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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system that operates to re-establish homeostasis to control
stress damage for repair and detoxification (Hernández and
Almansa 2002). However, in some halophytic plants, the
toxic effects might also result in the inducement of stress
defense mechanisms (Pál et al. 2006). Accumulation of
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
compatible solute, that plays a pivotal role in osmotic
adjustment in plants by helping to maintain sufficient cell
turgor for growth (Zimmermann 1978), and exogenous
proline is known to mitigate the detrimental effects of Na?
and improve growth and survival under various stresses
(Okuma et al. 2004; Harinasut et al. 1996). Citric acid is an
important organic acid for plant growth, that has relation-
ship with stress tolerance of heavy metal (Krotz et al. 1989;
Tesfaye et al. 2003; Zeng et al. 2008; Mailloux et al. 2008)
and salinity (Fougère et al. 1991). However, little infor-
mation has been reported about citric acid-related stress
defense mechanisms on plant growth and antioxidant
enzyme activities in plants, especially with regard to
exogenous citric acid, until now.
The halophyte Leymus chinensis (Trin.), a perennial
rhizomatous grass placed in the tribe Gramineae, is a plant
species with an extensive rhizome system that can bind soil
and sand, and tolerate to environmental stresses such as
salinity and drought (Anamthawat Jónsson et al. 1990). It is
widely distributed throughout northern China, Mongolia
and Siberia (Huang et al. 2004). Because of its intrinsic
adaptation to highly alkaline-sodic soil conditions (Jin
et al. 2006), L. chinensis is used as a soil-binding plant to
protect soil from desertification. During recent decades,
research on the stress responses of halophytic plants has
aided our understanding of the mechanisms of stress
adaption and stress tolerance in plants. Thus, the halophyte
L. chinensis has been considered as a perfect model plant
for the study of stress defense mechanisms. Expression
profiling of the genes involved in salt stress has been
carried out to understand the mechanisms of stress toler-
ance in the halophyte L. chinensis (Jin et al. 2006), but little
attention has been paid to the physiological responses to
saline stress and alkaline stress in this grass.
In our previous study, we have found that proline
accumulation occurs after 6 h with 200 mM NaCl and
100 mM Na2CO3 in 6-week-old LcWT07 and LcJS0107
plants and the antioxidant enzyme activities, especially
ascorbate peroxidase (APX) activity, decrease under stress
conditions (Sun and Hong 2010a). In the present study, we
further investigate the physiological responses to saline
stress (200 mM NaCl) and alkaline stress (100 mM
Na2CO3) in two halophyte L. chinensis model genotypes,
LcWT07 and LcJS0107. We also analyzed the effects of
exogenous citric acid on plant growth and antioxidant
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Plant Growth Regul (2011) 64:129–139
enzyme activities. Based on our results, we are able to test
the hypotheseis that citric acid is a component of the stress
response and that exogenous citric acid can improve salt
tolerance by stimulating plant growth and increasing the
activities of antioxidant enzymes.
Materials and methods
Plant material and growth conditions
Mature seeds of LcWT07 were obtained from the natural
grasslands in Siping, Jilin, China, and mature seeds of
LcJS0107 were obtained from the Jisheng Chinese Wildrye
Excellent Seed Station, Changchun, Jilin, China. The
grassland located on the Songnen Plain, Jilin province is
the largest natural L. chinensis grassland in the Eurasian
continent (Guo 1994). The climate is semi-arid, with windy
and dry winters and springs, and warm but comparatively
rain-rich summers, followed by short and cool autumns.
LcWT07 plants have adapted to these natural conditions as
the result of many years of natural evolution. LcJS0107
is a new, cultivated variety that exhibits drought resistance,
saline-alkali resistance and vegetative productivity
(National Key New Product Project, No. 98G041D6600
013). Seeds of both genotypes were de-husked and surface-
sterilized using the method from Sun and Hong (2010b),
with 70% ethanol for 1 min and then 5% sodium hypo-
chlorite for 20 min. And the surface-sterilized seeds were
submerged in sterile water in 4°C for 3 days, and those
sinking to the bottom were selected and germinated in
dishes (20 cm in diameter, 15 mm in height) with water-
saturated filter papers. The seedlings were grown in pots
(20 cm in diameter, 20 cm in height) containing clay/ver-
miculite (3/1, v/v) at a density of one seedling per pot. The
plants were grown at 25°C in greenhouse conditions using
a 16/8 h (day/night) photoperiod and a relative humidity of
45–70%. The pots were watered with Hoagland nutrient
solution (Hoagland and Arnon 1950) on alternate days and
watered daily with distilled water to replace water loss.
