (O2) leads to the formation of ozone (Andreae and Crutzen
1997; Renaut et al. 2009; Fig. 7.1). Molecules like volatile organic compounds
(VOC) also intervene in the complex reactions. Background ozone concentrations
in the troposphere (the layer of air that expands from the earth’s surface up to an
altitude of about 10 km) have increased by 500 % during the past century (Marenco
1994). Even though, in recent years, steps have been taken to reduce the emission of
ozone-forming pollutants, results are inconclusive (Jonson et al. 2006).
An ozone-enriched atmosphere induces a situation of stress in plants. Ozone is
easily absorbed through stomata and instantly fragments in contact with the plant
Fig. 7.1 Simplified scheme

of the formation of the
secondary pollutant ozone
in the troposphere. NO2
nitric oxide; O3 ozone; O•−
activated oxygen atom; O2
atmospheric dioxygen
7 Interactive Effects Between Ozone and Drought: Sorrow or Joy? 149
tissue. The highly energetic ozone molecule reacts with cell wall components and
causes the formation of reactive oxygen species (ROS). These highly reactive mole-
cules in turn diffuse into the cells where they can damage proteins, genes, lipids and
other biomolecules. The high oxidative potential of ROS interferes with cell signal-
ling and regulation, including induction of programmed cell death (Kangasjärvi
et al. 2005), and may eventually lead to the death of exposed plants. Leaf chlorosis,
formation of necrotic patches and an increase in the number of senescing leaves are
the most visible symptoms of ozone stress (Bohler et al. 2007, 2013).
7.3 Drought
Drought is the prolonged absence of rain that leads to a transient water deficit in the
soil and concomitantly a stress situation for plants. Not only does drought depend
on precipitation but also on the speed of water evaporation from the soil (Sherwood
and Fu 2014). The occurrence of drought is variable among the different regions
of the earth and dependent strongly on climatic regions. Droughts are common in
arid regions; but in recent years, occurrences have also become more frequent in
moderate climate, posing a threat for crops and forests (Kreuzwieser and Gessler
2010; Ciais et al. 2005). Recent considerations show that background dryness is
as important for hydrological changes as acute occurrences of drought, and needs
to be given more importance in the evaluation of the effect of climate change on
hydrological changes (Sherwood and Fu 2014).
In plants, drought induces a decrease in the internal water potential. The first
response is usually a reduction in stomatal conductance to reduce evaporation and
save water (Warren et al. 2007). Further effects involve an accumulation of os-
motically active solutes, to increase internal osmotic potential and improve water
retention and absorption (Evers et al. 2010). Visible symptoms of drought include
stunted leaves and an increase in leaf senescence (Bohler et al. 2013; Munn-Bosch
and Alegre 2004).
7.4 Co-Occurrence of Ozone and Drought
Due to the meteorological conditions favouring both ozone formation and drought
(i.e. a succession of warm days free of cloud cover), both are very likely to occur
simultaneously. This can have drastic consequences for vegetation, if the effects of
both stresses are synergistic. However, the effects of ozone and drought can also be
antagonistic, in which case a simultaneous occurrence might be beneficial to plants.
It has been postulated that a stomatal closure induced by drought may reduce the
flux of ozone into the plant and thus be protective. In this chapter, a closer look is
taken on the current state of understanding of the physiological, biochemical and
molecular effects that ozone and drought in combination have on plants.
7.5 Stomatal Conductance: Protection Against

Ozone by Drought
One of the common characteristics between ozone and drought exposure is the sig-
nificant involvement of stomata. While ozone enters plants through the stomata,
water vapour escapes through them; therefore, stomatal closure would theoretically
protect plants against both stresses (Fig. 7.2). While observations have shown that
stomatal conductance is an excellent marker for the severity of drought (Medrano
et al. 2002), the situation is less evident for ozone. Reports have concluded that
stomatal behaviour is not consistent, but dependant on many factors (Wittig et al.
2007). This has been mainly attributed to stomatal sluggishness in multiple publica-
tions (Hoshika et al. 2012, 2014; Paoletti and Grulke 2010; Dumont et al. 2013).
It has been shown that this delayed response time of stomata varies among species
(Hoshika et al. 2012; Paoletti and Grulke 2010), severity of stress (Hoshika et al.
2012) and on seasonal changes (Hoshika et al. 2014). It has furthermore been con-
cluded that the sluggish behaviour of stomata under ozone exposure can lead to
perturbations in the response to water deficit (Hoshika et al. 2014).
Stomatal closure during drought has been proposed as a protective measure
against ozone exposure if both stresses are present simultaneously. However, this
phenomenon has not been consistently observed. It has been shown that the interac-
tive effect between ozone and drought is dependent on many factors, e.g. species
(Wagg et al. 2012; Ribas et al. 2005; Biswas and Jiang 2011; Pell et al. 1993), se-
quence of appearance (Bohler et al. 2013; Le Thiec et al. 1994), severity (Le Thiec
et al. 1994), time of day (Le Thiec et al. 1994), developmental stage (Alonso et al.
2001; Skärby et al. 1998) or season (Pell et al. 1993). Biswas and Jiang (2011)
showed, for instance, that, under conditions of combined ozone and drought stress,
the ozone-sensitive modern winter wheat cultivar ( Triticum aestivum L. cv. Xiaoyan
22) improved its tolerance against ozone, while the ozone-tolerant primitive wheat
( Turgidum ssp. durum) lost ozone tolerance. Le Thiec et al. (1994) and Bohler et al.
(2013) hypothesized that the order of occurrence could play an important role in the
combined effect. An early drought could lead to a decrease in stomatal conductance
Fig. 7.2 Stomata forming a physical barrier against ozone absorption and water vapour loss
and a subsequent protection against a later ozone exposure, while the appearance of
drought during pre-existing ozone stress would suffer under the appearing sluggish-
ness of stomata, initially caused by ozone.
The sluggishness of stomata under ozone stress is most likely due to a perturba-
tion of the abscisic acid (ABA)-induced stomatal regulation by ethylene (Fig. 7.3).
Wilkinson et al. (Wilkinson and Davies 2009) showed that ozone-treated Leontodon
hispidus present a reduced sensitivity to exogenously applied ABA and that stomata
display a decreased response to a gradual drought. They furthermore measured an
increase in ethylene production in ozone-exposed L. hispidus, while observing no
change in ABA concentrations. Most importantly, it was determined that the appli-
cation of 1-Methylcyclopropene (1-MCP), which prevents ethylene from binding
to its receptors, restored the sensitivity of stomata to externally applied ABA and
to soil drying (Wilkinson and Davies 2009). This shows that ozone-induced emis-
sion of ethylene is responsible for the sluggish behaviour of stomata, leading to
increased effects of drought, rather than protective effects against ozone. Ethylene-
mediated inhibition of ABA-induced stomatal closure was also shown by Tanaka et
al., independently of ozone exposure (Tanaka et al. 2005).
7.6 Biomass Changes and Visible Symptoms
Estimations predict that ozone may cause up to 30 % loss in biomass of crop plants,
and up to 10 % in forest species (Fuhrer 2009; Broadmeadow 1998). Drought may
lead to yield loss as well, as was shown by a 30 % decrease in plant productivity af-
ter the 2003 summer drought in Europe (Ciais et al. 2005). Decreases in biomass are
indeed to be expected as a consequence of both ozone and drought exposure, since
both phenomena may lead to a decrease in net photosynthetic rate (A) and thus in
the net CO2 fixation (Wittig et al. 2007; Biswas and Jiang 2011; Flexas et al. 2002).
A decrease in biomass production can include reduced seed weight and number
(Biswas and Jiang 2011; Flexas et al. 2002). Coinciding appearance of ozone and
drought has been shown to have a cumulative effect on the decrease of seed biomass
(Biswas and Jiang 2011).
Fig. 7.3 Simplified representation of the interactive effects of ethylene and abscisic acid on sto-
matal closure, drawn in PathVisio (van Iersel et al. 2008)
Visible symptoms of ozone and drought are quite characteristic and can allow
differentiation between both types of stress. Whereas ozone leads to the formation
of necrotic patches and irregular chlorotic discoloration of leaves, drought rather
induces a homogenous and gradual discoloration of leaves that in addition appear
stunted and droopy (Bohler et al. 2013). It has furthermore been shown that, in pop-
lar saplings, the combined effect of ozone and drought leads to an additive display
of both symptoms (Bohler et al. 2013). In contrast, a field survey by Showman
(1991) determined that in 1988 (a year with particularly high ozone levels in com-
bination with drought), less ozone-related injuries were observed as compared to
1989 (a year with lower ozone concentrations and less drought). Besides, Matyssek
et al. (2010) discussed that in 2003 (an exceptionally dry summer), the impact of
ozone on beech trees at a test site in Kranzberg forest (Germany) was most likely
reduced by drought, and the detrimental effects on radial and whole-stem volume
increment were most likely due to the water deficit. These are further indications
that protection manifested by drought may be very dependent on specific environ-
mental conditions and that even if drought has a protective effect against ozone, the
aftermath of drought itself may be equally or more detrimental than ozone.
7.7 Carbon Metabolism
Physiological measurements of ozone-exposed plants have shown that net pho-

tosynthetic rate, maximum rate of RuBisCO-mediated carboxylation and carbox-
ylation efficiency can be decreased (Biswas and Jiang 2011). This can partly be
attributed to a decrease in stomatal conductance, but there are indubitably further
reasons for these effects. It has been clearly shown on multiple occasions, and for
many species, that the enzyme RuBisCO is affected by ozone stress. Studies have
shown reduced enzyme activity and abundance of RuBisCO subunits, but also of
RuBisCO activase (Bohler et al. 2007, 2010, 2013; Pelloux et al. 2001; Brendley
and Pell 1998). It is likely that the enzyme itself is damaged during ozone exposure,
as illustrated by the increase in abundance of a degradation fragment of RuBisCO
in poplar (Bohler et al. 2013). In addition, a number of enzymes of the Calvin
cycle have been shown to be decreasing in abundance in response to ozone (Bohler
et al. 2007, 2010, 2013). The fact that these are mostly redox-regulated enzymes
(RuBisCO activase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphospha-
tase and ribulose-5-phosphokinase) is understandable in the context of oxidative
stress, since the Calvin cycle is activated during the day by the reduction of key
enzymes. In an oxidative environment, these enzymes can become inhibited, which
might also be the cause for lower abundances.
During periods of water deficiency, various observations have been made for
RuBisCO. Parry et al. suggested that the reduction in activity of RuBisCO in
droughted tobacco was due to increases in the inhibition of the enzyme (Parry et al.
2002), while Pelloux et al. (2001) determined that the abundance of both RuBisCO
and RuBisCO activase transcripts and proteins in Aleppo pine hardly changed dur-
ing drought. Nevertheless, Sergeant et al. detected increases in the abundance of the
previously discussed fragment of RuBisCO in droughted oak (Sergeant et al. 2011).

Similar results were found in the combined stress experiment from Bohler et al.,
although it was shown that the increase in fragment abundance was less strong for
drought than for ozone (Bohler et al. 2013).
Not many publications discuss the primary carbon metabolism of plants exposed
to both ozone and drought. Pelloux et al. detected that RuBisCO and RuBisCO acti-
vase abundance were not changed during double stress (Pelloux et al. 2001). Bohler
et al. (2013) reported that in poplar, a very similar set of proteins were differen-
tially abundant in combined stress compared to ozone alone, but that the differences
were less severe. This clearly shows the existence of an interactive effect between
both stresses, but not necessarily a protective effect, since visual and morphological
symptoms were reported to be either similar or cumulative.
On the level of the chloroplast electron transport chain, many subunits of pho-
tosystems and ATPase have been reported to be significantly lower in abundance
after ozone stress (Bohler et al. 2007, 2011) while drought appears to have an effect
only on ATPase (Flexas et al. 2002; Tezara et al. 1999). Furthermore, ozone ap-
pears to induce an early increase in ferredoxin–NADP+–oxidoreductase, indicating
a need for reducing power that is consistent with the appearance of oxidative stress
(Bohler et al. 2013). Nevertheless, this is only transient, since after longer exposure
to ozone levels of ferredoxin–NADP+–oxidoreductase decrease, possibly due to an
overwhelmed system. The consistent reduction in photosystem subunits is most
likely caused by the accumulation of ATP and NADPH in the chloroplast, in conse-
quence of the decrease in Calvin cycle activity. These observations have not been
made in drought; and according to the results of Bohler et al. (2013), the combined
stress causes a very similar response, albeit to a lesser extent.
7.8 Antioxidant Metabolism
One of the main differences between ozone and drought is the induction of oxida-
tive stress, which is predominant during ozone exposure but less characteristic of
drought. Whereas ozone itself fragments into ROS and leads to a strong accumula-
tion (Langebartels et al. 2002; Pellinen 1999), drought response mostly uses ROS as
internally produced signalling molecules (Yao et al. 2013), although severe drought
may lead to photo-oxidative stress as well (Foyer and Noctor 2000). Consequently,
accumulation of ROS is likely to be considerably higher during ozone stress, and
more closely located to chloroplasts in drought. Experiments show an increase in
activity and/or abundance of antioxidant enzymes like peroxidases, catalases and
superoxide dismutases in plants exposed to ozone (Alonso et al. 2001) and of glu-
tathione reductase and superoxide dismutase during drought (Alonso et al. 2001;
Huseynova et al. 2014). Alonso et al. (2001) detected decreases in antioxidant en-
zyme activities in the combined stress compared to ozone or drought separately,
deducing that the cumulative effects of both stressors may overwhelm defence
systems. Similar observations were made by Wellburn et al. (1996). Among anti-
oxidant molecules, ascorbate is particularly important during ozone response. The
apoplast is the first location of ozone attack, and consequently apoplastic ascorbate
and ascorbate peroxidase are the primary defence against ozone (Sanmartin et al.
2003; Luwe et al. 1993). In drought, cytosolic and chloroplastic ascorbate-depen-
dent detoxification is of more importance, but appears to be dependent on species
(Mittler and Zilinskas 1994; Zhang and Kirkham 1996). Nevertheless, Kronfuß
et al. showed that in Norway spruce, total needle ascorbate was increased signifi-
cantly by ozone, while apoplastic ascorbate was increased significantly by drought
and a combined exposure led to a significant increase in both. Combined stress may
therefore increase the reduction potential and improve protection against oxidative
stress (Kronfuß et al. 1998). Reduction potential is considerably dependent on plant
species, and it has been proposed that resistance to ozone is associated with both
ozone flux and reduction potential (Dizengremel et al. 2008). Similarly, the interac-
tive effect of ozone and drought may differ, depending on how much antioxidant
molecules and enzymes are induced by either of the stresses.
7.9 Conclusions
Since tropospheric ozone accumulation and soil drying are caused by similar me-
teorological conditions, both situations are likely to emerge in parallel in nature.
Even though the combination of two stress conditions very often causes cumulative
effects, it was proposed that the stomatal closure induced by drought may be able to
protect plants against the influx, and hence the detrimental effects of ozone. How-
ever, as is commonly the case, observations do not consistently corroborate these
expectations. It appears that the response caused by the combination of both stresses
is determined by many environmental and phenotypical factors.
One of the main relevant factors appears to be the sequence of events. The pri-
mary appearance of ozone is likely to cause disturbances in the reactivity of stoma-
ta. A subsequent drought will cause delayed and limited stomatal closure, allowing
continuous entry of ozone into the plant. Drought preceding ozone, on the other
hand, will cause stomatal closure early in the sequence and cause a natural barrier
against ozone absorption. The individual and combinatorial consequences of ozone
and drought exposure can be affected by a number of additional factors such as spe-
cies, ozone flux and antioxidant capacity, sensitivity to ozone and drought, time of
day and vegetative season.
Only few studies have investigated the effects of combined ozone and drought
exposure on plant metabolism (Bohler et al. 2013; Pelloux et al. 2001). Neither of
them (Bohler et al. 2013; Pelloux et al. 2001) identified any major synergistic or
antagonistic effects. In addition, the use of high throughput molecular approaches
is quite rare for this topic. This is regrettable, since high throughput techniques
like transcriptomics, proteomics and metabolomics present some considerable ad-
vantages versus targeted experiments. Where specific studies rely on prior knowl-
edge and a clearly stated hypothesis, high throughput techniques approach a sub-
ject without any prior bias. This approach may easily lead to new discoveries that
were previously unpredicted and therefore unconfirmed. In addition to the wealth
of information that can be obtained, bioinformatic methods exist that can analyse,
represent and combine high-throughput measurements to an extent where the inter-
pretation becomes highly intuitive.
In nature, plants are often exposed to multiple constraints, but often research is
carried out on a single stressor. This is unavoidable for understanding the response
of plants to any particular stress, but the results apply neither to natural conditions
nor to expectations. Therefore, the study of combinations of constraints that natu-
rally co-occur is of major importance, as is the use of new technologies, to unravel
the response of plants against environmental stresses, so that crops and forests can
be protected and maintained for future generations.
References
Alonso R, Elvira S, Castillo FJ, Gimeno BS. Interactive effects of ozone and drought stress on
pigments and activities of antioxidative enzymes in Pinus halepensis. Plant Cell Environ.
2001;24(9):905–16.
Andreae MO, Crutzen PJ. Atmospheric aerosols: biogeochemical sources and role in atmospheric
chemistry. Science. 1997;276:1052–8.
Ashton TS. The industrial revolution (1760-1830). Oxford:Oxford University Press; 1997.
Biswas DK, Jiang GM. Differential drought-induced modulation of ozone tolerance in winter
wheat species. J Exp Bot. 2011;62(12):4153–62.
Bohler S, Bagard M, Oufir M, Planchon S, Hoffmann L, Jolivet Y, et al. A DIGE analysis of
developing poplar leaves subjected to ozone reveals major changes in carbon metabolism.
Proteomics. 2007;7(10):1584–99.
Bohler S, Sergeant K, Lefèvre I, Jolivet Y, Hoffmann L, Renaut J, et al. Differential impact of
chronic ozone exposure on expanding and fully expanded poplar leaves. Tree Physiol.
2010;30(11):1415–32.
Bohler S, Sergeant K, Hoffmann L, Dizengremel P, Hausman J-F, Renaut J, et al. A difference gel
electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of
ozone exposure on membrane proteins. J Proteome Res. 2011;10(7):3003–11.
Bohler S, Sergeant K, Jolivet Y, Hoffmann L, Hausman J-F, Dizengremel P, et al. A physiological
and proteomic study of poplar leaves during ozone exposure combined with mild drought.
Proteomics. 2013;13:1737–54.
Brendley BW, Pell EJ. Ozone-induced changes in biosynthesis of Rubisco and associated compen-
sation to stress in foliage of hybrid poplar. Tree Physiol. 1998;18(2):81–90.
Broadmeadow M. Ozone and forest trees. New Phytol. 1998;139:123–5.
Ciais P, Reichstein M, Viovy N, Granier A, Ogee J, Allard V, et al. Europe-wide reduction in pri-
mary productivity caused by the heat and drought in 2003. Nature. 2005;437(7058):529–33.
Dizengremel P, Le Thiec D, Bagard M, Jolivet Y. Ozone risk assessment for plants: central role of
metabolism-dependent changes in reducing power. Environ Pollut. 2008;156(1):11–5.
Dumont J, Spicher F, Montpied P, Dizengremel P, Jolivet Y, Le Thiec D. Effects of ozone on sto-
matal responses to environmental parameters (blue light, red light, CO2 and vapour pressure
deficit) in three Populus deltoides × Populus nigra genotypes. Environ Pollut. 2013;173:85–96.
Evers D, Lefèvre I, Legay S, Lamoureux D, Hausman J-F, Rosales ROG, et al. Identification of
drought-responsive compounds in potato through a combined transcriptomic and targeted me-
tabolite approach. J Exp Bot. 2010;61(9):2327–43.
Flexas J, Bota J, Escalona JM, Sampol B, et al. Effects of drought on photosynthesis in grapevines
under field conditions: an evaluation of stomatal and mesophyll limitations. Funct Plant Biol.
2002;29(4):461–71.
Foyer CH, Noctor G. Oxygen processing in photosynthesis: regulation and signaling. New Phytol.
2000;146(3):359–88.
Fuhrer J. Ozone risk for crops and pastures in present and future climates. Naturwissenschaften.
2009;96(2):173–94.
Hoshika Y, Watanabe M, Inada N, Koike T. Ozone-induced stomatal sluggishness develops pro-
gressively in Siebold’s beech ( Fagus crenata). Environ Pollut. 2012;166:152–6.
Hoshika Y, Carriero G, Feng Z, Zhang Y, Paoletti E. Determinants of stomatal sluggishness in
ozone-exposed deciduous tree species. Sci Total Environ. 2014;481:453–8.
Hull J. The second industrial revolution: the history of a concept. Storia Della Storiografia.
1999;36:81–90.
Huseynova IM, Aliyeva DR, Aliyev JA. Subcellular localization and responses of superoxide dis-
mutase isoforms in local wheat varieties subjected to continuous soil drought. Plant Physiol
Biochem. 2014;81:54–60.
Jonson J, Simpson D, Fagerli H, Solberg S. Can we explain the trends in European ozone levels?
Atmos Chem Phys. 2006;6(1):51–66.
Kangasjärvi J, Jaspers P, Kollist H. Signalling and cell death in ozone-exposed plants. Plant Cell
Environ. 2005;28(8):1021–36.
Kreuzwieser J, Gessler A. Global climate change and tree nutrition: influence of water availability.
Tree Physiol. 2010;30:1221–34.
Kronfuß G, Polle A, Tausz M, Havranek WM, Wieser G. Effects of ozone and mild drought stress
on gas exchange, antioxidants and chloroplast pigments in current-year needles of young Nor-
way spruce [Picea abies (L.) Karst.]. Trees. 1998;12(8):482–9.
Langebartels C, Wohlgemuth H, Kschieschan S, Grün S, Sandermann H. Oxidative burst and cell
death in ozone-exposed plants. Plant Physiol Biochem. 2002;40(6–8):567–75.
Le Thiec D, Dixon M, Pierre Garrec J. The effects of slightly elevated ozone concentrations and
mild drought stress on the physiology and growth of Norway spruce, Picea abies (L.) Karst.
and beech, Fagus sylvatica L., in open-top chambers. New Phytol. 1994;128(4):671–8.
Luwe M, Takahama U, Heber U. Role of ascorbate in detoxifying ozone in the apoplast of spinach
( Spinacia oleracea L.) leaves. Plant Physiol. 1993;101(3):969–76.
Marenco A. Evidence of a long-term increase in tropospheric ozone from Pic du Midi data series.
Consequences: positive radiative forcing. J Geophys Res. 1994;99:16617–32.
Matyssek R, Wieser G, Ceulemans R, Rennenberg H, Pretzsch H, Haberer K, et al. Enhanced
ozone strongly reduces carbon sink strength of adult beech ( Fagus sylvatica) - Resume from
the free-air fumigation study at Kranzberg Forest. Environ Pollut. 2010;158(8):2527–32.
Medrano H, Escalona JM, Bota J, Gulias J, Flexas J. Regulation of photosynthesis of C3 plants
in response to progressive drought: stomatal conductance as a reference parameter. Ann Bot.
2002;89(7):895–905.
Mittler R, Zilinskas BA. Regulation of pea cytosolic ascorbate peroxidase and other antioxidant
enzymes during the progression of drought stress and following recovery from drought. Plant
J. 1994;5(3):397–405.
Munn-Bosch S, Alegre L. Die and let live: leaf senescence contributes to plant survival under
drought stress. Funct Plant Biol. 2004;31(3):203–16.
Paoletti E, Grulke NE. Ozone exposure and stomatal sluggishness in different plant physiognomic
classes. Environ Pollut. 2010;158(8):2664–71.
Parry MAJ, Andralojc PJ, Khan S, Lea PJ, Keys AJ. Rubisco activity: effects of drought stress.
Ann Bot. 2002;89(7):833–9.
Pell EJ, Sinn JP, Eckardt R, Vinten Johansen C, Winner WE, Mooney HA. Response of radish to
multiple stresses. New Phytol. 1993;123(1):153–63.
Pellinen R, Palva T, Kangasjärvi J. Subcellular localization of ozone-induced hydrogen peroxide
production in birch ( Betula pendula) leaf cells. Plant J. 1999;20(3):349–56.
Pelloux J, Jolivet Y, Fontaine V, Banvoy J, Dizengremel P. Changes in Rubisco and Rubisco acti-
vase gene expression and polypeptide content in Pinus halepensis M. subjected to ozone and
drought. Plant Cell Environ. 2001;24(1):123–31.
Renaut J, Bohler S, Hausman J-F, Hoffmann L, Sergeant K, Ahsan N, et al. The impact of at-
mospheric composition on plants: a case study of ozone and poplar. Mass Spectrom Rev.
2009;28(3):495–516.
Ribas À, Peñuelas J, Elvira S, Gimeno BS. Contrasting effects of ozone under different water sup-
plies in two Mediterranean tree species. Atmos Environ. 2005;39(4):685–93.
Sanmartin M, Drogoudi P, Lyons T, Pateraki I, Barnes J, Kanellis A. Over-expression of ascorbate
oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox
states and increased sensitivity to ozone. Planta. 2003;216(6):918–28.
Sergeant K, Spieß N, Renaut J, Wilhelm E, Hausman J-F. One dry summer: a leaf proteome study
on the response of oak to drought exposure. J Proteomics. 2011;74(8):1385–95.
Sherwood S, Fu Q. A drier future? Science. 2014;343(6172):737–9.
Shortle WC, Bondietti EA. Timing, magnitude, and impact of acidic deposition on sensitive forest
sites. Water Air Soil Poll. 1992;61(3):253–67.
Showman RE. A comparison of ozone injury to vegetation during moist and drought years. J Air
Waste Manag Assoc. 1991;41(1):63–4.
Skärby L, Ro-Poulsen H, Wellburn FAM, Sheppard LJ. Impact of ozone on forests: a European
perspective. New Phytol. 1998;139:109–22.
Solomon S, Garcia RR, Rowland FS, Wuebbles DJ. On the depletion of Antarctic ozone. Nature.
1986;321(6072):755–8.
Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, et al. editors. IPCC, 2007:
Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the
Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge:
Cambridge University Press; 2007.
Stocker TF, Qin D, Platter G-K, Tignor MMB, Allen SK, Boschung J, et al. editors. IPCC, 2013:
Climate Change 2013: The Physical Science Basis. Working Group I Contribution to the Fifth
Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge: Cam-
bridge University Press; 2014.
Tanaka Y, Sano T, Tamaoki M, Nakajima N, Kondo N, Hasezawa S. Ethylene inhibits abscisic
acid-induced stomatal closure in Arabidopsis. Plant Physiol. 2005;138(4):2337–43.
Tezara W, Mitchell VJ, Driscoll SD, Lawlor DW. Water stress inhibits plant photosynthesis by
decreasing coupling factor and ATP. Nature. 1999;401(6756):914–7.
van Breemen N, Driscoll CT, Mulder J. Acidic deposition and internal proton sources in acidifica-
tion of soils and waters. Nature. 1984;307(5952):599–604.
van Iersel M, Kelder T, Pico A, Hanspers K, Coort S, Conklin B, et al. Presenting and exploring
biological pathways with PathVisio. BMC Bioinformatics. 2008;9(1):399.
Wagg S, Mills G, Hayes F, Wilkinson S, Cooper D, Davies WJ. Reduced soil water availability
did not protect two competing grassland species from the negative effects of increasing back-
ground ozone. Environ Pollut. 2012;165:91–9.
Warren CR, Bleby T, Adams MA. Changes in gas exchange versus leaf solutes as a means to cope
with summer drought in Eucalyptus marginata. Oecologia. 2007;154(1):1–10.
Wellburn FAM, Lau K-K, Milling PMK, Wellburn PMK. Drought and air pollution affect nitrogen
cycling and free radical scavenging in Pinus halepensis (Mill.). J Exp Bot. 1996;47(9):1361–7.
Wilkinson S, Davies WJ. Ozone suppresses soil drying- and abscisic acid (ABA)-induced stomatal
closure via an ethylene-dependent mechanism. Plant Cell Environ. 2009;32(8):949–59.
Wittig VE, Ainsworth EA, Long SP. To what extent do current and projected increases in surface
ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the
last 3 decades of experiments. Plant Cell Environ. 2007;30(9):1150–62.
Yao Y, Liu X, Li Z, Ma X, Rennenberg H, Wang X, et al. Drought-induced H2O2 accumulation in
subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves. Planta.
2013;238(1):217–27.
Zhang J, Kirkham MB. Enzymatic responses of the ascorbate-glutathione cycle to drought in sor-
ghum and sunflower plants. Plant Sci. 1996;113(2):139–47.
Chapter 8
Effect of High Temperature and Water Stress
on Groundnuts Under Field Conditions
Vijaya Gopal Kakani, Timothy R. Wheeler, Peter Q. Craufurd

and Rao C. N. Rachaputi
8.1 Introduction
In the semiarid environments of the world, which contribute to 90 % of global

groundnut production, high temperature and water stress often occur together
(Nix 1975; Kramer 1980). The effects of drought under field situations are well
established in groundnut (e.g. Williams et al. 1986; Nageswara Rao et al. 1988;
Chapman et al. 1993a). Reports of the effects of increased temperature, both air and
soil, in groundnut fields are available in the literature (e.g. Williams et al. 1975b;
Sivakumar et al. 1993). However, high-temperature studies on groundnut growth
and development are confined to controlled environment conditions (e.g. Wheeler
et al. 1997; Prasad et al. 1999a, 1999b, 2000; Craufurd et al. 2003).
High-temperature studies conducted on groundnut by Prasad et al. ( 1998,
1999a, 1999b, 2000) use a high-temperature treatment for a period of 12 h, with
temperature changing as a square wave. Such uniform temperature fluctuations do
not occur in natural environments. Temperatures under field conditions follow a
more sinusoidal pattern, reaching the peak during the afternoons (Fig. 8.1). To con-
firm the findings of studies of high-temperature effects on groundnuts conducted in
a controlled environment, field studies in natural, hot environments are essential.
Studies evaluating the effects of both drought and high temperature in groundnut
V. G. Kakani ()
Department of Plant and Soil Sciences,
Oklahoma State University, Stillwater, OK, USA
e-mail: v.g.kakani@okstate.edu
T. R. Wheeler
Department of Agriculture, University of Reading, Reading, UK
P. Q. Craufurd
Global Conservation Agriculture Program, CIMMYT, Nairobi, Kenya
R. C. N. Rachaputi
Center for Plant Science, University of Queensland, Kingaroy, Australia

160 V. G. Kakani et al.
Fig. 8.1 Diurnal temperature cycle under natural (−) hot environment at ICRISAT, India, and
controlled (−) high temperature treatment (40/22 °C—day/night with 12 h photo-thermoperiod)
have not been conducted so far under field conditions. Such studies under con-
trolled environment did not result in any definite conclusions (Craufurd et al. 1999).
This study evaluated the effects of high-temperature stress on groundnut yield,
and its interaction with drought, under field conditions. The objectives of this study
are: (1) to investigate the effects of water stress and high temperature on growth,
development and yield of groundnut grown in the semiarid tropics; (2) to test the
possible interaction between water stress and high temperature observed under con-
trolled environment, on yield and yield components under field conditions.
8.2 Materials and Methods
8.2.1 Location, Weather and Soil
An experiment to study the interaction between high temperature and water stress
was conducted at two sowing dates, at the International Crops Research Institute
for Semiarid Tropics (ICRISAT), Patancheru, Hyderabad, India. The ICRISAT is
located in semiarid tropics at an altitude of 545 m above sea level (ASL), 17°32’ N
latitude, 78°16’ E longitude.
A mini weather station (Fig. 8.2a) was set up to record daily values of t emperature
and incident solar radiation. Air and soil temperatures were measured using copper–
constantan thermocouples. Air temperatures were measured at canopy level and soil
temperatures at a 10-cm depth (i.e. in the podding zone; Fig. 8.2b). Solar radiation
8 Effect of High Temperature and Water Stress on Groundnuts … 161
Fig. 8.2 Pictures showing a broad bed and furrow system with mini—weather station; b Line
quantum sensor and thermocouples (TC) for measuring air (inside the cup) and soil temperature
(10 cm below soil surface), c Layout of heat tunnels in the field, and d Inside of the high tempera-
ture × irrigation treatment heat tunnel
received above the crop canopy was measured in each treatment using line quantum
sensors (LI-191SB, LI-COR Ltd). Measurements were logged at 10-s intervals and
averaged every 15 min throughout the crop-growth period. Daily weather was also
collected from a meteorological observatory located within 500 m of the experi-
mental site.
The soil at the experimental site was a reddish-brown alfisol, a member of isohy-
perthermic family of Udic Rhodustalf. Soil pH was 6.5. Depth of soil in the site was
120 cm. The soil moisture was 20 % w/w at field capacity and 8 % w/w at permanent
wilting point. These soils are well drained with moderate permeability.
8.2.2 Field Preparation and Experimental Design
The field site was ploughed to a depth of 30 cm with mouldboard and disc ploughs 15
days before sowing. The ploughed field was then laid into broad beds (1.2-m wide)
and furrows (0.3-m wide), in an East–West direction (Fig. 8.2a). The beds were then
levelled and compacted. Four furrows at 30-cm spacing and 5-cm depth were then
opened on the bed surface along the length of the bed. The whole area was then
divided into two halves, one for each sowing. Each sowing composed ten beds of
60 m length. The main irrigation treatments had a bulk bed in between to restrict
water seepage between treatments.
The experiment was planned at two sowing dates to ensure that the crop was
exposed to high temperature during the sensitive period of flowering. Sowing 1 was
on 21st January and sowing 2 was on 26th February. The experimental design was
split–split plot with two irrigation regimes as main plots—irrigated (IR) and fully
irrigated—replacing 100 % of crop evapotranspiration (ETC) and water-stress (WS)
irrigation with only 40 % of ETC from flowering to harvest otherwise fully irrigated;
temperature treatments as subplots—ambient temperature (T1), high-temperature
sowing 1 (T2), ambient temperature sowing 2 (T3) and high-temperature sowing 2
(T4); and genotypes TMV2 and ICGS11 as sub–subplot.
8.2.3 Cultivars and Sowing
TMV 2: This cultivar was released in 1940. TMV 2 is a Spanish botanical type, a
selection from ‘Gudhiatham Bunch’ and a local variety. It is widely adapted, well
suited for rainy and summer season cultivation in southern India. This cultivar is
moderately tolerant to water stress and high temperature.
ICGS 11/ICGV 87213: This cultivar was released in 1986. This cultivar is a Span-
ish botanical type, selection from natural hybrid population of Robut 33-1. It has
above-average tolerance to end-of-season drought. It is also photoperiod insensitive,
adapted to post-rainy season cultivation in India and performs well in West Africa.
Prior to sowing, seeds were treated with fungicide mixture, Thiram + Captan
(3:1). Seeds of cultivars TMV 2 and ICGS 11 were sown manually, 5 cm deep
and 10 cm apart in furrows made at 30 cm spacing on broad beds. An iron chain
with tags at 10 cm spacing was used to ensure that each plot received the required
number of plants. Soon after emergence, gaps were filled for ungerminated seeds.
Appropriate weed, pest and disease control measures were taken to maintain a
healthy crop stand.
8.2.4 Stress Treatments
8.2.4.1 Irrigation
Immediately after sowing, all plots were irrigated using an overhead sprinkler sys-
tem. A second sprinkler irrigation was given after 7 days to help seedlings emerge.
A drip irrigation system was then installed to provide adequate irrigation to the
growing seedlings. The drip irrigation system is shown in Fig. 8.2b and c. The drip
emitters were calibrated so that each supplied 10 L h−1 of water to crop plants. This
ensured that all plants in the plot were supplied with equal amount of water. Plots
were irrigated at 3-day intervals. Fully irrigated plots were replaced with water
equal to that lost through evaporation. Water stress (WS) plots were irrigated with
40 % of that given to fully irrigated plots, from anthesis to harvest. The amount of
water supplied to an irrigated plot ( L) was calculated using
L = Plot area × ETC (8.1)
ETC = Evaporation × K c , (8.2)
where Kc is the crop coefficient with a value of 0.8 for groundnut (Doorenbos and
Pruitt 1992) crop during the reproductive development period. The evaporation data
were obtained from the weather station at ICRISAT, which is given as:
Evaporation = Open − pan evaporation × Kpan (8.3)
Open-pan evaporation was obtained from United States Department of Agriculture

