Original Paper
Open Access
A simple, fast and accurate screening method to estimate maize
(Zea mays L) tolerance to drought at early stages
Lorena Álvarez-Iglesias1,3*, Begoña de la Roza-Delgado2, Manuel J Reigosa1,3, Pedro Revilla3,4, Nuria Pedrol1,3
Department of Plant Biology and Soil Science. University of Vigo. Campus As Lagoas-Marcosende, 36310, Vigo, Spain
Department of Animal Nutrition, Grasslands and Forages. Regional Institute for Research and Agro-Food Development
(SERIDA), 33300, Villaviciosa, Spain
3
Agrobiología Ambiental. Calidad de Suelos y Plantas. University of Vigo - Misión Biológica de Galicia (CSIC) Associated
Unit, Spain
4
Misión Biológica de Galicia (CSIC), Apartado 28, 36080 Pontevedra, Spain
*Corresponding author: E-mail: lorena.alvarez@uvigo.es.it
1
2
Abstract
There is a great need for the selection of plants with higher drought tolerance, so that fast and effective
techniques to identify variations in drought tolerance are mandatory for screening large numbers of genotypes.
This work presents a protocol for easy and reliable assessment of responses of maize genotypes to water stress
conditions imposed during early stages of development. Three experiments using 11 commercial maize hybrids
under four levels of water stress were carried out: i) germination, ii) seedling growth, and iii) early growth bioassays. Constant and uniform water stress was imposed using solutions of polyethylene glycol 6000 (PEG 6000).
Plant material was evaluated for several morphological, physiological and biochemical traits and monitored for
photosynthetic efficiency. Principal component analysis (PCA) of these joint experiments revealed that germination percentage, early root development and stomatal conductance were the most useful traits for discriminating
maize hybrids according to their tolerance to water stress. A subsequent greenhouse assay performed with two
hybrids with contrasting responses under soil drying conditions validated the previous results. According to our results, the key of drought tolerance was a rapid response of stomatal conductance, which allowed a longer survival
to stress even under severe desiccation. This work provides the researcher with a simple and reliable screening
method that could be implemented as a decision support tool in the selection of the most suitable genotypes for
cultivation in areas where water availability is a problem, as well as for the selection of tolerant genotypes to early
drought in breeding programs.
Keywords: Zea mays, germination, seedling growth, early growth
Introduction
Abiotic stresses, especially drought, are one of
the main challenges of agriculture at global scale
as they strongly affect the potential productivity of
crops, being responsible for large yield losses worldwide (Boyer et al, 2013). Drought is an endemic problem in large agricultural areas. Furthermore, inter-annual variations in water availability can compromise
crop yields even in humid areas, such as the Atlantic
coast of Spain, where maize is grown under rain-fed
conditions. According to current models, drought is
expected to worsen with ongoing climate changes,
becoming a great challenge for future maize production (Olesen et al, 2011; Supit et al, 2012; Pachauri
and Meyer, 2014).
Large screenings and identification of crop genotypes with high drought tolerance are one of the main
pillars of studies on drought tolerance. In the search
for an appropriate screening protocol, several strategies have been proposed. Direct measurement of
drought tolerance traits under realistic conditions,
Maydica 62-2017
by means of soil drying through natural drought or
exclusion of throughfall or irrigation, appears to be
the most unbiased approach. But field environmental conditions are unpredictable and heterogeneous,
and thus these techniques introduce remarkable errors and difficulties for interpreting and comparing
results. Moreover, replicated field trials performed on
a large enough scale and during long enough periods
would be in most cases prohibitive in terms of cost
and space requirement. Results from greenhouse
pot experiments can be easier to interpret but, still,
the temporal and spatial variability of soil water loss
results in unpredictable and heterogeneous conditions surrounding the root system (Passioura, 2006;
Verslues et al, 2006; Whitmore and Whalley, 2009;
Munns et al, 2010). More recently, high-throughput
phenotyping techniques have revolutionized comprehensive studies on plant performance and responses
to stresses, but their expansion is being hampered by
their high cost and technical complexity so they are
still far from being easily accessible to growers and
breeders worldwide. Thus, none of these approaches
RECEIVED 11/16/2017
Álvarez-Iglesias et al
are feasible at the large scale that routine evaluations
or breeding programs require. There is still a need for
simple, reliable and affordable laboratory screening
tests that allow predicting drought tolerance for large
genotype collections, as well as comparing and integrating results among institutions. Ideally this protocol should serve to agronomists, plant physiologists
and breeders, by being «suitable for genetic studies
and rapid screening while still being relevant to stress
conditions in the real world», as said in Verslues et al
(2006).
When implementing an appropriate screening
protocol, very controlled conditions are needed in
order to ensure the maximum reliability and repeatability (Vanhove et al, 2012). Hydroponic solutions in
combination with ionic or non-ionic osmotica have
been frequently used to simulate water stress effects
in plants. Hydroponics allows the maintenance of uniform conditions, and osmotica can lower the water
potential of the solution in a controlled and precise
manner. Polyethylene glycol (PEG), a neutral, nonionic and non-toxic polymer with high water solubility, is the most widely used osmoticum to mimic decreases in soil water potential. High molecular weight
PEG (6000 or above) cannot penetrate the cell wall
pores, resulting in conditions closely matching the
effect of reduced matric potentials and thus causing a loss of water from both the protoplast and the
cell wall and the collapse of the entire cell, including
the wall (cytorrhysis). As plants subjected to longterm soil water deficits also experiment cytorrhysis,
the use of PEG solutions avoid metabolic interferences associated to the use of ionic or low molecular
weight osmotica that penetrate into the cells causing
plasmolysis (Lawlor, 1970; Oertli, 1985; Verslues et
al, 2006). However, the use of PEG is a controversial issue. The main problem is that PEG solutions
are highly viscous, limiting O2 diffusion to the roots
(Munns et al, 2010), but this issue can be overcome
by replacing solutions frequently. The use of PEG
solutions is the most feasible option for simulating
drought conditions in short-term experiments, which
would be useful for evaluating the suitability of new
sources of germplasm, and also for screening of large
populations from the early generations in breeding
programs. In a more practical sense, these experiments could be easily implemented by scientific advisory institutions for the routine-based screening of
commercial hybrids for drought tolerance, and for the
creation of recommended lists that would help growers in the selection of the most suitable varieties for
cultivation.