Six-week-old plants cultured under these conditions were
used to determine morphological characteristics.
Measurement of the CO2 assimilation rate
The CO2 assimilation rate of normal expanded leaves of
6-week-old plants was measured under various stress
treatments from 11:00 to 14:00 in a sunny day using a
Li-Cor 6400 portable photosynthesis system. The leaf
temperature was maintained at 25 ± 0.2°C during the
measurements using a photosynthetic photon flux density
(PPFD) of 1,500 lmol photons m-2 s-1. The ambient CO2
concentration was maintained at 400 lmol CO2 mol-1 in
Plant Growth Regul (2011) 64:129–139
the cuvette using a CO2 mixer, and the relative humidity
was maintained at 20%. Data were recorded after the
system had equilibrated to a steady state (10 min after the
start of each measurement). The leaf areas were calculated
based on the measured part of the labeled area. We used the
leaf area of 1.40 cm2 (0.7 cm in width 9 2.0 cm in length)
in this study, and further experiments were also conducted
using the same leaf area.
Saline and alkaline stress treatments
Six-week-old LcWT07 and LcJS0107 plants were sub-
jected to saline stress (200 mM NaCl) or alkaline stress
(100 mM Na2CO3) for various treatment times (0, 1, 3, 6,
12, 24 and 72 h) until the pH value of the outflow
approached equilibrium. At this time, growth indexes,
including the plant growth rate, the photosynthesis evalu-
ated from the CO2 assimilation rate and the relative water
content were examined. Fresh leaf tissues were harvested
from stressed 6-week-old plants to determine the citric acid
concentrations and the activities of antioxidant enzymes.
To evaluate the effects of exogenous proline and citric
acid, 6-week-old plants were also watered under saline
stress or alkaline stress conditions combined with
50 mg l-1 proline or 50 mg l-1 citric acid until the pH
value of the outflow approached equilibrium, and the plants
were sampled after 72 h of stress. Three independent rep-
etitions were performed for each experiment, and ten plants
were analyzed in each experiment.
Estimation of the citric acid concentration
The citric acid concentrations were estimated using the
lead tetraacetate method (IFJU method, No. 22, 1973) with
some modifications. Fresh leaf tissues or roots (0.5 g) of
6-week-old LcWT07 and LcJS0107 were triturated using
liquid nitrogen and homogenized with extraction solution
(methanol/chloroform/water, 12/5/1, v/v/v, MCW). The
extraction solution was then centrifuged at 5,000 rpm for
5 min. We then added 1 ml of ammonia solution and 1 ml
of 20% BaCl2 solution to 5 ml of the supernatant and the
reaction was mixed completely. After incubating at room
temperature for 2 min, we added 15 ml of 95% ethanol
solution to the mixture and mixed the reaction completely
again. After a further incubation at room temperature for
exactly 5 min, the mixture was centrifuged at 8,000 rpm
for 5 min, and the supernatant was carefully decanted. The
pellet was suspended and washed with 2 ml of washing
solution (20% ethanol solution), and then centrifuged at
8,000 rpm for 5 min. The supernatant was decanted again,
and the pellet was washed another 2–3 times, as described
previously. To cover the pellet, we added 10 ml of 7.1%
Na2SO4 and incubated the tube for 10 min in a boiling
131
water bath, mixing completely. The solution, including the
BaSO4 pellet, was transferred to a 100 ml volumetric flask
and adjusted to 50 ml with 7.1% Na2SO4 solution. Acti-
vated charcoal (0.2 g) was added to the solution to help
with sedimentation. After incubation at room temperature
for 5 min, the supernatant was filtrated with the fine-mesh
filter paper. The filtered solution was diluted (1:10) and the
resulting solution was used as sample solution.