(USDA) class A type pan and Kpan with a value of 0.7 as the pan coefficient. Wa-
ter use efficiency (WUE) was calculated as the ratio of above-ground biomass dry
weight (including pod weight) to the amount of water supplied.
8.2.4.2 Temperature Treatments
Plants were exposed to high temperatures by covering them with plastic tunnels
supported by an iron frame, referred to from now on as ‘heat tunnel’. Plants in the
high-temperature treatment were covered with heat tunnel from flowering to 20
days after flowering (DAF), the most sensitive period for temperature stress (Prasad
et al. 1999a). Temperature inside the heat tunnel was controlled so as not to exceed
42–43 °C by opening and closing the flaps of the heat tunnel. This also ensured that
humidity did not build up in the heat tunnel. The polythene sheet (400 µ thick) used
allowed 80 % transmittance of light for plants in the heat tunnel, and the surface was
cleaned regularly for any settled dust to maintain transmittance levels.
8.2.5 Crop Development
The time of the key reproductive stages (R1, R2, R3 and R8; Boote 1982) were re-
corded in each plot. Observations were made daily on ten plants per plot. The crop
was considered to have reached a particular reproductive stage when 50 % or more
of the plants were at that stage of development.
8.2.6 Growth Analysis
Sampling of plants was done once in the vegetative stage, before flowering, and
at weekly intervals after imposition of water and temperature stress treatments.
An area of 0.6 m2 (0.5 × 1.2 m) from each plot was sampled at each harvest. A
s ubsample of five plants was tagged at flowering in each of the harvest areas. Daily
flower production was recorded on these plants from flower appearance for a period
of 30 days. These plants were also used to determine leaf area and partitioning of
dry matter to leaves, stems, and pods. Observations were also made on plant height,
node and leaf number, peg and pod number on plants of the subsample. To deter-
mine dry weights, plant components of the subsample and the remaining part of the
large sample was oven dried at 80 °C for 3–4 days and weighed. Total dry matter
and pod yields were recorded at harvest maturity in all replications of the experi-
ment using an area of 4 × 1.2 m.
8.2.7 Statistical Analysis
All the data were analyzed using an analysis of variance procedure (ANOVA) for
split–split plot design in Genstat 5 (Genstat 5 Committee, 1997). All percentage val-
ues were angular transformed before analysis to ensure homogeneity of variances.
Pod dry weight values were multiplied by a factor of 1.65 to account for energy
spent to synthesize oil in the seed (Duncan et al. 1978). Statistical significance was
tested by applying F-test at < 0.05, < 0.01 and < 0.001 level of probability, represent-
ed by *, ** and ***, respectively.
8.3 Results
8.3.1 Weather (Temperature and Relative Humidity)
A range of temperatures was imposed during flowering by using two sowing dates
combined with heat tunnels. As photoperiod did not vary much at the experimental
site (mean 12 h ± 45 min), and the genotypes used were insensitive to photoperiod,
results are described in terms of differences in mean temperature between treat-
ments, rather than by sowing dates. Daily maximum and minimum temperatures
recorded during the crop period in all the four treatments are presented in Fig. 8.3.
Temperatures to which different development phases were exposed to in each of the
temperature treatments are given in Table 8.1.
A combination of sowing dates and heat tunnels gave mean temperatures from
sowing to maturity of 26.3° (T1), 27.3° (T2), 29.0°(T3) and 29.7 °C (T4). The heat
tunnels were capable of raising day temperature by > 10 °C compared to ambient
(Fig. 8.3). During the 20-day high-temperature treatment at flowering, mean tem-
peratures were 33.8° (T1), 41.6° (T2), 38.7° (T3) and 43.5 °C (T4). Increase in soil
temperature was also observed with increase in air temperature (Table 8.1). Temper-
ature of the soil was highest in the T4 treatment where air temperature was highest.
Average daily relative humidity (RH) in the ambient treatments T1 (sowing 1) and
T3 (sowing 2) was 48.4 % (SE ± 0.95) and 44.3 (SE ± 0.98), respectively (Fig. 8.3).
Fig. 8.3 Daily maximum and minimum air temperatures recorded under ambient- and high-tem-
perature conditions and relative humidity in a early and b late sown groundnut crop
The calculated vapour pressure deficit (VPD) values were 1.82 and 2.26 kPa in T1
and T3, respectively. It was not possible to record RH in the T2 heat tunnel due to
lack of instruments and therefore VPD could not be estimated in T2. The RH level
in T4 during the 20-day period of high temperature averaged to 57 % (SE ± 1.12),
slightly above that of the ambient T3 treatment. VPD was therefore slightly lower
in T4, 2.06 kPa than in T3 (2.26 kPa).
Due to lower ambient temperature in T1 (sowing 1), heat tunnels for T2 were
kept closed during the greater part of the day to achieve the target temperature
of > 40 °C. This led to a buildup of humidity in the heat tunnel near to saturation,
which must have reduced the VPD. A better control of humidity was achieved in
the T4 heat tunnel treatment (sowing 2), keeping the heat tunnel open to reduce the
maximum temperature which at times was > 48 °C. These very high temperatures
were achieved because ambient temperatures were much higher at the second sow-
ing (> 38 °C).
Table 8.1 Average maximum (Max), minimum (Min) and mean air temperatures (°C), soil
temperatures (°C) and relative humidity (%) recorded during different developmental stages of
groundnut in the four temperature treatments to which the crop was exposed in the field
Devel- Treatments
opmen- T1 T2 T3 T4
tal Stage
Max Min Mean Max Min Mean Max Min Mean Max Min Mean
Max Min Mean Max Min Mean Max Min Mean Max Min Mean
Air temperature (°C)
Sowing– 30.2 14.8 22.5 30.7 15.2 23.0 35.2 18.1 26.7 35.3 18.1 26.7
R1
R1–R3a 33.9 16.5 25.2 41.6 17.4 29.5 38.7 20.3 29.5 43.5 18.05 30.8
R3–R8 37.8 20.4 29.1 38.6 20.3 29.5 38.6 23.7 31.2 38.6 23.7 31.2
Soil temperature (°C)
Sowing– 25.8 25.3 25.5 29.4 26.2 27.8 25.9 25.2 25.5 32.3 25.2 30.8
R8
Relative humidity (%)
Sowing 87.5 30.0 58.7 87.5 30.0 58.7 71.3 22.8 47.0 71.3 22.8 47.0
to R1
R1–R3a 73.3 22.2 47.7 NA NA NA 64.4 22.3 43.3 70.3 44.2 57.1
R3–R8 64.3 23.4 43.8 64.3 23.4 43.8 58.9 26.2 42.5 58.9 26.2 42.5
Developmental stages: R1 = Beginning flower; R3 = Beginning pod; R8 = Harvest maturity
NA not available
a
High temperature period
8.3.2 Analysis of Variance
The ANOVA table (Table 8.2) for 2 × 3 × 2 (WS × Temp × Geno) split–split plot anal-
ysis with three replications at final harvest shows the main effects and interactions
between the treatments. No significant interaction could be recorded at final harvest
for temperature and water stress treatments. However, a significant interaction for
water stress and temperature was recorded for only peg and pod number in the
harvest made immediately after imposing high-temperature treatments (i.e. at 54
DAS). Otherwise, only main effects of temperature and water stress, and their inter-
action with genotypes, could be observed in the various harvests made for growth
analysis in the study. Hence, results recorded only at final harvest are presented.
8.3.3 Water Use and Water Use Efficiency
The cumulative amount of water supplied to IR (100 % of ETC) and water stress
(40 % of ETC) treatments is presented in Table 8.3. No monitoring was possible of
evaporation in the high-temperature treatments T2 (sowing1) and T4 (sowing 2).
Hence, similar amounts were supplied to ambient- (T1 and T3) and high-tempera-
Table 8.2 Analysis of variance with mean square and treatment significance for growth and development parameters recorded at final harvest
Source Df VWT BM PWT FLN PGN PDN HI WUE
Replicate 2 6269 10997 986 173 85 5 0.0007 0.03
WS 1 30514* 94008* 17403 108* 356 156* 0.0114 0.05
Residual 2 891 3803 1259 3 114 5 0.0024 0.008
Temp 2 3768 20915* 16570** 180 375* 3 0.0640*** 0.305*
WS × Temp 2 5276 670 2553 103 214 29 0.0139 0.044
Residual 8 2176 4516 1179 69 86 18 0.0032 0.019
Geno 1 681 21776** 14754*** 2342*** 3589*** 668*** 0.0648*** 0.070*
WS × Geno 1 657 5487 2344** 289* 23 84* 0.0022* 0.007
Temp × Geno 2 564 1572 3448*** 181* 1171*** 175** 0.0225*** 0.002
WS × Temp × Geno 2 1088 2953 457 17 222 5 0.0001 0.0097
Residual 12 833 1554 218 38 93 17 0.0004 0.0094
8 Effect of High Temperature and Water Stress on Groundnuts …
All weights are g m−2 and all numbers are per m2

df degrees of freedom, BM biomass, FLN flower number, PGN peg number, PDN pod number, PWT pod weight, VWT vegetative weight, WUE water use
efficiency
*; **; *** indicate significance at 0.05, 0.01 and 0.001 levels of probability, respectively
167
Table 8.3 Cumulative Development stage TI and T2 IR WS T3 and T4 IR WS

amounts of irrigation
(mm) supplied to irrigated Sowing–R1 121 121 204 204
(IR—100 % of ETC) and R1–R3 98 43 201 89
water stress (WS—40 % of R3–R8 355 183 234 91
ETC) plots during different
stages of development
Table 8.4 Effect of Temperature WUE VPD WUE normalized

temperature treatments treatments for VPD
on WUE (g L−1) and VPD
(kPa) and normalized T1 0.88 1.82 1.6
WUE (WUE × VPD T2 1.21 NA NA
(g kPa L−1) T3 0.58 2.26 1.3
T4 0.64 2.06 1.3
SED 0.055***
*** indicates significance at 0.001 level of probability
ture (T2 and T4) treatments irrespective of irrigation treatment. Amount of irrigation
given was higher in T3 and T4 treatments due to greater ET demand associated with
the increase in ambient temperature in the second sowing.
Sowing date and temperature treatments significantly affected WUE (Table 8.4).
At ambient temperature, WUE was higher in T1 (sowing 1) than T3 (sowing 2), and
this was associated in part with a lower VPD. The highest WUE, 1.21 g m−2 L−1, was
recorded in T2, and both high-temperature treatments, T2 and T4, increased WUE
compared to their respective ambient controls.
WUE was strongly affected by VPD, which was lower at sowing 1 (T1) than
sowing 2 (T3). The normalized values of WUE for T1 and T3 were 1.6 and 1.3 g
kPa L−1, respectively. The higher WUE at sowing 1 was probably due to cooler
mean temperatures (Table 8.1). The higher WUE in T4 compared to T3 is accounted
for by the lower VPD in T4, which in turn is due to the high RH in the heat tunnel.
Although RH was not measured in T2, RH was very high in the heat tunnel, and
the high WUE in T2 is undoubtedly due to a higher RH. Accordingly, T2 has been
excluded from further analysis.
No interaction between these factors could be recorded for WUE. Water stress
treatments did not influence WUE. Main effects of temperature and cultivar were
significantly affected by WUE. Genotype ICGS 11 recorded significantly ( p < 0.01)
higher WUE of 0.74 g m−2 L−1 compared to 0.65 g m−2 L−1 in TMV 2.
8.3.4 Effects of Temperature × Water Stress Interactions
Table 8.5 shows the interaction effects for temperature and water stress treatments.
The effects of temperature and water stress interaction were apparent only in the
Table 8.5 Effects of Water stress treatments Temperature treatments

temperature (mean of
20-day high temperature) T3 (29 °C) T4 (31 °C)
and water stress Peg number
treatments on peg and Irri 15.81 7.96
pod number (plant−1)
WS 6.60 8.09
recorded in the harvest
made immediately after SED 2.35*
the withdrawal of high- Pod number
temperature treatments
Irri 3.72 1.74
WS 1.03 2.84
SED 0.76*
Irri irrigated, WS water stress
* indicates significance at 0.05 level of probability
harvests made immediately after ending the 20-day high-temperature treatment.

This interaction disappeared as the crop reached maturity. The interaction was sig-
nificant (< 0.05) between T3 and T4 for both peg and pod number at 54 DAS. High
temperature imposed in the irrigated treatment (IR) decreased the peg (50 %) or
pod (54 %) number. Water stress (WS) treatmentals reduced peg (68 %) and pod
(72 %) number under ambient temperature conditions. However, a combination of
high temperature (T4) and water stress (WS) increased peg, and in particular pod,
number relative to WS or T4. In general, water stress effects were more severe than
high-temperature effects.
8.3.5 Effects of Water Stress and Its Interaction with Genotypes
Water stress treatments significantly ( p < 0.05) altered the specific leaf area (SLA)
of the plants. There was no water stress x sowing interaction for SLA values, but
within a sowing there were differences between water stress treatments for SLA.
The SLA values recorded in irrigated conditions was lower (180.3 cm−2 g) in sowing
2 compared to the values in sowing 1 (192.2 cm−2 g). Treatment WS (40 % ETC)
increased the SLA in sowing 1 (201.2 cm−2 g), while it decreased the SLA in sowing
2 (163.8 cm−2 g).
Seasonal time course of biomass and pod weight in T1 is shown in Fig. 8.4. There
was no immediate effect on biomass or pod weights of the 20-day high-temperature
period. However, water stress treatment decreased biomass and pod weight through-
out the stress period.
The main effects of water stress were recorded only for biomass due to signifi-
cant ( p < 0.05) reduction in vegetative and pod weight. Vegetative (283.9 g m−2) and
pod weight (120.2 g m−2) in irrigated treatments (100 % ETC) were reduced by 20
and 37 %, respectively, due to water stress treatment (40 % ETC).
Fig. 8.4 Seasonal time course of biomass ( diamond) and pod weight ( circle) recorded in water
stress treatments, Irri (100 % ETC—closed) and WS (40 % ETC—open) in T1 treatment; red
inverted triangle indicates start and end of high-temperature treatment, while blue inverted tri-
angle indicates start of water stress (WS—40 % ETC) treatment
Cultivars differed in their response to water stress treatments (Table 8.6). The in-
teractions persisted until the final harvest. Cultivar ICGS 11 recorded significantly
( p < 0.05) higher values for flower number (40 %), pod number (50 %), pod yield
(37 %) and harvest index (HI; 31 %), than TMV 2 under irrigated conditions (100 %
ETC). When the genotypes were supplied with 40 % ETC, the differences for toler-
ance to water stress were clear between the genotypes. Flower number, biomass,
pod yield and HI decreased by 14, 31, 42 and 14 % in ICGS 11 and by 0, 23, 28 and
4 % in TMV 2, respectively, compared to those obtained in the irrigated treatment.
There was no effect of water stress treatments or its interaction with genotypes on
peg and pod number and pod set.
8.3.6 Effects of Temperature and Its Interaction with Genotypes
Main effects of temperature were significant for biomass (Fig. 8.4). High tempera-
ture decreased biomass in T3 and T4 by 21 and 12 %, respectively, compared to
T1. The smaller decrease in biomass in T4 compared to T3 can be attributed to
lower VPD in T4. Similar trend was also recorded for vegetative weight (data not
presented).
The interaction of temperature treatments with water stress disappeared with
advance in crop age, but temperature interactions with cultivar persisted until the
Table 8.6 Interaction Water stress Cultivar

between genotype and
water stress treatments TMV 2 ICGS 11
for flower number Flower number
(plant−1) at 30 DAA, pod Irri 32 53
number (plant−1), pod
WS 34 44
yield (g m−2) and harvest
index as observed at final SED 2.1*
harvest Pod number
Irri 12 24
WS 11 17
SED 1.5*
Pod yield
Irri 91.9 148.5
WS 66.1 88.4
SED 6.4**
Harvest index
Irri 0.23 0.33
WS 0.21 0.28
SED 0.017*
IR irrigated, WS water stress, SLA specific leaf area.
*, ** indicate significance at 0.05 and 0.01 levels of probability,
respectively
final harvest. A temperature x cultivar interaction was recorded for flower number,
pod number, pod yield and HI (Table 8.7).
Of the two cultivars, ICGS 11 was more tolerant to high temperature. In both
cultivars, a decrease in pod yield and HI was recorded under high-temperature treat-
ments, but the decrease was significantly less in ICGS 11 compared to the decrease
in TMV 2. Cultivar ICGS 11 maintained a high pod yield and high HI under high-
temperature treatments (T3 and T4). On the other hand, a severe decrease in pod
yield and HI were recorded in TMV 2. The higher pod yield and HI in ICGS 11
can be attributed to greater flower fruit-set (i.e. ratio of pod to flower number) and
pod number. In contrast, in TMV 2, reduction in flower number and fruit set was
recorded, and so pod number was decreased on exposure to high temperature.
8.4 Discussion
Studies to identify temperature × water stress interactions (Craufurd et al. 1999) or

to screen genotypes for heat tolerance have been conducted mainly in controlled
environments (Prasad et al. 1999a, 1999b, 2000; Wheeler et al. 1997). Under these
conditions, the temperature increase follows a square-wave pattern (Fig. 8.1).
Table 8.7 Interaction between genotype and temperature treatments for flower number (plant−1)
at 30 DAA, pod number (plant−1), pod yield (g m−2) and harvest index as observed at final harvest
Cultivar Mean temperature treatments (°C) SED
T1(27) T3 (29) T4 (30)
Flower number
TMV 2 35 35 28 4.2*
ICGS 11 42 55 50
Pod number
TMV 2 16 10 10 2.4**
ICGS 11 15 24 22
Pod yield
TMV 2 140.0 51.0 42.8 15.26***
ICGS 11 142.2 103.8 109.4
Harvest index
TMV 2 0.36 0.18 0.14 0.024***
ICGS 11 0.34 0.29 0.28
TMV 2 Spanish botanical type, a selection from ‘Gudhiatham Bunch’ and a local variety
*, **, *** indicate significance at 0.05, 0.01 and 0.001 levels of probability, respectively
SED Standard Error of Difference of Means
Hence, an interaction between temperature and water stress occurs on plant growth
during the entire 12 h of photo-thermo period, providing a longer period for the
interaction to influence the growth and development of the crop plant under study.
However, under field conditions, increase in day temperature follows a more or less
sinusoidal pattern (Fig. 8.1), and high air temperature effects on plant in field occur
for a short duration of only 3–4 h. Furthermore, the temperature of plant canopy can
be higher than that in controlled environment under similar air temperatures due to
associated radiative heating in semiarid tropic (SAT) regions (Guilioni et al. 2000).
Hence, the interaction between the stress events that occur under controlled envi-
ronment might be different from those occurring in the field. If true, this would have
important implications for using controlled environment facilities for screening for
water and temperature stress.
Temperature increase across the treatments, T1 to T4, (Table 8.1) was achieved
by using plastic heat tunnels in the field. Humidity was controlled in these heat
tunnels by opening the heat tunnel doors for brief periods during the day; nonethe-
less, an increase of humidity in these heat tunnels did occur, particularly at sowing
1 (i.e. T2). The normalized WUE values observed in this study were less (1.6–1.3 g
kPa L−1) than recorded by other researchers (e.g. 3.5 g kPa L−1 by Ong et al. 1987;
1.9 g kPa L−1 by Mathews et al. 1988) due to the higher temperatures to which the
crop was exposed. This experiment was conducted during the hot summer season
of India and warmer temperatures would have caused an increase in evaporation
with less water available for transpiration by the plants. Water stress inhibits leaf
expansion and stem elongation through a reduction of relative turgidity (Slatyer
1955; Allen et al. 1976; Vivekanandan and Gunasena 1976), thus altering both leaf
and stem morphology as observed in this study thus causing a reduction in growth,
resulting in lower WUE.
Increase in WUE of the plants in T2 and T4 indicates that less water was lost
from the soil through evaporation due to higher humidity in the heat tunnels. Lee
et al. (1972) recorded that increase in humidity from 50 to 95 % increased the flower
number, peg number and vegetative weight. Similar observations were made in this
study, notably in T2 where the RH was near saturation compared with 48 % under
ambient conditions. The use of heat tunnels resulted in clear temperature differ-
ences across treatments. These heat tunnels can thus be used in the field to screen
groundnut genotypes for high temperature tolerance, as humidity control can be
achieved with experience in using the heat tunnels (T2 vs. T4).
The effects of temperature and water stress on various components of groundnut
as recorded at final harvest are shown in the flow diagram (Fig. 8.5). The field study
also confirms the observations made under controlled environment studies (Kakani
2001) that the interaction for temperature and moisture stress is transient and disap-
pears with release of a stress treatment. The interaction between temperature and
water stress treatments was recorded in the harvests made immediately after the
withdrawal of high-temperature treatment (T4).
The interaction between water and temperature stress was significant only for
peg and pod number. This interaction is due to the sensitivity of the reproductive
processes such as pollen germination and fertilization to high temperature. In a
controlled environment with a maximum temperature of 37 °C for 10 days, a de-
crease in pod number of 43 % was recorded at 50 DAS (Kakani 2001). On the
other hand, in field, a temperature of 43.5 °C was imposed for 20 days that caused a
reduction of only 46 % in pod numbers. This lesser decrease in pod number can be
attributed to the greater tolerance to high temperature of the genotypes used in the
field (ICGS 11 and TMV 2) study compared to those in a controlled environment
(ICGV 86015 and ICG 796). Observations made on membrane thermostability and
cardinal temperatures for pollen germination and tube growth (Kakani et al. 2002)
also show that genotypes tested in field were more tolerant than those tested in a
controlled environment.
The reasons for the existence or disappearance of the interaction can be attrib-
uted to the moisture level at that particular stage of crop growth. In the controlled
environment study, the interaction with high temperature occurred when the mois-
ture content in water stress treatment was 60 % available soil moisture (ASM).
Similar to controlled environment pots, WS plots in field were at 100 % ASM until
the initiation of water stress at 30 DAS. Time was required to bring down the mois-
ture level to 40 %, which can be seen from the trends in biomass and pod yields
(Fig. 8.4). Estimates of soil water content by simple water balance as shown below
in WS × T2 treatment; assuming water loss of ETC from soil, indicate that the water
content of soil at the end of the high-temperature treatment was about 62 % ASM.
It can also be seen that biomass or pod yields in the water stress treatments are
lower than irrigated treatments only after 50 DAS and remain less until the final
harvest. This suggests that the interaction of water stress with high temperature
would also have occurred at a moisture level of 60 % ASM, as observed from the
Fig. 8.5 Summary of the combined high temperature and water stress effects on growth and devel-
opment of groundnut in SAT. ( Thick arrows = main routes for assimilate translocation; Thin black
arrows = routes for minor use of assimilates; broken arrow = information flow; red arrow = tem-
perature effects; blue arrow = water stress effects; red and green arrow = interaction of tempera-
ture and genotype; blue and green arrow = interaction of water stress and genotype; WT weight;
Labile = current and stored assimalte pool). Direction of red/blue arrows opposite to assimilate
route indicates negative effects. Pod number ( PDNO); peg number ( PGNO); flower number
( FLNO); pod weight ( PODWT); root weight ( ROOTWT); stem weight ( STEMWT)
pot studies. Model PNUTGRO was run to simulate the ASM in the irrigated and WS
treatments and presented in Fig. 8.6.
The simulations concur with soil water calculations and both confirm that the
soil moisture was around 60 % ASM at the end of high-temperature treatment. The
ASM averaged to 70 % from sowing to harvest in irrigated plots. In the case of WS
plots, ASM averaged to 40 % during the stress period, even though the plants expe-
rienced a severe stress of around 25 % towards harvest.
Controlled environment and field studies also suggest that when soil moisture is
around or less than 40 % ASM, critical for groundnut (Wright and Nageswara R ao
1994), water stress dominated the stress effects. Water is a reactant or substrate for
many reactions in plant (Kramer and Boyer 1995), and the rate at which these reac-
Fig. 8.6 Simulated values of percentage soil moisture in irrigated (● received 100 % ETC) and
water stress (○ received 40 % of ETC from flowering) treatments in sowing 1 from sowing to
harvest
tions occur is affected by temperature (Johnson and Thornley 1985). Thus, when
water stress goes below 40 % ASM, available substrate is limited, and hence the
role of the temperature is reduced on the reaction rates in the plant. The reduction
in dry weight of groundnut plants on exposure to water stress was due to severe
decrease in the amount of water available for transpiration (Fig. 8.7), as evapora-
tion was constant irrespective of the water stress treatment.
The increase in SLA value in 40 % ETC treatment of sowing 1 could be attrib-
uted to a decrease in biomass causing a decrease in leaf weight but not in leaf area.
On the other hand, decrease in SLA of 40 % ETC treatment in sowing 2 could be
attributed to decrease in both leaf weight and leaf size. To account for a decrease
in SLA, the transpiration efficiency ( TE) and transpiration ( T) values were derived
from the equations of Wright et al. (1996). The values of TE (Table 8.8) are similar
in response irrespective of water stress treatment and sowing date.
The decrease in SLA in 40 % ETC of sowing 2 can be attributed to the severe
reduction in transpiration, which could cause a decrease in leaf size along with a
decrease in leaf weight. The decrease in leaf weight and biomass due to reduced
transpiration can be attributed to reduced CO2 assimilation (Hsiao 1973). Similar
decrease in dry matter due to reduced photosynthesis under water stress conditions
were reported in groundnuts by Hubick et al. (1986).
There were differences in the values for T obtained ( Tsla) from SLA in the above
table using the equations (8.4–8.6) and T values ( Tsim) obtained from the simulations
made using the PNUTGRO model. The total ETC during the crop growth from 30
DAS, when 40 % ETC treatment was initiated, Tcalc and Tsim are depicted in Fig. 8.8.
Fig. 8.7 Simulated values of cumulative soil evaporation ( ES) and transpiration ( T) values in
irrigated—IR (supplied with 100 % ETC) and water stress (supplied with 40 % of ETC from flower-
ing) treatments in sowing 1 from sowing to harvest
Table 8.8 Observed specific leaf area (SLA, cm2 g−1) and vapour pressure deficit (VPD, kPa) in
water stress treatments, carbon isotope discrimination (Δ), transpiration efficiency (TE, g−1 kg−1),
transpiration during stress periods (T, L) derived from SLA values using the equations described
by Wright et al. (1996)
Water stress SLA Δ = 0.03(SLA) + 14 K = 14.4–0.53(Δ) VPD TE = k/ VPD T (mm)
treatment
Sowing 1
IR 192 19.76 3.92 1.82 2.16 208
WS 201 20.04 3.78 1.82 2.08 116
SED 1.14*
Sowing 2
IR 180 19.41 4.11 2.26 1.82 165
WS 164 18.91 4.37 2.26 1.94 99
SED 1.28*
IR irrigated, WS water stress, SLA specific leaf area
* indicates significance at 0.05 level of probability
(8.4)
k = − 0.53 ∆ + 14.4 (Pa)
(8.5)
∆ = 0.03 SLA + 14.0
T = above-ground biomass ( leaf + stem ) / TE ( L )

(8.6)
Fig. 8.8 Amount of water

received by the crop ( ETC)
and cumulative transpira-
tion values derived using
SLA ( Tsla) and simulated by
PNUTGRo model ( Tsim) in
irrigated (100 % ETC) and
water stress (40 % of ETC)
treatments in sowing 1 and
sowing 2 from flowering to
harvest
The Tsim values obtained from the PNUTGRO model are greater than Tsla values.
The greater Tsim values can be attributed to the greater amounts of biomass pre-
dicted by crop model under these conditions. The TE values are lower compared
to those obtained by Wright et al. (1996). This could be due to the high VPD of
1.82–2.26 under field conditions. These values are comparable to those obtained
by Hubick et al. (1986) when groundnut studies were conducted in glasshouse at a
VPD of 2.2. Similar to those reported here were obtained by Azam ali et al. (1989)
at VPD of 2.1 kPa in drying soil; Mathews et al. (1988) at 1.9 kPa in dry season with
occasional irrigation.
The results from this field study clearly show that both temperature and water
stress decrease pod yields in groundnut, but the cultivars used in this study differed
in their responses to temperature and water stress. Temperature moderately reduced
total biomass or vegetative weight (leaf + stem). In contrast, a severe decrease
in pod yield was recorded due to high temperature. However, under water stress
conditions, a greater decrease in biomass and vegetative yield occurred along with
a decrease in pod yield. This provides evidence to suggest that crop plants react
differently to environmental stresses and adopt different strategies to overcome the
stress events occurring at a particular location.
Pod yield decrease under water stress conditions can be attributed to a decreased
source (vegetative weight), and in one cultivar to a slight decrease in partitioning.
Such decrease in vegetative weight has been recorded in many experiments (Wright
et al. 1991; Sarma and Sivakumar 1989, 1990). There exists evidence in literature
for this decrease in pod yield under water stress conditions (Nageswara Rao et al.
1988; Ravindra et al. 1990; Williams et al. 1986). Thus, under water stress condi-
tions, pod yield is source limited. Decrease in partitioning was also recorded in
earlier studies by Greenberg et al. (1992).
Genotypes used in this study differed in their response to temperature treatments.

Genotypes did not differ in their vegetative weight, indicating that source was not
limiting. Thus, processes like photosynthesis or respiration, responsible for source,
are not much altered. In contrast, pod yield was reduced in both the genotypes.
A greater reduction in pod yield of > 70 % occurred in TMV 2, while it was
only around 23 % in ICGS 11. This indicates that ICGS 11 is more tolerant to high
temperature than TMV 2. The greater tolerance of ICGS 11 to high temperature can
be attributed to maintenance of a significantly higher partitioning under increas-
ing temperature conditions. This higher partitioning is due to greater sink strength
in ICGS 11 than in TMV 2. Such genotypic differences for reduction in pod yield
when exposed to high temperature were reported in several independent studies
(Talwar et al. 1999; Prasad et al. 1999a, 2000; Wheeler et al. 1997). In a screening
study conducted in 1991 in Sahelian region of Africa, Ntare et al. (2001) demon-
strated that groundnut genotypes significantly differ in their pod yields in hot envi-
ronments due to the effects on partitioning.
Under water stress conditions, a greater reduction in vegetative weight and pod
yield occurred in ICGS 11 than in TMV 2. Although the reductions were greater
in ICGS 11 under water stress, this genotype had higher vegetative and pod yield
under irrigated conditions. This is due to greater accumulation of assimilates and
higher partitioning of these assimilates to pod yield (Table 8.6). Under water stress,
only a slight decrease in flower number occurred in ICGS 11, which did not sig-
nificantly influence the peg and pod number. No such decrease in flower number
occurred in TMV 2. In addition, the genotype ICGS 11 had a higher WUE when
compared to TMV 2. This allowed the genotype to accumulate greater biomass even
under water stress conditions. Hence, genotype ICGS 11 was tolerant to both high
temperature and water stress conditions over TMV 2.
8.5 Conclusions
It can be inferred from this study that genotypes that are tolerant to water stress are
also tolerant to high temperature under field conditions. Mechanisms that a geno-
type adopts to overcome stresses differ. However, genotypes with the ability to
establish greater biomass and with a significantly greater partitioning of biomass
to pod yield would be suitable for sustaining higher yields in SAT areas with high
temperature and water stress. Genotypes with greater WUE are also more useful
for the SAT. Thus, screening of groundnut genotypes for both temperature and wa-
ter stress tolerance in field conditions are essential before recommending them for
SAT and before using them for further breeding of new genotypes to these stresses.
Controlled environments can be used for screening genotypes to high temperature
for specific processes and experiments under field conditions need to be adopted to
identify the various mechanisms for tolerance involved.
References
Allen LH Jr, Boote KJ, Hammond LC. Peanut stomatal diffusion resistance affected by soil water
and solar radiation. Proc Soil Crop Sci Soc Fla. 1976;35:42–46.
Azam-Ali SN, Simmonds LP, Nageswara Rao RC, Williams JH. Population, growth and water use
of groundnut maintained on stored water. III. Dry matter, water use and light interception. Exp
Agric. 1989;25:77–86.
Boote KJ. Growth stages of peanut ( Arachis hypogaea L.). Peanut Sci. 1982;9:35–40.
Chapman SC, Ludlow MM, Blamley FPC. Effect of drought during early reproductive develop-
ment on the dynamics of yield development of cultivars of groundnut Arachis hypogaea L.
Field Crops Res. 1993a;32:227–42.
Craufurd PQ, wheeler tr, ellis rh, summerfield rj, williams jh. Effect of temperature and water
deficit on water use efficiency, carbon isotope discrimination, and specific leaf area in peanut.
Crop Sci. 1999;39:136–142.
Craufurd PQ, Prasad PVV, Kakani VG, Wheeler TR, Nigam SN. Heat tolerance in groundnut.
Field Crops Res. 2003;80:63–77.
Doorenbos J, Pruitt WO. Crop water requirements. FAO Irrigation and Drainage. Paper, No. 24,
FAO, Rome, 1992, 144 pp.
Duncan WG, McCloud DE, McGraw RL, Boote KJ. Physiological aspects of peanut yield im-
provement. Crop Sci 1978;18:1015–1020.
Genstat 5 Committee. 1997. Genstat 5 Release 3 Reference Manual. Clarendon Press, Oxford, UK.
Greenberg DC, Williams JH, Ndunguru BJ. Differences in yield determining processes of
groundnut Arachis hypogaea L. genotypes in varied drought environments. Ann Appl Biol.
1992;120:557–66.
Guilioni L, Cellier P, Ruget F, Nicoullaud B, Bonhomme R. A model to estimate the temperature
of a maize apex from meteorological data. Agric For Syst. 2000;100:213–30.
Hsiao CT. Plant responses to water stress. Annu Rev Plant Physiol. 1973;24:519–70.
Hubick KT, Farquhar GD, Shorter R. Correlation between water-use efficiency and carbon isotope
discrimination in diverse peanut ( Arachis) germplasm. Aust J Plant Physiol. 1986;13:803–16.
Johnson IR, Thornley JHM. Temperature dependence of plant and crop processes. Ann Bot.
1985;55:1–24.
Kakani VG. Quantifying the effects of high temperature and water stress in groundnut ( Arachis
hypogaea L.). PhD thesis, The University of Reading; 2001.
Kakani VG, Prasad PVV, Craufurd PQ, Wheeler TR. Response of in vitro pollen germination and
pollen tube growth of groundnut ( Arachis hypogaea L.) genotypes to temperature. Plant Cell
Environ. 2002;25:1651–61.
Kramer PJ. Drought, stress, and origin of adaptations. In: Turner NC, Kramer PJ, editors. Adapta-
tion of plants to water and high temperature stress. New York: Wiley; 1980. p. 7–20.
Kramer PJ, Boyer JS. Water relations of plants and soils.London: Academic; 1995. 482 p.
Lee TA, Ketring DL, Powell RD. Flowering and growth response of peanut plants ( Arachis hypo-
gaea L. var. Starr) at two levels of relative humidity. Plant Physiol. 1972;49:190–3.
Mathews RB, Harris D, Nageswara Rao RC, Williams JH, Wadia KDR. The physiological basis
for yield differences between four genotypes of groundnut ( Arachis hypogaea L.) in response
to drought. I. Dry matter production and water use. Exp Agric. 1988;24:191–202.
Nageswara Rao RC, Williams JH, Sivakumar MVK, Wadia KDR. Effect of water deficit at dif-
ferent growth phases of peanut. II. Response to drought during re-flowering phase. Agron J.
1988;80:431–8.
Nix HA. The Australian climate and its effects on grain yield and quality. In: Lazenby A, Matheson
EM, editors. Australian field crops. 1. Wheat and other temperate cereals. Sydney, Angus and
Robertson; 1975. p. 183–226.
Ntare BR, Williams JH, Dougbedji F. Evaluation of groundnut genotypes for heat tolerance under
field conditions in a Sahelian environment using a simple physiological model for yield. J
Agric Sci. 2001;136:81–8.
Ong CK, Simmonds LP, Mathews RB. Response to saturation deficit in a stand of groundnut ( Ara-
chis hypogaea L.). 2. Growth and development. Ann Bot. 1987;59:121–8.
Prasad PVV, Craufurd PQ, Summerfield RJ. Sensitivity of peanut to timing of heat stress during
reproductive development. Crop Sci. 1999a;84:381–6.
Prasad PVV, Craufurd PQ, Summerfield RJ. Fruit number in relation to pollen production and vi-
ability in groundnut exposed to short episodes of heat stress. Ann Bot. 1999b;84:381–6.
Prasad PVV, Craufurd PQ, Summerfield RJ, Wheeler TR. Sensitivity of fruit-set to heat stress in
groundnut. J Exp Bot. 1998;40:30.
Prasad PVV, Craufurd PQ, Summerfield RJ, Wheeler TR. Effects of short episodes of heat stress on
flower production and fruit-set of groundnut ( Arachis hypogaea L.). J Exp Bot. 2000;51:777–
84.
Ravindra V, Nautiyal PC, Joshi YC. Physiological analysis of drought resistance and yield in
groundnut Arachis hypogaea L. Trop Agric. 1990;67:290–6.
Sarma PS, Sivakumar MVK. Response of groundnut to drought stress in different growth phases.
Agric Water Manage. 1989;15:301–10.
Sarma PS, Sivakumar MVK. Evaluation of groundnut response to early moisture stress during the
rainy and the post-rainy seasons. Agric For Meteorol. 1990;49:123–33.
Sivakumar MVK, Maidoukia A, Stern RD. Agroclimatology of West Africa: Niger. Information
bulletin no. 5. Patancheru, India: ICRISAT; 1993.
Slatyer RO. Studies on water relation of crop plants grown under natural rainfall in North Austra-
lia. Aust J Agric Res. 1955;6:365–77.
Talwar HS, Takeda H, Yashima S, Senboku T. Growth and photosynthesis responses of groundnut
genotypes to high temperature. Crop Sci. 1999;39:460–6.
Vivekanandan AS, Gunasena HPM. Lysimetric studies on the effect of soil moisture tension on
the growth and yield of maize Zea mays L. and groundnut Arachis hypogaea L. Beitrage zur
Tropischen Landwirtschaft Veterinaermedizin. 1976;14:269–378.
Wheeler TR, Chatzialoiglou A, Craufurd PQ, Ellis RH, Summerfield RJ. Dry matter partitioning in
groundnut exposed to high temperature stress. Crop Sci. 1997;37:1507–13.
Williams JH, Wilson JH, Bate GC. The growth and development of four groundnut Arachis hypo-
gaea L. cultivars in Rhodesia. Rhod J Agric Res. 1975b;13:131–44.
Williams JH, Rao RCN, Mathews R, Harris D. Responses of groundnut genotypes to drought. In:
Agrometeorology of groundnut. Proceedings of an international symposium, ICRISAT Sahe-
lian Center, 21–26 August 1985; 1986. p. 99–106.
Wright GC, Nageswara Rao RC. Groundnut water relations. In: Smart J, editors. The groundnut
crop: a scientific basis for crop improvement. London, Chapman and Hall; 1994. p. 281–325.
Wright GC, Hubick KT, Farquhar GD. Physiological analysis of peanut cultivar response to timing
and duration of drought stress. Aust J Agric Res. 1991;42:453–70.
Chapter 9
The Response of Plants to Simultaneous Biotic
and Abiotic Stress
Nicky J Atkinson, Ritushree Jain and Peter E Urwin
9.1 Introduction
Plants have developed specific mechanisms that allow them to detect precise envi-
ronmental changes and respond to complex stress conditions, minimising damage
whilst conserving valuable resources for growth and reproduction. Plants activate
a specific and unique stress response when subjected to a combination of multiple
stresses (Atkinson et al. 2013; Suzuki et al. 2014), and consequently the imposition
of single stresses individually may be suboptimal for developing and testing stress-
tolerant plants (Mittler and Blumwald 2010). This is particularly true for signalling
pathways that can act antagonistically such as the combinations of biotic and abiotic
stresses (Anderson et al. 2004; Asselbergh et al. 2008a). There is an urgent need to
understand the nature of multiple stress responses in plants and to create avenues
for developing plants that are resistant to multiple stresses yet maintain high yields.
In this chapter, we consider the effects of biotic and abiotic stresses acting simul-
taneously on plants, with an emphasis on elucidating the molecular mechanisms
involved.
Evidence in the literature from field, laboratory and molecular studies suggests
that plants respond to a specific combination of stresses in a manner distinctly dif-
ferent from the additive response to the individual stresses (Atkinson et al. 2013;
Prasch and Sonnewald 2013; Rasmussen et al. 2013; Rizhsky et al. 2004; Suzuki
et al. 2014; Iyer et al. 2013). Plants must produce an appropriate response to spe-
cific multiple stress conditions, as often the individual stresses may elicit opposing
reactions. For example, heat stress often causes plants to open their stomata in order
to cool the leaves, but under drought conditions this would be disadvantageous as
N. J. Atkinson () · R. Jain · P. E. Urwin

