Plant Growth Regul (2011) 64:129–139

DOI 10.1007/s10725-010-9547-9

ORIGINAL PAPER

Effects of citric acid as an important component of the responses

to saline and alkaline stress in the halophyte Leymus
chinensis (Trin.)
Yan-Lin Sun • Soon-Kwan Hong

Received: 28 June 2010 / Accepted: 3 November 2010 / Published online: 18 November 2010
Ó Springer Science+Business Media B.V. 2010

Abstract Some plants accumulate some compatible sol- enzymes. To compare with the mitigative effects of
utes and exude various organic acids when exposed to exogenous citric acid on stress, exogenous application of
environmental stress. These compatible solutes including proline was also performed under same conditions, and
proline have been suggested to be involved in stress tol- similar effects on the improvement of growth were
erance by maintaining sufficient cell turgor for growth, observed. Based on these results, we suggested that citric
thereby improving plant growth, protecting enzymes, and acid is an important component of the stress response in
membranes. However, less evidence exists regarding the L. chinensis, and exogenous application of 50 mg l-1 citric
protective roles of organic acids under stress conditions. acid might play a positive role on stress tolerance.
Here, we investigate the effects of citric acid as a com-
ponent of the response to stress on plant growth and anti- Keywords Alkaline stress
Citric acid accumulation

oxidant enzyme activities in two genotypes of halophyte Exogenous proline
Leymus chinensis
Saline stress
Leymus chinensis (Trin.) genotypes, LcWT07 and
LcJS0107. Data showed that both saline stress (200 mM
Abbreviations
NaCl) and alkaline stress (100 mM Na2CO3) reduced plant
PPFD Photosynthetic photon flux density
growth on the relative growth rate and CO2 assimilation
MCW Methanol/Chloroform/Water
rate, but increased the citric acid concentrations in 6-week-
MDA Malonyldialdehyde
old plants over the 72 h experimental period. When
TCA Trichloroacetic acid
50 mg l-1 citric acid was exogenously applied under stress
TBA Thiobarbituric acid
conditions, it significantly improved the plant growth and
PVP Polyvinylpyrrolidone
internal citric acid concentration, and also induced defense
SOD Superoxide dismutase
mechanisms by increasing the activities of antioxidant
NBT Nitroblue tetrazolium
CAT Catalase
Electronic supplementary material The online version of this APX Ascorbate peroxidase
article (doi:10.1007/s10725-010-9547-9) contains supplementary ANOVA Analysis of variance
material, which is available to authorized users.

Y.-L. Sun
S.-K. Hong (&)

Department of Bio-Health Technology, College of Biomedical
Science, Kangwon National University, Chuncheon,
Kangwon-Do 200-701, Korea Introduction
e-mail: soonkwan@kangwon.ac.kr
Y.-L. Sun Adverse environmental conditions, such as high salinity,
e-mail: ylsun04@mails.gucas.ac.cn high alkalinity, the presence of heavy metals, and drought
limit plant growth and drastically reduce plant productivity
S.-K. Hong
Institute of Bioscience and Biotechnology, Kangwon National (Boyer 1982). The injurious osmotic effects lead to the
University, Chuncheon, Kangwon-Do 200-701, Korea generation of oxidative stress and/or to inhibition of the

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130 Plant Growth Regul (2011) 64:129–139