The first objective of this work was to establish
a practical and reliable experimental method able to
group maize genotypes in relation to their tolerance
to drought at early stages. For this objective, we used
maize commercial hybrids and solutions of PEG 6000
to simulate different drought levels. Our second objective was to verify the reliability of this method by
62 ~ M24
2
evaluating, in a soil drying experiment under greenhouse conditions, two hybrids with contrasting tolerance to drought according to the previous method.
The proposed protocol would allow the screening of
large numbers of maize genotypes, leading to more
reproducible results and enabling unification of criteria used for classification of maize germplasm according to its drought tolerance at early stages.
Materials and Methods
Screening of drought tolerance in maize at germination, seedling growth and early growth
Eleven commercial single cross hybrids were
evaluated for their tolerance to early growth. These
hybrids were chosen among a set of maize hybrids
that were widely grown in Northwestern Spain, and
evaluated by SERIDA (Spain) for and their good performance under temperate climate (with typically
mild-humid summers), rainfed conditions. Hybrids
belong to different developers and FAO cycles. Commercial names of the chosen maize hybrids have
been omitted, and each hybrid was assigned a number randomly between 1 and 11 (Supplementary Table 1).
Drought tolerance was evaluated at three stages: seed germination, seedling growth (pre-germinated seeds) and early growth (young plants at V3
stage). Drought stress was imposed using aqueous
solutions of PEG 6000 at concentrations simulating
slight, moderate and severe stress conditions (Table
1). Concentrations were chosen according to our experience in previous assays, and considering that the
germination process is more sensitive to drought, i.e.,
imbibition in solutions with reduced water potential
delays the seed osmotic water uptake, thus lowering
seed water content below a «critical» (minimum) value
required for radicle emergence and growth (Bradford,
1995). All solutions were adjusted at pH 6.0. Osmotic
potentials were calculated following the equations
of Michel and Kaufmann (1973), and verified using a
cryoscopic osmometer (Gonotec OSMOMAT 030).
Germination assays
Screening for seed germination under water
stress conditions was conducted on 14 cm diameter Petri dishes by placing 30 seeds of each maize
hybrid on a Whatman No. 2 filter paper layer moistened with 10 ml of the corresponding solution. Petri
dishes were sealed with Parafilm and incubated in the
dark at 27ºC in a growth chamber. The number of
germinated seeds was counted every 12 h until no
new germination events were observed. A seed was
considered to be germinated when the seed coat was
ruptured and the root emerged > 1 mm. Total germination index (Gt) was calculated from the cumulative germination data as described in Chiapusio et al
(1997). Additionally, other germination indices were
derived from primary germination data to obtain information about the effects on the ontogeny of germination: speed of germination (S), speed of accumulated
Maydica electronic publication - 2017
screening for maize tolerance to drought
3
Table 1 - PEG concentrations, and their corresponding
osmotic potentials (o), used to evaluate responses to
induced drought stress for maize commercial hybrids.
Stress level
Germination
Control
Slight
Moderate
Severe
PEG 6000 (g l-1)
o (MPa)†
0
100
150
200
0
- 0.15
- 0.30
- 0.49
Seedling establishment / Early growth
Control
0
0
Slight
150
- 0.30
Moderate
200
- 0.49
Severe
300
- 1.03
†
Osmotic potentials of PEG 6000 solutions at 25 ºC calculated according to Michel and Kauffman (1973).
germination (AS), coefficient of rate of germination
(CRG) and mean germination time (MGT), following
Chiapusio et al (1997) and de Bertoldi et al (2009). For
each hybrid and treatment, five replicates, randomly
distributed in the growth chamber, were used.
Seedling growth assays
For the evaluation of seedling growth, seeds of
each hybrid were pre-germinated on containers with
moistened filter paper at 27ºC in the dark. Then 20
pre-germinated seeds of each hybrid (radicle length
1-3 mm and no coleoptiles emerged) were placed on
14 cm diameter Petri dishes with the corresponding
solution (Table 1) and incubated in a growth chamber
as described above. After 72 h, primary root and coleoptile lengths, as well as the number of secondary
roots, were recorded for all seedlings on each Petri
dish. Then the primary roots, coleoptiles and secondary roots harvested from each Petri dish were collected and, for each of these three samples, the fresh
weight (FW) and dry weight (DW) after drying 72 hours
at 60ºC were obtained. The dry weight/fresh weight
(DW/FW) ratios for each sample, total root weight and
shoot/root ratio were also calculated. Five replicates
per hybrid and treatment were used.
Early growth assays
Early growth assays were performed in a greenhouse located at the SERIDA experimental station ‘La
Mata’ in Grado, Asturias (43°32’N;7°00’W, 65 masl)
under natural light conditions (14 h of natural light and
10 h without light) and controlled temperatures (10 35ºC) and relative humidity (80 ± 10%). Maize seeds
were sown in 1 liter pots containing a mixture of perlite and vermiculite (2:1 v:v), and pots were placed in
large plastic trays for optimization of irrigation. Pots
were irrigated with tap water until coleoptile emergence, and thereafter with half-strength Hoagland
nutrient solution. All pots were irrigated daily by adding nutrient solution to each tray, and replacing the
solution every 2 days. When plants reached the V3
stage (three collared leaves), drought stress treatments were imposed by adding the corresponding
concentration of PEG 6000 to the Hoagland solution
(Table 1). Control plants were maintained in the Hoa-
62 ~ M24
gland solution. Treatments were maintained for 72 h,
and solutions were replaced daily to maintain constant concentrations.
From the beginning of treatments and every 24
h, net photosynthetic rate (measured as CO2 assimilation, µmol CO2 m-2 s-1), stomatal conductance to
water vapor (mol H2O m-2 s-1) and transpiration rate
(mol H2O m-2 s-1) were recorded in the third leaf of five
leaves per hybrid and treatment by using a LI-6400XT
portable photosynthesis system (Li-Cor Inc, Lincoln,
NE, USA). Water use efficiency (WUE) was calculated
as the ratio CO2 assimilation/stomatal conductance
(Xu and Hsiao, 2004). Measurements were performed
under constant light conditions (photon flux 1,000
μmol m-2 s-1) and CO2 concentrations (400 µmol CO2
m-2 s-1) to minimize effects of environmental variations. Irrigation and daily measurements were made
at the same hour to minimize interactions with circadian cycles and environmental conditions (Berger et
al, 2010).