Faculty of Biological Sciences, Centre for Plant Sciences,
University of Leeds, LS2 9JT, UK
e-mail: nickysj@hotmail.com

182 N. J. Atkinson et al.
more water would be lost (Rizhsky et al. 2004). Further, increased transpiration
caused by heat stress could enhance the uptake of salt or heavy metals, heighten-
ing the damage from these factors (Mittler and Blumwald 2010). The cost of plant
defence is likely to be reduced if specific genes have more general roles in different
stress responses, thus explaining the overlap between stress response pathways (As-
selbergh et al. 2008a; Bergelson and Purrington 1996; Herms and Mattson 1992).
This hypothesis is supported by studies showing that certain molecular signalling
pathways (AbuQamar et al. 2009; Dubos et al. 2010; Mengiste et al. 2003; Naru-
saka et al. 2004; Vannini et al. 2006; Zhang et al. 2006).
Plants exposed to a pest or pathogen often show increased susceptibility to an
abiotic stress such as water deficit (Audebert et al. 2000; Cockfield and Potter 1986;
English-Loeb et al. 1997; English-Loeb 1990; Khan and Khan 1996; Smit and Vam-
erali 1998). Conversely, the long-term abiotic stress can weaken defences and cause
enhanced susceptibility to pathogen attack (Amtmann et al. 2008; Goel et al. 2008;
Mittler and Blumwald 2010). The number of reports in the literature that have fo-
cussed on the interaction between biotic and abiotic stresses is growing, but is still
limited: this chapter reviews that literature, with additional in-depth analysis of rice,
an increasingly important crop plant in the study of stress tolerance.
9.2 The Challenge of Simultaneous Biotic and Abiotic

Stresses in Agriculture
Crops in field environments experience a wide range of environmental perturba-

tions during development that could limit their productivity. When plants are grown
under suboptimal environmental conditions, a yield gap is observed and thus the ac-
tual average yield obtained is much lower than the maximum yield potential of the
particular crop (Lobell et al. 2009). The yield gaps for three major cereal crops—
wheat, rice and maize—are 40, 75 and 30 % respectively, in major growing areas
of the world (Fischer et al. 2009). The major factors responsible for the yield gap
in crop species can be classed as: (i) abiotic factors, such as temperature extremes,
insufficient water or minerals or (ii) biotic factors, such as bacterial, viral, fungal or
insect attack (Gaspar et al. 2002). These environmental stresses are responsible for
large-scale crop loss each year and with the predicted climate change, such losses
are expected to increase. Nearly 50 % of crop yield losses each year are comprised
of abiotic stresses (Wang et al. 2003). The predicted climate change, characterised
by an increase in temperature, an increase in concentration of greenhouse gases,
an intensified hydrological cycle and an increase in troposhperic ozone levels, will
have a multifaceted effect on crop growth and productivity. The results from free-air
carbon dioxide (CO2) experiments (FACE) have established that an increase in CO2
levels in the atmosphere will lead to photosynthetic carbon gain, increased nitrogen-
use efficiency and decreased water use in the leaves, but the yield gain in crop spe-
cies will be much smaller than anticipated (Leakey et al. 2009). Also, the change in
hydrological cycle will cause frequent extreme events of floods and storms in coast-
9 The Response of Plants to Simultaneous Biotic and Abiotic Stress 183
al areas accompanied by drought and reduced soil moisture in the drier regions,
resulting in reduced productivity (Schmidhuber and Tubiello 2007). The anticipated
rise in temperature will lead to a shorter life cycle and increased biomass in plants.
Temperature changes outside the typical range during the major growth stages of
crop plants will highly affect the productivity (Moriondo et al. 2011). Currently,
pests and pathogens account for 15 % of the annual crop loss across the globe (Max-
men 2013). The increase in temperature and precipitation will alter the geographic
distribution and host range of various pests and pathogens (Newton et al. 2011). The
predicted changes will leave crop plants vulnerable to a large number of biotic and
abiotic environmental stresses, acting upon them simultaneously.
Traditional molecular studies designed to explore plant stress responses have been
driven by systems that artificially impose one particular stress or exogenous ap-
plication of hormones on model plant species grown in laboratory conditions. The
results of such studies have enhanced our understanding of the signalling cascades
and hormonal pathways that mediate plant responses towards various stresses and
have been used in achieving tolerance to biotic and abiotic stresses. However, the
plants engineered for tolerance to a single biotic or abiotic stress in the laboratory
have repeatedly failed to attain similar results in the fields (Atkinson and Urwin 2012;
Mittler 2006). This is because the crops in the field encounter more than one type of
stress at any given point in time, and with the prophesied climate change model the
incidences of simultaneous biotic and abiotic stresses on plants are bound to increase.
The effect of climate change on plant–pest interactions has been widely re-
viewed in recent years (Chakraborty 2005; Garrett et al. 2006; Gregory et al. 2009;
Luck et al. 2011; Newton et al. 2011; Scherm 2004). The response of plants to
a combination of biotic and abiotic stresses is tailored to the exact nature of the
stresses and there can be additive, negative or interactive effects of each of the
individual responses (Atkinson and Urwin 2012). Evidence suggests that increased
CO2 levels in the atmosphere will lead to suppression of plant defence responses
by the manipulation of the hormonal signalling pathways. Soybean plants show the
down-regulation of jasmonic acid (JA) and ethylene (ET) pathways resulting in the
reduction of cysteine protease inhibitors under increased CO2 levels that in turn re-
duce the plants’ defence against coleopteran pathogens (Zavala et al. 2008). At the
same time, the increased CO2 levels also result in the increased global expression
of salicylic acid (SA) in soybean plants (Casteel et al. 2012). The increased CO2
levels are likely to provide legumes with a photosynthetic advantage and protection
against drought-induced loss in N2 (Rogers et al. 2009). In tomato plants, elevated
CO2 levels have resulted in decreased resistance to the root-knot nematode (RKN)
Meloidogyne incognita (Sun et al. 2010). Apart from elevated levels of CO2, tem-
perature plays an important role in plant–pathogen interactions (Fu et al. 2009; Zhu
et al. 2010). Temperature-dependent resistance is seen towards blast disease in rice,
broomrape in sunflower and clover, downy mildew in musk melon and stripe rust
in wheat (Balass et al. 1993; Eizenberg et al. 2004; Eizenberg et al. 2009; Fu et al.
2009; Webb et al. 2010). An increase in temperature will also lead to more rapid
development, increased reproductive potential and more generations of pests and
pathogens in a season. These changes in pest life cycle and productivity could cause
unprecedented damage to the crops in one season (Scherm 2004).
Drought can aid pest and pathogen outbreaks in fields, at the same time patho-
gens can severely influence plant water relations and lead to low water potential
in plant cells (Mattson and Haack 1987). The bacterium Xylella fastidiosa causes
pathogen-induced drought in grape by severe reduction of water potential (Choi
et al. 2013). In the case of foliar pathogens, stomatal closure is the first physi-
ological barrier in the defence response. Stomatal closure is also a drought avoid-
ance strategy, thus drought-induced stomatal closure reduces pathogen entry into
the plant tissue. Similarly, pathogen-induced stomatal closure helps the plant in
efficient use of water (Sawinski et al. 2013). Drought enhances the symptoms of
fungal charcoal rot disease in common bean (Mayek-Perez et al. 2002), and leads to
reduction in plant water status and in turn increasing concentration of metabolites in
the plant tissue. Increased concentration of defence compounds in drought-stressed
tomato plants results in reduced susceptibility towards the herbivore Spodoptera
exigua (English-Loeb et al. 1997). However, the change in herbivore’s feeding be-
haviour also depends on the nature of the pest and its specificity towards the plant
species (Gutbrodt et al. 2011). Drought stress can influence the interaction between
two pathogens acting on the same plant and vice versa. Root-feeding herbivores can
also enhance resistance against foliar herbivores by abscisic acid (ABA)-mediated
hydraulic changes (Erb et al. 2011). The plant response towards simultaneous in-
festation by a foliar herbivore (aphids), their parasitoids and a root herbivore is also
altered by drought stress (Tariq et al. 2013).
Drought-induced changes in roots can interact or counteract root-specific patho-
gens. In water-dependent agricultural ecosystems, drought can increase the inci-
dence of soil-borne disease, especially plant-parasitic nematodes (PPNs). Drought
and PPN infection are the two biotic and abiotic stresses that are often encountered
simultaneously by rice plants in the fields. Drought can increase susceptibility of
rice to root-knot nematode infection in all ecosystems, especially in aerobic rice
cultivation. Cyst nematodes (CNs) can contribute to the drought-related losses in
rice by causing reduced stomatal conductance and reduced leaf water potential
(Audebert et al. 2000). A study on simultaneous drought and CN infection on Ara-
bidopsis has revealed that under simultaneous biotic and abiotic stress, the plant re-
sponses are dominated by abiotic stress-responsive changes (Atkinson et al. 2013).
An integrated approach should be used to test resistance traits under a range of
stress treatments (Mittler and Blumwald 2010). It is crucial to impose the stresses
simultaneously and treat each set of environmental conditions as an entirely new
stress to truly characterise the response of plants to multiple stresses (Mittler 2006).
9.3 Transcriptomic Studies of Simultaneous Biotic

and Abiotic Stresses
Traditionally, plant molecular responses to multiple stresses have been predicted by

comparing the results from two or more individual transcriptomic studies conducted
independently by exposing plants to a singular stress. The results obtained by these
comparisons identify the genes that might be involved in general stress responses of
a plant, but fail to highlight the genes that might play an important role when plants
are simultaneously exposed to a combination of biotic and abiotic stresses. Evidence

suggests that the response towards a pair of simultaneous biotic and abiotic stress is
not always additive of the responses seen towards these stresses individually. Plants
treat each set of simultaneous stresses as a different environmental condition and
tailor their response specifically to it (Atkinson and Urwin 2012). This may involve
differential regulation of a new set of genes that were not induced or repressed by
any of the stresses individually and vice versa (Mittler 2006). A systematic study
performed in Arabidopsis exploring transcriptomic response to simultaneous appli-
cation of flagellin and change in temperature determines that nearly 49.3 % of the
changes seen as a response to combinatorial stress could not have been predicted
by just studying the response to each of these stresses singly. The number of differ-
entially expressed genes increases with severity and complexity of the combination
of stresses (Rasmussen et al. 2013). When Arabidopsis plants are subjected to virus
infection in combination with drought and/or heat, the transcriptomic responses are
much more severe in the triple stress, followed by simultaneous virus and heat and
then simultaneous virus and drought stress treatment (Prasch and Sonnewald 2013).
By comparing the response of Arabidopsis plants under single, double and triple
stress, down-regulation of primary carbon metabolism was seen as plant’s general
response to stress. The abiotic stresses can significantly influence R-gene-mediated
defence in plants by significantly reducing the expression of defence-related genes
and in turn making plants highly susceptible to pathogen attack (Prasch and Son-
newald 2013). The study identified 11 genes that were differentially regulated in
all stress combinations and 23 genes that were specifically regulated when plants
were subjected to simultaneous heat, drought and virus infestation. When virus-
infected plants were subjected to drought or heat stress, 175 and 309 genes were
differentially regulated, respectively. In some cases, the transcriptomic response
to combinatorial stress can be dominated by one of the stresses. Transcriptomic
investigations of the combined effect of a biotic stress, Aspergillus parasiticus, and
an abiotic stress, drought, in peanut, showed that the response to the combinatorial
stress was more similar to the drought response alone with a very small proportion
of multiple stress-specific responses (Luo et al. 2005). Similar results were seen
in Arabidopsis plants simultaneously exposed to dehydration and infection with
the CN Heterodera schachtii. Ninety-seven percent of the genes differentially ex-
pressed in leaves and roots under multiple stress treatment were also differentially
expressed in drought-only treatment. Only 50 genes were expressed specifically in
response to simultaneous drought and nematode infection (Atkinson et al. 2013).
9.3.1 Case Study: Rice Transcriptomic Responses

to Simultaneous Biotic and Abiotic Stresses
A comprehensive investigation of systemic and local transcriptomic responses of

rice towards drought and nematode stress, in isolation as well as in combination,
was conducted using Affymetrix Rice GeneChip® arrays that provide maximum
coverage of the rice genome, representing 57,381 transcripts from both japonica-
and indica-type cultivars (Jain et al. unpublished). The replicate arrays for drought
and simultaneous drought and nematode stresses cluster in one group, whereas the
control and nematode stress arrays form the other group. The experimental model
was designed to mimic realistic stress conditions encountered by rice plants in the
fields.
The transcriptome response to the application of simultaneous stresses was
dominated by changes also observed in response to drought stress alone (95 %),
with some additional unique transcript changes (5 %). Nearly 10 % (4480) of the
genes on the chip had a twofold expression change at a significant level ( p ≤ 0.05)
in the roots, and a similar level was observed for drought stress. The transcrip-
tomic changes were tissue specific with only 5 % overlap between the roots and the
leaves. A total of 297 genes showed multiple stress-specific regulation. Of these,
75 % were up-regulated genes whilst 25 % were repressed. The changes unique to
simultaneous stress included novel members of gene families such as lipid-transfer
protein genes (LTPLs) and cytochrome P450s, known to be involved in crosstalk
between abiotic and biotic stresses. One of the genes highly induced specifically
under multiple stresses was LTPL 11, a previously uncharacterised member of this
stress-responsive protein family was known to be involved in pathogenesis as well
as abiotic stress response in rice (Atkinson et al. 2013; Vignols et al. 1997). In
Arabidopsis, LTPLs impart SA-mediated response and signal transduction during
fungal and bacterial pathogen attack (Maldonado et al. 2002; Molina and García-
Olmedo 1997). Four cytochrome P450 genes were differentially regulated in re-
sponse to simultaneous stress, two in leaves and two in roots (Jain et al. unpub-
lished). Cytochrome P450s in Arabidopsis mediate crosstalk between the abiotic
and biotic stress-responsive hormone pathways. They are involved in catabolism of
ABA, the major abiotic stress-responsive hormone, deactivation of gibberellic acid
and negative regulation of jasmonate pathway (Koo et al. 2011). The up-regulation
of the α-amylase responsible for the degradation of sucrose and the down-regula-
tion of starch synthase in multiple stressed plants indicate that multiple stresses
significantly modulate carbohydrate metabolism. Drought stress affects α-amylase
in leaves and thus modulates sugar metabolism (Jacobsen et al. 1986). Sucrose is
required for plant growth, and it also acts as a signalling molecule by modulating a
proton–sucrose symporter (Gupta and Kaur 2005).
The simultaneous stress response in rice is characterised by a unique set of genes
that is not differentially regulated when any of the two stresses act individually on
the plant, emphasising that the response to a combination of stresses is not additive
but is interactive of the responses seen under the influence of any of the stresses
singly.
9.4 Hormone Signalling and Master Regulators

in Stress Interaction
Due to the complex interacting nature of plant stress responses, research aimed at
developing stress-tolerant crops is increasingly focusing on the points of crosstalk
between pathways, or master regulators (Denancé et al. 2013; Miller et al. 2010).
Fig. 9.1 The multifaceted role of abscisic acid ( ABA) in plant biotic and abiotic stress responses.
This figure summarises the main interactions of ABA with components of the pathogen defence
pathway. ABA has both a positive and negative effect on various hormones and events involved in
the response to biotic stress, as well as orchestrating the abiotic stress response. Positive regulation
is shown by solid arrows, whilst negative regulation or inhibition is shown by dashed bars. JA
jasmonic acid, SA salicylic acid, SAR systemic acquired resistance
Plant hormones are at the hub of this interaction, in particular ABA (Atkinson and
Urwin 2012; Ton et al. 2009). ABA is central in the fine-tuning of stress responses
and is now considered a global regulator that can control the switch in priority be-
tween the response to biotic or abiotic stress, allowing plants to respond to the most
severe threat (Fig. 9.1; Anderson et al. 2004; Asselbergh et al. 2008a; Mauch-Mani
and Mauch 2005; Ton et al. 2009). This dominant role of ABA may arise from its
involvement in both the biotic and abiotic stress-regulatory networks.
Traditionally, ABA has been connected primarily with the response to abiotic
stress, whilst defence against pathogens and other biotic stresses is determined by
the mutual antagonism between SA, JA and ethylene signalling. New evidence sug-
gests that ABA acts both synergistically and antagonistically with these defence
pathways, with crosstalk at different levels (Asselbergh et al. 2008a; Atkinson and
Urwin 2012; Fujita et al. 2006; Yasuda et al. 2008). Its influence depends on the
timescale of infection and the nature of the pathogen (Ton et al. 2009). In the early
stages of defence against microbial invasion, ABA acts through the SA signalling
pathway as a key strategy to induce stomatal closure and thus reduce infection
(Melotto et al. 2006). After penetration, ABA is necessary for β-amino-butyric acid
(BABA)-induced callose deposition as a defence against fungal pathogens (Ton
and Mauch-Mani 2004), whilst during bacterial infection ABA can block callose
production or indeed has a positive effect, a balance that depends on the external en-
vironmental factors such as light and glucose levels (De Torres-Zabala et al. 2007;
Luna et al. 2011). Induced protection against the bacteria Ralstonia solanacearum
in Arabidopsis is unexpectedly independent of SA, JA and ethylene and is instead
dependent on ABA signalling and synthesis (Feng et al. 2012).
In the later stages of a pathogen infection, the hormones SA, JA and ethylene are
induced by pathogen-associated molecular patterns (PAMPs) to regulate a broad
spectrum of defensive compounds, processes that are generally inhibited by ABA
(Asselbergh et al. 2008b; Ton et al. 2009). Treatment with ABA actually increases
susceptibility to fungal and bacterial pathogens, a phenomenon demonstrated in
Arabidopsis, tomato and potato (Asselbergh et al. 2008b; Audenaert et al. 2002;
Henfling et al. 1980; Mohr and Cahill 2003) and in rice, where ABA treatment has
been shown to cause a reduction in plant defence against the blast fungus Magna-
porthe grisea (Koga et al. 2004). Furthermore, disruption of the ABA signalling
pathway can improve defence against pathogens (Anderson et al. 2004; Asselbergh
et al. 2007; Audenaert et al. 2002; Mohr and Cahill 2003). For example, Arabidop-
sis mutants with impaired ABA biosynthesis or signalling are more resistant to the
necrotrophic fungi Plectosphaerella cucumerina (Sánchez-Vallet et al. 2012). On
the analysis of transcription patterns in these mutants compared to wild-type plants,
it was found that defence genes regulated by SA, JA and ethylene were specifically
down-regulated by the ABA pathway. ABA treatment can repress the SA-mediated
systemic acquired resistance (SAR) pathway in Arabidopsis and tobacco, and in-
hibits the accumulation of important defence compounds such as lignins and phen-
ylpropanoids (Kusajima et al. 2010; Mohr and Cahill 2007; Yasuda et al. 2008).
In contrast, SA is known to obstruct abiotic stress signalling, leading to drought
susceptibility in maize when applied exogenously (Németh et al. 2002). In rice,
resistance to the rice blast fungus M. grisea is mediated by the balance between
ABA and SA (Jiang et al. 2010). ABA also antagonises JA and ethylene defence
signalling through the repression of defence genes such as PDF1.2 (Anderson et al.
2004), although JA production can contribute positively to tolerance against cer-
tain abiotic stresses such as chilling, salt, drought and osmotic stress (Santino et al.
2013).
This close association of ABA with defence signalling pathways may allow a
subtle shift in environmental conditions to cause a dramatic difference in stress
response, as any increase in ABA due to abiotic stress could repress the SA, JA and
ethylene defence responses. As abiotic stress conditions such as drought tend to be
a much greater threat to survival than biotic stresses, this would then allow plants to
prioritise the response to the more urgent stress.
The fine-tuning in the regulation of stress responses by ABA may be partially

controlled by the diversity amongst downstream signalling elements (Lee and Luan
2012). There are 14 members of the PYR/PYL/RCAR ABA receptor family, which
in turn activate 6–9 members of the A-type PP2C phosphatases and at least 3 mem-
bers of the SnRK2 kinases, known to carry out downstream protein phosphorylation
and dephosphorylation events (Lee and Luan 2012; Ma et al. 2009; Wasilewska
et al. 2008). Between them, these provide more than 200 signalling combinations
that may activate similar or different downstream targets. These molecular compo-
nents of the ABA signalling pathway may additionally provide opportunities for
genetic engineering of stress tolerance in crop plants.
Points of crossover between hormone signalling pathways include several influ-
ential TFs, such as MYC2. This is activated by ABA (Abe et al. 2003), is a posi-
tive regulator of JA-responsive defence genes (Anderson et al. 2004; Pieterse et al.
2009), and in addition represses the SA pathway (Laurie-Berry et al. 2006). Mem-
bers of the MYB and NAC TF family are also crucial controlling factors in multiple
stress responses, and have been fully reviewed recently (Atkinson and Urwin 2012).
Large multi-protein mediator complexes may function to integrate downstream
stress response signals from multiple sources (Balderas-Hernández et al. 2013).
These are central components of transcription complexes in eukaryotes, which in-
teract with ribonucleic acid (RNA) PolII and promote the assembly of TFs on pro-
moter sequences (Bourbon 2008). In Arabidopsis, mediator is made up of at least 27
subunits, one of which is Med25, encoded by the phytochrome and flowering time
1 ( PTF1) gene. It regulates a multitude of signalling pathways by interacting with
TFs central to the ABA and JA/ethylene cascades, such as MYC2 and ABA insensi-
tive 5 (ABI5) which transcriptionally activates ABA-responsive genes (Balderas-
Hernández et al. 2013).
Heat shock factors (HSFs) have also been identified as potential master regula-
tors of the response to multiple stresses (Atkinson and Urwin 2012). These are TFs
that act as molecular sensors of cellular stress-responsive reactive oxygen species
(ROS) and induce the expression of heat shock proteins (Miller and Mittler 2006).
As different stresses elicit different combinations of HSFs, they may contribute
to the fine-tuning of stress response outcomes (Rizhsky et al. 2004; von Koskull-
Döring et al. 2007; Yoshida et al. 2011). Recently, HSFA1b has attracted attention
as a target for engineering stress tolerance in crops. Post-transcriptionally regulated
during stress conditions, HSFA1b itself regulates 509 genes. When over-expressed
in Arabidopsis it confers dehydration tolerance, resistance to bacterial pathogens
and oomycetes, and improved seed yield under water-limited conditions. (Bechtold
et al. 2013). In oilseed rape, its over-expression led to improved productivity char-
acterised by an increased harvest index and seed yield. This is of particular interest
given that many stress-tolerant Arabidopsis mutants over-expressing the ABA or
SA signalling pathways show a diminished fecundity (Bechtold et al. 2013; van
Hulten et al. 2006). Clearly to attain impact in the development of broad-spectrum
stress-tolerant crop plants, improved disease and abiotic stress responses must go
hand in hand with the maintenance of growth and yield characteristics.
9.5 Interaction of Volatile Compounds

in Simultaneous Biotic and Abiotic Stresses
Plants interact with each other by emitting a unique blend of volatile organic com-
pounds (VOCs). The intensity and chemical composition of VOCs emitted by a
plant can define the physiological state of a plant and is an indication of the na-
ture of the stress acting upon them. The ratio of various compounds in the volatile
blend can hint to herbivorous insects or parasitic plants about the location of their
potential host (Runyon et al. 2006; Tumlinson 2014). Some of the VOCs are spe-
cific to certain plant species. For example isothoicynates, volatile catabolites of
the glucosinolates, are characteristic of the brassicaceous plants. Specialist brassica
pests like the cabbage aphid Brevicoryne brassicae and the cabbage seed weevil
Ceutorhynchus assimilis use isothiocyanates for host location (Bruce et al. 2005).
However, as plants in nature may suffer from more than one stress at a time, it can
be hypothesised that the multiple stresses will have a VOC signature different to
any of the stresses acting individually on the plants (Blande et al. 2014). Abiotic
stresses like heat, water stress, high-intensity light, ozone and salt stress lead to
increased emission of volatile compounds including isoprene, monoterpenes and
sesquiterpenes (Holopainen and Gershenzon 2010; Loreto and Schnitzler 2010).
The emission under a biotic stress is dominated by terpenes and green leaf volatiles
(GLVs), C6 aldehydes, alcohols and esters of lipoxygenase cleavage of fatty acids
(Holopainen and Gershenzon 2010). Two different stresses, two biotic or two abi-
otic stresses, are capable of initiating emissions of similar types of compounds that
might suggest an underlying common signature for the biotic and abiotic stresses. In
lima beans, exposure to ozone and spider mite infestation triggered the emission of
( E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and ( E, E)-4,8,12-trimethyl-1,3,7,11-
tridecatetraene (TMTT; Vuorinen et al. 2004).
Similar to the molecular and physiological effects, simultaneous application of
a biotic and an abiotic stress can have additive or opposing effects on the VOCs
emission. Additive effects can result in an increase in emitted VOCs and also can
increase susceptibility towards other stresses. Simultaneous exposure to ozone and
infection with spider mites in lima beans gave a 31 % increase in the emission of
VOCs compared to plants exposed to single stress and also made plants more sus-
ceptible to secondary herbivore attack by predatory mites. In behavioural assays,
the predatory mites preferred plants under dual stress over the plants that were just
exposed to high levels of ozone. This preference was a result of increased ratio of
( E)-β-ocimene in the emission blend of dual stressed plants (Vuorinen et al. 2004).
An additive effect on emitted VOCs was also observed in the deciduous tree Alnus
glutinosa during drought stress and simultaneous infection with the larvae of green
alder sawflies. Concurrent application of the two stresses increased the emission
of GLVs, monoterpenes and the markers of herbivory, ( E)-β-ocimene and methyl
jasmonate (Copolovici et al. 2014). The mild drought stress before larval attack in
this case showed a priming effect and made plants less susceptible to herbivory, in
contrast to the effect seen in lima beans under simultaneous ozone exposure and
spider mite attack. Perhaps the ozone dose used was insufficient to initiate a prim-
ing effect similar to drought stress. Brassica napus (oilseed rape) plants subjected
to herbivory under elevated levels of ozone or CO2 show contrasting interactions
between the biotic and the two abiotic stresses. Terpenoid emission was increased
in plants under elevated CO2 and subjected to herbivory, but reduced in the elevated
ozone and herbivory group. However, under both stress combinations plants be-
came susceptible to herbivory as determined by olfactory tube assays (Himanen
et al. 2009).
A detailed study to elucidate the effect of simultaneous biotic and abiotic stresses
in maize plants was conducted using inoculation of caterpillar regurgitant in com-
bination with changes in soil humidity, air humidity, temperature, light and mineral
dosage. The amount and the composition of the VOCs emitted by the maize plants
did not change with the abiotic conditions, but on simultaneous induction of bi-
otic stress there was an increase in the VOCs emission under all stresses except
the change in soil humidity. The composition of the emission blend also changed
with simultaneous application of biotic and abiotic stresses. Table 9.1 gives a de-
tailed overview of changes in VOCs under pairs of simultaneous biotic and abiotic
stresses in different species. In most cases, simultaneous stresses change the com-
position and increase the amount of VOCs emitted by a plant, depending on the
nature of the stresses applied. The VOCs emitted by stressed plants play a vital role
in plant–pathogen interaction. A better understanding of VOCs emission under mul-
tiple stresses may be valuable for managing insect pests of crop species.
9.6 Points of Convergence Between Biotic and Abiotic

Stress Signalling Pathways
Biotic and abiotic stress signal transduction is characterised by a complex arrange-

ment of interacting factors. Certain gene products are now known to be central to
both biotic and abiotic stress signalling, and may therefore control the specificity of
the response to multiple stresses (Fujita et al. 2006; Mauch-Mani and Mauch 2005).
Transcriptomic and genetic analyses have highlighted a number of putative candi-
dates that might act as points of convergence, including TFs, map kinases, HSFs,
ROS and small RNAs, and these discoveries have been fully reviewed recently
(Atkinson and Urwin 2012).
9.6.1 Rice as a Case Study
As one of the most important crop plants worldwide and a model monocotyledon,
rice is increasingly becoming a focus for applied plant stress research in the field
and laboratory. Discoveries of key stress response genes in rice will provide direct
opportunities for translational work to improve stress tolerance in cereal crops. Key
Table 9.1 Impact of simultaneous biotic and abiotic stresses on the emission of volatile compounds
192
Plant species
Biotic stress
Abiotic stress
Total VOCs in dual stress
( Z)-3-hexen-1-yl acetate
β-myrcene
( E, E)-α-farnesene
( E)-β-farnesene
linalool
DMNT
Indole
α-bergamontene
TMTT
Geranyl acetate
( E)-nerolidol
β-caryophyllene
( E)-β-ocimene
Methyl salicylate
1-penten-3-ol
( Z)-Hexen-1-ol
( E)-2-Hexenal
( E)-3-Hexenal
LOX products
α-Thujene
α-Pinene
Sabinene
Limonene
β-Elements
Β-sesquiphellandrene
Zea SL Increased ↓ ↑ n.s n.s n.s ↑ ↕ ↕ n.s n.s ↕ n.s ↕ – – – – – – – – – – – – –

mays soil humid-
ity (20 to
60–80 %)
Zea SL Increased ↑ n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s n.s – – – – – – – – – – – – –
mays air humid-
ity (20 to
45–65 %)
Zea SL Increased ↑ ↑ n.s n.s n.s n.s n.s n.s n.s ↑ n.s ↑ ↑ – – – – – – – – – – – – –
mays temperature
(17–37 °C)
Zea SL High light ↑ ↓ n.s ↓ n.s ↑ n.s ↑ n.s ↑ ↑ ↑ ↑ – – – – – – – – – – – – –
mays intensity (0
to 15,000–
20,000 lm/
m2)
Zea SL Reduced ↓ n.s n.s ↓ n.s ↕ ↓ ↓ n.s ↓ ↓ ↓ ↓ – – – – – – – – – – – – –
mays fertilisation
Zea SF Increased ↑ ↑ – – ↑ n.s n.s n.s ↑ n.s – n.s n.s – – – ↑ ↑ n.s – – – – – – ↑
mays copper
N. J. Atkinson et al.
(0–80 µM)
Alnus MP Drought ↑ n.s – – – – ↑ – – – – – – ↑ ↑ – – – – ↑ – – – – ↑ –
gluti-
nosa
Plant species
Biotic stress
Abiotic stress
Total VOCs in dual stress
( Z)-3-hexen-1-yl acetate
β-myrcene
( E, E)-α-farnesene
( E)-β-farnesene
linalool
DMNT
Indole
α-bergamontene
TMTT
Geranyl acetate
( E)-nerolidol
β-caryophyllene
( E)-β-ocimene
Methyl salicylate
1-penten-3-ol
( Z)-Hexen-1-ol
( E)-2-Hexenal
( E)-3-Hexenal
LOX products
α-Thujene
α-Pinene
Sabinene
Limonene
β-Elements
Β-sesquiphellandrene
Bras- PX Elevated O3 n.s – n.s – – – n.s – – – – – – – – – – – – – n.s n.s n.s n.s n.s –
sica (100 nL/L)
napus
Bras- PX Elevated ↑ – ↑ – – – ↑ – – – – – – – – – – – – – ↑ ↑ ↑ ↑ ↑ –
sica CO2
napus (750 µL/L)
Phase- TU Elevated ↑ n.s – – – – ↑ – – n.s – – – ↑ – – – – – – – – – – – –
olus O3 (150–
lunatus 200 nL/L)
Plant pathogens: SL Spodoptera littorali, SF Spodoptera frugiperda, MP Monsoma pulveratum, PX Plutella xylostella, TU Tetranychus urticae
n.s. not significant, – not determined in particular study, ↕ no regular pattern but fluctuates significantly with the stresses, ↑ significant increase, ↓ significant
9 The Response of Plants to Simultaneous Biotic and Abiotic Stress
decrease, DMNT ( E)-4,8-dimethyl-1,3,7-nonatriene, TMTT ( E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene

193
components of the pathways in rice responding to multiple environmental stresses

have already been elucidated. Members of the WRKY family of TFs are responsive
to both biotic and abiotic stresses and play a vital role in fine-tuning plants’ response
to simultaneous stress. In rice, WRKY13 antagonistically regulates the response to
drought and bacterial disease by selectively binding to the cis-acting elements and
specific sequences in the promoters of SNAC1 and WRKY45–1. It can also auto-
regulate its own expression by binding to its promoter (Xiao et al. 2013). WRKY45
imparts resistance against the fungal and bacterial pathogens in rice by differential
mechanisms (Shimono et al. 2012). The WRKY45-1 allele negatively regulates ABA
signalling and also increases plant susceptibility to bacterial pathogens, whilst the
WRKY45-2 allele positively regulates ABA signalling and increases resistance to
bacterial pathogens (Tao et al. 2011). Both alleles positively regulate resistance to
fungal blast disease (Tao et al. 2009). WRKY76 transcription repressor plays op-
posite role in response to rice blast disease and cold stress; over-expression of the
WRKY76 results in increased susceptibility towards blast infection but increases
tolerance to cold stress (Yokotani et al. 2013a). WRKY82 enhances defence against
biotic pathogens and tolerance against abiotic stress via the JA/ET pathways (Peng
et al. 2011).
Several disease-resistant cultivars have different natural expression levels of
OsMYB4 leading to varying degrees of resistance to sheath blight and leaf blight
diseases in rice (Singh et al. 2013). Ectopic expression of the rice OsMYB4 TF
enhances abiotic and biotic stress tolerance in many plants including Arabidopsis,
tomato and apple (Pasquali et al. 2008; Vannini et al. 2006, 2007). The JA-induced
MYB gene, JAmyb, is induced by high salinity, osmotic stress and ROS and its over-
expression results in induction of JA-induced TFs that play an important role in
biotic stress response (Yokotani et al. 2013b).
The OsNAC6 gene acts as a transcription inducer for biotic and abiotic stress
responses in rice. Constitutive over-expression of OsNAC6 results in increased tol-
erance to dehydration and salt stress along with greater resistance to blast disease,
but with growth and yield penalty (Nakashima et al. 2007). OsNAC5 also enhanc-
es abiotic stress tolerance in rice and is responsive to JA, but does not cause any
negative effect on plant growth (Takasaki et al. 2010). A plant-specific TF family,
ethylene-responsive factor TFs, bind to the GCC sequence specifically found in the
PR genes. These TFs are mainly involved in abiotic stress responses in plants. Four
ethylene-responsive genes, BIERF1-4, are up-regulated by salt, drought, wounding
and fungal pathogens (Cao et al. 2006).
In addition to TFs, various protein kinases (PKs) also act as the convergence
points in biotic and abiotic stress pathways in rice. Out of the 17 known rice
MAPK genes, five are induced by both biotic and abiotic stresses (Rohila and
Yang 2007). OsMAPK5 is the most studied rice MAPK; it confers ABA-mediated
tolerance to abiotic stress and resistance to brown spot, whilst negatively regulat-
ing the response to rice blast fungus (Sharma et al. 2013). Members of the rice
CDPK family are also involved in crosstalk between biotic and abiotic stresses.
OsCDPK12 regulates genes involved in ROS scavenging in stressed plant cells
resulting in reduced accumulation of H2O2. The over-expression of OsCDPK12

leads to positive regulation of salt tolerance and negative regulation of blast resis-
tance (Asano et al. 2012). OsCDPK13 is involved in the gibberellic acid-mediated
response in rice leaf sheath and cold tolerance (Abbasi et al. 2004). Four CIPK
PKs ( OsCIPK 2, OsCIPK 10, OsCIPK 11 and OsCIPK 14) also play important
roles in the crosstalk between biotic and abiotic stresses (Chen et al. 2011). Anoth-
er family of PKs, known as dual specificity PKs ( OsDPK), also shows response
to biotic and abiotic stresses. OsDPK1, OsDPK2 and OsDPK3 are all induced by
exogenous application of ABA, drought, salinity and in response to the rice blast
fungus (Gu et al. 2005). Involvement of these rice gene families in biotic as well
as abiotic stress responses presents them as candidates for transgenic improvement
of multiple stress tolerance.
9.7 Future Perspectives
Studies describing the effects of individual and combinatorial stresses have facili-
tated an initial understanding of the molecular interactions controlling plant stress
responses. Plants respond to the exact set of conditions they encounter by activat-
ing both specific and non-specific stress responses. Signal specificity is achieved
through the precise interplay between components of each pathway, particularly the
hormones ABA, SA and JA, TFs, HSFs, ROS and small RNAs. In the past, individ-
ual plant stress factors, which trigger linear signalling pathways, have been studied
in isolation. It seems that this model is no longer sufficient, as both biotic and abi-
otic stress pathways are inextricably linked in a network of molecular interactions.
The development of new crop varieties will depend on understanding crucial
stress-regulatory networks and the potential effects of different combinations of ad-
verse conditions. Studies of multiple stress responses in the model plants Arabidop-
sis and rice, as well as work on other species, have greatly increased our knowledge.
Plant efficiency in sensing and responding to each unique set of environmental
conditions means that different methods of imposing stress can lead to drastically
different transcriptional profiles (Bray 2004). Commonalities between biotic and
abiotic signalling pathways that have been identified may lead to their antagonistic
nature. Nodes that act in both biotic and abiotic stress response systems are excel-
lent candidates for manipulating stress tolerance (Baena-González and Sheen 2008;
Miller et al. 2010). To provide a model for crop stress responses, an integrated ap-
proach should be adopted, whereby future experiments are carried out in conditions
that reproduce natural or field conditions as accurately as possible (Deyholos 2010;
Mittler and Blumwald 2010; Suzuki et al. 2014).
The impacts of climate change pose further challenges for plant breeding and
biotechnology. Crops must be developed that can cope with multiple concurrent
stresses whilst still fulfilling their genetic potential to provide maximum yields and
thus ensure future global food security.
References
Abbasi F, Onodera H, Toki S, Tanaka H, Komatsu S. OsCDPK13, a calcium-dependent protein

kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol Biol.
2004;55(4):541–52.
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2
(bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.
Plant Cell Online. 2003;15(1):63–78.
AbuQamar S, Luo H, Laluk K, Mickelbart MV, Mengiste T. Crosstalk between biotic and
abiotic stress responses in tomato is mediated by the AIM1 transcription factor. Plant J.
2009;58(2):347–60.
resistance in plants. Physiol Plant. 2008;133(4):682–91.
Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ, Ehlert C, et al. An-
tagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways
modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell Online.
2004;16(12):3460–79.
Asano T, Hayashi N, Kobayashi M, Aoki N, Miyao A, Mitsuhara I, et al. A rice calcium-dependent
protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resis-
tance. Plant J. 2012;69(1):26–36.
Asselbergh B, Curvers K, França SC, Audenaert K, Vuylsteke M, Van Breusegem F, et al. Resis-
tance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely
production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol.
2007;144(4):1863–77.
Asselbergh B, De Vleesschauwer D, Höfte M. Global switches and fine-tuning-ABA modulates
plant pathogen defense. Mol Plant-Microbe Interact. 2008a;21(6):709–19.
Asselbergh B, Achuo AE, Höfte M, Van Gijsegem F. Abscisic acid deficiency leads to rapid activa-
tion of tomato defence responses upon infection with Erwinia chrysanthemi. Mol Plant Pathol.
2008b;9(1):11–24.
Atkinson NJ, Urwin PE. The interaction of plant biotic and abiotic stresses: from genes to the field.
J Exp Bot. 2012;63(10):3523–43.
to simultaneous biotic and abiotic stresses. Plant Physiol. 2013;162(4):2028–41.
Audebert A, Coyne D, Dingkuhn M, Plowright R. The influence of cyst nematodes ( Heterodera
sacchari) and drought on water relations and growth of upland rice in Côte d’Ivoire. Plant Soil.
2000;220(1–2):235–42.
Audenaert K, De Meyer GB, Höfte MM. Abscisic acid determines basal susceptibility of tomato to
Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms. Plant Physiol.
2002;128(2):491–501.
Baena-González E, Sheen J. Convergent energy and stress signaling. Trends Plant Sci.
2008;13(9):474–82.
Balass M, Cohen Y, Bar-Joseph M. Temperature-dependent resistance to downy mildew in musk-
melon: structural responses. Physiol Mol Plant Pathol. 1993;43(1):11–20.
Balderas-Hernández VE, Alvarado-Rodríguez M, Fraire-Velázquez S. Conserved versatile master
regulators in signalling pathways in response to stress in plants. AoB Plants. 2013;5:plt033.
Bechtold U, Albihlal WS, Lawson T, Fryer MJ, Sparrow PA, Richard F, et al. Arabidopsis HEAT
SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resis-
tance to drought, and infection. J Exp Bot. 2013;64(11):3467–81.
Bergelson J, Purrington CB. Surveying patterns in the cost of resistance in plants. Am Nat.
1996;148:536–58.
Blande JD, Holopainen JK, Niinemets Ü. Plant volatiles in polluted atmospheres: stress responses
and signal degradation. Plant Cell Environ. 2014:37:1892–904.
Bourbon H-M. Comparative genomics supports a deep evolutionary origin for the large, four-
module transcriptional mediator complex. Nucleic Acids Res. 2008;36(12):3993–4008.
Bray EA. Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. J Exp Bot.
2004;55(407):2331–41.
Bruce TJA, Wadhams LJ, Woodcock CM. Insect host location: a volatile situation. Trends Plant
Sci. 2005;10(6):269–74.
Cao Y, Song F, Goodman RM, Zheng Z. Molecular characterization of four rice genes encoding
ethylene-responsive transcriptional factors and their expressions in response to biotic and abi-
otic stress. J Plant Physiol. 2006;163(11):1167–78.
Casteel CL, Segal LM, Niziolek OK, Berenbaum MR, Delucia EH. Elevated carbon dioxide in-
creases salicylic acid in Glycine max. Environ Entomol. 2012;41(6):1435–42.
Chakraborty S. Potential impact of climate change on plant-pathogen interactions. Australas Plant
Pathol. 2005;34(4):443–8.
Chen X-f, Gu Z-m, Liu F, Ma B-j, Zhang H-S. Molecular analysis of rice CIPKs involved in both
biotic and abiotic stress responses. Rice Sci. 2011;18(1):1–9.
Choi H-K, Iandolino A, da Silva FG, Cook DR. Water deficit modulates the response of Vitis
vinifera to the Pierce’s disease pathogen Xylella fastidiosa. Mol Plant Microbe Interact.
2013;26(6):643–57.
Cockfield S, Potter D. Interaction of euonymus scale (Homoptera: Diaspididae) feeding dam-
age and severe water stress on leaf abscission and growth of Euonymus fortunei. Oecologia.
1986;71(1):41–6.
Copolovici L, Kännaste A, Remmel T, Niinemets Ü. Volatile organic compound emissions
from Alnus glutinosa under interacting drought and herbivory stresses. Environ Exp Bot.
2014;100:55–63.
De Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW, Rodriguez Egea P,
et al. Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling path-
way to cause disease. EMBO J. 2007;26(5):1434–43.
Denancé N, Sánchez-Vallet A, Goffner D, Molina A. Disease resistance or growth: the role of plant
hormones in balancing immune responses and fitness costs. Front Plant Sci. 2013;24:4.
Deyholos MK. Making the most of drought and salinity transcriptomics. Plant Cell Environ.
2010;33(4):648–54.
Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L. MYB transcription factors
in Arabidopsis. Trends Plant Sci. 2010;15(10):573–81.
Eizenberg H, Colquhoun J, Mallory-Smith CA. The relationship between temperature and small
broomrape ( Orobanche minor) parasitism in red clover ( Trifolium pratense). Weed Biol Ecol.
2004;52(5);735–741.
Eizenberg H, Hershenhorn J, Plakhine D, Kleifeld Y, Shtienberg D, Rubin B. Effect of tempera-
ture on susceptibility of sunflower varieties to sunflower broomrape ( Orobanche cumana) and
Egyptian broomrape ( Orobanche aegyptiaca). Weed Sci. 2009;51(3):279–86.
English-Loeb GM. Plant drought stress and outbreaks of spider mites: a field test. Ecology.
1990;71:1401–11.
English-Loeb G, Stout MJ, Duffey SS. Drought stress in tomatoes: changes in plant chemistry and
potential nonlinear consequences for insect herbivores. Oikos. 1997;79:456–68.
Erb M, Köllner TG, Degenhardt J, Zwahlen C, Hibbard BE, Turlings TC. The role of abscisic acid
and water stress in root herbivore-induced leaf resistance. New Phytol. 2011;189(1):308–20.
Feng DX, Tasset C, Hanemian M, Barlet X, Hu J, Trémousaygue D, et al. Biological control
of bacterial wilt in Arabidopsis thaliana involves abscissic acid signalling. New Phytol.
2012;194(4):1035–45.
Fischer R, Byerlee D, Edmeades GO, editors. Can technology deliver on the yield challenge to
2050. Expert meeting on how to feed the world in 2009.
Fu D, Uauy C, Distelfeld A, Blechl A, Epstein L, Chen X, et al. A kinase-START gene confers
temperature-dependent resistance to wheat stripe rust. Science. 2009;323(5919):1357–60.
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, et al. Cross-
talk between abiotic and biotic stress responses: a current view from the points of conver-
gence in the stress signaling networks. Curr Opin Plant Biol. 2006;9(4):436–42. doi:10.1016/j.
pbi.2006.05.014.
Garrett K, Dendy S, Frank E, Rouse M, Travers S. Climate change effects on plant disease: ge-
nomes to ecosystems. Annu Rev Phytopathol. 2006;44:489–509.
Gaspar T, Franck T, Bisbis B, Kevers C, Jouve L, Hausman J, et al. Concepts in plant stress physi-
ology. Application to plant tissue cultures. Plant Growth Regul. 2002;37(3):263–85.
Goel AK, Lundberg D, Torres MA, Matthews R, Akimoto-Tomiyama C, Farmer L, et al. The
Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants.
Mol Plant-Microbe Interact. 2008;21(3):361–70.
Gregory PJ, Johnson SN, Newton AC, Ingram JS. Integrating pests and pathogens into the climate
change/food security debate. J Exp Bot. 2009;60(10):2827–38.
Gu Z, Wang J, Huang J, Zhang H. Cloning and characterization of a novel rice gene family encod-
ing putative dual-specificity protein kinases, involved in plant responses to abiotic and biotic
stresses. Plant Sci. 2005;169(3):470–7.
Gupta A, Kaur N. Sugar signalling and gene expression in relation to carbohydrate metabolism
under abiotic stresses in plants. J Biosci. 2005;30(5):761–76.
Gutbrodt B, Mody K, Dorn S. Drought changes plant chemistry and causes contrasting responses
in lepidopteran herbivores. Oikos. 2011;120(11):1732–40.
Henfling J, Bostock R, Kuc J. Effect of abscisic acid on rishitin and lubimin accumulation and
resistance to Phytophthora infestans and Cladosporium cucumerinum in potato tuber tissue
slices. Phytopathology. 1980;70(11):1074–8.
Herms DA, Mattson WJ. The dilemma of plants: to grow or defend. Q Rev Biol. 1992;67:283–335.
Himanen SJ, Nerg A-M, Nissinen A, Pinto DM, Stewart CN, Poppy GM, et al. Effects of elevated
carbon dioxide and ozone on volatile terpenoid emissions and multitrophic communication of
transgenic insecticidal oilseed rape ( Brassica napus). New Phytol. 2009;181(1):174–86.
Holopainen JK, Gershenzon J. Multiple stress factors and the emission of plant VOCs. Trends
Plant Sci. 2010;15(3):176–84.
Iyer NJ, Tang Y, Mahalingam R. Integrative analysis of combined water-deficit and ozone stress in
Medicago truncatula. Plant Cell Environ. 2013;36:706–20.
Jacobsen JV, Hanson AD, Chandler PC. Water stress enhances expression of an α-amylase gene in
barley leaves. Plant Physiol. 1986;80(2):350–9.
Jiang C-J, Shimono M, Sugano S, Kojima M, Yazawa K, Yoshida R, et al. Abscisic acid interacts
antagonistically with salicylic acid signaling pathway in rice-Magnaporthe grisea interaction.
Mol Plant-Microbe Interact. 2010;23(6):791–8.
Khan MR, Khan MW. Interaction of Meloidogyne incognita and coal-smoke pollutants on tomato.
Nematropica. 1996;26(1):47–56.
Koga H, Dohi K, Mori M. Abscisic acid and low temperatures suppress the whole plant-specific
resistance reaction of rice plants to the infection of Magnaporthe grisea. Physiol Mol Plant
Pathol. 2004;65(1):3–9.
Koo AJK, Cooke TF, Howe GA. Cytochrome P450 CYP94B3 mediates catabolism and inactivation
of the plant hormone jasmonoyl-L-isoleucine. Proc Natl Acad Sci U S A. 2011;108(22):9298–
303.
Kusajima M, Yasuda M, Kawashima A, Nojiri H, Yamane H, Nakajima M, et al. Suppressive
effect of abscisic acid on systemic acquired resistance in tobacco plants. J Gen Plant Pathol.
2010;76(2):161–7.
Laurie-Berry N, Joardar V, Street IH, Kunkel BN. The Arabidopsis thaliana JASMONATE IN-
SENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during
infection by Pseudomonas syringae. Mol Plant Microbe Interact. 2006;19(7):789–800.
Leakey ADB, Ainsworth EA, Bernacchi CJ, Rogers A, Long SP, Ort DR. Elevated CO2 effects
on plant carbon, nitrogen, and water relations: six important lessons from FACE. J Exp Bot.
2009;60(10):2859–76.
Lee SC, Luan S. ABA signal transduction at the crossroad of biotic and abiotic stress responses.
Plant Cell Environ. 2012;35(1):53–60.
Lobell DB, Cassman KG, Field CB. Crop yield gaps: their importance, magnitudes, and causes.
Ann Rev Environ Res. 2009;34(1):179.
Loreto F, Schnitzler J-P. Abiotic stresses and induced BVOCs. Trends Plant Sci. 2010;15(3):154–
66.
Luck J, Spackman M, Freeman A, Griffiths W, Finlay K, Chakraborty S. Climate change and dis-
eases of food crops. Plant Pathol. 2011;60(1):113–21.
Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J. Callose deposition: a multifaceted plant
defense response. Mol Plant Microbe Interact. 2011;24(2):183–93.
Luo M, Liang XQ, Dang P, Holbrook CC, Bausher MG, Lee RD, et al. Microarray-based screening
of differentially expressed genes in peanut in response to Aspergillus parasiticus infection and
drought stress. Plant Sci. 2005;169(4):695–703.
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, et al. Regulators of PP2C phos-
phatase activity function as abscisic acid sensors. Science. 2009;324(5930):1064–8.
Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK. A putative lipid transfer protein
involved in systemic resistance signalling in Arabidopsis. Nature. 2002;419(6905):399–403.
[10.1038/nature00962]
Mattson WJ, Haack RA. The role of drought in outbreaks of plant-eating insects. BioScience.
1987;37(2):110–8.
Mauch-Mani B, Mauch F. The role of abscisic acid in plant-pathogen interactions. Curr Opin Plant
Biol. 2005;8(4):409–14.
Maxmen A. Crop pests: under attack. Nature. [Outlook]. 2013;501(7468):S15–7.
Mayek-Perez N, GarcÍa-Espinosa R, LÓpez-CastaÑeda C, Acosta-Gallegos JA, Simpson J. Wa-
ter relations, histopathology and growth of common bean ( Phaseolus vulgaris L.) during
pathogenesis of Macrophomina phaseolina under drought stress. Physiol Mol Plant Pathol.
2002;60(4):185–95.
Melotto M, Underwood W, Koczan J, Nomura K, He SY. Plant stomata function in innate immu-
nity against bacterial invasion. Cell. 2006;126(5):969–80.
Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTIBLE1 gene encodes an
R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in
Arabidopsis. Plant Cell Online. 2003;15(11):2551–65.
Miller G, Mittler R. Could heat shock transcription factors function as hydrogen peroxide sensors
in plants? Ann Bot. 2006;98(2):279–88.
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. Reactive oxygen species homeostasis and signal-
ling during drought and salinity stresses. Plant Cell Environ. 2010;33(4):453–67.
2006;11(1):15–9.
Mittler R, Blumwald E. Genetic engineering for modern agriculture: challenges and perspectives.
Annu Rev Plant Biol. 2010;61:443–62.
Mohr PG, Cahill DM. Abscisic acid influences the susceptibility of Arabidopsis thaliana to Pseu-
domonas syringae pv. tomato and Peronospora parasitica. Funct Plant Biol. 2003;30(4):461–9.
Mohr PG, Cahill DM. Suppression by ABA of salicylic acid and lignin accumulation and the
expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato.
Funct Integr Genomics. 2007;7(3):181–91.
Molina A, García-Olmedo F. Enhanced tolerance to bacterial pathogens caused by the transgenic
expression of barley lipid transfer protein LTP2. Plant J. 1997;12(3):669–75.
Moriondo M, Giannakopoulos C, Bindi M. Climate change impact assessment: the role of climate
extremes in crop yield simulation. Clim Change. 2011;104(3-4):679–701.
Nakashima K, Tran L-SP, Van Nguyen D, Fujita M, Maruyama K, Todaka D, et al. Functional
analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-
responsive gene expression in rice. Plant J. 2007;51(4):617–30.
Narusaka Y, Narusaka M, Seki M, Umezawa T, Ishida J, Nakajima M, et al. Crosstalk in the re-
sponses to abiotic and biotic stresses in Arabidopsis: analysis of gene expression in cytochrome
P450 gene superfamily by cDNA microarray. Plant Mol Biol. 2004;55(3):327–42.
Németh M, Janda T, Horváth E, Páldi E, Szalai G. Exogenous salicylic acid increases polyamine
content but may decrease drought tolerance in maize. Plant Sci. 2002;162(4):569–74.
Newton AC, Johnson SN, Gregory PJ. Implications of climate change for diseases, crop yields and
food security. Euphytica. 2011;179(1):3–18.
Pasquali G, Biricolti S, Locatelli F, Baldoni E, Mattana M. Osmyb4 expression improves adaptive
responses to drought and cold stress in transgenic apples. Plant Cell Rep. 2008;27(10):1677–
86.
Peng X-x, Tang X-k, Zhou P-l, Hu Y-j, Deng X-b, He Y, et al. Isolation and expression patterns of
rice WRKY82 transcription factor gene responsive to both biotic and abiotic stresses. Agric Sci
China. 2011;10(6):893–901.
Pieterse CM, Leon-Reyes A, Van der Ent S, Van Wees SC. Networking by small-molecule hor-
mones in plant immunity. Nat Chem Biol. 2009;5(5):308–16.
Prasch CM, Sonnewald U. Simultaneous application of heat, drought, and virus to Arabidopsis
plants reveals significant shifts in signaling networks. Plant Physiol. 2013;162(4):1849–66.
Rasmussen S, Barah P, Suarez-Rodriguez MC, Bressendorff S, Friis P, Costantino P, et al. Transcrip-
tome responses to combinations of stresses in Arabidopsis. Plant Physiol. 2013;161(4):1783–
94.
2004;134(4):1683–96.
Rogers A, Ainsworth EA, Leakey AD. Will elevated carbon dioxide concentration amplify the
benefits of nitrogen fixation in legumes? Plant Physiol. 2009;151(3):1009–16.
Rohila JS, Yang Y. Rice mitogen-activated protein kinase gene family and its role in biotic and
abiotic stress response. J Integr Plant Biol. 2007;49(6):751–9.
Runyon JB, Mescher MC, De Moraes CM. Volatile chemical cues guide host location and host
selection by parasitic plants. Science. 2006;313(5795):1964–7.
Sánchez-Vallet A, López G, Ramos B, Delgado-Cerezo M, Riviere M-P, Llorente F, et al. Disrup-
tion of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotro-
phic fungus Plectosphaerella cucumerina. Plant Physiol. 2012;160(4):2109–24.
Santino A, Taurino M, De Domenico S, Bonsegna S, Poltronieri P, Pastor V, et al. Jasmonate
signaling in plant development and defense response to multiple (a) biotic stresses. Plant Cell
Rep. 2013;32(7):1085–98.
Sawinski K, Mersmann S, Robatzek S, Böhmer M. Guarding the green: pathways to stomatal im-
munity. Mol Plant Microbe Interact. 2013;26(6):626–32.
Scherm H. Climate change: can we predict the impacts on plant pathology and pest management?
Can J Plant Pathol. 2004;26(3):267–73.
Schmidhuber J, Tubiello FN. Global food security under climate change. Proc Natl Acad Sci U S
A. 2007;104(50):19703–8.
Sharma R, De Vleesschauwer D, Sharma MK, Ronald PC. Recent advances in dissecting stress-
regulatory crosstalk in rice. Mol Plant. 2013;6(2):250–60.
Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, et al. Rice WRKY45 plays impor-
tant roles in fungal and bacterial disease resistance. Mol Plant Pathol. 2012;13(1):83–94.
Singh P, Siva R, Gothandam KM, Babu S. Naturally existing levels of Osmyb4 gene expression
in rice cultivars correlate with their reaction to fungal and bacterial pathogens. J Phytopathol.
2013;161(10):730–4.
Smit AL, Vamerali T. The influence of potato cyst nematodes ( Globodera pallida) and drought
on rooting dynamics of potato ( Solanum tuberosum L. ). Eur J Agron. 1998;9(2–3):137–46.
Sun Y, Cao H, Yin J, Kang L, Ge F. Elevated CO2 changes the interactions between nematode and
tomato genotypes differing in the JA pathway. Plant Cell Environ. 2010;33(5):729–39.
New Phytol. 2014;203(1):32–43.
Takasaki H, Maruyama K, Kidokoro S, Ito Y, Fujita Y, Shinozaki K, et al. The abiotic stress-
responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress
tolerance in rice. Mol Genet Genomics. 2010;284(3):173–83.
Tao Z, Liu H, Qiu D, Zhou Y, Li X, Xu C, et al. A pair of allelic WRKY genes play opposite roles
in rice-bacteria interactions. Plant Physiol. 2009;151(2):936.
Tao Z, Kou Y, Liu H, Li X, Xiao J, Wang S. OsWRKY45 alleles play different roles in abscisic acid
signalling and salt stress tolerance but similar roles in drought and cold tolerance in rice. J Exp
Bot. 2011;62(14):4863–74.
Tariq M, Wright DJ, Bruce TJ, Staley JT. Drought and root herbivory interact to alter the response
of above-ground parasitoids to aphid infested plants and associated plant volatile signals. PLoS
ONE. 2013;8(7):e69013.
Ton J, Mauch-Mani B. β-amino-butyric acid-induced resistance against necrotrophic pathogens is
based on ABA-dependent priming for callose. Plant J. 2004;38(1):119–30.
Ton J, Flors V, Mauch-Mani B. The multifaceted role of ABA in disease resistance. Trends Plant
Sci. 2009;14(6):310–7.
Tumlinson JH. The importance of volatile organic compounds in ecosystem functioning. J Chem
Ecol. 2014;40(3):212–3.
van Hulten M, Pelser M, Van Loon L, Pieterse CM, Ton J. Costs and benefits of priming for de-
fense in Arabidopsis. Proc Natl Acad Sci U S A. 2006;103(14):5602–7.
Vannini C, Iriti M, Bracale M, Locatelli F, Faoro F, Croce P, et al. The ectopic expression of
the rice Osmyb4 gene in Arabidopsis increases tolerance to abiotic, environmental and biotic
stresses. Physiol Mol Plant Pathol. 2006;69(1):26–42.
Vannini C, Campa M, Iriti M, Genga A, Faoro F, Carravieri S, et al. Evaluation of transgenic to-
mato plants ectopically expressing the rice Osmyb4 gene. Plant Sci. 2007;173(2):231–9.
Vignols F, Wigger M, Garcı́a-Garrido JM, Grellet F, Kader J-C, Delseny M. Rice lipid transfer
protein (LTP) genes belong to a complex multigene family and are differentially regulated.
Gene. 1997;195(2):177–86.
von Koskull-Döring P, Scharf K-D, Nover L. The diversity of plant heat stress transcription fac-
tors. Trends Plant Sci. 2007;12(10):452–7.
Vuorinen T, Nerg A-M, Holopainen JK. Ozone exposure triggers the emission of herbivore-induced
plant volatiles, but does not disturb tritrophic signalling. Environ Pollut. 2004;131(2):305–11.
Wang W, Vinocur B, Altman A. Plant responses to drought, salinity and extreme temperatures:
towards genetic engineering for stress tolerance. Planta. 2003;218(1):1–14.
Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, et al. An update on abscisic
acid signaling in plants and more…. Mol Plant. 2008;1(2):198–217.
Webb K, Ona I, Bai J, Garrett K, Mew T, Cruz V, et al. A benefit of high temperature: increased ef-
fectiveness of a rice bacterial blight disease resistance gene. New Phytol. 2010;185(2):568–76.
Xiao J, Cheng H, Li X, Xiao J, Xu C, Wang S. Rice WRKY13 regulates cross talk between abi-
otic and biotic stress signaling pathways by selective binding to different cis-elements. Plant
Physiol. 2013;163(4):1868–82.
Yasuda M, Ishikawa A, Jikumaru Y, Seki M, Umezawa T, Asami T, et al. Antagonistic interaction
between systemic acquired resistance and the abscisic acid-mediated abiotic stress response in
Arabidopsis. Plant Cell Online. 2008;20(6):1678–92.
Yokotani N, Sato Y, Tanabe S, Chujo T, Shimizu T, Okada K, et al. WRKY76 is a rice transcrip-
tional repressor playing opposite roles in blast disease resistance and cold stress tolerance. J
Exp Bot. 2013a;64(16):5085–97.
Yokotani N, Ichikawa T, Kondou Y, Iwabuchi M, Matsui M, Hirochika H, et al. Role of the rice
transcription factor JAmyb in abiotic stress response. J Plant Res. 2013b;126(1):131–9.
Yoshida T, Ohama N, Nakajima J, Kidokoro S, Mizoi J, Nakashima K, et al. Arabidopsis HsfA1
transcription factors function as the main positive regulators in heat shock-responsive gene
expression. Mol Genet Genomics. 2011;286(5–6):321–32.
Zavala JA, Casteel CL, DeLucia EH, Berenbaum MR. Anthropogenic increase in carbon di-
oxide compromises plant defense against invasive insects. Proc Natl Acad Sci U S A.
2008;105(13):5129–33.
Zhang T, Liu Y, Yang T, Zhang L, Xu S, Xue L, et al. Diverse signals converge at MAPK cascades
in plant. Plant Physiol Biochem. 2006;44(5):274–83.
Zhu Y, Qian W, Hua J. Temperature modulates plant defense responses through NB-LRR proteins.
PLoS Pathog. 2010;6(4):e1000844.
Chapter 10
Impact of Concurrent Drought Stress
and Pathogen Infection on Plants
Prachi Pandey, Ranjita Sinha, Kirankumar S. Mysore

and Muthappa Senthil-Kumar
10.1 Introduction
In the field conditions, plants are constantly exposed to concurrent abiotic and biotic
stresses that affect their overall growth and development (Mittler 2006; Atkinson
and Urwin 2012). Plant responses to individual biotic and abiotic stresses have been
well explored and a number of genes conferring tolerance to the individual stresses
have been identified. Some of the genes have also been reported to impart tolerance
to multiple independent abiotic and biotic stress conditions (Wang et al. 2010, 2013;
Senthil-Kumar et al. 2013; Tamirisa et al. 2014). A few recent studies suggest that
the combined effect of two or more abiotic stresses cause greater reduction in crop
yield when compared with the losses incurred by individual stresses (Rizhsky et al.
2002, 2004; Mittler 2006; Suzuki et al. 2014). Environmental factors like drought,
extreme temperature, and salinity potentially alter the occurrence and intensity of
a particular disease by modulating the plant responses to pathogen (Szittya et al.
2003; Wiese et al. 2004; Achuo et al. 2006; Amtmann et al. 2008; Goel et al. 2008;
Madgwick et al. 2011; Atkinson and Urwin 2012). The importance of different
M. Senthil-Kumar () · P. Pandey · R. Sinha

National Institute of Plant Genome Research, JNU Campus, Aruna Asaf Ali Marg,
P.O. Box No. 10531, New Delhi 110 067, India
e-mail: skmuthappa@nipgr.ac.in
P. Pandey
e-mail: prachipndy@gmail.com
R. Sinha
e-mail: sinharanjita83@gmail.com
K. S. Mysore
Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway,
Ardmore, OK 73401, USA
e-mail: ksmysore@noble.org

204 P. Pandey et al.
p redisposing abiotic stress factors on plant–pathogen interactions has also been

recently reviewed (Bostock et al. 2014).
The data from a number of individual stress studies have been analyzed using
bioinformatics tools to find the common genes altered under biotic and abiotic stress
conditions. For example, the response of thale cress ( Arabidopsis thaliana, hereaf-
ter referred to as Arabidopsis) to a variety of abiotic and biotic stresses was stud-
ied by the comparison and cluster analysis of differentially expressed genes from
publicly available microarray datasets (Ma and Bohnert 2007). Similarly, the gene
expression profiles of chickpea plant under different abiotic (drought, cold, and
high salinity) and biotic stress ( Ascochyta rabiei; causal agent of blight in chickpea)
conditions were compared (Mantri et al. 2010). Meta-analysis of transcriptomic
data from rice ( Oryza sativa) and Arabidopsis plants each exposed to independent
drought and bacterial stresses revealed the commonality of 38.5 and 28.7 % dif-
ferentially expressed genes between two stress conditions in the respective plants
(Shaik and Ramakrishna 2013, 2014). Yet, in another study, the molecular response
of rice plants to multiple biotic and abiotic stress conditions was compared and
genes responsive to both the stresses and to exclusively biotic stresses were identi-
fied (Narsai et al. 2013). Several other studies also support the existence of cross
talk between the abiotic and biotic stress pathways (Narusaka et al. 2004; Fujita
et al. 2006; Fraire-Velázquez et al. 2011). However, in all these studies, the plants
were not concurrently exposed to biotic and abiotic stresses, but only the data from
independently stressed plants were compared. Although the biotic and abiotic stress
response pathways have common elements, plant-“tailored” responses to the actual
concurrent abiotic and biotic stress cannot be predicted using the data from indi-
vidual stress studies (Mittler 2006).
The physiological and molecular responses against concurrent abiotic and biotic
stresses are beginning to be studied (Atkinson et al. 2013; Rasmussen et al. 2013;
Bostock et al. 2014; Kissoudis et al. 2014; Prasch and Sonnewald 2014). The avail-
able literature provides evidence that plants perceive concurrent stresses as a “new
stress” leading to reprogramming of their responses. Gene expression studies in
Arabidopsis plants exposed to concurrent stress conditions like cold and high light,
salt and heat, salt and high light, heat and high light, heat and flagellin, and cold
and flagellin also revealed that on an average 61 % of the transcripts expressed
during concurrent dual stresses were not observed in the single stress treatments
(Rasmussen et al. 2013). Likewise, drought and concurrent nematode infection in
Arabidopsis plants led to the induction of 50 unique genes (Atkinson et al. 2013).
Drought is one of the most important and frequently occurring abiotic factors
and can potentially alter the end result of plant–pathogen interaction. Hence, this
chapter is focused on the impact of drought stress on plant–pathogen relations and
the different ways through which drought modulates the plant–pathogen (fungi,
oomycete, bacteria, and virus) relations. We also speculate various aspects involved
in the concurrent stress-responsive signaling network of plants by reviewing recent
studies.
Table 10.1 A few examples of drought-mediated modulation of plant–pathogen interaction in plants
S. No. Pathogen Name of the Host plant Effect on plant– References
disease pathogen interaction
Fungi
Thielaviopsis basicola Black root rot Tobacco Susceptibility decreased Bateman 1961
Cephalosporium gramineum Stripe Wheat Bruehl 1968
Sclerotinia sclerotiorum White mold Nicotiana benthamiana Ramegowda et al. 2013
Sclerotinia sp. White mold Soybean, sunflower, Markell et al. 2008
canola, peanut
Fusarium solani f. sp. pisi Root and stem rot Sweet pea Susceptibility increased Krafts and Roberts 1969
Macrophomina phaseoli Charcoal rot Soybean, sorghum, cotton Mayek-Perez et al. 2002
Uncinula necator Powdery mildew Grapes Hartman and Beale 1998
Penicillium sp. and Aspergil- Seed decay Wheat Griffin 1966
lus sp.
Rhizoctonia sp. Stem canker Potato Lootsma and Scholte 1997
Verticillium sp. Early dying Potato Markell et al. 2008
Drechslera tritici-repentis Tan spot Wheat Janda et al. 2008a
Ascochyta sp. Ascochyta blight Pea, lentil, chickpea Markell et al. 2008
Oomycetes
Pythium sp. Root rot Pea Susceptibility decreased Kerr 1964
10 Impact of Concurrent Drought Stress and Pathogen Infection on Plants
Aphanomyces sp. Root rot Sunflower Markell et al. 2008

Plasmopara sp. Downy mildew Sunflower Markell et al. 2008
Phytophthora sp. Root rots Soybean, safflower, rho- Susceptibility increased McDonald and Cahill 1999; Duni-
dodendron, tomato way 1977; Blaker and MacDonald
1981; Ristaino and Duniway 1989
205
206
S. No. Pathogen Name of the Host plant Effect on plant– References

disease pathogen interaction
Virus
Pineapple mealybug wilt- Pineapple Pineapple Susceptibility increased Sether and Hu 2001
associated virus-1 Mealybug Wilt
Maize dwarf mosaic virus Mosaic Sweet corn Olson et al. 1990
Turnip mosaic virus Growth Arabidopsis Prasch and Sonnewald 2013
retardation
Bacteria
Xylella fastidiosa Pierce’s disease Vine Susceptibility increased McElrone et al. 2001
Pseudomonas syringae pv. Bacterial speck Arabidopsis Mohr and Cahill 2003
tomato 1065 disease
Streptomyces scabies Common scab Potato Cook and Papendick 1972
Pseudomonas syringae pv. Bacterial speck Nicotiana benthamiana Susceptibility decreased Ramegowda et al. 2013
Tabaci disease
a
PEG-mediated osmotic stress
PEG Polyethylene glycol
P. Pandey et al.
10 Impact of Concurrent Drought Stress and Pathogen Infection on Plants 207
10.2 Drought Modulates Plant–Pathogen Interaction
The net effect of concurrent drought and pathogen infection on plants depends on
duration and intensity of the two stresses. Based on these factors, the combination
of drought and pathogen infection can have two outcomes. In the first scenario, both
the stresses when occurring concurrently can act in unison to hamper plant growth
and development. For example, drought stress has been shown to aggravate many
fungal (Mayek-Perez et al. 2002), bacterial (McElrone et al. 2001; Mohr and Ca-
hill 2003), and viral (Olson et al. 1990; Prasch and Sonnewald 2013) infections in
plants. On the contrary, in the second case, the drought stress has been shown to en-
hance the tolerance of the plants toward pathogens (Ramegowda et al. 2013; Achuo
et al. 2006). The nature and outcome of plant–pathogen interaction under drought
stress differs with the type of pathogens (fungi, oomycete, bacteria, and viruses) as
they employ different strategies for infection. The different ways by which drought
modulates plant’s interactions with these pathogens are discussed. Apart from the
above-mentioned two scenarios, pathogens can enhance the resistance of plants to
drought (Reusche et al. 2012; Xu et al. 2008). However, this aspect is not discussed
in this chapter.
10.2.1 Plant–Fungal/Oomycete Pathogen Interactions