system that operates to re-establish homeostasis to control enzyme activities. Based on our results, we are able to test
stress damage for repair and detoxification (Hernández and the hypotheseis that citric acid is a component of the stress
Almansa 2002). However, in some halophytic plants, the response and that exogenous citric acid can improve salt
toxic effects might also result in the inducement of stress tolerance by stimulating plant growth and increasing the
defense mechanisms (Pál et al. 2006). Accumulation of activities of antioxidant enzymes.
compatible solutes and exudation of organic acids are the
regular defense responses among them (Flowers et al.
1977; Godbold et al. 1984). Proline is a well-known Materials and methods
compatible solute, that plays a pivotal role in osmotic
adjustment in plants by helping to maintain sufficient cell Plant material and growth conditions
turgor for growth (Zimmermann 1978), and exogenous
proline is known to mitigate the detrimental effects of Na? Mature seeds of LcWT07 were obtained from the natural
and improve growth and survival under various stresses grasslands in Siping, Jilin, China, and mature seeds of
(Okuma et al. 2004; Harinasut et al. 1996). Citric acid is an LcJS0107 were obtained from the Jisheng Chinese Wildrye
important organic acid for plant growth, that has relation- Excellent Seed Station, Changchun, Jilin, China. The
ship with stress tolerance of heavy metal (Krotz et al. 1989; grassland located on the Songnen Plain, Jilin province is
Tesfaye et al. 2003; Zeng et al. 2008; Mailloux et al. 2008) the largest natural L. chinensis grassland in the Eurasian
and salinity (Fougère et al. 1991). However, little infor- continent (Guo 1994). The climate is semi-arid, with windy
mation has been reported about citric acid-related stress and dry winters and springs, and warm but comparatively
defense mechanisms on plant growth and antioxidant rain-rich summers, followed by short and cool autumns.
enzyme activities in plants, especially with regard to LcWT07 plants have adapted to these natural conditions as
exogenous citric acid, until now. the result of many years of natural evolution. LcJS0107
The halophyte Leymus chinensis (Trin.), a perennial is a new, cultivated variety that exhibits drought resistance,
rhizomatous grass placed in the tribe Gramineae, is a plant saline-alkali resistance and vegetative productivity
species with an extensive rhizome system that can bind soil (National Key New Product Project, No. 98G041D6600
and sand, and tolerate to environmental stresses such as 013). Seeds of both genotypes were de-husked and surface-
salinity and drought (Anamthawat Jónsson et al. 1990). It is sterilized using the method from Sun and Hong (2010b),
widely distributed throughout northern China, Mongolia with 70% ethanol for 1 min and then 5% sodium hypo-
and Siberia (Huang et al. 2004). Because of its intrinsic chlorite for 20 min. And the surface-sterilized seeds were
adaptation to highly alkaline-sodic soil conditions (Jin submerged in sterile water in 4°C for 3 days, and those
et al. 2006), L. chinensis is used as a soil-binding plant to sinking to the bottom were selected and germinated in
protect soil from desertification. During recent decades, dishes (20 cm in diameter, 15 mm in height) with water-
research on the stress responses of halophytic plants has saturated filter papers. The seedlings were grown in pots
aided our understanding of the mechanisms of stress (20 cm in diameter, 20 cm in height) containing clay/ver-
adaption and stress tolerance in plants. Thus, the halophyte miculite (3/1, v/v) at a density of one seedling per pot. The
L. chinensis has been considered as a perfect model plant plants were grown at 25°C in greenhouse conditions using
for the study of stress defense mechanisms. Expression a 16/8 h (day/night) photoperiod and a relative humidity of
profiling of the genes involved in salt stress has been 45–70%. The pots were watered with Hoagland nutrient
carried out to understand the mechanisms of stress toler- solution (Hoagland and Arnon 1950) on alternate days and
ance in the halophyte L. chinensis (Jin et al. 2006), but little watered daily with distilled water to replace water loss.
attention has been paid to the physiological responses to Six-week-old plants cultured under these conditions were
saline stress and alkaline stress in this grass. used to determine morphological characteristics.
In our previous study, we have found that proline
accumulation occurs after 6 h with 200 mM NaCl and Measurement of the CO2 assimilation rate
100 mM Na2CO3 in 6-week-old LcWT07 and LcJS0107
plants and the antioxidant enzyme activities, especially The CO2 assimilation rate of normal expanded leaves of
ascorbate peroxidase (APX) activity, decrease under stress 6-week-old plants was measured under various stress
conditions (Sun and Hong 2010a). In the present study, we treatments from 11:00 to 14:00 in a sunny day using a
further investigate the physiological responses to saline Li-Cor 6400 portable photosynthesis system. The leaf
stress (200 mM NaCl) and alkaline stress (100 mM temperature was maintained at 25 ± 0.2°C during the
Na2CO3) in two halophyte L. chinensis model genotypes, measurements using a photosynthetic photon flux density
LcWT07 and LcJS0107. We also analyzed the effects of (PPFD) of 1,500 lmol photons m-2 s-1. The ambient CO2
exogenous citric acid on plant growth and antioxidant concentration was maintained at 400 lmol CO2 mol-1 in