At the end of the assay (after 72 h under stress
conditions), FW of roots and aerial parts were immediately recorded, as well as the DW after drying each
part during 72 h at 60°C. The third leaf of each plant
was detached and weighed separately, and for each
of them the leaf area (LA) was recorded with a Leaf
Area Meter CI-202 (CID Bio-Science Inc, Camas, WA,
USA), and the specific leaf area (SLA) was calculated
as the ratio LA/DW of the leaf (Garnier et al, 2001).
Leaf relative water content (RWC) was determined
in one leaf per plant following the equation [(fresh
weight – dry weight)/(turgid weight – dry weight)] ×
100 (Turner, 1981). The remaining upper leaves were
used to calculate free proline content using a modified Bates method (Ramos and Pedrol, 2001) and
total protein concentration according to a modified
Bradford method (Pedrol and Ramos, 2001). For each
maize hybrid and treatment, five replicate pots with
one plant per pot were evaluated.
Soil drying experiment
Based on the previous results, we identified two
maize hybrids with contrasting responses to PEG
treatment: the tolerant hybrid named 5 and the susceptible hybrid named 6. These two hybrids were
evaluated in a greenhouse under natural light conditions (14 h natural light/10 h dark) and controlled
temperatures (10 - 35ºC) and relative humidity (80
± 10%). Each seed was sown in a 1 liter pot filled
with commercial substrate (Gramoflor, GmbH & Co,
Vechta, Germany) at 100% water availability (WA),
previously calculated by the gravimetric method.
Pots were maintained under these conditions by
daily top irrigation until target weight until plants
reached V3 stage. At this moment, two treatments
were established for each hybrid: half of the pots
were maintained at 100% WA (control) throughout
the experiment, and the other half was subjected to
drought stress by withholding irrigation. All pots were
weighed every day to monitor WA; in control pots,
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Álvarez-Iglesias et al
4
water lost was replaced by watering every two days
until target weight. From here on, measurements of
gas exchange and chlorophyll a fluorescence were
carried out every 24 h to monitor the photosynthetic
performance of plants. Gas exchange-related parameters were measured with a LI-6400XT portable photosynthesis system, as described before. Parameters
related to chlorophyll a fluorescence (efficiency of the
photosynthetic system PSII [Y(II)], dissipated energy
as heat [Y(NPQ)], dissipated energy as fluorescence
[Y(NO)], non-photochemistry quenching [qN], photochemistry quenching [qL], and electron transport rate
[ETR]) were measured using a modulated pulse fluorimeter Maxi Imaging PAM (Walz, Effeltrich, Germany). A detailed review of these parameters and their
biological significance can be found in Maxwell and
Johnson (2000) and Baker (2008). All these measurements were made in the third leaf of the same plants,
in at least three plants per hybrid and treatment, and
at the same hour.
When the drought stressed pots reached 50% WA
three plants per hybrid, along with three control pots,
were harvested. Subsequent harvests were made
when the non-irrigated plants reached 35%, 25%,
and finally < 5% WA, which was considered the point
of maximum stress. Immediately after each harvest,
FW of aerial parts was recorded, as well as the DW
after drying at 60°C 72 h. LA, SLA, and RWC were
determined on the third leaf of each harvested plant
as described before. Material from the remaining upper leaves of at least four plants per hybrid and treatment were used to determine free proline and total
protein contents as described before. Using the same
material, cellular osmolarity measurements were performed with a Gonotec OSMOMAT 030 cryoscopic
osmometer (Gonotec, Berlin, Germany).
Statistical analyses
The statistical package IBM SPSS Statistics
v.22 (IBM Corp, NY, USA) was used for all statistical
analyses. For each hybrid, all data was expressed in
percentage with respect to the corresponding control
in order to allow a standardized comparison among
hybrids, beyond differences in plant growth and productivity. Two-way analyses of variance were made
with stress and hybrids as main sources and variation and the corresponding interactions. Repetitions
and their interactions were considered random effects. Data were tested for normality by Kolmogorov-
Table 2 - Summary statistics with minimum (Min), maximum (Max), mean (Mean) and standard deviation (SD) values of parameters measured on 11 maize commercial hybrids after germination, seedling growth and early growth bioassays.
Min
Max
Mean
SD
Germination
Percentage of germinated seeds (Gt)
Speed of germination (S)
Speed of accumulated germination (AS)
Coefficient of rate of germination (CRG)
Mean germination time (MGT)
10.00
0.60
0.94
0.83
24.00
100.00
15.00
47.79
1.56
128.57
76.99
6.18
17.75
1.19
64.15
23.44
3.09
10.70
0.15
22.28
Seedling establishment
Root length (mm)
Root dry weight (mg)
Root dry weight/fresh weight
Shoot length (mm)
Shoot dry weight (mg)
Shoot dry weight/fresh weight
Secondary roots (number)
Secondary roots dry weight (mg)
Secondary roots dry weight/fresh weight
Total root dry weight (mg)
Shoot/root ratio
8.14
1.52
0.08
0.00
0.00
0.07
0.00
0.00
0.08
1.52
0.00
144.78
14.95
0.27
78.83
26.64
0.30
6.36
18.10
0.36
30.66
3.02
53.31
5.99
0.16
25.63
8.50
0.15
2.88
5.42
0.17
11.40
0.72
31.70
3.05
0.05
21.48
7.07
0.05
1.45
3.62
0.06
5.94
0.57
2.69
0.17
2.69
0.14
1.859
95.48
180.49
988.83
90.29
8.81
9.89
0.39
1.86
14.25
1.48
0.11
1.46
0.09
1.01
86.73
90.56
627.27
43.00
2.17
3.56
0.07
0.65
5.90
0.45
0.50
0.03
0.01
0.30
7.08
28.70
91.60
17.08
1.20
2.04
0.08
0.35
2.76
Early growth
Total root dry weight (mg)
Total root dry weight/fresh weight
Shoot dry weight (mg)
Shoot dry weight/fresh weight
Shoot/root ratio
Relative water content (RWC) (%)
Leaf area (LA) (cm2)
Specific leaf area (SLA) (cm2 g dw-1)
Protein content (mg g dw-1)
Proline content (µmol g dw-1)
Photosynthetic rate (72h) (µmol CO2 m-2 s-1)
Stomatal conductance (72h) (mol H2O m-2 s-1)
Transpiration rate (72h) (mmol H2O m-2 s-1)
Water use efficiency (WUE) (72h)
62 ~ M24
0.55
0.07
0.43
0.07
0.36
66.73
28.95
463.99
10.24
0.36
-0.31
-0.03
0.10
-1.34
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screening for maize tolerance to drought
Smirnov test and homogeneity of variances by Levene’s test. When variances were homogeneous, the
effects of treatments on each parameter were determined with one-way analyses of variance, and least
significant differences (LSD) test was used for post
hoc mean comparisons. In the case of heteroscedasticity, variance was analyzed by Kruskall-Wallis H
test and Tamhane’s T2 for post hoc multiple comparisons. Gas exchange variables were subjected
to covariance analyses (ANCOVA) with environmental parameters previous to their analysis. A principal
component analysis (PCA) was performed with data
from the PEG 6000 screening assays, in order to
synthesize all the measured variables into a limited
number of principal components (PCs). Percentage
data were first standardized. PCs with eigenvalues
above 1 were selected and used for a hierarchical
cluster analysis (CA), allowing hybrids to be organized into distinct groups with similar responses to
early drought. Data from soil drying experiments were
analyzed by independent samples t-test, comparing
each drying treatment with the 100% WA control for
each hybrid.