During Drought Stress
The availability of moisture is crucial for the establishment of fungal/oomycete

infections on plants (Agrios 2005). The effect of concurrent drought and fungal/
oomycete pathogen infection on plant growth has been fairly investigated in the
past (Table 10.1). Drought stress can affect the plant–pathogen interaction by in-
creasing or decreasing plant’s propensity for infection. For soil-borne pathogens,
the outcome of drought and fungal/oomycete pathogen interaction also depends on
the effect of drought on the pathogen per se. So, under drought conditions, the de-
gree of infection caused by a soil-borne fungi/oomycete on plants varies depending
on whether the pathogen is favored by wet or dry soils (Cook and Papendick 1972).
Drought can also influence the plant–pathogen interactions by inducing changes in
the host physiology. The drought-induced changes in host physiology can be direct
or indirect. The direct effects include the modulation of plant defense mechanisms
against the pathogen. The indirect effects consist of changes in the nutritional status
of plants brought about by drought stress.
10.2.1.1 Negative Effect of Concurrent Drought Stress

and Fungal/Oomycete Infection on Plants
Fungal pathogens like Sclerotium cepivorum (causal agent of root rot in onions), Strep-
tomyces scabies (causal agent of common scab in potato), Fusarium sp. (causal agent
of wilt in crop plants), and Urocystis agropyri (causal agent of smut on cereals), whose
infections are known to be favored in dry soils, show more aggressive pathogenesis
under drought conditions (Colhoun 1973). Edmunds (1964) observed that Macroph-
omina phaseoli (causal agent of charcoal stalk rot in sorghum) infection on sorghum
plants under drought conditions caused more damage compared to nonstressed condi-
tions. Drought conditions also enhanced the susceptibility of safflower and rhododen-
dron to oomycete pathogen Phytophthora sp. (causal agent of root rot; Duniway 1977;
Blaker and MacDonald 1981). Similarly, disease-resistant wheat plants were shown
to become susceptible to Fusarium roseum f. sp. cerealis under drought stress (Papen-
dick and Cook 1974). In all the above cases, the semidry conditions in soil apparently
favored the fungal infection. The successful infection by fungal pathogens in dry soils
can be possibly due to the fact that infection by these fungi depends on volatile root
exudates that diffuse more rapidly through dry soil (Kerr 1964).
The altered physiology of plants due to drought stress can also favor the pathogen
infection. For example, drought stress leads to nutrition deficiency in some plants
and this secondary effect along with drought-induced physiological changes can
aggravate the pathogen infection (Lawlor and Cornic 2002; Lawlor 2002). Drought
stress-induced changes like the accumulation of osmolytes and nutrient leakage
have been reported to lead to enriched nutrient supply for the pathogen. Drought
stress-mediated exacerbation of infection under this category is best exemplified by
pathogenesis of Macrophomina phaseolina (causal agent of charcoal rot in common
bean) in common bean (Mayek-Perez et al. 2002). The stress-related amino acids
like proline and asparagine have recently been shown to be utilized efficiently by
M. phaseolina (Ijaz et al. 2013). The impact of drought was found to be more severe
on a number of wilt and root-rot diseases. The wilt- and root-rot-causing fungi are
known to interfere with the water relations of plants by colonizing the xylem vessels
(Yadeta and Thomma 2013). Thus, the drought along with the pathogen imposes ad-
ditional stress on plants and causes severe impact on plant growth.
10.2.1.2 Positive Effects of Concurrent Drought Stress

and Fungal/Oomycete Pathogen Infection on Plants
The root-infecting oomycetes like Pythium sp. (causal agent of root rot in crops),
Aphanomyces sp. (causal agent of root rot in sunflower and sugar beets), and Plas-
mopara sp. (causal agent of downy mildew) need adequate soil moisture for their
survival in soil and for plant infection. Hence, the occurrence of downy mildew of
sunflower and Aphanomyces root rot of sugar beets was less severe under drought
stress conditions (Markell et al. 2008). Similar to soil-borne oomycete pathogens, less
moisture in the atmosphere during drought is also shown to affect the pathogenesis of
foliar fungal and oomycete pathogens. Many foliar pathogens such as those causing
leaf spots are able to infect plants only when leaves are moist. Additionally, many fo-
liar fungal pathogens produce spores that are dispersed by rain splash and germinated
under high-humidity conditions. Pathogens that need rain to spread are unlikely to
cause epidemics under drought conditions (Markell et al. 2008). The above-men-
tioned reports exemplify the effect of atmospheric water on the pathogen infection.
Drought acclimation in plants is known to combat some fungal pathogen infection

during the combined stress. Ramegowda et al. (2013) showed that upon infection
with Sclerotinia sclerotiorum (causal agent of white mold in beans), the well-wa-
tered Nicotiana benthamiana plants showed severe cell death, whereas the drought-
acclimated plants exhibited reduced cell death. Thus, moderate drought was found to
enhance plant’s defense against pathogens by inducing expression of defense-related
genes. The drought-mediated suppression of infection can also be attributed to the
accumulation of abscisic acid (ABA). For example, drought-stressed tomato plants
which showed the accumulation of ABA exhibited enhanced resistance against
Botrytis cinerea (causal agent of grey mould in tomato; Achuo et al. 2006).
Taken together, drought can be favorable to either the pathogen or the host de-
fense response. However, the consequences of concurrent drought on pathogen in-
fection depend on the host, type of pathogen as well as the severity of drought
stress. The ability of some fungi to interfere with the water relations of the plants
and utilize the stress-induced molecules as nutrient source gives them an advan-
tage under water stress conditions. On the other hand, plants can also fine-tune
their defense responses under drought conditions to combat the pathogen infec-
tion. Thus, the modulation of plant–fungal/oomycete pathogen interaction during
drought stress involves many facets, which can be interpreted by more systematic
studies in this direction.
10.2.2 Plant–Bacterial Interaction During Drought Stress
Like fungi/oomycete, bacterial pathogens also depend on water for infection. The
majority of the bacterial diseases are favored by the conditions of high humidity. A
high water content in the apoplast facilitates bacterial growth. Incubation of plants
at high relative humidity was shown to promote the growth of avirulent bacteria
on plants (Freeman and Beattie 2009). Water-soaked lesions are typical charac-
teristics of many bacterial leaf spot diseases and are known to be important for
bacterial multiplication (Rudolph 1984). This reflects the importance of water in
bacterial infections on plants. Thus, water scarcity should reduce bacterial infec-
tion on plants. This is true for the majority of cases. However, drought in few cases
enhances plant’s susceptibility to bacterial infections. Thus, drought can modulate
plant–pathogen interactions for either the benefit of the host plant or the bacterium.
A detailed discussion of both the scenarios is provided below.
10.2.2.1 Negative Effect of Concurrent Drought Stress

and Bacterial Infection on Plants
Drought stress was found to enhance the susceptibility of grapevines to Xylella

fastidiosa (causal agent of Pierce’s disease; Thorne et al. 2006). X. fastidiosa has
been reported to spread in plants by causing damage to intra-vessel pit membranes
(Newman et al. 2003). The exposure of plants to drought conditions has also been
shown to lead to the disruption of pit membranes (Stiller and Sperry 2002). Drought
stress, thus, facilitates the spread of X. fastidiosa in the plant. Drought-stressed
Arabidopsis plants were found to be susceptible to an avirulent bacterial patho-
gen, Pseudomonas syringae pv. tomato 1065 (Mohr and Cahill 2003). In this study,
the susceptibility induced by drought was attributed to ABA. The exogenous ABA
treatment is shown to render Arabidopsis plants susceptible to P. syringae infec-
tion by probably suppressing the salicylic acid (SA)-mediated defense responses
(Mohr and Cahill 2003). Bacteria also modulate ABA-mediated responses for their
infection and survival inside the plants. HopAM1, a type III effector of P. syringae,
increases the virulence of a weak pathogen ( P. syringae pv. maculicola M6 CE) un-
der drought stress condition by enhancing the ABA-mediated suppression of basal
defense responses in plants (Goel et al. 2008).
Drought stress has also been found to contribute to enhanced susceptibility of
plants to vascular wilt causing bacteria. In combination with drought stress, X. fas-
tidiosa (causal agent of Pierce’s disease) increases the severity and progression of
leaf scorch in Parthenocissus quinquefolia vine, reducing the total leaf area and
number of nodes (McElrone et al. 2001). The dual stress caused increased reduction
in stomatal conductance, leaf water potential, hydraulic conductivity, and xylem
vessel length (McElrone et al. 2003) compared to individual stresses.
Another factor responsible for severe occurrence of disease under drought condi-
tion is reduction in the population of antagonistic bacteria in dry soils. For example,
drought conditions are known to increase infection caused by S. scabies (causal agent
of common scab in potatoes) in potatoes (Lapwood 1966). The decreased abundance
of antagonistic bacteria in dry soil which otherwise limit lenticels infection by S.
scabies leads to enhanced infection under drought conditions (Lewis 1970).
10.2.2.2 Positive Effect of Concurrent Drought Stress

and Bacterial Infection on Plants
Moderate drought stress can enhance the tolerance of plants to bacterial infection by
activating the stress response machinery. The acclimation of N. benthamiana plants
to moderate drought stress (40–60 % field capacity [FC] of soil) increased its toler-
ance to bacterial pathogen P. syringae pv. tabaci (causal agent of wildfire disease
in tobacco) (Ramegowda et al. 2013). The degree of disease tolerance in drought-
stressed plants was correlated to the extent of reactive oxygen species (ROS) ac-
cumulation (Ramegowda et al. 2013). The relation of increased ROS content to
defense against bacterial infection was further substantiated by the application of
methyl viologen (MV), a compound that provokes ROS production by disrupting
electron transport chain in chloroplast. The MV-treated plants had high ROS and
showed decreased bacterial growth (Ramegowda et al. 2013).
Drought stress can also help prevent pathogen multiplication and spread. At
cellular level, water-deficit conditions help the plant to prevent bacterial survival
and progression. In fact, Arabidopsis plants are known to promote effector-medi-
ated signaling for localized desiccation of site of pathogen infection (Freeman and
Beattie 2009). Plants employ this effector-mediated localized desiccation possibly

by one of the three ways, namely programmed cell death (PCD) of the vascular
tissues, pectin-mediated occlusion of vessels, and reduction in aquaporin-mediated
water exchange from xylem to surrounding tissues (Beattie 2011).
10.2.3 Plant–Viral Interaction During Drought Stress
The majority of the available reports on the effect of concurrent drought on viral
infection suggest the negative impact of the concurrent stresses on plants (Olson
et al. 1990; Clover et al. 1999; Sether and Hu 2001; Prasch and Sonnewald 2013).
Drought stress has been shown to affect susceptibility of plants to viral infection.
Moderate drought (0–15 %) increases the susceptibility of bean plants to tobacco
mosaic virus (TMV) by fourfold (Yarwood et al. 1955). Furthermore, the simul-
taneous infection of Pineapple mealybug wilt-associated virus-1 (PMWaV-1) and
drought stress in pineapple has been reported to cause more loss in fruit produc-
tion than that caused by the individual stresses (Sether and Hu 2001). Similarly,
the concurrent drought stress and Maize dwarf mosaic virus (MDMV) infection in
sweet corn during vegetative and reproductive stages were found to additively re-
duce the growth and yield of plants (Olson et al. 1990). This may be due to the fact
that viral infections under drought stress can subvert plants’ metabolic machinery
toward viral multiplication and stress responses. Recently, Prasch and Sonnewald
(2013) studied the molecular responses of Arabidopsis plant subjected to concur-
rent turnip mosaic virus (TuMV) infection, heat, and drought stress. The concurrent
drought and viral infection led to greater reduction in biomass. However, the TuMV
level was not altered in the dually stressed plant (Prasch and Sonnewald 2013). The
combined stress was found to alter the circadian rhythm of plant by increasing the
expression of circadian clock-associated 1 ( CCA1) gene that is known to regulate a
wide array of genes including genes involved in photosynthesis. The combination
of viral infection and drought stresses down-regulated the genes involved in pho-
tosynthesis, adenosine triphosphate (ATP) synthesis, glycolysis, and tricarboxylic
acid (TCA) cycle. In contrast, the expression of genes involved in photorespiration,
such as glycolate oxidase and glucose–glyoxylate aminotransferase, was up-regu-
lated. This possibly resulted in reduction in biomass (Prasch and Sonnewald 2013).
Thus, the concurrent drought and viral infection possibly force plant machinery to
divert its energy toward defense responses, thereby leading to the down-regulation
of photosynthesis and other primary metabolic pathway genes.
Drought has also been shown to negatively affect virus translocation in plants
(Liu et al. 2009). For example, drought inhibits the systemic spread of tomato spot-
ted wilt virus in tomato (Cordoba et al. 1991). Moreover, in the study of Yarwood
et al. (1955), increased drought intensity was found to decrease the viral infection in
bean leaves. This signifies that the intensity of drought has a role to play in decid-
ing the outcome of plant–viral interactions. Unlike bacteria, fungus, and oomycete,
virus does not require nutrients for its growth, so drought-driven alleviation of viral
infection apparently occurs by some other mechanisms that are not yet known.
10.3 Plant–Pathogen Interactions During Drought Stress:

Current Understanding of the Underlying
Molecular Mechanisms
The signaling mechanisms involved in plant responses to biotic and abiotic stress
conditions have been well elucidated. Various studies in this direction have led to
the identification of a number of genes that are co-regulated under abiotic and bi-
otic stress conditions. The occurrence of cross talk between signaling pathways of
abiotic and biotic stresses is well known (Fujita et al. 2006; Tippmann et al. 2006;
Fraire-Velázquez et al. 2011). A couple of reports on the molecular mechanisms of
plant’s resistance against concurrent drought–nematode and drought–viral infec-
tion (Atkinson et al. 2013; Prasch and Sonnewald 2013) revealed the occurrence
of “shared” and “tailored” responses in plants exposed to the concurrent stresses.
The shared response consists of genes commonly expressed in abiotic and biotic
stress conditions. The tailored response, on the other hand, implies the genes ac-
tivated/repressed exclusively in response to the concurrent stress conditions. The
“shared response” can be largely understood from the molecular mechanisms of
plant response under independent and concurrent stress conditions. However, the
inferences drawn from the individual stress studies cannot be extrapolated to ex-
plain the tailored response of plants under concurrent stresses. In this section, we
describe the molecular basis of plant responses to concurrent drought and patho-
gen stresses based on our understanding from independent and the combined stress
studies (Fig. 10.1).
10.3.1 Clues from Studies on Independent Stresses
As already stated, the abiotic and biotic stress response machinery of plants shares
some common elements (Fig. 10.1a). The various elements of abiotic and biotic
stress signaling are known to interact with each other leading to a cross talk between
the signaling components of the two stress response pathways. Among the common
elements, the most important are ROS and Ca2 + . Independent exposure of plants
to drought and pathogen stress leads to a rapid increase in the levels of Ca2 + and
ROS in the cells (Takahashi et al. 2011; Miller et al. 2010). The further downstream
components of the signaling cascades, namely calcium-dependent protein kinases
(CDPKs) and mitogen-activated protein kinases (MAPKs), are also known to play
a synergistic role in drought and pathogen stress response of plants. For example,
SA-induced MAPK (SIPK) is known to be activated by both SA and osmotic stress
(Mikolajczyk et al. 2000; Hoyos and Zhang 2000). However, the modulation of
MAPK expression also confers antagonistic effects on different stress responses
(Xiong and Yang 2003; Shi et al. 2011). Also, silencing of OsMAPK5 in rice leads
to constitutive up-regulation of pathogenesis-related (PR) proteins and enhanced
pathogen resistance. However, these plants were sensitive to salt, cold, and drought
stress (Xiong and Yang 2003).
The response of plants to drought and pathogen infection is known to be largely

regulated by phytohormones. The exogenous application of drought-responsive
hormone, ABA, has been shown to increase the disease susceptibility in a number of
studies (Thaler and Bostock 2004; Mohr and Cahill 2003; Audenaert et al. 2002; de
Torres-Zabala et al. 2007). The ABA-deficient tomato ( sitiens mutant) plants have
been found to exhibit enhanced resistance to B. cinerea infection due to enhanced
PR proteins and repression of SA response (Thaler and Bostock 2004; Audenaert
et al. 2002). The enhanced resistance to pathogen infection in ABA-deficient mu-
tants can be attributed to reduced cuticle thickness and enhanced H2O2 production
in response to B. cinerea in tomato (Asselbergh et al. 2007) and altered cell wall
composition in Arabidopsis (Sanchez-Vallet et al. 2012). Contrastingly, the role
of ABA as a positive regulator of defense has also been reported (Mauch-Mani
and Mauch 2005; Melotto et al. 2006; Ton et al. 2009). ABA is shown to regulate
plant defense responses against pathogens through a number of ways like modify-
ing callose deposition, promoting stomatal closure, and regulating the expression of
defense genes. For example, ABA is necessary for β-aminobutyric acid (BABA)-
induced callose deposition during defense against fungal pathogens (Ton and
Mauch-Mani 2008). However, it blocks the callose deposition induced by bacterial
infection (de Torres-Zabala et al. 2007). ABA activates stomatal closure that acts as
a barrier against bacterial infection (Melotto et al. 2006). Moreover, transcriptome
and meta-analyses of gene expression profiles of Arabidopsis plants infected with
Pythium irregular led to the identification of ABA-responsive element (ABRE) in
the promoters of many of the defense genes (Adie et al. 2007; Wasilewska et al.
2008). Thus, ABA acts as a global switch regulating response toward biotic and
abiotic stresses (Asselbergh 2008). However, the mechanism of action of ABA is
still not completely deciphered. The identification of the molecular mechanisms
involved in phytohormone-mediated cross talk between biotic and abiotic stress
signaling needs to be done in order to elucidate the exact molecular mechanism by
which different phytohormones modulate plant defense responses against different
pathogens under drought conditions.
Together with the phytohormones, transcription factors (TF) like ABA-responsive
element-binding protein (AREB), MYC, NAM//ATAF1/CUC2 (NAC), ethylene-
responsive element-binding protein (EREB), WRKY, and coronatine insensitive
1 (COI1) are activated by pathogen challenge and drought stress (Atkinson et al.
2013). MYC2 has been found to be important in the interaction between the abiotic
and biotic stress pathways. It is activated by ABA (Abe et al. 2003) and positively
regulates jasmonic acid (JA)-induced defense genes, but represses the combined JA-
and SA-mediated gene expression (Laurie-Berry et al. 2006; Pieterse et al. 2009).
NAC and AP2/ERF TFs have also been associated with both abiotic and biotic
stress signaling. NAC TFs like OsNAC6 ( O. sativa NAC), tobacco stress-induced1
(TSI1), RD26, and botrytis-susceptible1 (BOS1) induce tolerance to both abiotic
and biotic stresses, others like A. thaliana activating factor 1 (ATAF1) impart toler-
ance to either of the stresses (Mengiste et al. 2003). Apart from these, ribosome pro-
duction factor 1 (RPF1), WRKY82, and WRKY85 have been shown to play roles in
conferring stress tolerance to both biotic and abiotic stresses (Asselberg et al. 2008;
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Fig. 10.1 Molecular understanding of the effect of concurrent drought on plant–pathogen

interactions. a Schematic representation of cross talk between key players of plant defense response
against concurrent drought and pathogen infection. The figure shows the signaling cascades and
Qiu and Yu 2009; Peng et al. 2011). Genes that confer tolerance to both biotic and
abiotic stress can form a part of the shared response exhibited by plants under con-
current drought and pathogen infection. However, their function under concurrent
stress conditions needs to be validated. The above-described independent single
stress studies are not useful for understanding the tailored response. Clear under-
standing can be obtained only from combined stress studies.
10.3.2 Clues from Combined Stress Studies
A recent study by Atkinson et al. (2013) on concurrent drought and nematode infec-
tion revealed that in addition to the overlapping transcript changes, the combined
stress treatment induced a set of genes that were not differentially regulated by
either of the single stresses. This study thus points toward the activation of a tai-
lored response which consists of unique program of gene expression in response to
the combined stresses. The genes differentially expressed under combined stress
included those involved in cell wall modification, carbohydrate metabolism, re-
dox regulation, and transcriptional regulation. A characteristic down-regulation of
disease-resistance genes (e.g., azelaic acid induced 1; AZI1) was also observed un-
der concurrent stress treatment. This may be due the suppression of SA-mediated
signaling by ABA. In order to understand the effect of concurrent stress on plants,
Prasch and Sonnewald (2013) subjected Arabidopsis plants to concurrent drought,
heat stress, and viral infection. The analyses of the microarray profiles of the stressed
plants revealed the expression of 11 genes under all the stress (single, double, and
triple stress combinations) conditions. These common genes are the ones encoding
transcription factors like Rap2.9 and G-box binding factor 3 (GBF3), a transmem-
brane receptor and a lipase. The transcript analysis also showed 23 stress-specific
genes that were differentially expressed in the triple stress condition. This consisted
a few representative proteins. The dotted arrows indicate the induction or suppression of abiotic
stress response elements by the biotic stress response elements, whereas the bold arrows indicate
the modulation by the ABA on biotic stress response elements. b Schematic representation of the
hypothetical response of plants to concurrent stress conditions. The first line of defense in plants
exposed to concurrent drought and pathogen infection presumably consists of Ca2 + -dependent
ROS production ( 1). The nature, localization, and intensity of ROS and Ca signals can define
the downstream events. The overall response of plants to concurrent stress is a combination of
shared ( 2) and tailored responses ( 3) and this defines increased or decreased plant susceptibility to
pathogen infections under drought stress. The question mark signifies the unexplored events of the
tailored mechanism. The response ( 6) of the plants to the concurrent stress conditions depends on
the intensity of the two stresses ( 4/5) as well as the nature of host and plant. The small triangles
represent the intensity of drought stress ( D) and the pathogen load ( P). ROS reactive oxygen spe-
cies, ABA abscisic acid, JA jasmonic acid, SA salicylic acid, Et ethylene, SAR systemic acquired
resistance, PR genes pathogen-related genes, CDPKs calcium-dependent protein kinases, MAPK
mitogen-activated protein kinase, AREB, ABA-responsive element-binding protein, NAC NAM//
ATAF1/CUC2, COI1 coronatine insensitive 1, MYB myeloblastosis, EREBP ethylene responsive
element binding protein, WRKY stands for the first four amino acids (tryptophan [W], arginine [R],
lysine [K] and tyrosine [Y] of the heptapeptide WRKYGQK, which is the hall mark of WRKY
proteins, transcription factors
of three transcription factors including DREB2A, and two zinc finger proteins
together with other stress-responsive proteins like cold-regulated 47, ABI5 binding
protein (AFP1), a pentatricopeptide repeat-containing protein, and a universal stress
protein family protein. The gene list also shows the presence of positive and nega-
tive regulators of a particular pathway. For example, AFP1 is a negative regulator
of ABA, whereas Arabidopsis Toxicos en Levadura (ATL4) is a positive regulator.
Major factors that can decide responses under concurrent stress conditions include
the severity and complexity of the stresses imposed. For example, in the above
study, the number of significantly regulated genes corresponding to drought alone,
virus alone, and stress combinations varied and corresponded to 518, 682, and 1744
respectively (Prasch and Sonnewald 2013).
On the basis of both the cross talk and concurrent stress studies, we hypothesize
a mechanism of plants response to concurrent stress conditions (Fig. 10.1b). Like
the individual stress conditions, under concurrent stress conditions, the Ca2 + -depen-
dent ROS production forms the first line of defense. We hypothesize a preferential
role for ABA in governing the concurrent stress responses than the other hormones.
However, this certainly needs to be validated and there may be exceptions. The
regulation mediated by JA, SA, and ET, however, also seems to be important and
this can be a key feature in the differentiation of response of plants against various
pathogens (necrotrophic/biotrophic).
10.4 Conclusions and Future Perspectives
The global climate change is leading to the emergence of new and complex stress
combinations and the impact of these stress combinations on crop productivity is
evolving as a major concern. Considering the impact of abiotic and biotic stress
conditions on crop yield, enormous efforts have been made over the past three de-
cades, to understand the independent effect of these stress conditions on plants. The
concurrent drought and pathogen infection can either increase the susceptibility of
plants to the pathogen or it can suppress the pathogen infection depending on vari-
ous factors like type of the pathogen, host species, and severity of drought stress. For
example, drought aggravates the diseases caused by wilt/rot-causing pathogens. On
the other hand, drought acclimation has been shown to confer resistance to patho-
gen infection in some cases. Drought environment can also affect the pathogen per
se. Although a number of reports reflect on the physiological effect of concurrent
drought stress on plant–pathogen interactions (Table 10.1), the understanding of
molecular mechanism imparting combined stress tolerance in plants is in its infancy.
As is evident from the two reports on molecular responses of plants to concurrent
stresses, the combat mechanisms of plants to concurrent abiotic and biotic stresses
are characterized by a combination of shared and tailored responses. Whereas the
shared responses are nearly well deciphered, the molecular events leading to and
explaining the tailored responses are yet to be understood. The detailed analysis of
the plant responses under concurrent drought and pathogen infection is needed to
unravel the intricate regulatory network involved in plant–pathogen interactions

under such conditions. The candidate genes differentially expressed under the con-
current stress conditions can be the potential targets for the manipulation in order to
develop plants with improved resistance under concurrent drought–pathogen infec-
tion. These genes can also serve as important markers for selecting the concurrent
stress-resistant crops.
However, the experimental evaluation of the effects of the combined drought and
pathogen stress on plants is a challenging task owing to the difficulties in accurate
concurrent stress imposition on plants. For example, compared to imposition of heat
stress, coinciding drought stress conditions that occur gradually in soil-drying ex-
periments with pathogen infection is difficult. The other hurdle of combined stress
studies is the optimization of inoculum concentration and drought intensity that
would not be lethal to the plant when imposed concurrently. These two factors are
important deciding factors of the outcome of combined stresses. Owing to these
complexities, physiological, molecular, and biochemical changes in plants exclu-
sively exposed to concurrent stress conditions are yet to be identified. We need to
develop standardized protocols for the imposition of drought stress and concurrent
pathogen infection in order to assess the impact of drought on plant–pathogen in-
teraction.
Effective categorization of the pathogens on the basis of their dependence on
water for infection needs to be done. The pathogen which is more infective under
drought conditions can be a possible threat to crops in the areas prone to drought
stresses. Thus, understanding the effect of drought on pathogen can help in the pre-
diction of emerging diseases under drought condition. This would be particularly
helpful in case of predicting the effect of pathogens causing wilts and rot on plants
under drought conditions. Overall, unraveling of physiological and molecular basis
of plant responses to concurrent drought and pathogen infection will be a crucial
step forward for the development of stress-resistant crops that can survive under the
field conditions.
Acknowledgments Projects on “understanding combined stress tolerance” at MS-K laboratory

are supported by National Institute of Plant Genome Research core funding and DBT-Ramal-
ingaswami reentry fellowship grant (BT/RLF/re-entry/23/2012). KSM laboratory projects are
supported by The Samuel Roberts Noble Foundation, National Science Foundation, and Bill and
Melinda Gates Foundation.
References
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis AtMYC2

(bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.
Plant Cell. 2003;15:63–78.
Achuo EA, Prinsen E, Höfte M. Influence of drought, salt stress and abscisic acid on the resistance
of tomato to Botrytis cinerea and Oidium neolycopersici. Plant Pathol. 2006;55:178–86.
Adie BAT, Pérez-Pérez J, Pérez-Pérez MM, Godoy M, Sánchez-Serrano J-J, Schmelz EA, Solano
R. ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the
activation of defenses in Arabidopsis. Plant Cell. 2007;19:665–81.
Agrios GN. Plant pathology, 5th ed., Burlington: Academic; 2005.
resistance in plants. Physiol Plant. 2008;133:682–91.
Asselbergh B, Curvers K, Francxa SC, Audenaert K, Vuylsteke M, Breusegem FV, Hofte M. Re-
sistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely
production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol.
2007;144:1863–77.
Asselbergh B, De Vleesschauwer D, Höfte M. Global switches and fine-tuning-ABA modulates
plant-pathogen defense. Mol Plant-Microbe In. 2008;21:709–19.
Atkinson NJ, Urwin PE. The interaction of plant biotic and abiotic stresses: from genes to the field.
J Exp Bot. 2012;63:3523–43.
to simultaneous biotic and abiotic stresses. Plant Physiol. 2013;162:2028–41.
Audenaert K, De Meyer GB, Höfte MM. Abscisic acid determines basal susceptibility of tomato to
Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms. Plant Physiol.
2002;128:491–501.
Bateman DF. The effect of soil moisture upon development of poinsettia root rots. Phytopathology.
1961;51:445–51.
Beattie GA. Water relations in the interaction of foliar bacterial pathogens with plants. Annu Rev
Phytopathol. 2011;49:533–55.
Blaker NS, MacDonald JP. Predisposing effects of soil moisture extremes on the susceptibility of
rhododendron to Phytophthora root and crown rot. Phytopathol. 1981;71:831–4.
Bostock RM, Pye MF, Roubtsova TV. Predisposition in plant disease: exploiting the nexus in
abiotic and biotic stress perception and response. Annu Rev Phytopathol. 2014;52:517–49.
Bruehl GW. Ecology of cephalosporium stripe disease of winter wheat in Washington. Plant Dis
Reptr. 1968;52:590–4.
Clover GRG, Smith HG, Azam-Ali SN, Jaggard KW. The effects of drought on sugar beet growth
in isolation and in combination with beet yellows virus infection. J Agric Sci. 1999;133:251–61.
Colhoun J. Effects of environmental factors on plant disease. Annu Rev Phytopathol.
1973;11:343–64.
Cook RJ, Papendick RI. Influence of water potential of soils and 3555 plants on root disease. Ann
Rev Phytopathol. 1972;10:349–74.
Cordoba AR, Taleisnik E, Brunotto M, Racca R. Mitigation of tomato spotted wilt virus infection
and symptom expression by water stress. J Phytopathol. 1991;133:255–63.
de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW, Egea PR, Bogre L,
Grant M. Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signaling
pathway to cause disease. EMBO J. 2007;26:1434–43.
Duniway JM. Changes in resistance to water transport in safflower during the development of
Phytophthora root rot. Phytopathol. 1977;67:331–7.
Edmunds LK. Combined relation of plant maturity, temperature and soil moisture to charcoal stalk
rot development in grain sorghum. Phytopathol. 1964;54:514–7.
Fraire-Velázquez S, Rodríguez-Guerra R, Sánchez-Calderón L. Abiotic and biotic stress response
crosstalk in plants. In: Shanker AK, Venkateswarulu B, editors. Abiotic stress response in
plants—physiological, biochemical and genetic perspectives. Hyderabad: InTech; 2011.
pp. 3–26.
Freeman BC, Beattie GA. Bacterial growth restriction during host resistance to Pseudomonas
syringaeis associated with leaf water loss and localized cessation of vascular activity in Arabi-
dopsis thaliana. Mol Plant Microbe Interact. 2009;22:857–67.
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K.
Crosstalk between abiotic and biotic stress responses: a current view from the points of conver-
gence in the stress signaling networks. Curr Opin Plant Biol. 2006;9:436–42.
Goel AK, Lundberg D, Torres MA, Matthews R, Akimoto-Tomiyama C, Farmer L, Dangl JL,
Grant SR. The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-
stressed plants. Mol Plant Microbe Interact. 2008;21:361–70.
Griffin DM. Fungi attacking seeds in dry seed beds. Proc Linnean Soc NSW. 1966;91:84–9.
Hartman J, Beale J. Powdery mildew of grape. Plant pathology fact sheet. University of Kentucky
College of Agriculture. 1998; PPFS-FR-S–12.
Hoyos ME, Zhang S Calcium-independent activation of salicylic acid-induced protein kinase and
a 40-kilodalton protein kinase by hyperosmotic stress. Plant Physiol. 2000;122:1355–64.
Ijaz S, Sadaqat HA, Khan AN. A review of the impact of charcoal rot ( Macrophomina phaseolina)
on sunflower. J Agr Sci. 2013;151(2):222–7.
Janda T, Cseplo M, Nemeth CS, Vida GY, Pogany M, Szalai G, Veisz O Combined effect of water
stress and infection with necrotrophic fungal pathogen Drechslera tritici-repentis on growth
and antioxidant activity in wheat. Cereal Res Commun. 2008;36(1):53–64.
Kerr A. The influence of soil moisture on infection of peas by Pythium ultimum. Aust J Biol Sci.
1964;17:676–85.
Kissoudis C, van de Wiel C, Visser RGF, van der Linden G. Enhancing crop resilience to combined
abiotic and biotic stress through the dissection of physiological and molecular crosstalk. Front
Plant Sci. 2014;5:207.
Kraft JM, Roberts DD. Influence of soil water and soil temperature on the pea root rot complex
caused by Pythium ultimum and Fusarium solani f. sp. pisi. Phytopathology.1969;59:149–52.
Lapwood DH. The effects of soil moisture at the time potato tubers are forming on the incidence
of common scab ( Streptomyces sabies). Ann Appl Biol. 1966;58:447–54.
Laurie-Berry N, Joardar V, Street IH, Kunkel BN. The Arabidopsis thaliana JASMONATE IN-
SENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during
infection by Pseudomonas syringae. Mol Plant Microbe Interact. 2006;19:789–800.
Lawlor DW. Limitation to photosynthesis in water stressed leaves: stomata vs. metabolism and the
role of ATP. Ann Bot. 2002;89:1–15.
Lawlor DW, Cornic G. Photosynthetic carbon assimilation and associated metabolism in relation
to water deficits in higher plants. Plant Cell Environ. 2002;25:275–94.
Lewis BG. Effects of water potential on the infection of potato tubers by Streptomyces scabies in
soil. Ann Appl Biol. 1970;66:83–8.
Liu JZ, Richerson K, Nelson RS. Growth conditions for plant virus-host studies. Curr Protoc Mi-
crobiol. 2009 Chapter 16:Unit16A.1.
Lootsma M, Scholte K. Effect of soil moisture content on the suppression of Rhizoctonia stem
canker on potato by the nematode Aphelenchus avenae and the springtail Folsomia fimetaria.
Plant Pathol. 1997;46(2):209–15.
Ma S, Bohnert HJ. Integration of Arabidopsis thaliana stress-related transcript profiles, promoter
structures, and cell-specific expression. Genome Biol. 2007;8:R49.
Madgwick JW, West JS, White RP, Semenov MA, Townsend JA, Turner JA, Fitt BDL. Impacts of
climate change on wheat anthesis and fusarium ear blight in the UK. European J Plant Path.
2011;130:117–31.
Mantri NL, Ford R, Coram TE, Pang ECK. Evidence of unique and shared responses to major
biotic and abiotic stresses in chickpea. Environ Exp Bot. 2010;69:286–92.
Markell S, Khan M, Secor G, Gulya T, Lamey A (2008) Row crop diseases in drought years
NSDU-PP1371. http://www.ag.ndsu.edu/publications/landing-pages/crops/row-crop-diseases-
in-drought-years-pp-1371. Accessed 24 July 2014.
Mauch-Mani B, Mauch F. The role of abscisic acid in plant-pathogen interactions. Curr Opin Plant
Biol. 2005;8:409–14.
Mayek-Perez N, Garcia-Espinosa R, Lopez-Castaneda C, Acosta-Gallegos JA, Simpson J. Wa-
ter relations, histopathology and growth of common bean ( Phaseolus vulgaris L.) during
pathogenesis of Macrophomina phaseolina under drought stress. Physiol Mol Plant Pathol.
2002;60:185–95.
McDonald KL, Cahill DM. Influence of abscisic acid and the abscisic acid biosynthesis inhibitor,
norflurazon, on interactions between Phytophthora sojae and soybean (Glycine max). Euro-
pean Journal of Plant Pathol. 1999;105:651–8.
McElrone AJ, Sherald JL, Forseth IN. Effects of water stress on symptomatology and growth of
Parthenocissus quinquefolia infected by Xylella fastidiosa. Plant Dis. 2001;85:1160–4.
McElrone AJ, Sherald JL, Forseth IN. Interactive effects of water stress and xylem-limited bacte-
rial infection on the water relations of a host vine. J Exp Bot. 2003;54:419–30.
Melotto M, Underwood W, Koczan J, Nomura K, He SY. Plant stomata function in innate immu-
nity against bacterial invasion. Cell. 2006;126:969–80.
Mengiste T, Chen X, Salmeron J, Dietrich R. The BOTRYTIS SUSCEPTABLE1gene encodes an
R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in
Arabidopsis. Plant Cell. 2003;15:2551–65.
Mikolajczyk M, Awotunde OS, Muszynska G, Klessig DF, Dobrowolska G. Osmotic stress induc-
es rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase
ASK1 in tobacco cells. Plant Cell. 2000;12:165–78.
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. Reactive oxygen species homeostasis and signal-
ling during drought and salinity stresses. Plant Cell Environ. 2010;33:453–67.
2006;11:15–9.
Mohr PG, Cahill DM. Abscisic acid influences the susceptibility of Arabidopsis thaliana to Pseu-
domonas syringae pv.tomato and Peronospora parasitica. Funct Plant Biol. 2003;30:461–9.
Narsai R, Wang C, Chen J, Wu J, Shou H, Whelan J. Antagonistic, overlapping and distinct re-
sponses to biotic stress in rice ( Oryza sativa) and interactions with abiotic stress. BMC Ge-
nomics. 2013;14:93.
Narusaka Y, Narusaka M, Seki M, Umezawa T, Ishida J, Nakajima M, Enju A, Shinozaki K. Cross-
talk in the responses to abiotic and biotic stresses in Arabidopsis: analysis of gene expression
in cytochrome P450 gene superfamily by cDNA microarray. Plant Mol Biol. 2004;55:327–42.
Newman KL, Almeida RPP, Purcell AH, Lindow SE. Use of a green fluorescent strain for analysis
of Xylella fastidiosa colonization of Vitis vinifera. Appl Environ Microbiol. 2003;69:7319–27.
Olson AJ, Pataky JK, D’Arcy CJ, Ford RE. Effects of drought stress and infection by maize dwarf
mosaic virus on sweet corn. Plant Dis. 1990;74:147–51.
Papendick RI, Cook RJ. Plant water stress and development of Fusarium foot rot in wheat sub-
jected to different cultural practices. Phytopathol.1974;64:358–63.
Peng XX, Tang XK, Zhou PL, Hu YJ, Deng XB, He Y, Wang HH. Isolation and expression patterns
of rice WRKY82 transcription factor gene responsive to both biotic and abiotic stresses. Agri
Sci China. 2011;10:893–901.
Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM. Networking by small-molecule
hormones in plant immunity. Nature Chem Biol. 2009;5:308–16.
Prasch CM, Sonnewald U. Simultaneous application of heat, drought and virus to Arabidopsis thali-
ana plants reveals significant shifts in signaling networks. Plant physiol. 2013;162(4):1849–66.
Prasch CM, Sonnewald U Signaling events in plants: stress factors in combination change the
picture. Environ Exp Bot. 2014. doi:10.1016/j.envexpbot.2014.06.020.
Qiu Y, Yu D. Over-expression of the stress-induced OsWRKY45 enhances disease resistance and
drought tolerance in Arabidopsis. Environ Exp Bot. 2009;65:35–47.
Ramegowda V, Senthil-Kumar M, Ishiga Y, Kaundal A, Udayakumar M, Mysore KS. Drought
stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in
Nicotiana benthamiana. Int J Mol Sci. 2013;14(5):9497–513.
Rasmussen S, Barah P, Suarez-Rodriguez MC, Bressendorff S, Friis P, Costantino P, Bones AM,
Nielsen HB, Mundy J. Transcriptome responses to combinations of stresses in Arabidopsis.
Plant Physiol. 2013;161:1783–94.
Reusche M, Thole K, Janz D, Truskina J, Rindfleisch S, Drübert C, Polle A, Lipka V, Teichmann T.
Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo
xylem formation and enhances drought tolerance in Arabidopsis. Plant Cell. 2012;24:3823–37.
Ristaino JB, Duniway JM. Effect of pre-inoculation and post-inoculation water stress on the sever-
ity of Phytophthora root rot in processing tomatoes. Plant Dis. 1989;73:349–52.
Rizhsky L, Liang HJ, Mittler R. The combined effect of drought stress and heat shock on gene
expression in tobacco. Plant Physiol. 2002;130:1143–51.
2004;134:1683–96.
Rudolph K. Multiplication of Pseudomonas syringae pv. phaseolicola “in planta” I. Relation
between bacterial concentration and water-congestion in different bean cultivars and plant spe-
cies. J Phytopathol. 1984;111(3–4):349–62.
Sánchez-Vallet A, López G, Ramos B, Delgado-Cerezo M, Riviere MP, Llorente F, Fernández PV,
Miedes E, Estevez JM, Grant M, Molina A. Disruption of abscisic acid signaling constitutively
activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina. Plant
Physiol. 2012;160:2109–24.
Senthil-Kumar M, Wang K, Mysore KS. AtCYP710A1 gene-mediated stigmasterol production
plays a role in imparting temperature stress tolerance in Arabidopsis thaliana. Plant Signal
Behav. 2013;8(2):e23142.
Sether DM, Hu JS. The impact of Pineapple mealybug wilt-associated virus-1 and reduced irriga-
tion on pineapple yield. Australasian Plant Pathol. 2001;30(1):31–6.
Shaik R, Ramakrishna W. Genes and co-expression modules common to drought and bacterial
stress responses in Arabidopsis and rice. PloS ONE. 2013;8:e77261.
Shaik R, Ramakrishna W. Machine learning approaches distinguish multiple stress conditions us-
ing stress-responsive genes and identify candidate genes for broad resistance in rice. Plant
Physiol. 2014;164(1):481–95.
Shi J, Zhang L, An HL, Wu CA, Guo XQ. GhMPK16, a novel stress-responsive group D MAPK
gene from cotton, is involved in disease resistance and drought sensitivity. BMC Mol Biol.
2011;12:22.
Stiller V, Sperry JS. Cavitation fatigue and its reversal in sunflower ( Helianthus annuus L.). J Exp
Bot. 2002;53:1155–61.
New Phytol. 2014;203(1):32–43.
Szittya G, Silhavy D, Molnár A, Havelda Z, Lovas A, Lakatos L, Bánfalvi Z, Burgyán J. Low
temperature inhibits RNA silencing-mediated defence by the control of siRNA generation.
EMBO J. 2003;22:633–40.
Takahashi F, Mizoguchi T, Yoshida R, Ichimura K, Shinozaki K. Calmodulin-dependent activation
of MAP kinase for ROS homeostasis in Arabidopsis. Mol Cell. 2011;41:649–60.
Tamirisa S, Vudem DR, Khareedu VR. Overexpression of pigeonpea stress-induced cold and
drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis. J
Exp Bot. 2014; doi:1093/jxb/eru224.
Thaler JS, Bostock RM. Interactions between abscisic-acid-mediated responses and plant resis-
tance to pathogens and insects. Ecology. 2004;1:48–58.
Thorne ET, Stevenson JF, Rost TL, Labavitch JM, Matthews MA. Pierce’s disease symptoms:
comparison with symptoms of water deficit and the impact of water deficits. Am J Enol Vitic.
2006;57:1–11.
Tippmann HF, Schluter US, Collinge DB. Common themes in biotic and abiotic stress signaling
in plants, floriculture, ornamental and plant biotechnology. In Teixeira da Silva JA, editor.
Floriculture, ornamental and plant biotechnology. Advances and topical issues, vol. 3. Ikenobe:
Global Science Books; 2006. pp. 52–67.
Ton J, Flors V, Mauch-Mani B. The multifaceted role of ABA in disease resistance. Trends Plant
Sci. 2009;14:10–317.
Ton J, Mauch-Mani B. b-Amino-butyric acid-induced resistance against necrotrophic pathogens is
based on ABA-dependent priming for callose. Plant J. 2008;38:119–30.
Wang GP, Hui Z, Li F, Zhao MR, Zhang J, Wang W. Improvement of heat and drought pho-
tosynthetic tolerance in wheat by overaccumulation of glycinebetaine. Plant Biotech Rep.
2010;4:213–22.
Wang C, Deng P, Chen L, Wang X, Ma H, Hu W, Yao N, Feng Y, Chai R, Yang G, He G. A wheat