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the cuvette using a CO2 mixer, and the relative humidity water bath, mixing completely. The solution, including the
was maintained at 20%. Data were recorded after the BaSO4 pellet, was transferred to a 100 ml volumetric flask
system had equilibrated to a steady state (10 min after the and adjusted to 50 ml with 7.1% Na2SO4 solution. Acti-
start of each measurement). The leaf areas were calculated vated charcoal (0.2 g) was added to the solution to help
based on the measured part of the labeled area. We used the with sedimentation. After incubation at room temperature
leaf area of 1.40 cm2 (0.7 cm in width 9 2.0 cm in length) for 5 min, the supernatant was filtrated with the fine-mesh
in this study, and further experiments were also conducted filter paper. The filtered solution was diluted (1:10) and the
using the same leaf area. resulting solution was used as sample solution.
After preparation of the sample solution, 10 ml of 27%
Saline and alkaline stress treatments sodium acetate solution, 2 ml of diazotate solution, and
5 ml of glacial acetic acid were added to 20 ml of sample
Six-week-old LcWT07 and LcJS0107 plants were sub- solution to prepare the reference solution, while 10 ml 27%
jected to saline stress (200 mM NaCl) or alkaline stress of sodium acetate solution, 2 ml of diazotate solution, and
(100 mM Na2CO3) for various treatment times (0, 1, 3, 6, 5 ml of 1% lead tetraacetate solution rather than glacial
12, 24 and 72 h) until the pH value of the outflow acetic acid were added to 20 ml sample solution as the test
approached equilibrium. At this time, growth indexes, solution. After exactly 13 min, we measured the absor-
including the plant growth rate, the photosynthesis evalu- bance at 420 nm (NanoPhotometerTM, IMPLEN, UK). The
ated from the CO2 assimilation rate and the relative water concentration of citric acid was calculated from a standard
content were examined. Fresh leaf tissues were harvested graph based on known concentrations.
from stressed 6-week-old plants to determine the citric acid
concentrations and the activities of antioxidant enzymes. Antioxidant activities
To evaluate the effects of exogenous proline and citric
acid, 6-week-old plants were also watered under saline Fresh leaf tissues (1.0 g) were ground in a mortar with
stress or alkaline stress conditions combined with liquid nitrogen and then homogenized in 10 ml buffer
50 mg l-1 proline or 50 mg l-1 citric acid until the pH containing 50 mM potassium phosphate (pH 7.8), 0.5 mM
value of the outflow approached equilibrium, and the plants EDTA, 1% polyvinylpyrrolidone (PVP), 1 mM ascorbic
were sampled after 72 h of stress. Three independent rep- acid and 10% glycerol. The homogenates were centrifuged
etitions were performed for each experiment, and ten plants at 12,000 rpm for 20 min at 4°C. The supernatants of the
were analyzed in each experiment. homogenates were used as enzyme extracts and were
stored at 4°C until use.
Estimation of the citric acid concentration Superoxide dismutase (SOD, EC 1.15.1.1) activity was
determined according to the method of Beauchamp and
The citric acid concentrations were estimated using the Fridovich (1971) by monitoring the photo-reduction of
lead tetraacetate method (IFJU method, No. 22, 1973) with nitroblue tetrazolium (NBT). The reaction mixture con-
some modifications. Fresh leaf tissues or roots (0.5 g) of tained: 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA,
6-week-old LcWT07 and LcJS0107 were triturated using 13 mM methionine, 75 lM NBT, 2 lM riboflavin, and
liquid nitrogen and homogenized with extraction solution 100 ll enzyme extract. Riboflavin was added as the last
(methanol/chloroform/water, 12/5/1, v/v/v, MCW). The component, and the reaction was initiated by placing the
extraction solution was then centrifuged at 5,000 rpm for tubes under 15 W fluorescent lamps. The reaction was
5 min. We then added 1 ml of ammonia solution and 1 ml stopped after 10 min by removing the reaction tubes from the
of 20% BaCl2 solution to 5 ml of the supernatant and the light source. Non-illuminated and illuminated reactions
reaction was mixed completely. After incubating at room without enzyme extract served as calibration standards. SOD
temperature for 2 min, we added 15 ml of 95% ethanol activity was calculated based on the absorbance at 560 nm.
solution to the mixture and mixed the reaction completely Catalase (CAT, EC 1.11.1.6) activity was determined by
again. After a further incubation at room temperature for monitoring the decrease in absorbance at 240 nm. The
exactly 5 min, the mixture was centrifuged at 8,000 rpm reaction medium contained 50 mM potassium phosphate
for 5 min, and the supernatant was carefully decanted. The buffer (pH 7.0), 12.5 mM H2O2, and 50 ll enzyme extract
pellet was suspended and washed with 2 ml of washing (Beers and Sizer 1952).
solution (20% ethanol solution), and then centrifuged at Ascorbate peroxidase (APX, EC 1.11.1.11) activity was
8,000 rpm for 5 min. The supernatant was decanted again, determined using the method of Nakano and Asada (1981).
and the pellet was washed another 2–3 times, as described The reaction medium contained 50 mM Tris–HCl (pH 7.8),
previously. To cover the pellet, we added 10 ml of 7.1% 0.5 mM ascorbate, 0.1 mM EDTA, 0.1 mM H2O2 and
Na2SO4 and incubated the tube for 10 min in a boiling 50 ll enzyme extract for a total volume of 1 ml. The