Results
Screening of drought tolerance at germination,
seedling growth and early growth
The commercial maize hybrids used in this study
showed large variability for their responses to drought
stress, particularly for S, AS, root length, shoot length,
DW of secondary roots, total root DW, photosynthetic
rate, and stomatal conductance. Conversely, variability was limited for RWC, CRG, specific leaf area, and
the ratios root DW/FW and shoot DW/FW (Table 2).
Traits with wide or narrow variability were distributed
among the three stages of development evaluated.
Differences among hybrids were highly significant
for all traits (data not shown) and differences among
stress levels were as well highly significant except for
shoot DW. Analyses of variance for the parameters
measured along time (photosynthetic rate, stomatal
conductance, transpiration rate, and water use efficiency) showed significant differences among hybrids
only for water use efficiency, while differences among
stress levels or among times were always highly significant (data not shown). Genotype × stress level interactions were highly significant for all traits except
MGT; interactions were generally of rank rather than
of magnitude; therefore, results and discussion will
focus on genotype × stress level when pertinent.
Total germination at slight water deficit was not
significantly reduced except for the hybrid 10 (Supplementary Table 2). Significant reductions of germination began at moderate stress level, and reached
50% at the higher stress level, except for hybrids 2, 4
and 5, which were able to maintain germination levels around 100% throughout the stress conditions.
Kinetics of germination, measured by the indices S,
AS, CRG and MGT was also different among hybrids,
62 ~ M24
5
being affected by all stress levels for all genotypes
except for hybrid 5.
Seedling growth of all hybrids was significantly affected at all stress levels, particularly for coleoptile-related traits as coleoptile growth was strongly affected
by all stress treatments, being completely inhibited at
300 g l-1 (Supplementary Table 2). Slight stress conditions strongly affected the seedling development,
with reductions in growth and weight of primary roots
of around 50-65%. Exceptions were hybrids 2, which
was less affected, and 3, in which the coleoptile
growth was stimulated. At severe stress conditions,
all hybrids were negatively affected. Conversely,
secondary root development was stimulated for all
hybrids at slight stress conditions, not significantly
affected at moderate stress and strongly inhibited
under severe stress conditions. Roots were strongly
reduced in hybrids 6 and 10 at the slight stress level.
Total root biomass was generally balanced between
the reduction of main root growth and the stimulation
of secondary roots development due to water stress.
Hybrids 1, 2, 5, and 8 had outstanding root development while hybrid 6 showed the worst response. On
the other hand, root biomass was stimulated in hybrid
3 at slight and moderate stress levels, whereas hybrid
5 had higher secondary root biomass at all stress levels when compared to control.
In the early growth assay, morphological measurements revealed that stress conditions stimulated
root growth for all hybrids (Supplementary Table 2),
showing increases in root biomass (with the exception of hybrids 7, 10, and 11) and decreases in shoot/
root ratios (excepting hybrids 5, in which this ratio
remained constant, and 6, in which aerial growth prevailed). No clear trends were found for aerial biomass.
The DW/FW ratios for roots and aerial parts also increased with stress intensity, again with the exceptions of hybrids 5 (for which shoot DW/FW remained
constant), 10 and 11. Variations in leaf area did not
show specific trends, although in most cases reductions were found at the higher stress levels. Punctual
exceptions were found for hybrids 2, 3, and 6, while
hybrid 4 showed consistent increases at all stress levels. A general reduction in the SLA was also observed
at all stress levels compared to the control. The leaf
RWC decreased with PEG 6000 treatments in a dosedependent manner in all cases, but the magnitude of
these reductions differed among genotypes. Hybrids
1, 4, and 7 showed reductions around 25% at the
highest PEG concentration, whereas these reductions were lower (around 10%) for hybrids 5 and 6.
The concentrations of stress-related metabolites
(proline and soluble proteins) had a negative relationship with stress level for most hybrids, particularly for
moderate and severe stress levels. Proline increased
clearly with stress intensity in hybrid 2, whereas hybrid 5 had low and stable proline levels. The total concentration of soluble proteins generally decreased
as stress intensity increased, although at slight and
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Álvarez-Iglesias et al
6
Table 3 - Principal component analysis of germination. seedling establishment and early growth parameters measured on 11
maize commercial hybrids. Eigenvalues and variance of the first 6 principal components (PC) are given.