WRKY transcription factor TaWRKY10 confers tolerance to multiple abiotic stresses in trans-
genic tobacco. PLoS ONE. 2013;8(6):e65120.
Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, Frey NFd, Leung J. An update
on abscisic acid signaling in plants and more. Mol Plant. 2008;1:198–217.
Wiese J, Kranz T, Schubert S. Induction of pathogen resistance in barley by abiotic stress. Plant
Biol (Stuttg.). 2004;6:529–36.
Xiong L, Yang Y. Disease resistance and abiotic stress tolerance in rice are inversely modulated
by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell. 2003;15:745–59.
Xu P, Chen F, Mannas JP, Feldman T, Sumner LW, Roossinck MJ. Virus infection improves
drought tolerance. New Phytol. 2008;180:911–21.
Yadeta KA, Thomma BPHJ. The xylem as battleground for plant hosts and vascular wilt pathogens.
Front Plant Sci. 2013;4:97.
Yarwood CE. Deleterious effects of water in plant virus inoculation. Virology. 1955;1:268–85.
Chapter 11
Combined Stresses in Forests
Patrick Mitchell, Tim Wardlaw and Libby Pinkard
11.1 What Is Stress to a Tree or Forest Ecosystem?
Environmental stress can be viewed as the physical, chemical, and biological con-
straints on the productivity and development of ecosystems. For plants, Grime
(1977) hypothesized that stress is one of the three fundamental drivers shaping plant
strategies and he defined stress as a set of external constraints limiting the rate
of resource acquisition, growth, or reproduction (Grime 1977). Stress, in a broad
sense, is the major force limiting species distribution and ecosystem structure and
function. Forest ecosystems are maintained in a dynamic equilibrium by continu-
ous stress-inducing factors, as well as stochastic disturbance events. For example,
primary climatic stress factors can be broadly categorized as light, temperature,
and water and largely explain the distribution of biomes and forest types globally
(Boisvenue and Running 2006). Competitive and other biotic interactions are also
important in limiting species and population distribution and function. Thus, the
role of stress in triggering and shaping plant functioning is complex and can be bet-
ter understood by considering responses that arise when a particular individual or
population is exposed to conditions outside its normal operating range.
The impacts of stress on growth and development are evident at different tem-
poral scales for both individuals and populations (Fig. 11.1a). At the whole-plant
level, the initial stress response or period of decline in a process such as growth or
photosynthesis, happens within seconds to days. Acclimation can follow the ini-
tial response and involves compensation or enhanced resistance to the initial stress
P. Mitchell () · L. Pinkard
CSIRO Land and Water Flagship, Private Bag 12, Hobart,
Tasmania 7001, Australia
e-mail: patrick.mitchell@csiro.au
T. Wardlaw
Forestry Tasmania, 79 Melville Street, Hobart, Tasmania 7000, Australia

224 P. Mitchell et al.
Fig. 11.1 Representation of the different scales at which stress defines the response of plant func-
tioning in individuals and populations. a Describes changes in plant function or system state at
both the individual and population level across a range of temporal scales. The initiation of stress
in the individual results in a stress response, followed by a period of acclimation. Over generations,
adaptation can allow further recovery from stress and some return to normal conditions or the
steady state ( solid line) or a new steady state ( dashed line; modified from Lambers et al. 2008). b
An individual exposed to a permanent stress exhibits three phases of stress as proposed by Selye
(1936). The initial decline in plant function or distress can induce acute damage and may result
in loss of biomass or plant injury (disturbance). The period of recovery, termed eustress, results
from acclimation processes and may enable recovery back to the normal range ( dashed horizontal
line) or enhanced resistance to subsequent stress ( solid line). If stress persists, exhaustion occurs
whereby the plant shows chronic damage or death. (Modified from Steinberg et al. 2008)
response over days to weeks. Over longer time-scales, adaptation involves evolu-
tionary responses arising from genetic changes in the population that can alleviate
the impact of the stress (Lambers et al. 2008). Selye (1936) summarized the response
of the individual to continuous or permanent stress into a three-phase stress model
(Fig. 11.1b). The alarm phase is characterized by distress or a decline in physiologi-
cal function. The resistance phase involves recovery to the normal range in function-
ing and may include a period of acclimation that increases resistance to subsequent
stress. Finally, the exhaustion phase occurs if the stress continues or intensifies so
11 Combined Stresses in Forests 225
that chronic distress dominates any acquired resistance. In this generalized model of
stress, improved stress resistance in response to the initial stress involves energetic
costs and changes and the expression of different genes to trigger a suite of acclima-
tion processes (e.g., heat shock proteins, osmoregulatory compounds) that enhance
resistance to subsequent stress (Steinberg et al. 2008). If the stress is maintained,
exhaustion eventuates, causing chronic damage and a collapse of cellular functions
(e.g., membrane integrity, photosynthetic apparatus). While Selye’s three-phase
stress model was originally formulated to describe human physiology, it provides
a simple model of how stress-defense systems might develop in individual plants.
Because tree species are long-lived, they may be exposed to multiple cycles
of stress and/or various types of stress that act in concert to bring about changes
in plant functioning and survival. In response to a myriad of stress combinations,
trees have evolved many strategies to resist, tolerate, and recover during periods of
stress. Climate change and other human influences and disturbance have the poten-
tial to introduce novel combinations of stressors that make predicting impact from
multiple stressors exceedingly difficult. For example, changes in temperature and
atmospheric [CO2] will modify the range of “normal conditions” at which species
will operate, which could have implications for recovery rates and effectiveness of
acclimation processes during acute or chronic stress events.
To date, the study of forest stress within the fields of forest pathology, entomol-
ogy, ecology, and tree physiology has taken different perspectives regarding the sig-
nificance of multiple stressors. Forest pathology and entomology have sometimes
assumed that epidemics of insects or fungi and the associated stress were dominated
by single causal factors (Mueller-Dombois 1986). This perspective has often failed
to explain the causes and consequences of major pest outbreaks in forests, because
it tended to ignore other contributing factors such as stand-level dynamics and cli-
matic variation (Mueller-Dombois 1987; Akashi and Mueller-Dombois 1995). Plant
physiologists tend to explore stress by minimizing inherent complexities of stress
events through careful experimental manipulation that focuses on specific respons-
es to stressors such as drought/water deficit or salinity. These studies provide an
important mechanistic basis for how plants respond and cope with stress, but are
rarely of sufficient scale and design to properly consider the impact of multiple
stressors and changes in their intensity, duration, or frequency. Ecologists attempt to
explore the impacts of one or multiple stressors in the field through observation of
natural and human-induced gradients in environmental conditions. However, these
studies are often retrospective and must disentangle layers of complexity from ob-
served impacts and scant mechanistic information. A more holistic picture of forest
responses to stress involves an appreciation of the mechanistic and physiological in-
sights within the context of complex trophic interactions, spatial and temporal vari-
ation in the landscape, and their role in triggering a hierarchy of responses within a
population or ecosystem. To start gaining a deeper understanding of environmental
stress and its multifaceted nature, it is important to consider these challenges using
conceptual frameworks through which the system can be viewed.
Understanding changes in forest health in the face of rapid climate change pres-
ents further challenges surrounding how we utilize the wealth of climate projections
to predict potential stress dynamics and responses in biological systems such as for-
ests (Bonan 2014). For example, projections of reduced water availability and con-
comitant increases in temperature might be predicted with reasonable certainty for a
particular region or landscape. Yet, predicting impacts on a forest ecosystem is diffi-
cult, given that co-occurring tree species respond very differently to drought, owing
to differences in factors such as rooting patterns, water management strategies, and
ontogeny (Koepke 2010; Fensham and Fairfax 2007; Engelbrecht and Kursar 2003;
Mitchell et al. 2008). In the case of drought and many other potential environmental
drivers, the resultant physiological stress and the associated impact is not purely
defined by the exposure (i.e., climatic drivers) to stress, but also by how exposure
interacts with the sensitivity of the organism or system to produce an impact on the
system. Sensitivity encompasses many factors, including genetic/phenotypic traits,
soil conditions, and stress history for a particular site. Thus, it is important to con-
sider physiological stress for an individual as an interaction between components of
exposure and sensitivity in determining what factors are important for understand-
ing vulnerability of forests to potential stress-inducing factors (Mitchell et al. 2013).
In this chapter, we examine how different abiotic and biotic factors combine
to induce stress in trees, and its impacts on forest health more broadly. Some rel-
evant conceptual frameworks are introduced that help to disentangle interrelations
between the drivers of stress and interpret the range of impacts often described
and observed in forests under stress. Examples of combined stresses are used to
emphasize that physiological stress commonly arises through the joint contribution
of primary, secondary, anthropogenic, and conditioning factors. The relevance of
intensity, frequency, and duration of the individual and combined stress is discussed
in conjunction with how they moderate physiological distress and recovery. A large
focus of this chapter concerns stressors associated with global climate change, with
a particular emphasis on associated increases in drought. However, insights gleaned
from these examples are pertinent to many other types of stresses in natural and
managed forest ecosystems.
11.2 Conceptualizing Multiple Stressors and Their

Consequences for Forests
The causes and consequences of changes in forest health and condition can be
viewed as a continuum of responses that are related to the temporal scale at which
they impact on forest health (Fig. 11.2). At one end of this continuum lie forest
declines or diebacks, which can be characterized as a protracted malfunction of tree
health and a progressive decline in stand vigor and productivity over time (Mueller-
Dombois 1988). Forest declines tend to occur over decades or even generations
(Fig. 11.2). Forest declines tend to be driven by a combination of biotic and abi-
otic stressors, often involving multiple trophic-level interactions and a strong role
from human influences (Jurskis 2005; Manion 1981). An example of forest decline
involving complex trophic interactions is the phenomenon known as Bell miner
associated dieback in Australia. This form of forest decline became common in the
Fig. 11.2 Responses of forest ecosystems to stress can be described as a continuum based on the
duration over which the forest is impacted and the role of different factors in mediating the stress
early 1990s, and by the early 2000s was estimated to threaten 2.5 million ha of rem-
nant eucalypt forests in northern New South Wales and south-eastern Queensland
(Wardell-Johnson et al. 2005). Bell miner associated dieback is attributed to the
exclusion of natural enemies of leaf-feeding psyllids by high densities of bell miner
( Manorina melanophrys) populations. Dieback is triggered by increases in defolia-
tion from psyllids that may increase the susceptibility of trees to additional biotic
attack. Dieback in several eucalypt species was thought to be driven by multiple
feedbacks between forest structure and site conditions, physiological responses
that alter foliar chemistry, the abundance of sap-sucking psyllids, and bell miner
populations (Stone 2005). At the other end of the forest stress continuum lie those
event-driven changes in forest health caused by acute stress. These stress events
tend to operate at much shorter temporal scales (months to years) and tend to be
driven primarily by climatic factors (Jurskis 2005). As discussed in Box 1, episodic
stress events such as droughts are frequently characterized by multiple climatic and
biotic stressors; however, they tend to involve a less complicated set of feedbacks.
It is also worth pointing out that the initial trigger for both types of responses may
be quite similar, i.e., long-term drought, yet differences in the intensity, frequency,
and duration of the primary driver influence the rate at which forests are impacted.
Box 1 A surge in the awareness and study of drought impacts on forests in the last
decade is providing a glimpse of how multiple stressors might combine to affect tree
health and survival as a consequence of global environmental change (Allen et al.
2010; van Mantgem et al. 2009). Drought-induced tree die-off events are examples
of extreme stress events in forest ecosystems reflecting conditions beyond the toler-
ances of the affected tree species. A survey of published studies documenting epi-
sodic tree die-off events highlights the universal role that drought plays across a
diverse range of forest types, including semi-arid shrub lands through to tropical
rainforests (from Allen et al. 2010, Mitchell et al. 2014 and unpublished data). A
clear pattern emerging from these studies is that these extreme drought events gen-
erally coincide with elevated temperatures and heat-wave events (Allen et al. 2010;
Mitchell et al. 2014). This is a well-documented climatological phenomenon that
occurs at regional and continental scales (Vautard et al. 2007; Lyon 2011). In addi-
tion to heat stress, a large proportion of drought die-off events are associated with
biotic agents (Fig. 11.3). In this survey, 25 of the 67 die-off events had some evi-
dence of biotic agents with defoliating and wood-boring insects being most common
228
Fig. 11.3 Location of drought-induced forest die-off events across the world in relation to 12 different climatic zones. Those events associated with biotic
agents, e.g., defoliating insects, stem borers are highlighted in red
P. Mitchell et al.
and a small number of observations involving fungal infection ( unpublished data).

The majority of die-off events associated with biotic agents occurred at sites where
mean annual temperature was < 20 °C, a pattern suggesting a greater likelihood of
drought–biotic agent interactions in relatively cooler environments. It is impor-
tant to note that this survey only involves die-off events that were characterized as
episodic; relatively sudden incidences of canopy collapse and mortality during or
directly after a drought event. As we discuss in this chapter, protracted tree declines
are almost always associated with biotic agents as tree health is diminished over
several years or decades, exposing them to greater incidence of infection or attack.
Studies of protracted forest decline from many different ecosystems demonstrate
that a complete etiology of these types of stress involves a consideration of climatic,
soil, stand dynamics, physiological, genetic, and ecological factors and their distri-
bution through time and space (Mueller-Dombois 1986; Manion 1981; Landsberg
1983). Manion (1981) presented the concept of a decline syndrome that involved
three or more sets of factors to explain the complex interactions of biotic and abi-
otic factors. The first set of factors is termed predisposing; those long-term factors
that are relatively static, such as climate, site conditions, and genotypic/phenotypic
traits Manion (1981) suggested that predisposing factors weaken a plant growing
in a particularly hostile location. The second set of factors, termed inciting, induces
short-term stress on trees associated with events such as insect defoliation, frost,
drought, or air pollution. It is assumed that these events result in drastic injury to the
plant. The third set of factors is termed contributing factors, tending to operate over
the long term, because they persist on the host for extended periods. While infesta-
tion by contributing agents such as canker fungi, bark beetles, or viruses is often
thought to be the key agent inducing dieback or mortality, they may merely occur in
association with stress caused by predisposing and inciting factors. Manion’s model
provides a useful approach for clarifying the contribution of different factors when
there are multiple biotic agents present during protracted forest decline.
Episodic or acute stress arises from relatively discrete events that induce short-
(minutes to days) and long-term (months to years) responses involving plant defens-
es, productivity, and survival. One prominent framework describes severe drought
events that are associated with tree mortality and forest collapse (McDowell et al.
2008). McDowell and colleagues proposed two interrelated pathways through
which tree water and carbon balance influence the process of tree death (McDowell
et al. 2008, 2011). One physiological pathway resulting in mortality is termed hy-
draulic failure. This is caused by severe declines in tree water balance that produce
large tensions on the water column in the xylem, and cavitation involving entry of
air into the water-transport system (Tyree and Sperry 1988, 1989). The process of
cavitation is well documented and is thought to result in cell death through dehy-
dration (Brodribb and Cochard 2009). Alternatively, carbon starvation is a process
involving the exhaustion of available carbohydrates. The exhaustion of carbohy-
drates comes about through an imbalance between carbohydrate supply from pho-
tosynthesis, and demand from growth and respiration (McDowell et al. 2008). This
pathway for mortality during drought has been postulated, because water deficit can
reduce photosynthesis via stomatal closure, while respiration continues to deplete
stored carbohydrates. Additional factors such as biotic agents can amplify declines
in carbon balance, if carbon supply or transport is compromised through stressors
such as defoliators or wood borers (Galiano et al. 2011). While trees can deplete
carbohydrates during drought (Mitchell 2013; Hartmann et al. 2013; Poyatos et al.
2013), there is limited evidence for implicating carbon starvation solely for tree
mortality, because trees rarely exhaust measurable stores of carbon. However, given
our current knowledge of how plants store, translocate, and utilize carbohydrates
during drought (Sala et al. 2012), it is likely that low carbohydrate availability can
effect water transport and heighten physiological stress. This framework has helped
to stimulate much research into how primary drivers such as water deficit facilitate
the action of multiple stressors associated with plant hydraulics, carbohydrate dy-
namics, and plant defensive systems.
The other important element of McDowell’s mortality framework is that it links
exposure or the attributes of drought intensity and duration with the plant’s life-
support system (McDowell et al. 2011). For example, short and intense droughts
will reduce plant water balance, rapidly leading to hydraulic failure, and have little
effect on the availability of carbohydrates. Conversely, because carbohydrate uti-
lization is rate-limited through processes such as respiration, droughts that induce
extended periods of zero or negative carbon balance will deplete carbohydrates
(Mitchell 2013; Poyatos et al. 2013). Elevated temperatures may not only contribute
to heat stress and increased evaporative demand but also increase respiration and
the rate at which carbohydrates are depleted during long duration droughts (Adams
et al. 2009). This framework also highlights the need to understand the dynamics of
intensity and duration in defining the mechanisms underlying the observed stress.
Both of the frameworks outlined above describe interactions of multiple stress-
ors using different perspectives and levels of detail. So, how can we develop a more
generalized picture of the triggers and relationships between different factors across
the entire continuum of responses identified in Fig. 11.2? One way to view physi-
ological stress is to partition the influence of primary, secondary, anthropogenic,
and conditioning factors in influencing plant health and physiological stress (Mitch-
ell et al. 2013; Fig. 11.3). Primary factors such as drought tend to affect a forest
over large areas and at the regional scale can operate independently of other biotic
and abiotic factors. Secondary factors are dependent on the occurrence of primary
factors, but may be the sole source of stress or act in concert with the primary fac-
tor. These are typically biotic agents and their impact can be related to: changes in
host physiology or condition, climatic conditions, disturbance events, and food web
dynamics (Garrett et al. 2006). Conditioning factors include soil depth and type, the
size and age distribution of the stand and the site’s stress history. These factors have
a large influence on the spatial and temporal patterns of stress across the landscape
and can introduce considerable variation in the impacts of stress events, even within
monospecific stands. For example, meteorological drought conditions across forest
landscapes can be relatively homogenous, yet the magnitude of physiological stress
may be greater for stands on ridge top sites, where water availability is diminished
by the shallow, porous nature of the soils (Matusick et al. 2013). Over longer time-
scales, these conditioning factors promote adaptation within the populations. Ac-
climation is triggered by changes in physiological condition at a range of scales
Fig. 11.4 A generalized framework for understanding the roles of primary and secondary stress-
ors, conditioning factors, and human influences to define physiological stress. Acclimation repre-
sents a feedback on conditioning factors such as stress history and stand dynamics
and includes: changes in gene expression, biochemical changes in photosynthesis,

changes in allocation patterns (e.g., reduction in leaf area) and an upregulation of
plant defense compounds (Peñuelas et al. 2013; Breda et al. 2006). Acclimation
may also induce feedbacks affecting the activity of secondary factors that can am-
plify the physiological stress further (see examples below). Anthropogenic factors
or human influences play a role in influencing forest conditions in almost every
ecosystem through deforestation, pollution, altered fire regimes, and the introduc-
tion of invasive species. These processes alter the presence and abundance of biotic
agents at multiple trophic levels and play a major role in affecting the sensitivity of
the forest to stress through changes in soil conditions, stand density, and structure.
The overview presented in Fig. 11.4 helps to highlight some key features in un-
derstanding the dynamics of stress in forest stands. First, unraveling the sequence of
triggers for different stressors is crucial for understanding how stress comes about.
Second, it is important to consider the impact of any given stress event as part of a
longer-term regime of stressors that continually shape the sensitivity of the forest
stand to subsequent stressors (Dreesen et al. 2014). High severity events that reduce
stand density through mortality or canopy collapse can have a stabilizing effect on
forests and reduce the impact of future drought through acclimation and reductions
in competition for soil water (Lloret et al. 2012). Patterns in stress response and
recovery reflect the current state and conditioning of the system thereby influencing
the severity of future stresses (Loehle and LeBlanc 1996; Niinemets 2010). Third,
the contribution from primary, secondary, conditioning, and anthropogenic factors
will vary according to their magnitude (intensity, frequency, and duration) and how
they overlap in time and space. Some relevant examples are presented below that
highlight how plants respond to different combinations of the primary, secondary,
anthropogenic, and conditioning factors presented in Fig. 11.4.
11.2.1 Combinations of Primary Factors
At their extreme, low air temperatures can cause freezing injury including cell burst,
damage to foliar and stem tissues, and death (Clements and Ludlow 1977). At sub-
lethal levels, low temperatures capable of causing frosts inhibit rates of photosyn-
thesis through limiting the rates of the biochemical reactions of photosynthesis.
There can also be a light-dependent decrease in photosynthetic efficiency termed
cold-induced photoinhibition, which can amplify the impacts of frost (Davidson
2004). Successive sublethal frost events reduced photosynthesis of Eucalyptus
globulus and E. nitens saplings growing in southern Australia between 9 and 17 %.
High early morning light conditions following a frost event contributed to pho-
tosynthetic reduction via photoinhibition, but only before midmorning (Davidson
2004). Many tree species can acclimate to frosts which reduces photoinhibitoin
effects (Long et al. 1983), although the effectiveness of acclimation varies between
species. For example, photosynthesis of cold-acclimated E. nitens recovered within
a day following a frost event, whereas in E. globulus it took 3 days to recover to
pre-frost levels (Davidson 2004).
The combined stressors of waterlogging and salinity are common to many re-
gions where disturbance has led to an increase in dryland salinity from increased
water tables or increases in soil sodicity and reductions in infiltration (Barrett-Len-
nard and Shabala 2013). Waterlogging restricts plant growth by inducing hypoxia
in the roots resulting in diminished carbon metabolism and nutrient supply (Trought
and Drew 1980). Responses to salinity involve osmotically mediated changes in
water status and toxic effects associated with salt accumulation in tissues (Munns
and Termaat 1986). Under waterlogging and saline conditions, hypoxia exacer-
bates these toxic effects and affects plant K+ nutrition (Barrett-Lennard and Shabala
2013). The co-occurrence of waterlogging and salinity can induce similar or larger
reductions in gas exchange in eucalypt species depending on species tolerances to
either of these stressors (van der Moezel et al. 1989).
The increase in atmospheric [CO2] is thought to increase water-use efficiency
during drought due to decreases in stomatal conductance, a common response ob-
served in tree species exposed to elevated [CO2] (Ainsworth and Rogers 2007).
However, this leaf-level response may be negated where increases in leaf growth
and vegetation cover under favorable conditions enhance stress impacts during ad-
verse conditions. This has been demonstrated at the stand-level, where those stands
of Liquidambar styraciflua exposed to elevated [CO2] experienced larger declines

in leaf area index during water deficit than stands exposed to ambient conditions
(Warren et al. 2011). These patterns presumably arise because gains in plant growth
and tree size realized under elevated [CO2], increases inter-tree competition for wa-
ter during drought. Experiments assessing the interactive effect of drought, elevated
temperature, and [CO2] conclude that elevated temperature and [CO2] can amelio-
rate the effects of potentially stressful water deficits from higher temperature at
moderate drought intensities (Duan et al. 2013) but does not influence leaf water
relations and time to mortality when water deficit is extreme (Duan et al. 2014).
11.2.2 Primary and Secondary Factor Interactions
Drought affects multiple physiological pathways in trees that can influence both the
attractiveness of the host to particular pest species (e.g., increases in tissue carbo-
hydrate and nitrogen concentration) and constitutive and induced defense mecha-
nisms. Moderate water stress can promote defense through increases in secondary
metabolites in foliage (Ayres and Lombardero 2000), while severe water stress can
result in tougher foliage that reduces defoliation damage (Steinbauer 2001). How-
ever, acclimation to water stress can also enhance folivory activity in some species.
Rivas-Ubach et al. (2014) concluded that increased production of compounds as-
sociated with osmoprotection (potassium, sugars, and antioxidants), a response that
increases the tolerance of a low water potential in Quercus ilex, promoted more
severe attack from defoliating insects, highlighting the complexity of drought–pest
interactions (Fig. 11.5).
Moderate water stress can also promote secondary metabolite production in stems
(Jactel et al. 2012), resulting in increased resistance to damage from pests such as
stem borers and fungi (Fig. 11.6). Under severe water stress, the capacity of the host
to divert carbohydrates to production of defense compounds decreases (Rouault
et al. 2006), thereby reducing resistance to pests. For example, drought reduces the
capacity of Eucalyptus globulus to produce bark exudates as a defense against the
stem borer Phorocantha mastersi (Pook and Forrester 1984). Severe water deficit
can result in increased concentration of compounds favoring fungal development,
such as glucose which has been shown to stimulate growth of Armillaria spp and
enable them to grow in the presence of normally inhibitory phenols (Wargo 1996).
11.2.3 The Significance of Conditioning Factors
An important conditioning factor affecting the sensitivity of plants is stress his-

tory. Dreesen et al. (2014) examined the impacts of one-off and repeated periods
of drought and/or heat stress in herbaceous plant assemblages. Drought and heat
treatments reduced leaf survival to a larger extent than either heat or drought alone,
and the occurrence of successive drought and heat treatments with a low recovery
window (2 weeks) increased the leaf sensitivity to the combined stress treatment.
Fig. 11.5 Summary of

how acclimation to drought
stimulates increases in foli-
vory activity. An increase in
drought stress triggers (a) a
shift in the foliar metabolome
and a concomitant increase
in the concentrations of
antioxidants, nitrogen, and
sugars (b). These changes
in foliar chemistry promote
higher folivory activity on
drought-stressed trees (c).
Increases in folivory can also
stimulate an upregulation in
the concentration of plant
defensive compounds such as
terpenes and phenolics (d).
(Modified from Rivas-Ubach
et al. 2014)
Interestingly, treatments that had longer recovery times (3.5 or 6 weeks) did not af-
fect leaf and whole-plant survival (Dreesen et al. 2014). Studies in woody species
show that the impact of repeated drought on photosynthetic capacity is dependent
on the intensity and frequency of the drought regime (Liu et al. 2010). Incomplete
recovery in some species was only observed after the third severe drought cycle and
was attributed to stomatal limitations on photosynthesis (Liu et al. 2010). In another
study, the increasingly incomplete recovery of photosynthesis was associated with a
reduction in the maximum quantum efficiency relative to control plants, pointing to
significant metabolic impairment of the photosynthetic apparatus (Liu et al. 2010;
Gallé and Feller 2007).
Soil conditions can exert a strong control on the development of stress from pri-
mary climatic drivers by controlling how plants match water uptake with demand.
While a mismatch in water uptake and demand is characteristic in plants experienc-
ing drought stress, plant water uptake can be impeded in frozen soils. In boreal for-
est ecosystems, a delay in soil warming in frozen soils compared with air warming
at the end of winter can result in plant water deficit even when soils are wet (Repo
et al. 2005, 2008). The disjunct between root dormancy (or slowed metabolism)
and increased shoot growth can induce xylem cavitation (the process of air filling
and blocking xylem conduits), and lead to reductions in tree growth, photosynthetic
efficiency, and plant water potential (Larsen 1993). Thus, rapid climate change in
boreal forests that alters patterns in frost and/or soil and air temperatures has the po-
tential to introduce stress combinations via delays in thawing events and/or earlier
starts to spring-time growth.
11.2.4 Contribution from Human Influences
The effects of pollution such as nitrogen deposition can play a significant role in
forest declines in the northern hemisphere. High rates of tree mortality in Japanese
red pine ( Pinus densiflora) forests were found to be correlated with early pheno-
logical development in south-facing stands and exposure to extremely low air tem-
peratures (Shan 2000). Acid rain played a crucial role in reducing frost hardiness,
thereby increasing the sensitivity of foliage that had developed early in the growing
season (Shan 2000).
Trees in highly disturbed agricultural landscapes may succumb to stress from a
variety of sources. Landsberg and Wylie [25] proposed a conceptual model of the
initiation and development of rural dieback in Eucalyptus spp. Factors controlling
leaf nitrogen, populations of leaf feeding insects or defoliation can directly promote
dieback (Landsberg 1983). Changes in these factors arise from a variety of differ-
ent sources including: climate extremes, salinity, excessive nutrients, changes in
conditions for insects and their predators, soil compaction, and increased competi-
tion with agricultural crops. Interestingly, they showed that the nitrogen concentra-
tion of resprouting foliage produced by weakened defoliated trees made them more
attractive to leaf feeding insects (White 1984), leading to a cycle of defoliation and
incomplete recovery, progressive dieback, and sometimes mortality. The case of
rural dieback in Australian eucalypts demonstrates the multifaceted nature of some
tree declines and the problems with treating single causal factors when attempting
to manage 170 fragmented and highly disturbed forests and woodlands.
11.3 How Does the Contribution from Different Stressors

Affect the Magnitude and Direction of the Stress
Response?
It is helpful to view the impact of multiple stressors in terms of whether the com-
bination of two or more stressors produces antagonistic, additive, or synergistic
outcomes for plant function such as growth or photosynthesis. Antagonistic stress
combinations produce a response that is less than would be expected from add-
ing the impact of two hypothetical stressors, stress A and stress B (the additive
response). Synergistic stress combinations result in a response that is greater than
the additive response, implying an amplifying effect from the interaction of stress A
and stress B. Manipulative experiments that simulate two common stress combina-
tions, drought and defoliation, show that growth responses can range from antago-
nistic through to synergistic. In fast-growing E. globulus, treatments involving 50 %
defoliation at a low water availability enhanced growth relative to that of plants
in the undefoliated, low water stress treatment (Pinkard et al. 2011). One might
expect defoliation to reduce water loss under mild drought, thereby reducing the
decline in water deficit and plant growth. However, the interaction of defoliation
of Quercus robur and Pinus pinaster (85 and 50 % defoliation, respectively) and
water deficit tends to produce additive or synergistic growth outcomes (Gieger and
Thomas 2002; Jacquet et al. 2014). These conflicting results suggest that the host
physiology, conditioning factors such as tree age, and the simulated intensity of the
primary and secondary stressors can determine the magnitude and direction of the
response.
The intensity of the primary stressor has the potential to modulate the impact
of additional secondary and conditioning factors. As a primary stressor such as
drought progresses, cell expansion and growth are often the first casualties of wa-
ter deficit, followed by a decline in the rate of photosynthesis (Hsiao et al. 1976),
and the eventual breakdown of water and sugar transport (Hölttä et al. 2009). The
addition of a secondary stressor at a given drought intensity, can act to preserve or
further disrupt changes to these processes. Of the few studies to test for interactions
between stress type (drought and simulated herbivory) and intensity (moderate and
high), Bansal et al. (2013) reported a larger impact on growth in Pinus sylvestris in
the combined stress treatment than in the single stress treatments at the moderate
intensity (Bansal et al. 2013). However, under high intensity stress, the reduction
in growth was largest in the drought treatments, regardless of levels of herbivory
Fig. 11.6 Relationship between the level of damage ( effect size) associated with pests of woody
organs or foliage and a species-specific index of water stress (see text). For woody organs, condi-
tions of low water stress may promote allocation of carbohydrates produced by photosynthesis
to growth rather than defense; the host may demonstrate less resistance to pests of woody organs
under these conditions. Many studies show that under moderate levels of water stress, resistance
to pests of woody organs increases, but severely water-stressed trees are less likely to be resistant
to these pests. For foliar organs, low levels of water stress induce nutritional changes in the foli-
age that can leave trees less resistant to defoliation pests. Under moderate levels of water stress,
the host resistance to foliar pests may increase, due to the production of defense compounds in
the leaves. Under severe water stress conditions, while production of defense compounds may
be more limited, physical changes such as increases in leaf toughness may promote resistance.
(Adapted from Jactel 2012).
(Bansal et al. 2013). Recently, Jactel et al. (2012) conducted a meta-analysis of

drought studies that involved damage by insects and fungi. The study defined the
intensity of plant water deficit using a species-specific index of water stress using
plant water status and the stem hydraulic resistance to cavitation. They concluded
that the impact on tree growth or survival (derived using an effect size method)
increases with the intensity of water stress for foliar pests and pathogens, whereas
the effect from pests on woody organs was dependent on whether the insect or fun-
gal species was a primary (develop on healthy trees) or secondary (develop during
physiological stress) agent Jactel et al. (2012). Figure 11.6 summarizes some of the
key findings of the meta-analysis of Jactel et al., and shows how risk of damage
is strongly determined by the relationship between intensity of water stress and
changes in the resistance of different tree organs (foliar or woody).
Intensity-dependent responses may also arise because a highly sensitive process
such as growth ceases at a threshold water deficit (Mitchell et al. 2014) so that any
further reductions in water deficit combined with injury from biotic attack have
minimal effect on growth-related responses. Figure 11.7 illustrates a hypothetical
Fig. 11.7 A hypothetical scenario illustrating how the importance of primary, secondary, and mul-
tiple stressors changes across different stress intensities. The two stressors, herbivory ( solid black
line) and drought ( solid blue line), and their interaction for any given intensity are represented
by the drought × herbivory line ( gray, dot-dash). The trajectory of the additive impact of the
two stressors, i.e., drought + herbivory is given as a reference ( dashed line). At high intensities,
drought may reduce plant functioning to zero (death), whereas herbivory has less impact on func-
tion. The positioning of the drought x herbivory line below the additive impact line indicates a
synergistic effect (multiple stress impact is greater than the sum), and above the additive impact
line indicates an antagonistic effect (multiple stress impact is less than the sum). In this scenario,
the stress impacts of drought x herbivory on tree vigor are determined by “multiple stress effects”
at low to moderate intensities up until some threshold value ( vertical dashed line). Beyond this
threshold, the drought x herbivory line converges on the drought stress line, indicating tree func-
tion to be solely determined by the “primary stress effect” (drought in this case; after Mitchell et al.
2013). Copyright Oxford University Press.
response of a tree species to the combined stressors of drought and herbivory, and
demonstrates why predicting the impacts from stress combinations under chang-
ing intensities is notoriously complex and difficult. The impact of combined stress
on some element of plant function, e.g., growth, tends to be significant at low-to-
moderate intensities of the stress, whereas as drought stress intensifies, the primary
stressor becomes the dominant impact on plant function. This example helps to
emphasize why consideration of the appropriate physiological thresholds related
to water and carbon balance will help to differentiate between the effects of single
and multiple stressors. Thus, it is critical that researchers can measure and report
stress intensity using parameters such as soil and/or plant water potential, percent-
age change in leaf area or leaf temperature.
11.4 Recovery from Multiple Stressors
An essential component of characterizing the complete response to stress is defin-

ing recovery. Recovery can be defined as the ability of an individual to resume
prestress function, such as the return to some mean growth rate, canopy structure,
or level of productivity. Recovery from stress is often not considered in the study of
stress tolerance in herbaceous species, but for long-lived woody species, assessing
recovery from stress can provide a powerful insight into the resilience of a species
or forest ecosystem. Time to recovery can be a useful metric to understand the se-
verity of the stress event, and tends to increase with increasing impairment of plant
functioning, i.e., hydraulic dysfunction (Brodribb and Cochard 2009). Tracking the
trajectory of growth or carbon gain beyond their initial decline or distress period,
helps to reveal the degree to which the stress was transient, delayed, or sustained. It
can allow us to elucidate the key physiological recovery mechanisms involved, such
as remobilization of carbohydrates, hydraulic repair, recovery of leaf biochemistry,
or the action of heat shock proteins (Peñuelas et al. 2013). Mechanisms of recovery
are often associated with metabolic costs that delay a return to prestress levels.
Brodribb et al. (2010) found that the recovery of gas exchange via the restoration of
hydraulic conductance tracks the growth of new xylem tissues, suggesting that the
recovery imposes significant carbon costs after drought. Recovery strategies such
as resprouting, enables a canopy suffering severe damage from dieback or herbivo-
ry to be rebuilt, and for the re-establishment of prestress rates of growth and carbon
gain. While many species such as eucalypts, draw on a large store of nonstructural
carbohydrates for rapid recovery (Pinkard et al. 2011), canopy recovery in taxa
such as Pinus spp. is often limited due to a smaller pool of stored carbon (Galiano
et al. 2011; Mitchell et al. 2014), particularly following multiple stress events. Con-
trasting recovery strategies are reflected in differences in life history, ontogeny and
resource-use, and will have important consequences for water and carbon balance
during and after stress. For example, the ability to resist and recover from repeated
drought events was influenced by plant size in the resprouting Quercus ilex, pre-
sumably due to differences in rooting depth and the size of the carbohydrate store
(Lloret et al. 2004). Bansal et al. (2013), as well as others (Jacquet et al. 2013), have
shown that recovery is hampered to a greater extent by the presence of a primary
stressor such as drought, rather than the addition of a secondary agent such as her-
bivores.
11.5 How Do We Predict Responses and Impacts from