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activity was monitored by the decrease in absorbance at two accessions of L. chinensis used in this study, LcWT07
290 nm. APX activity was determined using an extinction and LcJS0107, have similar morphological characteristics.
coefficient of 2.8 mM-1 cm-1. Six-week-old LcJS0107 plants are stronger than LcWT07
Protein concentration was determined using the Brad- at the same age, with 3.26 and 11.63% increment in height
ford (1976) method. All of the antioxidant enzyme activi- and straw number, respectively (Table 1). And the mature
ties are expressed as units per mg protein. leaves of LcJS0107 were a bit wider than the leaves of
LcWT07.
Lipid peroxidation
Citric acid accumulation under saline
Lipid peroxidation was estimated by determining the and alkaline stress
malonyldialdehyde (MDA) content of the leaves (Heath
and Packer 1968). Fresh leaf tissues (0.5 g) were homog- NaCl and Na2CO3 markedly increased citric acid exuda-
enized in 2 ml of 0.1% trichloroacetic acid (TCA). A tion, irrespective of genotype and tissue. LcWT07 showed
0.5 ml volume of enzyme extract was mixed with 1.5 ml of a slight increase in citric acid exudation in the leaves after
0.5% thiobarbituric acid (TBA) prepared in 20% TCA and 1 h of saline stress and a significant increase after 6 h
the resulting mixture was incubated at 90°C for 30 min. under stress (Fig. 1a). The citric acid concentrations in the
After stopping the reaction in an ice bath, the test solution leaves of LcWT07 stress tended to decrease after 24 h of
was centrifuged at 8,000 rpm for 10 min, and the absor- NaCl exposure and reached a comparable level to the
bance of the supernatant was measured at 532 nm. After control at 72 h. LcJS0107 showed a similar pattern of citric
subtracting the non-specific absorbance at 600 nm, the acid exudation as LcWT07 but a bit lower citric acid
MDA concentration was determined using the extinction concentration in the leaves than LcWT07 (Fig. 1a).
coefficient 155 mM-1 cm-1. Both genotypes had faster exudation of citric acid in the
roots than that in the leaves, with a significant increase at
Statistical analysis 3 h after NaCl stress (Fig. 1b). The roots of both genotypes
had a maximum exudation of citric acid at 3 h of stress,
Statistically significant differences between the means and remained relatively high levels over a 24 h treatment
were determined by based on two-way analysis of variance period. After 72 h after stress, both genotypes showed a
(ANOVA) using Duncan’s multiple-range test (Duncan sudden and significant decrease in citric acid exudation,
1955). A P value of less than 0.05 was considered and the concentrations returned to the control level
significant. (Fig. 1b).
Under Na2CO3 stress, the citric acid exudation in the
leaves of both genotypes showed a similar pattern to that
Results under NaCl stress (Fig. 1c). LcWT07 showed a slight
increase in citric acid exudation after 1 h of stress, a sig-
Morphological characteristics of LcWT07 nificant increase after 3 h and a maximum after 6 h of
and LcJS0107 plants stress (Fig. 1c). After 12 h of stress, the citric acid con-
centrations in the leaves of LcWT07 began to decrease
The morphological characteristics of Leymus include stiff slightly declining to the control level after 72 h. The citric
leaves, leaf blades strongly ribbed on top but glabrous acid concentrations in the leaves of LcJS0107 also began to
underneath, awnless glumes and lemmas, two to seven increase after 1 h of Na2CO3 stress, and the concentrations
spikelets, and long anthers (Melderis 1980). Wang and Lou of the citric acid exuded increased significantly after 3 h of
(1987) have demonstrated that the differences between the stress, reaching a maximum at 12 h. After 72 h of stress,
divergent L. chinensis populations were significant in straw the citric acid concentrations in LcJS0107 leaves decreased
numbers, leaf colour, leaf types, and seed production. The significantly and returned to the control level.