After preparation of the sample solution, 10 ml of 27%
sodium acetate solution, 2 ml of diazotate solution, and
5 ml of glacial acetic acid were added to 20 ml of sample
solution to prepare the reference solution, while 10 ml 27%
of sodium acetate solution, 2 ml of diazotate solution, and
5 ml of 1% lead tetraacetate solution rather than glacial
acetic acid were added to 20 ml sample solution as the test
solution. After exactly 13 min, we measured the absor-
bance at 420 nm (NanoPhotometerTM, IMPLEN, UK). The
concentration of citric acid was calculated from a standard
graph based on known concentrations.
Antioxidant activities
Fresh leaf tissues (1.0 g) were ground in a mortar with
liquid nitrogen and then homogenized in 10 ml buffer
containing 50 mM potassium phosphate (pH 7.8), 0.5 mM
EDTA, 1% polyvinylpyrrolidone (PVP), 1 mM ascorbic
acid and 10% glycerol. The homogenates were centrifuged
at 12,000 rpm for 20 min at 4°C. The supernatants of the
homogenates were used as enzyme extracts and were
stored at 4°C until use.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was
determined according to the method of Beauchamp and
Fridovich (1971) by monitoring the photo-reduction of
nitroblue tetrazolium (NBT). The reaction mixture con-
tained: 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA,
13 mM methionine, 75 lM NBT, 2 lM riboflavin, and
100 ll enzyme extract. Riboflavin was added as the last
component, and the reaction was initiated by placing the
tubes under 15 W fluorescent lamps. The reaction was
stopped after 10 min by removing the reaction tubes from the
light source. Non-illuminated and illuminated reactions
without enzyme extract served as calibration standards. SOD
activity was calculated based on the absorbance at 560 nm.
Catalase (CAT, EC 1.11.1.6) activity was determined by
monitoring the decrease in absorbance at 240 nm. The
reaction medium contained 50 mM potassium phosphate
buffer (pH 7.0), 12.5 mM H2O2, and 50 ll enzyme extract
(Beers and Sizer 1952).
Ascorbate peroxidase (APX, EC 1.11.1.11) activity was
determined using the method of Nakano and Asada (1981).
The reaction medium contained 50 mM Tris–HCl (pH 7.8),
0.5 mM ascorbate, 0.1 mM EDTA, 0.1 mM H2O2 and
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
290 nm. APX activity was determined using an extinction
coefficient of 2.8 mM-1 cm-1.
Protein concentration was determined using the Brad-
ford (1976) method. All of the antioxidant enzyme activi-
ties are expressed as units per mg protein.
Lipid peroxidation
Lipid peroxidation was estimated by determining the
malonyldialdehyde (MDA) content of the leaves (Heath
and Packer 1968). Fresh leaf tissues (0.5 g) were homog-
enized in 2 ml of 0.1% trichloroacetic acid (TCA). A
0.5 ml volume of enzyme extract was mixed with 1.5 ml of
0.5% thiobarbituric acid (TBA) prepared in 20% TCA and
the resulting mixture was incubated at 90°C for 30 min.
After stopping the reaction in an ice bath, the test solution
was centrifuged at 8,000 rpm for 10 min, and the absor-
bance of the supernatant was measured at 532 nm. After
subtracting the non-specific absorbance at 600 nm, the
MDA concentration was determined using the extinction
coefficient 155 mM-1 cm-1.
Statistical analysis
Statistically significant differences between the means
were determined by based on two-way analysis of variance
(ANOVA) using Duncan’s multiple-range test (Duncan
1955). A P value of less than 0.05 was considered
significant.
Results
Morphological characteristics of LcWT07
and LcJS0107 plants
The morphological characteristics of Leymus include stiff
leaves, leaf blades strongly ribbed on top but glabrous
underneath, awnless glumes and lemmas, two to seven
spikelets, and long anthers (Melderis 1980). Wang and Lou
(1987) have demonstrated that the differences between the
divergent L. chinensis populations were significant in straw
numbers, leaf colour, leaf types, and seed production. The
Table 1 Morphological descriptions of LcWT07 and LcJS0107 Leymus chinensis used in this study
Genotype Plant age The height of plantsa (mm)
LcWT07 6-week-old 167.55 ± 19.47
LcJS0107 6-week-old 173.01 ± 10.18
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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Plant Growth Regul (2011) 64:129–139
two accessions of L. chinensis used in this study, LcWT07
and LcJS0107, have similar morphological characteristics.