Multiple Stressors?
As highlighted in the preceding sections, forest responses to stresses are complex

and extrapolation of experimental results to situations outside experimental condi-
tions may be inappropriate. Models can play an important role in quantifying the
impacts of stress under a range of conditions or scenarios. Some progress has been
achieved in simulating the impacts of single stress-related disturbances such as fire
and harvesting on net primary productivity in Canadian boreal forests (Li et al.
2003), spruce bark beetle outbreaks in Norway (Jönsson et al. 2012) and drought
impacts on biomass stocks in the Amazon (Rammig et al. 2010). However, even
single stresses are, in general, poorly represented in models, reflecting the difficulty
in representing complex, nonlinear responses to stress, a lack of mechanistic un-
derstanding of response processes and a lack of data for model validation (Jönsson
et al. 2012; Pinkard et al. 2011). The importance of incorporating physiological re-
sponses into modeling the impacts of biotic attack is demonstrated by a recent study
into carbon exchange in a conifer forest. The magnitude of change in ecosystem
carbon fluxes during spruce beetle infestation was influenced not only by the final
rates of mortality in the two dominant species but also by carbon losses incurred
during the period in which tree growth and gas exchange were declining (Frank
et al. 2014). Thus, plant physiological responses are foundational to the prediction
of broader ecological outcomes during stress.
The challenges in modeling stress are exacerbated when considering multiple
stress dynamics arising from interplay between primary, secondary, conditioning,
and anthropogenic factors. A comparison of the capacity of six models, ranging in
scale from tree to globe, to simulate drought mortality in pinyon pine-juniper wood-
lands, found that none of the tested models dealt well with multiple stress interac-
tions such as biotic agents and drought. The authors concluded that the models were
useful for defining key processes rather than quantifying impacts (McDowell et al.
2013). There is always a trade-off in models between the need to represent complex
systems appropriately and making the models so complex it is difficult to param-
eterize them. McDowell et al’s study illustrated that models of varying complexity
can be effective in determining the likely direction of change in a system. How-
ever, quantifying the level of change at the ecosystem-level will require modeling
frameworks that consider a hierarchy of stress responses and interactions distrib-
uted across the forest landscape. While there are few if any examples of models that
can achieve this, synthesizing data from a diversity of sources, such as controlled
experiments, environmental drivers (climatic, pest dynamics, soils), stand-level
responses, and process-based/spatially explicit models could be achieved using ap-

proaches such as hierarchical Bayesian models (Cable et al. 2009; Metcalf et al.
2009; Ogle and Barber 2008).
11.6 Conclusions
The extent and magnitude of impacts from multiple stressors in forests are likely to
become larger in response to future shifts in climate and land-use intensification.
Building on existing perspectives of stress dynamics, we have presented a concep-
tual framework that allows us to generalize about the nature of stress interactions
with regard to a range of impacts, such as protracted forest declines and episodic
forest collapse. This perspective highlights some key issues in understanding the
mechanisms underlying stress in forest ecosystems: (1) The specific sequence of
triggers for different stressors is crucial for defining their physiological response
and impact; (2) any single event needs to be viewed as part of a longer-term re-
gime of stressors that continually shapes the sensitivity of the forest stand to sub-
sequent stressors; (3) the contribution from primary, secondary, conditioning, and
anthropogenic factors will vary according to their magnitude (intensity, frequency,
and duration) and how they overlap in time and space. Increasing intensity of any
given stress can lead to threshold-type responses that exacerbate or diminish effects
from other stressors; and (4) recovery patterns are facilitated by changes in water
and carbon balance and can inform overall levels of stress that are not necessarily
apparent during the period of distress. While there is much progress to be made in
translating our mechanistic understanding of multiple stressors into models for pre-
dicting broad-scale impacts, this chapter raises some pertinent issues for researchers
dealing with combined stress in complex ecosystems.
References
Adams HD, Guardiola-claramonte M, Barron-gafford GA, Camilo J. Temperature sensitivity of

drought-induced tree mortality portends increased regional die-off under global-change-type
drought. Proc Natl Acad Sci U S A. 2009;106(17):7063–6.
Ainsworth EA, Rogers A. The response of photosynthesis and stomatal conductance to rising
[CO2]: mechanisms and environmental interactions. Plant Cell Environ. 2007;30(3):258–70.
Akashi Y, Mueller-Dombois D. A landscape perspective of the Hawaiian rain forest dieback. J Veg
Sci. 1995;6(4):449–64.
Allen CD, Macalady AK, Chenchouni H, Bachelet D, McDowell N, Vennetier M, et al. A global
overview of drought and heat-induced tree mortality reveals emerging climate change risks for
forests. For Ecol Manage. 2010;259(4):660–84. doi:10.1016/j.foreco.2009.09.001.
Ayres MP, Lombardero MaJ. Assessing the consequences of global change for forest disturbance
from herbivores and pathogens. Sci Total Environ. 2000;262(3):263–86. doi:10.1016/S0048-
9697(00)00528-3.
Bansal S, Hallsby G, Löfvenius MO, Nilsson M-C. Synergistic, additive and antagonistic impacts
of drought and herbivory on Pinus sylvestris: leaf, tissue and whole-plant responses and recov-
ery. Tree Physiol. 2013;33(5):451–63.
Barrett-Lennard EG, Shabala SN. The waterlogging/salinity interaction in higher plants re-
visited-focusing on the hypoxia-induced disturbance to K + homeostasis. Funct Plant Biol.
2013;40(9):872–82.
Boisvenue C, Running SW. Impacts of climate change on natural forest productivity-evidence
since the middle of the 20th century. Glob Change Biol. 2006;12:862–82.
Bonan GB. Connecting mathematical ecosystems, real-world ecosystems, and climate science.
New Phytol. 2014;202(3):731–3.
Breda N, Huc R, Granier A, Dreyer E. Temperate forest trees and stands under severe drought: a
review of ecophysiological responses, adaptation processes and long-term consequences. Ann
For Sci. 2006;63:625–44.
Brodribb TJ, Cochard H. Hydraulic failure defines the recovery and point of death. Plant Physiol.
2009;149:575–84.
Brodribb TJ, Bowman DJMS, Nichols S, Delzon S, Burlett R. Xylem function and growth rate
interact to determine recovery rates after exposure to extreme water deficit. New Phytol.
2010;188(2):533–42.
Cable JM, Ogle K, Tyler AP, Pavao-Zuckerman Ma, Huxman TE. Woody plant encroachment
impacts on soil carbon and microbial processes: results from a hierarchical Bayesian analysis
of soil incubation data. Plant Soil. 2009;320:153–67.
Clements RJ, Ludlow MM. Frost Avoidance and Frost resistance in Centrosema virginianum. J
Appl Ecol. 1977;14(2):551–66.
Davidson NJ, Battaglia M, Close DC. Photosynthetic responses to overnight frost in Eucalyptus
nitens and E. globulus. Trees. 2004;18(3):245–52.
Dreesen FE, De Boeck HJ, Janssens IA, Nijs I. Do successive climate extremes weaken the resis-
tance of plant communities? An experimental study using plant assemblages. Biogeosciences.
2014;11(1):109–21.
Duan H, Amthor JS, Duursma RA, O’Grady AP, Choat B, Tissue DT. Carbon dynamics of eucalypt
seedlings exposed to progressive drought in elevated [CO2] and elevated temperature. Tree
Physiol. 2013;33(8):779–92.
Duan H, Duursma RA, Huang G, Smith RA, Choat B, O’Grady AP, et al. Elevated [CO2] does
not ameliorate the negative effects of elevated temperature on drought-induced mortality in
Eucalyptus radiata seedlings. Plant Cell Environ. 2014;37:1598–613.
Engelbrecht B, Kursar T. Comparative drought-resistance of seedlings of 28 species of co-occur-
ring tropical woody plants. Oecologia. 2003;136(3):383–93.
Fensham RJ, Fairfax RJ. Drought-related tree death of savanna eucalypts: species susceptibility,
soil conditions and root architecture. J Veg Sci. 2007;18(1):71–80.
Frank JM, Massman WJ, Ewers BE, Huckaby LS, Negrón JF. Ecosystem CO2/H2O fluxes are ex-
plained by hydraulically limited gas exchange during tree mortality from spruce bark beetles. J
Geophys Res Biogeosci. 2014;119:1195–1215. doi:10.1002/2013JG002597.
Galiano L, Martínez-Vilalta J, Lloret F. Carbon reserves and canopy defoliation determine the
recovery of Scots pine 4 year after a drought episode. New Phytol. 2011;190(3):750–9.
Gallé A, Feller U. Changes of photosynthetic traits in beech saplings ( Fagus sylvatica) under se-
vere drought stress and during recovery. Physiol Plant. 2007;131(3):412–21.
Garrett KA, Dendy SP, Frank EE, Rouse MN, Travers SE. Climate change effects on plant disease:
genomes to ecosystems. Annu Rev Phytopathol. 2006;44(1):489–509.
Gieger T, Thomas FM. Effects of defoliation and drought stress on biomass partitioning and water
relations of Quercus robur and Quercus petraea. Basic Appl Ecol. 2002;3(2):171–81.
Grime JP. Evidence for the existence of three primary strategies in plants and its relevance to eco-
logical and evolutionary theory. Am Nat. 1977;111(982):1169–94.
Hartmann H, Ziegler W, Kolle O, Trumbore S. Thirst beats hunger-declining hydration during
drought prevents carbon starvation in Norway spruce saplings. New Phytol. 2013;200:340–9.
Hölttä T, Mencuccini M, Nikinmaa E. Linking phloem function to structure: Analysis with a cou-
pled xylem-phloem transport model. J Theor Biol. 2009;259(2):325–37.
Hsiao TC, Acevedo E, Fereres E, Henderson DW. Water stress, growth, and osmotic adjustment.
Philos Trans R Soc Lond B Biol Sci. 1976;273(927):479–500.
Jacquet J-S, Bosc A, O’Grady AP, Jactel. H. Pine growth response to processionary moth defolia-
tion across a 40 year age chronosequence. For Ecol Manage. 2013;293:29–38.
Jacquet JS, Bosc A, O’Grady A, Jactel H. Combined effects of defoliation and water stress on pine
growth and non-structural carbohydrates. Tree Physiol. 2014;34(4): 1–10.
Jactel H, Petit J, Desprez-Loustau M-L, Delzon S, Piou D, Battisti A, et al. Drought effects on dam-
age by forest insects and pathogens: a meta-analysis. Glob Change Biol. 2012;18(1):267–76.
Jönsson AM, Schroeder LM, Lagergren F, Anderbrant O, Smith B. Guess the impact of Ips ty-
pographus—an ecosystem modelling approach for simulating spruce bark beetle outbreaks.
Agric For Meteorol. 2012;166-167(0):188–200.
Jurskis V. Eucalypt decline in Australia, and a general concept of tree decline and dieback. For
Ecol Manage. 2005;215(1-3):1–20. doi:10.1016/j.foreco.2005.04.026.
Koepke D, Kolb T, Adams H. Variation in woody plant mortality and dieback from severe
drought among soils, plant groups, and species within a northern Arizona ecotone. Oecologia.
2010;163(4):1079–90.
Lambers H, Chapin FS, Pons TL. Assumptions and approaches. Plant physiological ecology. New
York: Springer; 2008. p. 154–204.
Landsberg J. Water stress, leaf nutrients and defoliation: a model of dieback in rural eucalypts.
Aust J Ecol. 1983;8:27–41.
Larsen JB. Testing winter desiccation resistance for species and provenance selection at timber-
lines. In: Alden J, editor. Forest development in cold climates. New York: Plenum Press; 1993.
p. 59–64.
Li Z, Apps MJ, Kurz WA, Banfield E. Temporal changes of forest net primary production and net
ecosystem production in west central Canada associated with natural and anthropogenic distur-
bances. Can J For Res. 2003;33(12):2340–51. doi:10.1139/x03-168.
Liu C-C, Liu Y-G, Guo K, Zheng Y-R, Li G-Q, Yu L-F, et al. Influence of drought intensity on the
response of six woody karst species subjected to successive cycles of drought and rewatering.
Physiol Plant. 2010;139(1):39–54.
Lloret F, Siscart D, Dalmases C. Canopy recovery after drought dieback in holm-oak Mediterra-
nean forests of Catalonia (NE Spain). Glob Change Biol. 2004;10(12):2092–9.
Lloret F, Escudero A, Iriondo JM, Martínez-Vilalta J, Valladares F. Extreme climatic events and
vegetation: the role of stabilizing processes. Glob Change Biol. 2012;18(3):797–805.
Loehle C, LeBlanc D. Model-based assessments of climate change effects on forests: a critical
review. Ecol Modell. 1996;90(1):1–31. doi:10.1016/0304-3800(96)83709-4.
Long SP, East TM, Baker NR. Chilling damage to photosynthesis in Young Zea mays: I. Ef-
fects of light and temperature variation on photosynthetic CO2 assimilation. J Exp Bot.
1983;34(2):177–88.
Lyon B. Southern Africa summer drought and heat waves: observations and coupled model behav-
ior. J Climate. 2011;22(22):6033–46. doi:10.1175/2009JCLI3101.1.
Manion PD. Decline diseases of complex biotic and abiotic origin. Tree disease concepts. Engle-
wood Cliffs: Prentice-Hall; 1981. p. 324–39.
Matusick G, Ruthrof K, Brouwers N, Dell B, Hardy GJ. Sudden forest canopy collapse corre-
sponding with extreme drought and heat in a Mediterranean-type eucalypt forest in southwest-
ern Australia. Eur J Forest Res. 2013;132(3):497–510.
McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, et al. Mechanisms of plant
survival and mortality during drought: why do some plants survive while others succumb to
drought? New Phytol. 2008;178(4):719–39.
McDowell NG, Beerling DJ, Breshears DD, Fisher RA, Raffa KF, Stitt M. The interdepen-
dence of mechanisms underlying climate-driven vegetation mortality. Trends Ecol Evol.
2011;26(10):523–32.
McDowell NG, Fisher RA, Xu C, Domec JC, Hölttä T, Mackay DS, et al. Evaluating theories of
drought-induced vegetation mortality using a multimodel-experiment framework. New Phytol.
2013;200(2):304–21.
Metcalf CJE, McMahon SM, Clark JS. Overcoming data sparseness and parametric con-
straints in modeling of tree mortality: a new nonparametric Bayesian model. Can J For Res.
2009;39(9):1677–87. doi:10.1139/X09-083.
Mitchell PJ, Veneklaas EJ, Lambers H, Burgess SSO. Using multiple trait associations to de-
fine hydraulic functional types in plant communities of south-western Australia. Oecologia.
2008;158:385–97.
Mitchell PJ, Battaglia M, Pinkard EA. Counting the costs of multiple stressors: is the whole greater
than the sum of the parts? Tree Physiol. 2013;33(5):447–50.
Mitchell PJ, O’Grady AP, Tissue DT, White DA, Ottenschlaeger ML, Pinkard EA. Drought re-
sponse strategies define the relative contributions of hydraulic dysfunction and carbohydrate
depletion during tree mortality. New Phytol. 2013;197(3):862–72.
Mitchell PJ, O’Grady AP, Hayes KR, Pinkard EA. Exposure of trees to drought-induced die-
off is defined by a common climatic threshold across different vegetation types. Ecol Evol.
2014;4(7):1088–101.
Mitchell PJ, O’Grady AP, Tissue DT, Worledge D, Pinkard EA. Co-ordination of growth, gas ex-
change and hydraulics define the carbon safety margin in tree species with contrasting drought
strategies. Tree Physiol. 2014;34:443–58.
Mueller-Dombois D. Perspectives for an etiology of stand-level dieback. Annu Rev Ecol Syst.
1986;17:221–43. (ArticleType: research-article/ Full publication date: 1986/ Copyright © 1986
Annual Reviews).
Mueller-Dombois D. Natural dieback in forests. Bioscience. 1987;37(8):575–83.
Mueller-Dombois D. Towards a unifying theory for stand-level dieback. Geojournal.
1988;17(2):249–51.
Munns R, Termaat A. Whole-plant responses to salinity. Funct Plant Biol. 1986;13(1):143–60.
Niinemets Ü. Responses of forest trees to single and multiple environmental stresses from seed-
lings to mature plants: Past stress history, stress interactions, tolerance and acclimation. For
Ecol Manage. 2010;260(10):1623–39.
Ogle K, Barber J. Bayesian data-model integration in plant physiological and ecosystem ecology.
In: Lüttge U, Beyschlag W, Murata J, editors. Progress in Botany. Springer Berlin Heidelberg;
2008. p. 281–311.
Peñuelas J, Sardans J, Estiarte M, Ogaya R, Carnicer J, Coll M, et al. Evidence of current
impact of climate change on life: a walk from genes to the biosphere. Glob Change Biol.
2013;19(8):2303–38.
Pinkard EA, Eyles A, O’Grady AP. Are gas exchange responses to resource limitation and defolia-
tion linked to source: sink relationships? Plant Cell Environ. 2011;34(10):1652–65.
Pinkard EA, Battaglia M, Roxburgh S, O’Grady AP. Estimating forest net primary production
under changing climate: adding pests into the equation. Tree Physiol. 2011;31(7):686–99.
Pook EW, Forrester R. Factors influencing dieback and drought-affected dry sclerophyll forest tree
species. Aust For Res. 1984;14:201–17.
Poyatos R, Aguadé D, Galiano L, Mencuccini M, Martínez-Vilalta J. Drought-induced defoliation
and long periods of near-zero gas exchange play a key role in accentuating metabolic decline
of Scots pine. New Phytol. 2013;200:388–401.
Rammig A, Jupp T, Thonicke K, Tietjen B, Heinke J, Ostberg S, et al. Estimating the risk of Ama-
zonian forest dieback. New Phytol. 2010;187(3):694–706.
Repo T, Kalliokoski T, Domisch T, Lehto T, Mannerkoski H, Sutinen S, et al. Effects of timing of
soil frost thawing on Scots pine. Tree Physiol. 2005;25(8):1053–62.
Repo T, Lehto T, Finér L. Delayed soil thawing affects root and shoot functioning and growth in
Scots pine. Tree Physiol. 2008;28(10):1583–91.
Rivas-Ubach A, Gargallo-Garriga A, Sardans J, Oravec M, Mateu-Castell L, Pérez-Trujillo M,
et al. Drought enhances folivory by shifting foliar metabolomes in Quercus ilex trees. New
Phytol. 2014;202(3):874–85.
Rouault G, Candau J-N, Lieutier F, Nageleisen L-M, Martin J-C, Warzée N. Effects of drought and
heat on forest insect populations in relation to the 2003 drought in Western Europe. Ann For
Sci. 2006;63(6):613–24.
Sala A, Woodruff DR, Meinzer FC. Carbon dynamics in trees: feast or famine? Tree Physiol.
2012;32(6):764–75.
Selye H. A syndrome produced by diverse nocuous agents. Nature. 1936;138:32.
Shan Y. Phenological disorder induced by atmospheric nitrogen deposition: original causes of
pine forest decline over Japan. Part II. Relationship among earlier phenological development,
extreme of minimum air temperature, and forest decline of pines over the Japan. Water Air Soil
Pollut. 2000;117(1-4):205–15.
Steinbauer MJ. Specific leaf weight as an indicator of juvenile leaf toughness in Tasmanian
bluegum ( Eucalyptus globulus ssp. globulus): implications for insect defoliation. Aust For.
2001;64(1):32–7. doi:10.1080/00049158.2001.10676158.
Steinberg CEW, Stürzenbaum SR, Menzel R. Genes and environment-striking the fine balance
between sophisticated biomonitoring and true functional environmental genomics. Sci Total
Environ. 2008;400(1-3):142–61.
Stone C. Bell-miner-associated dieback at the tree crown scale: a multi-trophic process. Aust For.
2005;68(4):237–41. doi:10.1080/00049158.2005.10674971.
Trought M, Drew M. The development of waterlogging damage in young wheat plants in anaero-
bic solution cultures. J Exp Bot. 1980;31:1575–85.
Tyree MT, Sperry JS. Do woody plants operate near the point of catastrophic xylem dysfunction
caused by dynamic water stress? Answers from a model. Plant Physiol. 1988;88:574–80.
Tyree MT, Sperry JS. Vulnerability of xylem to cavitation and embolism. Ann Rev Plant Physiol
Plant Mol Biol. 1989;40(1):19–36.
van der Moezel PG, Watson LE, Bell DT. Gas exchange responses of two Eucalyptus species to
salinity and waterlogging. Tree Physiol. 1989;5(2):251–7.
van Mantgem PJ, Stephenson NL, Fulé PZ, et al. Widespread increase of tree mortality rates in the
western United States. Science. 2009;323(5913):521–4.
Vautard R, Yiou P, D’Andrea F, de Noblet N, Viovy N, Cassou C, et al. Summertime European
heat and drought waves induced by wintertime Mediterranean rainfall deficit. Geophys Res
Lett. 2007;34(7):1–5.
Wardell-Johnson G, Stone C, Recher H, Lynch AJJ. A review of eucalypt dieback associated with
bell miner habitat in south-eastern Australia. Aust For. 2005;68(4):231–6. doi:10.1080/00049
158.2005.10674970.
Wargo P. Consequences of environmental stress on oak: predisposition to pathogens. Ann For Sci.
1996;53(2-3):359–68.
Warren JM, Norby RJ, Wullschleger SD. Elevated CO2 enhances leaf senescence during extreme
drought in a temperate forest. Tree Physiol. 2011;31(2):117–30.
White TCR. The abundance of invertebrate herbivores in relation to the availability of nitrogen in
stressed food plants. Oecologia. 1984;63(1):90–105.
Chapter 12
The Interactive Effects of Drought
and Herbivory on Ecophysiology of Trees
Sheel Bansal
12.1 Introduction
The impact of drought or herbivory on tree growth and physiology can range from
moderate and recoverable to severe and lethal depending on the intensity of either
stressor. When these two stressors occur simultaneously, their combined impact on
tree performance is assumed to be synergistic, i.e., greater than expected based on
simple additive effects from either stressor alone (Niinemets 2010). This assump-
tion is fueled from repeated observations of massive forest dieback following insect
outbreaks during years with extreme drought (Ayres and Lombardero 2000; Matt-
son and Haack 1987). Drought affects a broad set of physiological processes such as
transpiration and photosynthesis, hydraulic conductivity, and carbohydrate utiliza-
tion, while herbivory elicits a number of carbon- and nitrogen-expensive defense
mechanisms (Taiz and Zeiger 2002). Thus, the two stressors complement their
negative impacts on tree ecophysiology. However, tree responses to either stressor
may trigger physiological adjustments that protect against the effects of the second
stressor (Fujita et al. 2006), thereby leading to antagonistic (less than expected)
responses to co-occurring drought and herbivory. There are thousands of published
studies on the effects of drought or herbivory, yet very few have simultaneously
considered their combined impacts on tree performance (Bansal et al. 2013; Trow-
bridge et al. 2014). Unfortunately, studies on multiple stressors frequently show
non-additive effects (i.e., synergistic or antagonistic), and therefore the combined
effects cannot be predicted based on results from single-stressor studies. Given that
both drought events and biotic stressors (e.g., insect outbreaks) are expected to occur
with increased frequency and intensity with climate change (Mitchell et al. 2013),
research on the effects from these combined stressors on tree growth and physiol-
ogy is critical for predicting future forest health and productivity.
S. Bansal ()
USDA-Forest Service, Pacific Northwest Research Station,
3625 93rd Avenue SW, Olympia, WA 98512, USA
e-mail: sheelbansal9@gmail.com

246 S. Bansal
12.2 Drought Alone
The environmental condition known as “drought” can be defined simply as the

condition when soil moisture falls below a minimum threshold causing reduced
plant growth (Bréda et al. 2006). However, under natural scenarios, “drought” can
be episodic or chronic, moderate or severe, and caused by warm temperatures, low
precipitation, high vapor pressure deficit, intense solar radiation, salt accumulation,
and/or freezing soils (Larcher 2003). Also, the effects of drought on plant ecophysi-
ology are a function of innate adaptive plant traits to conserve water, current plant
health and vigor, and competitive interactions to acquire limited soil moisture s upply.
Consequently, the impacts of drought on growth and physiology are context-depen-
dent on a number of external and internal factors. Nevertheless, the physiological
responses of trees to drought have many similarities across species. This topic has
been well reviewed, particularly in recent years, because the traditional mechanisms
assumed to cause drought-induced tree mortality are being questioned and expanded
upon (McDowell et al. 2008; McDowell 2011; Ryan 2011; Sala et al. 2010).
The fast-acting responses of trees to drought occur at the genetic, biochemical,
and cellular levels (Fig. 12.1; Osakabe et al. 2014). Soil drying and desiccation
of root tissues trigger the expression of drought-induced genes that synthesize
various hormones, in particular abscisic acid (ABA; Fig. 12.2; Chaves et al. 2003).
Fig. 12.1 Genetic, biochemical, and hormonal signaling factors in stomatal closure and retrograde
signaling during water stress. (Figure from Osakabe et al. 2014)
12 The Interactive Effects of Drought and Herbivory on Ecophysiology of Trees 247
Fig. 12.2 Whole-plant responses to drought stress. Left, long-term or acclimation responses; right,
short-term response. (Figure from Chaves et al. 2003)
ABA is a ubiquitous plant hormone that activates several physiological processes

in response to environmental stress. In the case of drought stress, ABA is trans-
located to leaf tissues from roots, and there it binds to the plasma membrane of
stomatal guard cells (Taiz and Zeiger 2002). The result is a flux of ions across
the cell membrane, leading to rapid osmotic adjustments, shrinkage, and closure
of the stomatal guard cells. ABA-induced stomatal closure is a key physiologi-
cal mechanism to quickly limit water loss and to increase water-use efficiency,
particularly for isohydric species. However, stomatal closure may not be 100 %
effective at constraining water vapor loss (“leaky stomata”), and water vapor also
diffuses through the cuticular membranes that enclose leaf tissues (cuticular tran-
spiration; Burghardt and Riederer 2003; Kerstiens 1996; Schreiber and Riederer
1996). Moreover, stomatal closure comes at significant costs of reduced CO2 up-
take for photosynthesis, thus limiting the production of carbon assimilates that are
needed for growth, maintenance, reproduction, and/or the production of defense
compounds against herbivores (Kempel et al. 2011).
There are many longer-term morphological adjustments that occur in response
to drought stress. At the leaf level, reduced turgor pressure in cells decreases
growth rates, in particular the process of cellular elongation (Meier et al. 1992).
Reduced elongation is evident in droughted plant leaves as a decrease in the ratio
of leaf area to leaf mass (i.e., lower specific leaf area; Abrams et al. 1994). Lower
specific leaf area has adaptive value for plants exposed to chronic moisture stress
because there is less leaf area for water loss through stomatal or cuticular transpira-
tion (Grace 1990; Bansal et al. 2014). Unfortunately, lower specific leaf area also
248 S. Bansal
reduces leaf area for light interception and CO2 uptake, which has negative impacts
on photosynthetic carbon assimilation rates (Oren et al. 1986).
At the whole-plant level, large-scale redistribution of carbon assimilates can be
used to further minimize water loss and to increase soil moisture uptake. Specifi-
cally, plants typically undergo an increase in the ratio of root-to-shoot biomass,
an increase in rooting depth and root density, and leaf shedding or abscission in
response to drought (Larcher 2003). While these changes may be critical for plant
survival during periods of extreme drought stress, they also come at a severe cost to
carbon uptake and assimilation.
If drought conditions persist, even extreme physiological and morphological
adaptive responses may not adequately prevent dysfunction of basic processes
necessary for survival (Sevanto et al. 2014). If water loss continues via cuticular
transpiration, even after water uptake by roots has diminished, tension builds up
on the transpiration stream in the xylem (i.e., more negative xylem water pres-
sure), particularly for trees because of their high transpirational areas and long dis-
tances to transport water (Taiz and Zeiger 2002). With increasing tension, hydraulic
conductance of water to leaves from roots is eventually disrupted by cavitations and
embolisms of air bubbles into the xylem stream. These breaks in the water column
can quickly lead to 100 % loss of hydraulic conductivity, although the extent that
plants are vulnerable to cavitations under conditions of negative xylem water pres-
sure differs greatly among plant taxa (Cochard 1992; Maherali et al. 2004; Tyree
and Ewers 1991).
Hydraulic failure has been the traditional mechanism assumed to cause mortality
in trees exposed to frequent and severe drought events. However, as described above,
many of the ecophysiological responses to cope with drought stress reduce carbon
assimilation, which have led to the development of a newer “carbon-starvation”
hypothesis regarding drought-induced tree mortality (McDowell et al. 2008;

Sala et al. 2010). While hydraulic failure is expected to cause tree mortality rela-
tively quickly, carbon starvation is hypothesized to take place over longer periods
of time in which plants experience negative carbon balances (i.e., greater carbon
use than carbon gain). As trees become depleted in carbohydrates, they are unable
to meet metabolic demands for basic functioning, or to biosynthesize carbon-rich
defense compounds necessary against biotic agents (Fig. 12.3; Gutbrodt et al. 2011;
McDowell 2011). Clearly, these consequences of carbon starvation have direct
implications for tree–herbivore relationships.
12.3 Herbivory Alone
Herbivory can be defined as the consumption of plant material, often occurring on

living plants, but not always lethal (Ohgushi 2005). However, this simple defini-
tion is one of the only ubiquitous generalizations that can be made regarding the
impact of herbivory on plant performance. The reason being that the effects of her-
bivory are context-dependent on a number of factors, including the herbivore func-
Fig. 12.3 Theoretical relationship, based on the hydraulic framework, between the temporal
length of drought (duration), the relative decrease in water availability (intensity), and the three
hypothesized mechanisms underlying mortality. Carbon starvation is hypothesized to occur when
drought duration is long enough to curtail photosynthesis longer than the equivalent storage of car-
bon reserves for maintenance of metabolism. Hydraulic failure is hypothesized to occur if drought
intensity is sufficient to push a plant past its threshold for irreversible desiccation before carbon
starvation occurs. Biotic agents, such as insects and pathogens, can amplify or be amplified by
both carbon starvation and hydraulic failure. (Figure from McDowell et al. 2008)
tional group (e.g., insects, mites, mammals), feeding behavior (e.g., d efoliators,
phloem-feeders, cell-content feeders), and stage of host physiological develop-
ment (e.g., seed, juvenile, adult; Agrawal 1998; Karban and Myers 1989). In addi-
tion, the impacts from herbivory may be confounded by hitchhiker pathogens such
as parasites, bacteria, fungi, and viruses that are often introduced during feeding
(Hatcher 1995; Trapp and Croteau 2001). Among herbivores, trees are probably
most affected by phytophagous insects, and entire forests have been decimated
from beetles, moths, weevils, budworms, and caterpillars (Ayres et al. 2014). Her-
bivore-induced plant responses include the production of secondary metabolites,
physical deterrents, compensatory physiology and growth, and tissue abscission
(Agrawal 1998; Strauss and Agrawal 1999). Many of these responses come with
high carbon costs (Dungan et al. 2007), similar to the impacts from drought stress.
Like drought, plant responses to herbivory begin at the subcellular level
(Fig. 12.4; Wu and Baldwin 2009). Wounding of plant tissues from feeding or the
injection of foreign compounds from herbivores initiates the release of hormones
such as jasmonic acid (JA), elicits defense-related genes, and increases the produc-
tion and modification of secondary metabolites (Karban and Myers 1989; Kessler
and Baldwin 2002). The ultimate goal of these defense compounds is to reduce the
preference for the host plant or the performance of the herbivore. Secondary me-
tabolites are generally categorized as terpenoids or phenolics, which are carbon-rich
allelochemicals such as flavonoids, tannins, and lignins, or as nitrogen-containing
250 S. Bansal
Fig. 12.4 A model summarizing early signaling events in herbivore-attacked plants. After
h erbivore attack, herbivore elicitors (here FAC??) bind to putative receptors on plasma mem-
branes and activate further responses. Through an unknown mechanism, Ca2+ influx is initiated,
which depolarizes cell membranes. Increased Ca2+ (likely together with a CDPK) greatly enhances
NADPH oxidases located in cell membrane and leads to ROS production. MAPKs (at least SIPK
and WIPK) are quickly activated; they transcriptionally regulate many genes involved in JA and
ethylene biosynthesis, as well as NADPH oxidase and WRKY transcription factors (TFs). SIPK
is likely also involved in NO production; both ROS and NO modify amino acids in proteins and
induce transcriptional changes of various defense-related genes. A yet to-be-indentified pathway
triggers JA biosynthesis. JA is further converted to JA-Ile by JAR; binding of JA-Ile to SCFCOI1
initiates the degradation of JAZ proteins that negatively regulate JA-responsive genes. Without
phosphorylation, ACS is degraded through 26S proteasome pathway; after being phosphorylated
by SIPK, it gains higher stability and enhances ethylene biosynthesis. Red arrows represent phos-
phorylation; blue arrows represent transcriptional regulation. AOC allene oxide cyclase, AOS allene
oxide synthase, CDPK calcium-dependent protein kinase; FAC Fatty acid chains; JAZ jasmonate
ZIM-domain, LOX lipoxygenase, OPDA 12-oxo-phytodienoic acid, OPR3 OPDA reductase 3, NO
nitric oxide, NOA NO-associated protein, NR nitrate reductase, ROS reactive oxygen species, SCF
Skp, Cullin, F-box, SIPK salicylic acid-induced protein kinase, WIPK wound-induced protein
kinase. (Figure from Wu and Baldwin 2009)
compounds, which include alkaloids, cyanogenic glycoside, and lectins (Taiz and
Zeiger 2002). Because these compounds are carbon and nutrient expensive to plants,
they are only produced when necessary, especially those that have no a pparent func-
tion toward growth or development (Langenheim 1990; Poorter and Villar 1997).
Conifer trees utilize volatile monoterpenes as a primary defense against insect her-
bivory and exude oleoresins following wounding, which c onstitute large carbon
investments into compounds that are not recycled and u ltimately lost to the environ-
ment (Croteau and Johnson 1985; Trapp and Croteau 2001; Trowbridge et al. 2014).
Plants also undergo defensive, morphological adjustments to protect against
herbivory (Hanley et al. 2007). These changes can include the production of
external thorns, prickles, spines, and hairs (Myers and Bazely 1991) or an increase
in epicuticular waxes, cutins, and suberins (Eigenbrode and Espelie 1995). In addi-
tion, plants can cope with herbivory through repair of wounded tissues, abscission
of infected tissues, or compensatory regrowth of lost tissues (Neely 1970). Like
chemical defenses, morphological changes comes at large carbon investments for
the host plant, which increase survivorship but at the cost of reduced growth, repro-
duction, and carbohydrate storage (Fig. 12.5; Agrawal 2011; Dungan et al. 2007;
Orians et al. 2011).
Insect herbivores have additional impacts on plant carbon balance beyond the
induction of plant chemical and morphological defense responses. Consumption of
leaf tissues by defoliators reduces the amount of leaf area available for photosyn-
thetic carbon assimilation, while phloem-feeders (such as weevils or beetle larvae)
directly consume phloem sap sugars as they are transported through the stems from
leaves to roots (Karban and Myers 1989). Both of these impacts from herbivory
can have tremendous consequences on carbohydrate reserves that are needed for
growth, reproduction, and metabolic functions, thus reducing plant vigor and ulti-
mately leading to mortality.
12.4 Drought Combined with Herbivory
When trees experience more than one stressor simultaneously, complimentary

impacts on related physiological processes may turn an otherwise recoverable situ-
ation into catastrophic dysfunction and mortality. From the above descriptions on
the impacts of drought stress or herbivory alone, it is clear that there are several
ecophysiological mechanisms that would be negatively impacted if both stressors
co-occurred. Herbivory tends to increase carbon demands, while drought stress
decreases carbon gain, making it easy to assume that the two stressors combined
will have a synergistic, negative effect on plant carbon balance. Thus far, very few
studies have empirically tested the interactions of drought and herbivory on plant
performance, particularly for trees (Bansal et al. 2013; Trowbridge et al. 2014).
One study that explicitly tested for synergistic, additive, or antagonistic effects
from drought combined with herbivory (simulated phloem-feeding weevils in this
case) on ecophysiology of Pinus sylvestris seedlings found, contrary to expectations,
that many traits were affected antagonistically (Fig. 12.6; Bansal et al. 2013). Spe-
cifically, gas exchange and growth rates were sharply reduced when both stressors
co-occurred, although the total, combined effects were less than expected based
on additive effect of either stressor alone. While these findings were unantici-
252 S. Bansal
Fig. 12.5 Conceptual model for resource flows in plants. The labile resource pool is derived from
newly captured pools of carbon and nutrient pools or from remobilized storage reserves. The
labile carbon pool is generated from photosynthesis, primarily by mature source leaves. The labile
nutrient pool is obtained from roots. The resulting labile resource pool can then be allocated to
support the growth of sink tissues (roots, leaves, or reproductive tissues), to defense traits, and
to storage tissues. Herbivore-induced export of resources from leaves or from fine roots ( dashed
arrows) into stems and storage roots functions to sequester resources in tissues inaccessible to
the respective herbivores but may incur opportunity costs if resources allocated for storage limit
growth and reproduction or ecological costs if other enemies specialize on these storage tissues.
(Figure from Orians et al. 2011)
pated, there are a few biological mechanisms that could explain the results. First,
exposure to drought stress or herbivore wounding may have triggered a series
of stress-induced genes and physiological responses that “primed” or protected
the trees from the second, co-occurring stressor (Bowler and Fluhr 2000; Fujita
et al. 2006; Leshem and Kuiper 1996; Rennenberg et al. 2006). For example, some
studies have shown a short-term increase in resin acid concentrations in plants ex-
posed to moderate drought stress (Turtola et al. 2003), which could facilitate wound
healing and monoterpene emissions to cope with herbivory. Pinus taeda showed an
Fig. 12.6 The combined impacts from drought and herbivory on various plant traits were syner-
gist, additive, or antagonistic (greater than, equal to, or less than expected effects, respectively,
based on single stressor effect sizes). The bars represent the overall effect size difference (mean
±95 % CI) between the observed and expected additive effects from combined drought and her-
bivory on morphological traits at final harvest and second-year physiological traits of P. sylvestris
seedlings. The zero line represents the expected additive effects from combined stressors. When
the means (and their 95 % confidence limits) were greater than or less than the zero line, they were
considered synergistic or antagonistic, respectively. (Figure from Bansal et al. 2013)
increase in defense resin synthesis in response to soil moisture stress despite a de-
crease in growth rates (Lorio and Sommers 1986). This phenomenon of interacting
responses to multiple stressors has also been documented for the combined effects
of fire and herbivory on Pinus radiata, in which tree exposed to fire had increased
antiherbivore resin defenses that provided protection against subsequent bark beetle
attacks (Lombardero and Ayres 2011).
A second possibility for antagonistic effects from drought and herbivory on
tree ecophysiology was that the impact of drought stress overrode the effects of
herbivory. A study conducted in situ that monitored monoterpene emissions of
Pinus edulis found the influence of soil moisture was relatively strong compared
to herbivory during the midsummer drought (Trowbridge et al. 2014). However,
they also showed how herbivory played a dominant role in affecting plant defenses
during periods of the growing season with higher soil water availability, thus dem-
254 S. Bansal
onstrating the importance of stressor severity and temporal variability on the inter-
action of drought and herbivory.
Even though Bansal et al. (2013) generally found antagonistic effects of drought
and herbivory on tree physiology and growth, there were two key functional traits,
specific leaf area and water-use efficiency (ratio of carbon gain to water loss), that
exhibited relatively strong, synergistic effects from the combined impact of the two
stressors (Fig. 12.6). These two traits are particularly important to resource-use
efficiency, carbon gain and allocation, and survival (Reich et al. 1997). Drough-
ted seedlings had decreased shoot biomass and needle size, thus reducing water
demands disproportionately to water supply. Those morphological adjustments
allowed the seedlings to maintain relatively high water saturation for individual
needles, which in turn led to an increase in specific leaf area in response to drought.
The increase in water-use efficiency of droughted seedlings was driven by stomatal
closure in response to decreased soil moisture. Unlike with drought, the mecha-
nisms leading to an increase in specific leaf area and water-use efficiency from
herbivory were less clear, as were the mechanisms driving the synergistic effects
from both stressors combined. Regardless of the mechanisms involved, the syner-
gistic increase in specific leaf area increased light-capturing area for photosynthetic
carbon gain, while the increase in water-use efficiency decreased carbon gain but
conserved relatively more water under droughted conditions. Consequently, these
two synergistic effects aided in establishing seedlings and coping with multiple
stressors.
Intensity-dependent effects from multiple stressors have rarely been explored.
In Bansal et al. (2013), stressor intensity played an important role in determining
the impact of drought or herbivory alone but also affected how the two stressors
interacted. For example, the cumulative effects from the two stressors on height,
diameter, and shoot biomass were stronger (synergistic or additive) when both
stressors were of moderate intensity, but were antagonistic when either stressor was
severe. This suggests that co-occurring stressors at lower intensity could have a
disproportionate, negative impact on seedling growth (Mitchell et al. 2013). In con-
trast, the combined effects of drought and herbivory were stronger on needle length
and gas exchange when drought stress was severe, irrespective of herbivore intensi-
ty, thus demonstrating how the effects of multiple stressors are also trait-dependent.
12.5 Drought Effects on Herbivores and Plant–Herbivore