Table 1 Morphological descriptions of LcWT07 and LcJS0107 Leymus chinensis used in this study
Genotype Plant age The height of plantsa (mm) The leaf widthb (mm) Straw no. Leaf colour

LcWT07 6-week-old 167.55 ± 19.47 4.45 ± 0.22 48.67 ± 3.06 Grey-green

LcJS0107 6-week-old 173.01 ± 10.18 4.53 ± 0.64 54.33 ± 5.51 Yellow-green
a
The height of plants dose not mean the longest leaf length, but the average height of aerial parts of 6-week-old plants
b
The leaf width means the average width of mature leaves of 6-week-old plants. Values represented here are the mean ± standard error
(SE, n = 10)

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Fig. 1 Citric acid concentrations in the leaves (a and c) and roots known concentrations and determined as described in ‘‘Materials and
(b and d) of LcWT07 and LcJS0107 plants under saline stress methods’’. Values represented graphically in the form of a histogram
(200 mM NaCl, a and b) and alkaline stress (100 mM Na2CO3, c and are the mean ± standard error (SE, n = 10). Means followed by the
d) for various treatment times (0, 1, 3, 6, 12, 24 and 72 h). The same letter in the same series are not significantly different at
untreated leaves and roots served as experimental control. The P \ 0.05 according to two-way ANOVA using Duncan’s multiple-
concentration of citric acid was calculated using a standard graph of range test, and no letter written means insignificant difference

For the roots under Na2CO3 stress, a significant increase LcWT07 and LcJS0107 (Fig. 2). Exogenous proline has
of citric acid exudation occurred at 3 h in both genotypes been considered to play a role in mitigating the detrimental
(Fig. 1d). In succession, the exudation of citric acid slightly effects of Na? (Harinasut et al. 1996). We also investigated
decreased at 6 h after stress in LcWT07 and reached whether exogenous proline modifies the internal citric acid
the control levels after 72 h. LcJS0107 showed similar concentrations under NaCl and Na2CO3 stress. The exog-
responses to Na2CO3 stress as LcWT07, but, according enous application of citric acid under NaCl stress signifi-
to the statistical analysis, the exudation of citric acid cantly enhanced the exudation of citric acid in LcWT07
decreased at 6 h to levels significantly lower than the levels leaves, but the increased citric acid concentration was
observed at 3 h (Fig. 1d). comparable to that of the control (Fig. 2a). For LcWT07
To understand the exudation of citric acid in plants, the leaves after 72 h of NaCl stress, exogenous proline sig-
total citric acid concentrations were determined in whole nificantly induced the exudation of citric acid at levels
stressed plants including leaves and roots (Fig. S1). Under comparable to the control, as with exogenous citric acid
NaCl stress, both genotypes began to exude citric acid after (Fig. 2a). However, LcJS0107 exuded citric acid at a sig-
1 h, and this increased significantly after 3 h (Fig. S1A). nificantly high rate with either exogenous citric acid or
After 72 h of stress, the citric acid concentrations in whole exogenous proline, and the citric acid concentrations were
plants recovered to the control levels. Na2CO3 stress also remarkably higher than in the control. For the roots,
caused similar responses in citric acid exudation in whole both exogenous citric acid and exogenous proline induced
plants as NaCl stress (Fig. S1B). A remarkable increase in the exudation of citric acid significantly, irrespective of
the citric acid concentration was obtained after 3 h of stress genotype (Fig. 2b).
in whole plants. The exudation of citric acid in both Under Na2CO3 stress, both genotypes showed different
genotypes began to decrease after 24 h and almost disap- responses in respect of the internal citric acid concentra-
peared after 72 h of stress. tions of the leaves (Fig. 2c). For LcWT07 leaves, exoge-
nous application of both citric acid and proline caused a
The effect of exogenous citric acid on citric acid significant decrease compared to the control, but these
accumulation levels were significantly higher than that without exoge-
nous application. However, exogenous application caused a
To verify whether exogenous citric acid modifies the significant increase in the internal citric acid concentration
internal citric acid concentration, we investigated the in LcJS0107 leaves compared to the control (Fig. 2c). For
internal citric acid concentrations in the leaves and roots of the roots of both genotypes, the exogenous application of