Six-week-old LcJS0107 plants are stronger than LcWT07
at the same age, with 3.26 and 11.63% increment in height
and straw number, respectively (Table 1). And the mature
leaves of LcJS0107 were a bit wider than the leaves of
LcWT07.
Citric acid accumulation under saline
and alkaline stress
NaCl and Na2CO3 markedly increased citric acid exuda-
tion, irrespective of genotype and tissue. LcWT07 showed
a slight increase in citric acid exudation in the leaves after
1 h of saline stress and a significant increase after 6 h
under stress (Fig. 1a). The citric acid concentrations in the
leaves of LcWT07 stress tended to decrease after 24 h of
NaCl exposure and reached a comparable level to the
control at 72 h. LcJS0107 showed a similar pattern of citric
acid exudation as LcWT07 but a bit lower citric acid
concentration in the leaves than LcWT07 (Fig. 1a).
Both genotypes had faster exudation of citric acid in the
roots than that in the leaves, with a significant increase at
3 h after NaCl stress (Fig. 1b). The roots of both genotypes
had a maximum exudation of citric acid at 3 h of stress,
and remained relatively high levels over a 24 h treatment
period. After 72 h after stress, both genotypes showed a
sudden and significant decrease in citric acid exudation,
and the concentrations returned to the control level
(Fig. 1b).
Under Na2CO3 stress, the citric acid exudation in the
leaves of both genotypes showed a similar pattern to that
under NaCl stress (Fig. 1c). LcWT07 showed a slight
increase in citric acid exudation after 1 h of stress, a sig-
nificant increase after 3 h and a maximum after 6 h of
stress (Fig. 1c). After 12 h of stress, the citric acid con-
centrations in the leaves of LcWT07 began to decrease
slightly declining to the control level after 72 h. The citric
acid concentrations in the leaves of LcJS0107 also began to
increase after 1 h of Na2CO3 stress, and the concentrations
of the citric acid exuded increased significantly after 3 h of
stress, reaching a maximum at 12 h. After 72 h of stress,
the citric acid concentrations in LcJS0107 leaves decreased
significantly and returned to the control level.
The leaf widthb (mm) Straw no. Leaf colour
4.45 ± 0.22 48.67 ± 3.06 Grey-green
4.53 ± 0.64 54.33 ± 5.51 Yellow-green
Plant Growth Regul (2011) 64:129–139
Fig. 1 Citric acid concentrations in the leaves (a and c) and roots
(b and d) of LcWT07 and LcJS0107 plants under saline stress
(200 mM NaCl, a and b) and alkaline stress (100 mM Na2CO3, c and
d) for various treatment times (0, 1, 3, 6, 12, 24 and 72 h). The
untreated leaves and roots served as experimental control. The
concentration of citric acid was calculated using a standard graph of
For the roots under Na2CO3 stress, a significant increase
of citric acid exudation occurred at 3 h in both genotypes
(Fig. 1d). In succession, the exudation of citric acid slightly
decreased at 6 h after stress in LcWT07 and reached
the control levels after 72 h. LcJS0107 showed similar
responses to Na2CO3 stress as LcWT07, but, according
to the statistical analysis, the exudation of citric acid
decreased at 6 h to levels significantly lower than the levels
observed at 3 h (Fig. 1d).
To understand the exudation of citric acid in plants, the
total citric acid concentrations were determined in whole
stressed plants including leaves and roots (Fig. S1). Under
NaCl stress, both genotypes began to exude citric acid after
1 h, and this increased significantly after 3 h (Fig. S1A).