Eigenvalue
Explained variance (%)
Accumulated explained variance (%)
PC 1
PC 2
PC 3
PC 4
PC 5
PC 6
12.74
45.49
45.49
3.23
11.53
57.02
2.33
8.33
65.35
1.88
6.70
72.05
1.49
5.32
77.37
1.21
4.32
81.69
0.71
0.94
0.93
0.92
-0.81
0.31
-0.05
-0.09
-0.12
0.21
0.06
0.06
0.07
0.11
-0.19
0.09
0.04
0.07
0.03
0.11
0.44
0.10
0.08
-0.06
0.17
-0.15
-0.10
-0.09
-0.05
-0.06
0.84
0.70
-0.87
0.93
0.91
0.81
0.63
0.71
0.87
-0.06
0.27
0.22
-0.19
-0.06
0.23
0.38
0.36
-0.13
-0.18
-0.34
-0.11
-0.07
-0.13
0.08
-0.28
-0.33
0.08
-0.01
0.29
0.15
-0.12
-0.06
-0.17
0.25
0.29
-0.23
0.00
0.00
-0.04
-0.07
-0.08
0.06
0.15
0.11
-0.09
0.16
0.22
-0.18
-0.01
0.04
-0.06
0.25
0.26
-0.14
-0.32
-0.81
0.08
-0.71
0.39
0.88
0.06
0.08
0.61
0.32
0.14
-0.07
-0.18
0.55
0.43
0.26
-0.06
-0.24
-0.47
0.16
-0.19
-0.31
0.12
0.51
0.73
0.68
0.77
-0.01
-0.05
-0.38
0.40
0.06
0.49
0.01
0.58
0.38
0.06
-0.23
0.37
0.58
0.53
-0.22
0.73
-0.07
0.84
-0.05
0.35
-0.01
0.09
0.01
-0.10
-0.02
-0.18
-0.22
-0.16
-0.08
0.15
0.12
-0.28
0.08
-0.38
-0.15
0.58
0.71
0.06
0.06
-0.07
-0.20
-0.08
0.00
-0.13
-0.02
-0.04
0.26
0.06
-0.08
-0.05
0.19
-0.49
-0.46
0.34
0.08
0.05
0.43
Germination
Gt
S
AS
CRG
MGT
Seedling establishment
Root length
Root DW
Root DW/FW
Coleoptile length
Coleoptile DW
Secondary roots
Secondary roots DW
Total root DW
Shoot/Root ratio
Early growth
Root DW
Root DW/FW
Shoot DW
Shoot DW/FW
Shoot/Root ratio
RWC
LA
SLA
Protein
Proline
Photosynthetic rate (T3)
Conductance (T3)
Transpiration (T3)
WUE (T3)
The most important parameters contributing to each PC (|correlation| ≥ 0.4) are remarked.
moderate stress conditions protein concentration increased in all hybrids excepting 1, 7, and 9. Under
severe stress conditions, the protein concentration
decreased for all hybrids. Strong decreases with respect to their controls were found for hybrids 6 and 7.
Differences among hybrids for gas exchange
measurements increased with the time of exposure
to PEG, and data obtained 72 h after the beginning of
PEG treatments (T3) were clearly discriminant. All hybrids showed the highest values of CO2 assimilation
and stomatal conductance at moderate stress levels, whereas WUE decreased as stress intensity increased. The magnitude of these responses showed
a great variability among hybrids.
Principal components and cluster analyses were
carried out in order to synthesize all the measured
variables. The first six principal components (PCs)
had eigenvalues above 1 and explained 81.7% of
the variability. PC1 explained 45.5% and joined most
of the parameters related with germination, seedling
growth and plant water status (DW/FW, RWC, and
WUE); therefore, we can consider PC1 as a Growth
component (Table 3). PC2 explained 11.5% of the
62 ~ M24
variability, and had positive loadings for gas exchange parameters as well as root development in
young plants. Thus, PC2 can be considered a Photosynthesis component. PC3 to PC6 explained low
percentages of variability (less than 10% each), and
their significance was less clear. PC3 was associated
to aerial growth, leaf conductance and transpiration;
PC4 to early growth parameters; PC5 to leaf growth,
and finally PC6 had positive contributions from WUE
and negative from proline and protein contents.
To clarify the comparison between hybrids, PC
scores were used in a hierarchical cluster analysis,
allowing the identification of homogeneous groups of
population samples (Figure 1). Responses of hybrids
were grouped into five clusters, corresponding to four
functional units. The first cluster comprised the largest number of cases including all controls, but it was
not particularly similar to any functional profile. Clusters 2 and 5 distinguished those individuals tending
to maintain high productivities and high conductance
and transpiration rates even at high levels of stress.
The third cluster grouped the lowest number of cases, maintaining a good photosynthetic performance
Maydica electronic publication - 2017
screening for maize tolerance to drought
7
compared to hybrid 5. As expected, in both cases
the decrease in stomatal opening is coupled with a
progressive and sharp decrease in CO2 assimilation
rates. Transpiration rates also followed the same trend
than stomatal conductance for both hybrids (data not
shown). On the other hand, fluorescence-related parameters did not change significantly throughout the
experiment except for slight decreases of ETR at the
first days of water withholding, more evident for hybrid 6. Hybrid 6 also showed an occasional reduction
of Y(II), coupled with a significant increase of Y(NPQ)
and qN (Table 4).
For the early growth- and water status-related
parameters, no clear effects were shown on RWC
at 50% or 35% WA (data not shown), and deviations with respect to each control were not very high
at 25% WA. Effects were stronger at higher stress
levels, with RWC reductions of 10% and 40% with
respect to the control for hybrids 5 and 6, respectively. Both hybrids showed reductions in leaf growth,
greater in the case of hybrid 6. Significant reductions
in root and aerial DW were also found in hybrid 6 at
the higher stress level.
Increases in cellular osmolarity were observed
at 25% and <5% WA for both hybrids, being these
increases more pronounced in hybrid 5 (data not
shown). Proline contents were analyzed in order to
detect its possible role as an active osmolyte in cells.
5
4
2
10
2
PC 2
1,0
2
1
5
0,5
5
3
11
9
0,0
1
8
3
9
9
3
-0,5
1
4
8
10
1-11
4
6
1
4
11
-1,0
7
6
10
7
-1,5
6
2+5
7
PC 1
-2,0
-2,5
-2,0
-1,5
-1,0
-0,5
0,0
0,5
1,0
1,5
Figure 1 - Principal component analysis of germination,
seedling establishment and early growth parameters measured on 11 maize commercial hybrids. Hybrids are labelled
with numbers between 1 and 11. Symbols are means of
each experimental replicate. Samples were distributed according to the scores of the principal components 1 (PC 1)
and 2 (PC 2). Dashed lines combine homogeneous groups
of samples according to hierarchical cluster analysis.