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Fig. 2 Citric acid concentrations in the leaves (a and c) and roots known concentrations and determined as described in ‘‘Materials and
(b and d) of LcWT07 and LcJS0107 plants stressed with saline stress methods’’. Values represented graphically in the form of a histogram
(200 mM NaCl, a and b) and alkaline stress (100 mM Na2CO3, c and are the mean ± standard error (SE, n = 10). Means followed by the
d) in the presence or absence of either proline or citric acid for 72 h. same letter in the same series are not significantly different at
The untreated leaves and roots served as experimental control. The P \ 0.05 according to two-way ANOVA using Duncan’s multiple-
concentration of citric acid was calculated using a standard graph of range test, and no letter written means insignificant difference

citric acid and proline also induced citric acid exudation, relative water contents dropped by only 2% relative to the
and exogenous citric acid induced higher internal citric controls (Figs. 3, S3). With exogenous application of citric
acid concentrations than did exogenous proline (Fig. 2d). acid or proline under saline stress and alkaline stress, this
To understand the changes of citric acid exudation after situation was improved, mitigating the reduction in the
exogenous application, we investigated the citric acid relative water contents compared to those of plants stressed
concentrations in whole stressed plants (Fig. S2). Under with 72 h of saline or alkaline stress (Fig. 3).
saline stress, both the exogenous application of citric acid Photosynthesis, as evaluated from the CO2 assimilation
and proline resulted in a significant increase in citric acid rate, was significantly reduced in plants stressed with NaCl
exudation, irrespective of genotype (Fig. S2A). However, for 72 h to about 64.2 and 79.6% of the control in LcWT07
under alkaline stress, LcWT07 returned the level of citric and LcJS0107, respectively (Fig. 4a). However, with the
acid exudation to the control level with exogenous appli- addition of exogenous citric acid or proline, the 72 h saline
cation of both, whereas LcJS0107 showed higher levels of stress did not cause a significant decrease in the CO2
internal citric acid with exogenous application (Fig. S2B). assimilation rate in either genotype, but the levels returned
to the control level. Treatment with Na2CO3 for 72 h also
The effect of exogenous citric acid on the growth induced a decrease in the CO2 assimilation rate, with
of LcWT07 and LcJS0107 decrements of 46.6 and 52.6% of the control obtained for
LcWT07 and LcJS0107, respectively (Fig. 4b). The addi-
At the beginning of experimental period, treatment with tion of exogenous citric acid or proline to plants under
NaCl and Na2CO3 brought about a slight increase in the alkaline stress also did not cause an increase in the CO2
relative water contents of the leaves of LcWT07 and assimilation rate of either LcWT07 or LcJS0107, with the
LcJS0107 plants, which might be caused by the water levels remaining comparable to the control.
absorbability of the leaves (Fig. S3). Until 12 h of stress, a The biomasses, as evaluated from the height of the
visible decrease in the relative water contents caused by plants and their relative growth rates, were investigated in
dehydration was obtained in both genotypes. Even if the this study. We did not observe any remarkable differences
leaves were treated with NaCl or Na2CO3 for 72 h, that in the height of plants because of the short experimental

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Fig. 3 Relative water content (%) in the leaves of LcWT07 and Fig. 4 Photosynthesis evaluated by the CO2 assimilation rate in
LcJS0107 plants stressed with saline stress (200 mM NaCl, a) and LcWT07 and LcJS0107 plants stressed with saline stress (200 mM
alkaline stress (100 mM Na2CO3, b) in the presence or absence of either NaCl, a) and alkaline stress (100 mM Na2CO3, b) in the presence or
proline or citric acid for 72 h. The untreated plants served as absence of either proline or citric acid for 72 h. The untreated plants
experimental control 72 h. Single saline stress or alkaline stress, 72 h served as experimental control 72 h. Single saline stress or alkaline
Proline. Stress in the presence of proline, 72 h Citric acid. Stress in the stress, 72 h Proline. Stress in the presence of proline, 72 h Citric acid.
presence of citric acid. Values represented graphically in the form of a Stress in the presence of citric acid. Values represented graphically in
histogram are the mean ± standard error (SE, n = 10). Means the form of a histogram are the mean ± standard error (SE, n = 10).
followed by the same letter in the same series are not significantly Means followed by the same letter in the same series are not
different at P \ 0.05 according to two-way ANOVA using Duncan’s significantly different at P \ 0.05 according to two-way ANOVA
multiple-range test, and no letter written means insignificant difference using Duncan’s multiple-range test, and no letter written means
insignificant difference
period (72 h), but either stress treatment inhibited the
growth of plants to a certain extent (Fig. 5a, b). With the
exogenous application of citric acid or proline, the height exogenous citric acid to plants under alkaline stress, the
of the plants increased slightly compared to that without relative growth rates of both genotypes significantly
exogenous application. For the relative growth rates, saline increased compared to plants without exogenous applica-
stress for 72 h caused a significant decrease in LcWT07 tion, but the levels observed were comparable to the
and LcJS0107, with a 90.95 and 76.72% decline relative to control.
the controls, respectively (Fig. 5c). The addition of exog-
enous proline to plants under saline stress significantly Changes in lipid peroxidation and antioxidant
enhanced the relative growth rates in LcWT07 and enzyme activities
LcJS0107 over those without exogenous proline; the
addition of exogenous citric acid to plants under saline Membrane damage was caused by the membrane lipid
stress also significantly enhanced the relative growth rates, peroxidation, as estimated by the contents of MDA in this
but the levels were comparable to those of the controls. study (Fig. 6). For the control plants, the levels of MDA
Alkaline stress significantly inhibited plant growth, and the were higher in LcWT07 than in LcJS0107, but under saline
relative growth rates were reduced significantly almost to stress or alkaline stress, both in the presence or absence of
zero (Fig. 5d). However, the level of alkaline stress in the either citric acid or proline, LcJS0107 showed higher
presence of exogenous proline did not decrease, but concentrations of MDA than did LcWT07. Although the
the relative growth rates were increased compared to the saline and alkaline stresses used in this study were very
control, especially in LcWT07 plants. With the addition of effective in inhibiting plant growth as expressed by the