Interactions
Clearly, drought and herbivory has many overlapping consequences on tree eco-
physiology. However, from an ecological perspective, drought also has a direct
effect on herbivorous insect populations. In addition, the changes in plant chemis-
try that occur from drought can affect herbivore feeding preferences, thus altering
plant–insect interactions (Gutbrodt et al. 2011; Mattson and Haack 1987).
Fig. 12.7 Kaplan–Meier survivorship of Lochmaea suturalis larvae over time for plots with
elevated treatments ( N = 24 per time point, solid lines) and ambient plots ( N = 24 per time point,
broken lines). Gray shaded areas show 95 % confidence intervals of the Kaplan–Meier estimator.
Significance of interactions with time: a P = 0.057 and b P < 0.0001. c Warming was only significant
in a three-way interaction with CO2 and drought ( P = 0.019). (Figure from Scherber et al. 2013)
Drought can have either negative or positive impacts on herbivore populations,

depending on the severity and duration of the drought. Moisture stress typically has
negative impacts on the fitness of developing larvae (Fig. 12.7; Scherber et al. 2013).
However, drought is frequently associated with warmer temperatures, which accel-
erates insect metabolism, leading to faster growth, consumption, and developmental
rates (Jamieson et al. 2012). Warmer winter temperatures in particular tend to
enhance insect overwintering survival (Bale et al. 2002; Bentz et al. 2010), as well as
induce earlier emergence and phenological development (Parmesan and Yohe 2003).
Consequently, the effects of drought on herbivorous insect populations are not unidi-
rectional and context-dependent.
The direct effects of drought on herbivore populations are confounded by indirect
effects of drought on their forage quality, i.e., on host plant ecophysiology (Bau-
erfeind and Fischer 2013; Koricheva et al. 1998; Rouault et al. 2006). There has
been a long-standing “plant stress hypothesis” which states that plants under abiotic
stress have lower defensive capabilities and are therefore more suitable as a food
source for herbivorous insects (Mattson and Haack 1987; White 1974; White 1984).
Temporal correlations between drought events and insect outbreaks support this hy-
pothesis (Hart et al. 2014). Alternatively, the “plant vigor hypothesis” predicts that
insects will preferentially feed on faster-growing, healthier plants that have higher
nutritional content and lower defense compounds (Price 1991). A suite of meta-
analyses, modeling, and manipulative studies have shown that drought stress does
not consistently lead to increased or decreased insect consumption, and is often de-
pendent on species, feeding guild, and specialization of the herbivores (Bauerfeind
and Fischer 2013; Grinnan et al. 2013; Haynes et al. 2014; Huberty and Denno
2004; Larsson 1989; Rouault et al. 2006). A unique study conducted by Gutbrodt
et al. (2011) elegantly demonstrated how the effects of drought stress on plant tissue
256 S. Bansal
moisture content (health) and secondary metabolite concentrations (defense) led to

differing feeding preferences for a generalist compared to a specialist herbivore.
These changes in feeding behavior that occur on drought-stressed plants will likely
impact subsequent insect population dynamics, and further influence plant vigor
and chemistry, thus creating a feedback system and further complicating the interac-
tion of drought and herbivory.
12.6 Conclusions
In the environment, the co-occurrence of multiple environmental stressors is the rule

rather than the exception (Chapin et al. 1987; Niinemets 2010; Vierling and Kimpel
1992). Global climate change is expected to increase the frequency and intensity
of drought events and herbivorous insect outbreaks (Allen et al. 2010; Bale et al.
2002; Vinebrooke et al. 2004; Williams and Jackson 2007), thereby increasing the
probability that the two will co-occur in the future. Moreover, expected increases in
other stressors, such as extreme heat events and wildfires, could exacerbate condi-
tions beyond a critical threshold of plant tolerance. Consequently, forests worldwide
are at increased risk of extreme dieback. For drought and herbivory in particular,
their combined, negative impact on tree carbon balance has important implications
for forest productivity and carbon sequestration at global scales. Therefore, improv-
ing our understanding of the interacting effects of multiple stressors on tree growth
and physiology is crucial (but poorly investigated) for managing future forests.
References
Abrams MD, Kubiske ME, Mostoller SA. Relating wet and dry year ecophysiology to leaf struc-
ture in contrasting temperate tree species. Ecology. 1994;75:123–33.
Agrawal AA. Induced responses to herbivory and increased plant performance. Science.
1998;279(5354):1201–2.
Agrawal AA. Current trends in the evolutionary ecology of plant defence. Funct Ecol.
2011;25(2):420–32.
Allen CD, Macalady AK, Chenchouni H, Bachelet D, McDowell N, Vennetier M, et al. A global
overview of drought and heat-induced tree mortality reveals emerging climate change risks for
forests. For Ecol Manage. 2010;259(4):660–84.
Ayres MP, Lombardero MJ. Assessing the consequences of global change for forest disturbance
from herbivores and pathogens. Sci Total Environ. 2000;262(3):263–86.
Ayres MP, Hicke JA, Kerns BK, McKenzie D, Littell JS, B and LE, et al. Disturbance regimes
and stressors. In: Peterson DL, Vose JM, Patel-Weynand T, editors. Climate change and United
States forests. New York: Springer; 2014. pp. 55–92.
Bale JS, Masters GJ, Hodkinson ID, Awmack C, Bezemer TM, Brown VK, et al. Herbivory in
global climate change research: direct effects of rising temperature on insect herbivores. Glob
Change Biol. 2002;8(1):1–16.
Bansal S, Hallsby G, Löfvenius MO, Nilsson M-C. Synergistic, additive and antagonistic impacts
of drought and herbivory on Pinus sylvestris: leaf, tissue and whole-plant responses and recov-
ery. Tree Physiol. 2013;33(5):451–63.
Bansal S, Harrington CA, Gould PJ, St. Clair JB. Climate-related genetic variation in
drought-resistance of Douglas-fir (Pseudotsuga menziesii). Global Change Biol. 2014; doi:
10.1111/gcb.12719
Bauerfeind SS, Fischer K. Testing the plant stress hypothesis: stressed plants offer better food to
an insect herbivore. Entomol Exp Appl. 2013;149(2):148–58.
Bentz BJ, Régnière J, Fettig CJ, Hansen EM, Hayes JL, Hicke JA, et al. Climate change and
bark beetles of the Western United States and Canada: direct and indirect effects. Bioscience.
2010;60(8):602–13.
Bowler C, Fluhr R. The role of calcium and activated oxygens as signals for controlling
cross-tolerance. Trends Plant Sci. 2000;5(6):241–6.
Bréda N, Huc R, Granier A, Dreyer E. Temperate forest trees and stands under severe drought: a
review of ecophysiological responses, adaptation processes and long-term consequences. Ann
Sci For. 2006;63(6):625–44.
Burghardt M, Riederer M. Ecophysiological relevance of cuticular transpiration of deciduous
and evergreen plants in relation to stomatal closure and leaf water potential. J Exp Bot.
2003;54(389):1941–9.
Chapin FS, Bloom AJ, Field CB, Waring RH. Plant responses to multiple environmental factors.
Bioscience. 1987;37(1):49–57.
Chaves MM, Maroco JP, Pereira JS. Understanding plant responses to drought—from genes to the
whole plant. Funct Plant Biol. 2003;30(3):239–64.
Cochard H. Vulnerability of several conifers to air embolism. Tree Physiol. 1992;11(1):73–83.
Croteau R, Johnson MA. Biosynthesis of terpenoid wood extractives. In: Higuchi T, editor.
Biosynthesis and biodegradation of wood components. London: Academic; 1985. pp. 379–439.
Dungan RJ, Turnbull MH, Kelly D. The carbon costs for host trees of a phloem-feeding herbivore.
J Ecol. 2007;95(4):603–13.
Eigenbrode SD, Espelie KE. Effects of plant epicuticular lipids on insect herbivores. Annu Rev
Entomol. 1995;40(1):171–94.
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, et al. Crosstalk
between abiotic and biotic stress responses: a current view from the points of convergence in
the stress signaling networks. Curr Opin Plant Biol. 2006;9(4):436–42.
Grace J. Cuticular water loss unlikely to explain tree-line in Scotland. Oecologia. 1990;84(1):64–8.
Grinnan R, Carter T, Johnson MT. The effects of drought and herbivory on plant-herbivore interac-
tions across 16 soybean genotypes in a field experiment. Ecol Entomol. 2013;38(3):290–302.
Gutbrodt B, Mody K, Dorn S. Drought changes plant chemistry and causes contrasting responses
in lepidopteran herbivores. Oikos. 2011;120(11):1732–40.
Hanley ME, Lamont BB, Fairbanks MM, Rafferty CM. Plant structural traits and their role in
anti-herbivore defence. Perspect Plant Ecol Evol Syst. 2007;8(4):157–78.
Hart SJ, Veblen TT, Eisenhart KS, Jarvis D, Kulakowski D. Drought induces spruce beetle
( Dendroctonus rufipennis) outbreaks across northwestern Colorado. Ecology. 2014;95:930–9.
Hatcher PE. Three-way interactions between plant pathogenic fungi, herbivorous insects and their
host plants. Biol Rev. 1995;70(4):639–94.
Haynes KJ, Allstadt AJ, Klimetzek D. Forest defoliator outbreaks under climate change: effects
on the frequency and severity of outbreaks of five pine insect pests. Glob Change Biol.
2014;20:2004–18.
Huberty AF, Denno RF. Plant water stress and its consequences for herbivorous insects: a new
synthesis. Ecology. 2004;85(5):1383–98.
Jamieson MA, Trowbridge AM, Raffa KF, Lindroth RL. Consequences of climate warming and
altered precipitation patterns for plant-insect and multitrophic interactions. Plant Physiol.
2012;160(4):1719–27.
Karban R, Myers JH. Induced plant responses to herbivory. Annu Rev Ecol Syst. 1989;20(1):331–48.
Kempel A, Schädler M, Chrobock T, Fischer M, van Kleunen M. Tradeoffs associated with
constitutive and induced plant resistance against herbivory. Proc Natl Acad Sci U S A.
2011;108(14):5685–9.
258 S. Bansal
Kerstiens G. Cuticular water permeability and its physiological significance. J Exp Bot.
1996;47(12):1813–32.
Kessler A, Baldwin IT. Plant responses to insect herbivory: the emerging molecular analysis. Annu
Rev Plant Biol. 2002;53(1):299–328.
Koricheva J, Larsson S, Haukioja E. Insect performance on experimentally stressed woody plants:
a meta-analysis. Annu Rev Entomol. 1998;43(1):195–216.
Langenheim JH. Plant resins. Am Sci. 1990;78:16–24.
Larcher W. Physiological plant ecology: ecophysiology and stress physiology of functional groups.
4th ed. New York: Springer; 2003. p. 513.
Larsson S. Stressful times for the plant stress: insect performance hypothesis. Oikos.
1989;56:277–83.
Leshem Y, Kuiper P. Is there a GAS (general adaptation syndrome) response to various types of
environmental stress? Biol Plant. 1996;38(1):1–18.
Lombardero MJ, Ayres MP. Factors influencing bark beetle outbreaks after forest fires on the
Iberian Peninsula. Environ Entomol. 2011;40(5):1007–18.
Lorio PL, Sommers RA. Evidence of competition for photosynthates between growth processes
and oleoresin synthesis in Pinus taeda L. Tree Physiol. 1986;2:301–6.
Maherali H, Pockman WT, Jackson RB. Adaptive variation in the vulnerability of woody plants to
xylem cavitation. Ecology. 2004;85(8):2184–99.
Mattson WJ, Haack RA. The role of drought in outbreaks of plant-eating insects. Bioscience.
1987;37(2):110–8.
McDowell N. Mechanisms linking drought, hydraulics, carbon metabolism, and vegetation
mortality. Plant Physiol. 2011;155(3):1051–9.
McDowell N, Pockman WT, Allen CD, Breshears DD, Cobb N, Kolb T, et al. Mechanisms of plant
survival and mortality during drought: why do some plants survive while others succumb to
drought? New Phytol. 2008;178(4):719–39.
Meier C, Newton R, Puryear J, Sen S. Physiological responses of Loblolly pine ( Pinus taeda)
seedlings to drought stress: osmotic adjustment and tissue elasticity. J Plant Physiol.
1992;140(6):754–60.
Mitchell PJ, Battaglia M, Pinkard EA. Counting the costs of multiple stressors: is the whole greater
than the sum of the parts? Tree Physiol. 2013;33(5):447–50.
Myers JH, Bazely D. Thorns, spines, prickles, and hairs: are they stimulated by herbivory and
do they deter herbivores. In: Tallamy DW, Raupp MJ, editors. Phytochemical induction by
herbivores. New York: Wiley; 1991. pp. 325–44.
Neely D. Healing of wounds on trees. J Am Soc Hortic Sci. 1970;95(5):536–40.
Niinemets U. Responses of forest trees to single and multiple environmental stresses from
seedlings to mature plants: past stress history, stress interactions, tolerance and acclimation.
For Ecol Manage. 2010;260(10):1623–39.
Ohgushi T. Indirect interaction webs: herbivore-induced effects through trait change in plants.
Annu Rev Ecol Evol Syst. 2005;36:81–105.
Oren R, Schulze E-D, Matyssek R, Zimmermann R. Estimating photosynthetic rate and an-
nual carbon gain in conifers from specific leaf weight and leaf biomass. Oecologia.
1986;70(2):187–93.
Orians C, Thorn A, Gómez S. Herbivore-induced resource sequestration in plants: why bother?
Oecologia. 2011;167(1):1–9.
Osakabe Y, Osakabe K, Shinozaki K, Tran LSP. Response of plants to water stress. Front Plant
Sci. 2014;5:1–8.
Parmesan C, Yohe G. A globally coherent fingerprint of climate change impacts across natural
systems. Nature. 2003;421(6918):37–42.
Poorter H, Villar R. The fate of acquired carbon in plants: chemical composition and construction
costs. In: Bazzaz FA, Jones B, editors. Plant resource allocation. New York: Academic; 1997.
pp. 39–72.
Price PW. The plant vigor hypothesis and herbivore attack. Oikos. 1991;62:244–51.
Reich PB, Walters MB, Ellsworth DS. From tropics to tundra: Global convergence in plant
functioning. Proc Natl Acad Sci U S A. 1997;94(25):13730–4.
Rennenberg H, Loreto F, Polle A, Brilli F, Fares S, Beniwal RS, et al. Physiological responses of
forest trees to heat and drought. Plant Biol. 2006;8(5):556–71.
Rouault G, Candau J-N, Lieutier F, Nageleisen L-M, Martin J-C, Warzée N. Effects of drought and
heat on forest insect populations in relation to the 2003 drought in Western Europe. Ann For
Sci. 2006;63(6):613–24.
Ryan MG. Tree responses to drought. Tree Physiol. 2011;31(3):237–9.
Sala A, Piper F, Hoch G. Physiological mechanisms of drought-induced tree mortality are far from
being resolved. New Phytol. 2010;186(2):274–81.
Scherber C, Gladbach DJ, Stevnbak K, Karsten RJ, Schmidt IK, Michelsen A, et al. Multi-factor
climate change effects on insect herbivore performance. Ecol Evol. 2013;3(6):1449–60.
Schreiber L, Riederer M. Ecophysiology of cuticular transpiration: comparative investigation
of cuticular water permeability of plant species from different habitats. Oecologia.
1996;107(4):426–32.
Sevanto S, McDowell NG, Dickman LT, Pangle R, Pockman WT. How do trees die? A test of the
hydraulic failure and carbon starvation hypotheses. Plant Cell Environ. 2014;37(1):153–61.
Strauss SY, Agrawal AA. The ecology and evolution of plant tolerance to herbivory. Trends Ecol
Evol. 1999;14(5):179–85.
Taiz L, Zeiger E. Plant physiology. 3rd ed. Sunderland: Sinauer Associates Inc.; 2002. p. i-690.
Trapp S, Croteau R. Defensive resin biosynthesis in conifers. Annu Rev Plant Biol.
2001;52(1):689–724.
Trowbridge AM, Daly RW, Helmig D, Stoy PC, Monson RK. Herbivory and climate interact serial-
ly to control monoterpene emissions from pinyon pine forests. Ecology. 2014;95(6):1591–603.
Turtola S, Manninen A-M, Rikala R, Kainulainen P. Drought stress alters the concentration of wood
terpenoids in Scots pine and Norway spruce seedlings. J Chem Ecol. 2003;29(9):1981–95.
Tyree MT, Ewers FW. The hydraulic architecture of trees and other woody plants. New Phytol.
1991;119(3):345–60.
Vierling E, Kimpel JA. Plant responses to environmental stress. Curr Opin Biotechnol.
1992;3(2):164–70.
Vinebrooke RD, Cottingham KL, Norberg J, Scheffer M, Dodson SI, Maberly SC, et al. Impacts of
multiple stressors on biodiversity and ecosystem functioning: the role of species co-tolerance.
Oikos. 2004;104(3):451–7.
White T. A hypothesis to explain outbreaks of looper caterpillars, with special reference to
populations of Selidosema suavis in a plantation of Pinus radiata in New Zealand. Oecologia.
1974;16(4):279–301.
White T. The abundance of invertebrate herbivores in relation to the availability of nitrogen in
stressed food plants. Oecologia. 1984;63(1):90–105.
Williams JW, Jackson ST. Novel climates, no-analog communities, and ecological surprises. Front
Ecol Environ. 2007;5(9):475–82.
Wu J, Baldwin IT. Herbivory-induced signalling in plants: perception and action. Plant Cell
Environ. 2009;32(9):1161–74.
Index
A Brassica, 4, 98, 190, 191, 193

Abiotic stress, 9, 36, 38, 49, 93, 94, 97, 123, Broccoli, 4
188–190, 195, 212, 226, 255
in agriculture, 182 C
Accessions, 11 Cabbage, 4, 190
Albutilon theophrasti, 4 Cadmium, 72, 82, 85–87
Alnus fruticosa, 3 Callose, 121 188, 213
Antioxidant enzymes, 80, 103, 135, 136, 153 Candidate-gene, 13
Antioxidant(s), 8, 12, 58, 62, 102, 103 Carbon, 9, 28, 32, 35, 50–52, 61, 97, 100, 128,
activity in leaves, 74 148, 152, 153, 176, 182, 185, 238–240,
defense system, 77, 126–128 247–256
enzymes, 136 balance, 229, 230, 238, 240, 251, 256
Apple, 4, 194 starvation, 229, 230, 248, 249
Arabidopsis, 3–5, 8, 13, 36, 37, 45, 52, Carissa spinarum, 3, 8
101–112, 127, 184–189, 194, 195, 204, Chlorella, 5
206, 210–216 Chlorophyll, 11, 29, 100, 101, 105, 107, 109,
Artimesia, 3 126, 128, 132, 133
Aspen, 4, 34 fluorescence, 11, 101, 105, 132, 133
Assimilates, 9, 96, 174, 178, 247, 248 Chlorotic, 9, 29, 152
Association mapping, 12, 13, 109, 139 Chromatin, 9, 12
Avirulent, 9, 209, 210 Climate, 1, 51, 61, 72, 96, 124, 134, 183, 209,
229, 232–235, 240
B change, 2, 10, 39, 40, 94, 149, 182, 183,
Bacterial infection, 188, 209, 210, 213 195, 216, 225, 226, 234, 256
Barley, 3, 4, 8, 11, 29, 56, 58, 94, 95, 97–113 CO2, 1–5, 10
Bean, 4, 30, 184, 208, 211, Co-expression modules, 12
Beech, 3, 4, 80, 152 Combined stress, 1–3, 5–13, 36, 62, 65, 72,
Beet, 3 95–113, 124, 129, 137, 139, 153, 154,
Bell pepper, 4 209–217, 225–235, 240, 253
Biomass, 9, 39, 40, 88, 89, 107, 109, 151, 163, Computational, 12
167–178, 183, 211, 224, 239, 248, 254 Concurrent stress, 195, 204, 211, 212,
Biotic, 6, 9, 11–13, 102, 104, 112, 128, 215–217
182–195, 226–231, 237, 239, 248, 249 Copper, 72, 80–83, 160, 192
stress, 38, 124, 185–194, 204, 212–216 Cotton, 4, 28, 101
Birch, 3–5, 34 Cowpea, 5, 37
Boron, 11 Crithmum maritimum, 4
Crop improvement, 65, 95, 106, 113, 114

R. Mahalingam (ed.), Combined Stresses in Plants, DOI 10.1007/978-3-319-07899-1
262 Index
Crops, 6–12, 28–30, 36, 42, 72, 82, 87–89, 94, G

95, 101, 134, 137, 139, 149, 155, 160, Gene(s), 8
182–191, 195, 208, 217, 235 expression, 12
Cucumber, 3, 4 Genevestigator, 12
Cultivars, 9, 12, 56, 58, 65, 95, 112, 113, 132, Genome, 11
162, 170, 171, 177, 185, 194 Germanium, 11
Germplasm, 12
D Global warming, 2
Dactylis glomerata, 3 Grapes, 4
Defense genes, 12, 213 Greenhouse, 10
Desensitizing, 9 Groundnut, 159–173
Developmental stage, 8, 13, 137, 150, 166 Growth chamber, 10
Digital imaging, 11 Growth, 9
Drought, 1–11, 64, 94–114, 125, 127, 130, GWAPP, 13
134–139, 149–155, 160, 162, 183–195, Haberlea rhodopensis, 3
204–217, 225–239, 246–256 Heat, 2–10, 30–42, 111, 125–139, 148,
stress, 7, 36, 38, 51, 56, 94, 98–107, 111, 161–190, 211, 215, 217, 225, 227, 230,
112, 130, 150, 184–186, 190–194, 233, 238, 256
204–217, 234, 238, 247–256 stress, 181, 182, 215
Drought, 2 Hibiscus, 5
Dunaliella salina, 4 High
soil or water salinity, 49, 63, 65
temperature, 8
E
-performance computing, 11
Ecophysiology, 246, 247, 249, 251, 253–255
-throughput biomolecular (omic) analysis,
Edaphic, 10
50
Elevated CO2, 13
Histone, 9
Elymus athericus, 4
Homeostasis, 12
Epigenetic, 9
Hordeum maritimum, 4
Epoxidation, 11
Hormone, 12
Eucalyptus, 3–5, 232, 233, 235
signalling, 186, 189
Hydraulic failure, 229, 230, 248
F
FACE, 5, 10, 60, 63, 72–90, 182, 225
I
Field, 1, 2, 6–13, 32, 34, 41, 42, 50, 73, 89, 91,
Image analysis, 11
94, 103, 106, 111–113, 125, 129, 130,
Infrared thermography, 11
137, 138, 152, 160–162, 166, 172–174,
Infrastructure, 10
177, 178, 182, 183, 191, 195, 210, 217,
Insects, 5, 190, 225–228, 233–237, 249, 255
225
Integrating, 13
Flavonoid, 12, 60, 105
Foliar, 9, 32, 36, 180, 184, 208, 227, 232, 234,
236, 237 J
Food Jatropha, 3
safety, 72, 87
security, 1 K
Forest, 151, 152, 223, 225–231, 234–240, 256 Kentucky bluegrass, 4
decline, 226, 229
die-off, 228 L
ecosystems, 223, 226, 227, 234, 240 Legumes, 7, 28, 123–139, 183
stress, 225, 227 Lesions, 9, 209
Fungal infection, 208, 229 Light-harvesting, 11
Linseed, 4
Lipid, 11, 30, 38, 77, 104, 126, 128, 129, 186
Lotus, 3, 57, 123, 125, 129, 130
Index 263
M Pisum sativum, 4
Machine learning, 12 Plant systems biology, 52
Maize, 3 Pollutants, 11
Mapping populations, 11 Poplar, 3, 8
Mass spectrometry, 54, 109 Populus cathayana, 3
Medicago truncatula, 3 Populus, 3, 4
Membrane, 11 Portuluca oleracea, 8
Mercury, 8 Potato, 3
Metabolism, 12 Prosopis, 3
Metabolites, 35, 249 Proteome, 8
Metabolome, 8 Proteomic, 8
Metabolomics, 54, 56, 59, 62 Pyramiding, 12
Methylation, 9
MicroRNAs, 8 Q
Model plant, 11 QTL, 11, 106
Multiple stresses, 12
R
N Radish, 4
Near surface reflectance spectroscopy, 11 Rain-out shelters, 10
Necrotic, 9, 149, 152 Ranunculus acris, 3
Nerium oleander, 3 Reactive oxygen species, 8, 30, 77, 102, 103,
Nitrogen, 11, 29, 35–38, 60–64, 74, 106, 123, 126, 149, 189, 210, 215, 250
127, 233–235, 249 Receptor-like kinases, 12, 127
Non-host resistance, 12 Red maple, 3
Non-invasive imaging, 11, 13 Redox, 12, 52, 55, 127, 134, 139, 152, 215
Nutrients, 3–5, 10, 29, 96, 101–104, 211, 235 Remote sensing, 11
Reproductive stage, 8, 163
O Resistant, 9, 132, 188, 194, 208, 217, 236
Oak, 3, 153 R-genes, 12
Oats, 4, 94 Rice, 3, 4, 9, 28, 30, 54–57, 71–89, 95, 111,
Omics, 8, 12, 13, 52, 109 112, 182–191, 194, 195, 204, 212
Open top chambers, 10, 72 Robotics, 11
Osmolytes, 8, 36, 51, 56–59, 97, 104, 110, Root, 9, 50, 74, 82, 84, 96–102, 107–112, 125,
125, 128, 208 127, 134–138, 174, 183, 184, 205–208,
Oxidative stress, 31, 33, 49, 80, 100–103, 126, 234, 246, 248
130, 131, 136, 152–154 Rosa meillanda, 5
Ozone, 2–11, 148–155, 182, 190, 191
S
P Salinity, 3, 4, 8–11, 50–66, 94–113, 124,
Pathogen, 1 132–135, 195, 204, 225, 232, 235
Peanuts, 4 Screen Aided CO2 Control, 10
Pearl millet, 3 Screening, 11–13, 113, 139, 172, 178
Peas, 3 tool, 11
Pepper, 3 Seagrass, 4
Phaseolus vulgaris, 3 Secondary metabolites, 35–38, 55, 104, 105,
Phenomics, 11 233, 249
Phenotype, 9 Seed
Phloem, 9 quality, 8, 106, 107
Photosynthesis, 80, 86, 90, 111, 211, 223 yield, 8, 40, 41, 189
Phragmites australis, 4 Semi-arid tropics, 172
Physiological, 11 Senescence, 6, 11, 39, 42, 43, 109, 149
Picea asperata, 3 Sensitive, 7, 9, 31, 34, 41, 54–59, 100, 101,
Pine, 3 105, 125, 128, 134, 135, 150, 162, 163,
Pinus halpensis, 3 212, 237
264 Index
Sensitizing, 9 Toxicities, 11, 100

Sesuvium potulacastrum, 3 Transcription factors, 12, 110–113, 125, 213
Signaling, 12, 58–63, 97, 102, 104, 111, 204, Transcriptome, 7, 54, 186, 213
212–217, 246, 250 Transcriptomics, 54, 62, 65, 109, 154
Sorghum, 3, 28, 30, 42, 205, 208 Tree mortality, 229, 230, 235, 240, 248, 256
Soybean, 3–7, 11, 13, 28–42, 56, 123, 183, 205 Trees, 34, 62, 152, 225, 229, 232, 233
Spartina densiflora, 4 Trifolium subterraneum, 5
Spectroscopy, 11, 54, 56, 74
Spinach, 4 U
Spruce, 3, 4, 154, 239 UV, 11
Stackhosia tryonii, 3 UV-B, 11
Stomatal conductance, 8, 34, 35, 39, 51, 100,
127, 149–152, 184, 210, 232 V
Stress Vegetative growth
combination, 3–5, 129, 131, 135, 136, 139 Vigna radiata, 5
matrix, 6 Viguiera discolor, 3
Suaeda salsa, 8 Viral infection, 211, 212, 215
Sugarcane, 3
Sunflower, 4, 100, 183, 205, 208
Swietania macrophylla, 3 W
Symptoms, 9, 11, 149, 151–153, 184 Water stress, 34–36, 96–99, 125, 126, 129,
Synergy, 245 130
Water use efficiency, 163, 166, 169, 232
Watermelon, 3
T Wavelength, 11
Tailored response, 212, 215 Wheat, 3, 4, 9, 29, 71–79, 182, 183, 205
TASSEL, 13 Willows, 3
Tea, 4
Temperature, 2–11, 29–42, 62, 72, 99, 110,
111, 125–131, 136, 139, 160–185, 191, X
192, 223–226, 229, 233, 238 Xanthophyll, 11
Tobacco, 3, 4, 152, 188, 205, 210–213
Tolerance, 11, 126–131, 182–191, 210, 211 Y
Tomato, 3, 4, 105, 111, 183, 194, 206, 213 Yield, 8, 182, 189, 194, 211

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1 Consideration of Combined Stress: A Crucial Paradigm

2 The Impact of Enhanced Atmospheric CO2 Concentrations

3 Investigating the Effect of Elevated CO2 in the Growth

4 Combination of Elevated CO2 Levels and Soil Contaminants’

5 Tolerance to Combined Stress of Drought and Salinity in Barley....... 93

6 Combined Abiotic Stress in Legumes..................................................... 123

7 Interactive Effects Between Ozone and Drought: Sorrow or Joy?..... 147

8 Effect of High Temperature and Water Stress

9 The Response of Plants to Simultaneous Biotic

10 Impact of Concurrent Drought Stress and Pathogen

11 Combined Stresses in Forests................................................................ 223

12 The Interactive Effects of Drought and Herbivory

Imrul Mosaddek Ahmed Department of Agronomy, College of Agriculture and

Ritushree Jain Faculty of Biological Sciences, Centre for Plant Sciences,

Santiago Signorelli Laboratorio de Bioquímica, Departamento de Biología

A Net photosynthetic rate

DREB Dehydration response element binding protein

OPDA 12-Oxo-phytodienoic acid

© Springer International Publishing Switzerland 2015 1

1.2 Importance of Combined Stress

Table 1.1 (continued)

Table 1.1 (continued)

Here is an example to illustrate the importance of considering more than a single

1.3 Which Combination of Stresses to Study?

should be noted that this is a vast oversimplification of the complexity involved in

1.4 Omics of Combined Stress

A detailed review of the transcriptome studies on combined stresses in plants has

studies of combined stresses imposed during reproductive stages of crop develop-

1.5 Phenotypic Responses to Stresses

1.6 Contrasts Between Laboratory and Field Studies

'ƌŽǁƚŚ ĐŚĂŵďĞƌ 'ƌĞĞŶ ŚŽƵƐĞ &ŝĞůĚ ƐƚƵĚŝĞƐ

1.8 Strategies for Improving Tolerance to Combined

Transcription factors Defense Abiotic stress Elite germplasm Sequencing

Redox homeostasis Pre-invasion barriers Ion homeostasis

Incorporate into cultivars by marker assisted breeding

accessed through programs like Genevestigator (Zimmermann et al. 2004). Re-

Han Y, Wang L, Zhang X, Korpelainen H, Li C. Sexual differences in photosynthetic activity, ul-

Richard C. Sicher and James A. Bunce

The agricultural industry is uniquely dependent upon climate and a changing

© Springer International Publishing Switzerland 2015 27

2.2 Positive Effects of CO2 Enrichment on Plants

2.3 Negative Effects of CO2 Enrichment on Plants

2.4 Elevated Temperature Effects on Plant Growth

2.5 Heat Stress Responses of Plants

2.6 Heat Stress Effects on Photosynthesis

2.7 Effects of CO2 Enrichment and Heat Stress

Soybean, which possesses C3 photosynthesis, generally has a substantial, long-term

Fig. 2.1 Effects of elevated

of the suppression of photorespiration and increased carboxylation rates due to

of the species they examined, cultivating plants in atmospheres containing elevated

2.8 Effects of CO2 Enrichment and Heat Stress

Both CO2 enrichment and supraoptimal temperatures affect a number of metabolic

2.9 CO2 Enrichment Mitigates Metabolite Responses

1 Consideration of Combined Stress: A Crucial Paradigm

2 The Impact of Enhanced Atmospheric CO2 Concentrations

3 Investigating the Effect of Elevated CO2 in the Growth

4 Combination of Elevated CO2 Levels and Soil Contaminants’

5 Tolerance to Combined Stress of Drought and Salinity in Barley....... 93

6 Combined Abiotic Stress in Legumes..................................................... 123

7 Interactive Effects Between Ozone and Drought: Sorrow or Joy?..... 147

8 Effect of High Temperature and Water Stress

9 The Response of Plants to Simultaneous Biotic

10 Impact of Concurrent Drought Stress and Pathogen

11 Combined Stresses in Forests................................................................ 223

12 The Interactive Effects of Drought and Herbivory

1.2 Importance of Combined Stress

1.3 Which Combination of Stresses to Study?

1.4 Omics of Combined Stress

1.5 Phenotypic Responses to Stresses

1.6 Contrasts Between Laboratory and Field Studies

1.8 Strategies for Improving Tolerance to Combined

2.2 Positive Effects of CO2 Enrichment on Plants

2.3 Negative Effects of CO2 Enrichment on Plants

2.4 Elevated Temperature Effects on Plant Growth

2.5 Heat Stress Responses of Plants

2.6 Heat Stress Effects on Photosynthesis

2.7 Effects of CO2 Enrichment and Heat Stress

2.8 Effects of CO2 Enrichment and Heat Stress

2.9 CO2 Enrichment Mitigates Metabolite Responses

2.10 Effects of CO2 Enrichment and Heat Stress

2.11 Effects of CO2 Enrichment and Heat Stress

3.2 Physiological Characteristics of the Plant Response

3.2.1 High Soil and/or Water Salinity

3.2.2 Elevated CO2 in the Growth Environment of the Plants

3.2.3 Combined Application of High Salinity and Elevated CO2

3.3 Integrated High-Throughput Biomolecular Analyses

a sufficient condition for high in vivo protein production. Regulation of translation,

3.4 Omic Analyses of Salinity Stress on Plants

3.4.3 Results of Integrated Metabolomic and Transcriptomic

3.5 Omic Analyses of Elevated CO2 Stress on Plants

3.6 Omic Analysis of Plant Response to Combined

3.7 Experimental Design Specifications of Integrated

d erivative with relative concentrations depending on the derivatization conditions

4.2 Materials and Methods

4.2.3 Crop Cultivation and Sample Preparation

4.2.4 Analysis of Antioxidant Enzyme Activities in Leaves

4.2.6 Determination of Soil pH and Sequential Extraction of Soil

4.2.7 Scanning Electron Microscopy

4.3 Results and Discussion

4.3.1 Changes in Enzyme Activities

At p anicle-initiation growth stage, the activity of CAT in leaves of wheat grown

4.3.2 Copper Concentration in Plants

4.3.3 Cadmium Concentration in Plants

4.3.4 Variations in Soil pH, and Cu and Cd Fractionation in Soil

5.2 Drought Stress and Tolerance