μ
62 ~ M24
PPhotosynthetic rate (Control)
SStomatal conductance (Control)
PPhotosynthetic rate (Desiccation)
S Stomatal conductance (Desiccation)
Conductance Control
Conductance Dessication
S
25
μ
μ
under stress with a concomitant decrease in early
growth. Finally, cluster 4 included cases with high
photosynthetic performance and high productivities
under stress conditions. PC1 and PC2 were used for
graphical representation of hybrids and clusters (Figure 1). Hybrids with high scores in PC1 had a good
growth response, and those with high scores in PC2
maintained high photosynthetic activities at the corresponding stress level. Therefore, hybrids with high
scores in PC1 and PC2 (2, 3, 5, and 8) are considered
tolerant to early drought while those with low scores
(hybrids 6, 7 and 10) are considered sensitive.
Soil drying experiment
Hybrids 5 and 6 were considered as representatives of tolerant and susceptible hybrids based on
their responses to increasing concentrations of PEG
6000, and both had shown high yields in previous field
evaluations by SERIDA in northwestern Spain. Young
plants showed visible drought symptoms when water
availability (WA) dropped below 50%. The experiment
continued until WA fell below 5% and plant damage
was irreversible (i.e. CO2 uptake below 2.0 μmol CO2
m-2 s-1). This critical point was reached by hybrid 5 in
15 days and by hybrid 6 in 11 days. Both hybrids also
differed for the time required to reach 50%, 35% and
25% WA, which was shorter for hybrid 6 in all cases.
The higher rates of CO2 assimilation and stomatal conductance were found during the first days of
drought stress, when an increase of photosyntheticrelated parameters was observed for both hybrids
(Figure 2). Hybrid 5 showed a faster and more efficient response to drought stress, decreasing its stomatal conductance and consequently reducing CO2
assimilation, but increasing its WUE and thus the survival period. Conversely, a delayed reduction in stomatal conductance was observed in hybrid 6, leading
to a faster desiccation and a reduced number of days
at which plants reached the irreversible point when
0,30
**
0,25
20
***
15
0,20
μ
Severe stress
1,5
3
11
Photosynthetic rate (μmol CO2 m-2 s-1)
8
Moderate stress
***
**
***
***
0,15
10
*** ***
***
**
***
*
5
***
***
*
*** ***
0
0
0,10
***
***
Hybrid 5
2
4
6
8
10
Days
Conductance Control
12
14
0,05
***
**
0,00
16
Conductance Dessication
25
0,30
***
Photosynthetic rate (μmol CO2 m-2 s-1)
Slight stress
2,0
Stomatal conductance (mol H2O m-2 s-1)
Control
2,5
**
0,25
*
20
0,20
15
*
0,15
*
***
10
0,10
***
***
***
5
Stomatal conductance (mol H2O m-2 s-1)
3,0
0,05
Hybrid 6
*** *** ***
0
0
2
4
6
8
10
***
***
0,00
12
14
16
Days
Figure 2 - Evolution of photosynthetic rate (µmol CO2 m-2 s-1)
and stomatal conductance (mol H2O m-2 s-1), measured on
control plants (100% water availability) and plants exposed
to progressive desiccation in two maize commercial hybrids
with previous contrasted sensitivity to early drought. For
each parameter and time, asterisks denote significant differences between control and desiccation treatments: * - P ≤
0.05; ** - P ≤ 0.01; *** - P ≤ 0.001; t-test.
Maydica electronic publication - 2017
Álvarez-Iglesias et al
8
Table 4 - Evolution of parameters related to photosynthetic performance of on plants of maize commercial hybrids 5 and 6 exposed to progressive desiccation, based
on imaging measurements of chlorophyll a fluorescence.
Day
Y(II)
Y(NPQ)
Y(NO)
qN
qL
ETR
Hybrid 5
10
Hybrid 6
1
3
6
7
-
---++
++
---
For each parameter and time, signs denote significant
reductions (-) or stimulations (+) with respect to their respective controls: one sign - P < 0.05; two signs - P <
0.01; three signs - P < 0.001; t-test.
Its increase was clearly correlated with the stress severity in hybrid 6, with a sharp increase at the end of
desiccation. But in hybrid 5, proline levels remained
lower than control values throughout the desiccation
period, being significantly higher than control only at
35% WA. These values are not consistent with the
increase in cellular osmolarity at the higher stress
levels. Levels of soluble proteins followed a common
pattern with proline in hybrid 5, showing a significant
increase at 35% WA. In the case of hybrid 6, protein concentrations remain lower than in control from
35% WA on.
Discussion
Identification of genotypes with high drought
tolerance is one of the main pillars for the selection
of the most adequate genotype, or breeding of new
ones. Therefore, our main objective was to find a simple and reliable method that allows large scale evaluations under controlled conditions.
Drought can impact plants at every developmental stage and at multiple levels, and in consequence
plants have evolved complex strategies involving a
large number of responses at morphological, physiological and biochemical levels of organization (Blum,
1996; Tardieu et al, 2011). In the present study, the parameters selected to explore the responses of maize
hybrids to drought demonstrated to have good discriminating ability. Differences in stress response indicate that there are differences in the ability of maize
hybrids for their ability to detect the stress, to respond
and to tolerate the stress up to different stress levels
(Chaves et al, 2009). However, the abrupt appearance of the stress and the short period under stress
that characterizes this type of screenings affects the
ability of the plant to respond to stress, forcing the
sudden triggering of its response and allowing the
discrimination of those cultivars with more efficient
capacity to cope with drought stress. In the seedling
growth evaluations, differences in growth patterns
are due to the diverse ability for facing stress. Conversely, morphological differences found in the early
growth assay might be affected by the time of stress
imposition, as the influence of previous growth under
62 ~ M24
non-stressed conditions is underestimated.