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b Fig. 5 The height and relative growth rate of LcWT07 and LcJS0107
plants stressed with saline stress (200 mM NaCl, a and c) and alkaline
stress (100 mM Na2CO3, b and d) in the presence or absence of either
proline or citric acid for 72 h. The untreated plants served as
experimental control 72 h. Single saline stress or alkaline stress, 72 h
Proline. Stress in the presence of proline, 72 h Citric acid. Stress in
the presence of citric acid. Values represented graphically in the form
of a histogram are the mean ± standard error (SE, n = 10). Means
followed by the same letter (labeled on the right side for LcWT07 and
on the left side for LcJS0107 in c and d) in the same series are not
significantly different at P \ 0.05 according to two-way ANOVA
using Duncan’s multiple-range test, and no letter written means
insignificant difference

the experimental period (72 h, Fig. 7a, b). However, with

the exogenous application of either proline or citric acid,
CAT activity significantly increased compared to the con-
trol and stress conditions without exogenous application.
Leaf SOD activity remained nearly constant in both plants
under various stresses during the experimental period
(Figs. 7c, d). Either exogenous proline or citric acid
enhanced SOD activity significantly. However, the activity
was slightly higher in the present of exogenous application
than that in the absence. Leaf APX activity decreased
slightly in both plants after saline stress during the exper-
imental period, and increased significantly relative to the
levels seen in the controls under the exogenous application
of either proline or citric acid (Fig. 7e). Under alkaline
stress, leaf APX activity showed different responses in both
genotypes, as there was a significant decrease in the APX
activity of LcWT07 but not of LcJS0107 (Fig. 7f). The
exogenous application of both proline and citric acid with
stress also did not induce remarkable changes in APX
activity in LcWT07 but not in LcJS0107, compared to the
controls. However, it significantly enhanced the APX
activities compared with those of stressed plants in the
absence of any exogenous application.

Discussion

In the present study, the effects of citric acid on plant

growth and stress tolerance in stressed L. chinensis plants
were investigated. Due to the intrinsic alkaline-tolerance of
this grass, our previous studies suggested that stressing
with 200 mM NaCl and 100 mM Na2CO3 did not result in
the death of 6-week-old LcWT07 plants until 144 and
120 h, respectively; LcJS0107 often possessed stronger
vitality than LcWT07 but still died (albeit one experi-
relative growth rate, this effect was not able to promote leaf mental day later than LcWT07, data not shown). We
lipid peroxidation in either genotype. Neither saline stress therefore chose to use 72 h, a time point at which both
nor alkaline stress used in this study caused any changes in genotypes had strong vitality, as the experimental period in
the MDA contents (Fig. 6). this work. Under these stresses, plant growth was signifi-
Leaf CAT activity increased slightly in both genotypes cantly inhibited, with relative growth rates declining
stressed with either saline stress or alkaline stress during rapidly in 6-week-old LcWT07 and LcJS0107 L. chinensis