Right from the earliest crop stages, drought
causes a great decline in germination rates and increased seedling mortality (Anjum et al, 2011). In our
study, slight stress caused a delay in germination,
while moderate and severe levels also decreased the
percentage of germination. Total germination index
(GT) provides a good overall assessment of the effects of water stress on germination, but should not
be used as the only indicator. Indices S, AS, CRG,
and MGT revealed differences in the dynamics and
progress of the germination process between hybrids
in cases where GT, that considers only the final time,
did not show any effect. Hence, combination of GT
with other indices (S, AS, CRG, and MGT) allow an
efficient comparison of the effects of drought stress
on maize hybrids (Chiapusio et al, 1997).
A direct consequence of drought is cellular dehydration that leads to a reduced cell expansion. Due to
this early seedling growth, in which expansive growth
processes play a key role, is largely affected (Sharp
et al, 1988). This growth reduction was smaller for
hybrids 2, 3, and 5 (subsequently classified as tolerant). Mild or moderate stress conditions typically
reduce shoot growth in maize seedlings. In contrast,
roots are less sensitive than shoots to growth inhibition at low water potentials, and thus root elongation
and dry weight accumulation is less affected than
for shoots (Westgate and Boyer, 1985). As a consequence, the shoot/root ratios decrease. This balance
between root and shoot growth has a genetic regulation but also significant environmental effects (Ruta et
al, 2010). Under optimal conditions coleoptile growth
is considered a desirable trait as it is associated with
a further higher yield (Bruce et al, 2002; Rebetzke et
al, 2006). But under drought stress conditions, the
maintenance of growth of the main root is considered to be an adaptive mechanism for optimization of
soil water uptake (Sharp and Davies, 1989), enabling
roots to penetrate deeper in the soil and increasing
the possibilities of finding water sources. However,
the initial growth of secondary roots is also necessary
as they increase the surface area for water uptake
and could guarantee the subsequent water supply to
the main root before the water deficit reach severe
values. Thus, a good development of the root system can be critical for seedling establishment, and
increases the possibilities of survival under severe
drought conditions. According to this, hybrids classified as sensitive had a scarce root growth and higher
shoot/root ratios than the tolerant ones at all stress
levels. Under severe stress conditions, although there
is a strong overall reduction of seedling growth, the
maintenance of root growth is still observed, at least
for the primary root. Many studies of stress response
focus on the aerial parts of the plant given the difficulties in accessing the root system and in establishing
precise and uniform stress conditions in the environment surrounding the roots. However, in vitro evalu-
Maydica electronic publication - 2017
screening for maize tolerance to drought
ations of the first stages of development have been
proposed as a convenient and reliable approach
(Ruta et al, 2010).
In young plants, changes in biomass partitioning were also expected to occur in order to optimize
plant water uptake vs. water loss. The reduction of
vegetative growth (plant height and leaf area) and
biomass are well-known effects of drought stress
(Blum, 1996). Reduction in leaf growth is a direct
consequence of drought stress, but it can be also
considered an adaptive response to avoid water loss
by evapotranspiration. On the contrary, root growth
may be favored. Thus, under drought conditions and
as occurred in seedling growth, decreased shoot to
root ratios can be expected (Shao et al, 2008). For
those hybrids in which the strongest reductions in
seedling root growth were observed (6, 7, and 10),
young plants maintained high shoot/root ratios under
stress in greenhouse assays. Those sensitive hybrids
also suffered the strongest reductions in germination
rates under drought stress conditions. This relation
between morphological traits and drought stress tolerance from the early stages of development were
reported previously in maize (Bruce et al, 2002; Ruta
et al, 2010) but also in other species (Grzesiak et al,
1997; Lopes and Reynolds, 2011).
For other morphological traits such as aerial
biomass or LA, changes as a response to drought
stress are well known (Van Volkenburgh and Boyer,
1985; Edmeades et al, 1999). But in this experiment
these parameters did not show significant differences among hybrids and stress levels, although in
most cases reductions in leaf area were found at the
higher stress levels. Despite its suitability for evaluations under strictly controlled conditions, water stress
imposed by PEG is notably different from naturally
occurring drought stress. Water stress imposed by
PEG is interpreted by the plant as an instant stimulus rather than a natural long-term stimulus, allowing
the observation of the early responses of the plant
(Granda et al, 2011). But longer periods of stress
conditions would be needed for these morphological
responses to become obvious. On the other hand,
physiological parameters with a «water status» component (SLA and RWC) showed clear differences between treatments with an overall reduction as stress
level increased, although RWC was able to discriminate hybrid tolerance only under severe stress, as in
Sucre and Suárez (2011). For these parameters, and
as water loss caused by PEG treatment occur soon
after inducing the stress, significant differences appear after a short period under stress.
Drought also induces a metabolic re-programming
that results in changes in the whole transcriptome and
metabolome (Niinemets, 2016) and, consequently, in
an increased protein expression and accumulation
that can be quantified as an estimation of the magnitude of the response to drought. At a biochemical
level, osmotic adjustment mediated by the accumu-
62 ~ M24
9
lation of certain metabolites is considered one of the
most conspicuous responses to drought (Muscolo
et al, 2015). This osmotic adjustment helps plants
to maintain an adequate leaf turgor, and is mediated
by organic solutes such as proline, glycine betaine
or soluble carbohydrates that can also contribute to
other functions (Farooq et al, 2009). Although some
of the most sensitive and tolerant hybrids showed a
consistent response for these parameters, changes
in leaf proline and protein concentrations did not
show very clear trends among all hybrids and stress
levels, similarly to findings of Chimenti et al (2006) in
young and flowering maize populations.
Another well-known effect of drought is stomatal closure, which limits intercellular CO2 availability,
reducing photosynthetic carbon fixation. Stomatal
closure represents one of the earliest responses to
drought, protecting the plant from transpirative water loss and increasing its water use efficiency. On
the contrary, the primary photochemical events of
PSII are considered to be very resilient to drought,
so it is widely accepted that under moderate water
deficits photosynthetic capacity is maintained. Under severe drought conditions, non-stomatal constraints to photosynthesis appear as a consequence
of the prolonged decrease in CO2 availability, which
causes impairment between light energy captured
and conversion. Biochemical limitations, consisting
on the down-regulation of enzymes of the photosynthetic metabolism, appear as a consequence of the
metabolic impairment caused by the lower intercellular CO2, and result in a reduced but still reversible
photosynthetic capacity. Moreover, photochemical
limitations to photosynthesis appear when there is an
excess of energy that cannot be used for CO2 fixation
and that needs to be dissipated. If dissipation mechanisms fail, an irreversible damage to photosystems
can occur (Flexas and Medrano, 2002; Flexas et al,
2004; Chaves et al, 2009). In our first experiment,
consistent and significant results were obtained for
measurements made 48 and 72 h after the onset of
stress treatments. In general terms, hybrids able to
reduce stomatal conductance can, subsequently, reduce transpiration values and limit water loss. Values
for net photosynthetic rate follow the opposite trend,
as stomatal closure implies limited CO2 availability.
In the case of hybrid 10 stomatal opening did not involve higher CO2 availability, perhaps due to a damage of the photosynthetic machinery.
Drought tolerance is a complex trait. In consequence, a large number of morphological, physiological and agronomical traits can be used to assess
responses to drought (Farooq et al, 2009). For dealing with these complex data matrices, multivariate
analyses are the best approach. Multivariate analysis
methods such as PCA and CA are powerful tools for
the joint analysis of large sets of variables. PCA is
mainly used as a tool in exploratory data analysis and
for making predictive models, as this method allows
Maydica electronic publication - 2017
Álvarez-Iglesias et al
transforming a number of possible correlated variables into a limited number of uncorrelated variables
or principal components (PCs). In our work PCA provided a global perspective of hybrids response, and
allowed us to identify those traits with the highest
discriminating ability for drought tolerance. CA was
also a valuable method to classify maize genotypes
into groups that share similar responses and levels of
stress tolerance.
In the experiments included in our protocol, photosynthesis-related traits had the highest selective
value, suggesting promising opportunities for selection at this level. It would be worthwhile investigating
the mechanisms underlying the response of maize to
drought at the photosynthetic level with a more appropriate experimental design simulating real drought
conditions.
In the soil drying experiment, the study of the
response of photosynthesis-related traits was completed with the study of the response of chlorophyll
fluorescence parameters, as chlorophyll fluorescence
analysis is considered a reference method for studying stress response (Baker, 2008). Leaves capture
part of the light for photosynthesis, part of the light is
dissipated as heat and part returns as fluorescence;
thus, the increase of one of these fractions is associated with the reduction of the others (Maxwell and
Johnson, 2000). The efficiency of the photosynthetic
system PSII [Y(II)], the photochemistry quenching (qL)
and the electron transport rate (ETR) inform of the
photosynthetic activity that can be affected by CO2
availability. But these parameters showed a null predictive value of early drought conditions and a poor
discriminating ability, if compared to gas exchangerelated measurements. Our results are in agreement
with those reviewed in Baker and Rosenqvist (2004)
and Berger et al (2010), who pointed out that relevant
changes in fluorescence-related parameters are often
achieved only under mild or severe drought conditions, appearing as a consequence of the decrease of
intracellular CO2 concentration derived from stomatal
closure. These stomatal limitations are responsible
for the early decline in photosynthetic rate and stomatal conductance of hybrid 6, which can be related
to the punctual decreases detected in ETR (Flexas
et al, 2002), whereas subsequent decreases in these
parameters did not result in significant decreases in
ETR. No consistent differences were found for other
fluorescence parameters related to possible damages in the photosynthetic machinery. Thus, in this
experiment fluorescence-related parameters were
not good indicators of drought stress response. On
the contrary, the best indication of drought stress response was obtained by monitoring gas exchange.
The final increase in cell osmolarity found in both
hybrids can be partially related to the decrease in
RWC due to desiccation and, in the case of hybrid 6,
proline synthesis. But the magnitude of this increase
in hybrid 5, which is not associated with an increase
62 ~ M24
10
in proline levels, suggest that other metabolites different from proline may be contributing to this higher
osmolarity (Ashraf and Foolad, 2007).
Separately, PEG-based screening and soil drying experiments gave the same results as they both
considered hybrids 5 and 6 as tolerant and sensitive to drought, respectively. But when comparing
parameters measured in screening and soil drying
experiments, consistent relationships can be found
for several parameters, whereas trends are less obvious in other cases. For both hybrids, the same trends
were observed for LA, SLA, and RWC in both assays
but with different magnitudes. Same trends but different magnitudes were also observed in the case of
photosynthetic-related parameters (photosynthetic
rate and stomatal conductance). On the other hand,
different trends were found in the responses of root
DW, aerial DW and, consequently, shoot/root ratio.
Whereas at the end of the soil drying experiments reductions in both root and aerial biomasses can be
found as a consequence of drought, these reductions
are not observed in the PEG-based screening experiment. In the case of proline and soluble protein contents, no clear relations were found when comparing
the results of each hybrid between both assays. As
mentioned before, the different timing and intensity
of stress imposition in these two experiments can explain the differences when no clear trends are found.
As proline and protein contents had the lowest
discriminant ability in PEG-based experiments, the
utility of these parameters seem to be limited for
screening purposes. But when performing soil drying experiments for a better understanding of drought
responses, their utility have been demonstrated (Hare
and Cress, 1998). In the case of morphological parameters (root and aerial biomasses), although the
results may not be coincident in both experiments,
they have demonstrated to have modest discriminant
ability and, therefore, their measurement is useful for
screening purposes.
In this work, we describe a method based on the
use of PEG 6000 solutions to characterize a set of
maize hybrids according to their tolerance to drought
at early stages of development. Our results highlighted different responses of morphological, physiological and biochemical characters that, when considered together, allowed an efficient discrimination of
maize genotypes. The subsequent assay performed
under soil drying conditions validated our previous
results, but highlighted similarities and differences in
the response of the parameters evaluated. We can
conclude that this method is useful for screening
maize genotypes for drought tolerance. This method
also enables the rapid assessment and comparison
of the responses of morphological and physiological
traits potentially involved in drought stress tolerance
of germplasm, complementing more detailed physiological and agronomic studies.
Maydica electronic publication - 2017
screening for maize tolerance to drought
Acknowledgements
The authors acknowledge the companies shown
in table 1 as providers of the maize seed. This research was financed by the FICYT (Fundación para
el Fomento en Asuturias de la Investigación Científica Aplicada y la Tecnología, Principado de Asturias,
Spain). Lorena Álvarez-Iglesias acknowledges a grant
from FICYT.
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