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one of stress responses (Godbold et al. 1984; Shlizerman

et al. 2007). In particular, the exudation of citric acid
has been reported to be closely-related with aluminum
poisoning (De La Fuente et al. 1997; Ma and Furukawa
2003), iron stress (Shlizerman et al. 2007), alkaline stress
(Timpa et al. 1986) and high salinity (Fougère et al.
1991). In this study, citric acid also had expectedly
accumulated rapidly and prominently in the leaves and
roots of stressed plants after only 1 h of treatment. Then,
whether exogenous citric acid could improve stress
responses and tolerance becomes a question.
The reduced water contents that results from the saline
and alkaline stress were alleviated in part by the addition
of exogenous proline and citric acid. Photosynthesis was
maintained in the stressed plants by exogenous proline
and citric acid. Exogenous proline and citric acid did not
improve the height of plants, but significantly stimulated
the relative water contents of stressed plants. Exogenous
citric acid had the ability to increase the internal citric
acid concentrations in the leaves and roots of saline
stressed plants, and the internal citric acid concentrations
in roots of both genotypes showed a faster increase than
in leaves, that might result in direct contact with soil and
Fig. 6 Lipid peroxidation evaluated by MDA content in the leaves of direct exogenous citric acid absorption. In addition,
LcWT07 and LcJS0107 plants under saline stress (200 mM NaCl)
and alkaline stress (100 mM Na2CO3) in the presence or absence of exogenous proline also increased the accumulation of
either exogenous citric acid or proline for 72 h. The untreated plants citric acid in the leaves of saline stressed plants in vivo,
served as experimental control. Equal amounts of fresh leaf tissues irrespective of genotype. Therewith, pronounced increases
(0.5 g) for each of the experimental sets were sampled from more in antioxidant enzymes caused by increased internal citric
than three untreated or treated plants. The amount of MDA was
determined as described in ‘‘Materials and methods’’. Values acid were observed, suggesting exogenous citric acid
represented graphically in the form of a histogram are the activated defense mechanisms to avoid stress damage.
mean ± standard error (SE, n = 10). The MDA levels in the same The activities of CAT and APX, which plays a role in
series are analyzed according to two-way ANOVA using Duncan’s scavenging and neutralizing hydrogen peroxide, were
multiple-range test at P \ 0.05, and no letter written means insignif-
icant difference stimulated significantly by exogenous citric acid and
proline. However, the activity of SOD detoxifying O2-
was not changed in the presence of exogenous citric acid
plants. The photosynthesis was also declined significantly or proline in both genotypes.
and cooperatively with the inhibition of plant growth, Taken together, these results demonstrate saline stress
although the relative water contents were not significantly (200 mM NaCl) and alkaline stress (100 mM Na2CO3)
decreased by 72 h saline or alkaline stress. Lipid peroxi- used in this study induce stress defense mechanisms in
dation is an effective indicator of cellular oxidative damage 6-week-old L. chinensis plants, and exudation of citric acid
and was estimated here by measuring the levels of MDA. is one of stress-induced responses. Our results also suggest
No marked increase in the MDA contents of stressed plants that exogenous citric acid increases internal citric acid
was obtained, suggesting that at least, no extensive lipid concentrations in stressed plants, and this action might
peroxidation of cell-membrane components was caused by enhance stress tolerance by improving photosynthesis, and
salt-induced oxidative stress in this study. Antioxidant relative growth rate and increasing activities of antioxidant
enzymes including SOD, CAT, and APX, which promote enzymes. Further studies should focus on citric acid
the scavenging of reactive oxygen species, also did not metabolism-related gene expression and the relevant gene
show significant differences over 72 h experimental period, expression profiles of the halophyte L. chinensis in com-
suggesting that no oxidative damage was caused under bination with the exogenous application of citric acid. The
stressed conditions in this study. results might provide a more comprehensive understanding
To counteract environmental stresses, some plants, on the effect of exogenous citric acid on stress tolerance in
especially halophytic plants induce intrinsic stress defense L. chinensis and a new strategy for researching adaptive
mechanisms, of which the exudation of organic acids is mechanisms by examining halophyte plants as models.

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138 Plant Growth Regul (2011) 64:129–139

Fig. 7 Changes of the activities of antioxidant enzymes in the leaves and methods’’. Values represented graphically in the form of a
stressed with saline stress (200 mM NaCl, a, c, e) and alkaline stress histogram are the mean ± standard error (SE, n = 10). The levels in
(100 mM Na2CO3, b, d, f) in the presence or absence of either the same series are analyzed according to two-way ANOVA using
exogenous citric acid or proline for 72 h. The untreated plants served Duncan’s multiple-range test at P \ 0.05, and no letter written means
as experimental control. a, b CAT; c, d SOD; e, f APX. The activities insignificant difference
of antioxidant enzymes were determined as described in ‘‘Materials

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Plant Growth Regul (2011) 64:129–139 139

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