After 72 h of stress, the citric acid concentrations in whole
plants recovered to the control levels. Na2CO3 stress
caused similar responses in citric acid exudation in whole
plants as NaCl stress (Fig. S1B). A remarkable increase in
the citric acid concentration was obtained after 3 h of stress
in whole plants. The exudation of citric acid in both
genotypes began to decrease after 24 h and almost disap-
peared after 72 h of stress.
The effect of exogenous citric acid on citric acid
accumulation
To verify whether exogenous citric acid modifies the
internal citric acid concentration, we investigated the
internal citric acid concentrations in the leaves and roots of
133
known concentrations and determined as described in ‘‘Materials and
methods’’. Values represented graphically in the form of a histogram
are the mean ± standard error (SE, n = 10). Means followed by the
same letter 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
LcWT07 and LcJS0107 (Fig. 2). Exogenous proline has
been considered to play a role in mitigating the detrimental
effects of Na? (Harinasut et al. 1996). We also investigated
whether exogenous proline modifies the internal citric acid
concentrations under NaCl and Na2CO3 stress. The exog-
enous application of citric acid under NaCl stress signifi-
cantly enhanced the exudation of citric acid in LcWT07
leaves, but the increased citric acid concentration was
comparable to that of the control (Fig. 2a). For LcWT07
leaves after 72 h of NaCl stress, exogenous proline sig-
nificantly induced the exudation of citric acid at levels
comparable to the control, as with exogenous citric acid
(Fig. 2a). However, LcJS0107 exuded citric acid at a sig-
nificantly high rate with either exogenous citric acid or
exogenous proline, and the citric acid concentrations were
also remarkably higher than in the control. For the roots,
both exogenous citric acid and exogenous proline induced
the exudation of citric acid significantly, irrespective of
genotype (Fig. 2b).
Under Na2CO3 stress, both genotypes showed different
responses in respect of the internal citric acid concentra-
tions of the leaves (Fig. 2c). For LcWT07 leaves, exoge-
nous application of both citric acid and proline caused a
significant decrease compared to the control, but these
levels were significantly higher than that without exoge-
nous application. However, exogenous application caused a
significant increase in the internal citric acid concentration
in LcJS0107 leaves compared to the control (Fig. 2c). For
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
(b and d) of LcWT07 and LcJS0107 plants stressed with saline stress
(200 mM NaCl, a and b) and alkaline stress (100 mM Na2CO3, c and
d) in the presence or absence of either proline or citric acid for 72 h.
The untreated leaves and roots served as experimental control. The
concentration of citric acid was calculated using a standard graph of
citric acid and proline also induced citric acid exudation,
and exogenous citric acid induced higher internal citric
acid concentrations than did exogenous proline (Fig. 2d).
To understand the changes of citric acid exudation after
exogenous application, we investigated the citric acid
concentrations in whole stressed plants (Fig. S2). Under
saline stress, both the exogenous application of citric acid
and proline resulted in a significant increase in citric acid
exudation, irrespective of genotype (Fig. S2A). However,
under alkaline stress, LcWT07 returned the level of citric
acid exudation to the control level with exogenous appli-
cation of both, whereas LcJS0107 showed higher levels of
internal citric acid with exogenous application (Fig. S2B).
The effect of exogenous citric acid on the growth
of LcWT07 and LcJS0107
At the beginning of experimental period, treatment with
NaCl and Na2CO3 brought about a slight increase in the
relative water contents of the leaves of LcWT07 and
LcJS0107 plants, which might be caused by the water
absorbability of the leaves (Fig. S3). Until 12 h of stress, a
visible decrease in the relative water contents caused by
dehydration was obtained in both genotypes. Even if the
leaves were treated with NaCl or Na2CO3 for 72 h, that
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Plant Growth Regul (2011) 64:129–139
known concentrations and determined as described in ‘‘Materials and
methods’’. Values represented graphically in the form of a histogram
are the mean ± standard error (SE, n = 10). Means followed by the
same letter 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
relative water contents dropped by only 2% relative to the
controls (Figs. 3, S3). With exogenous application of citric
acid or proline under saline stress and alkaline stress, this
situation was improved, mitigating the reduction in the
relative water contents compared to those of plants stressed
with 72 h of saline or alkaline stress (Fig. 3).
Photosynthesis, as evaluated from the CO2 assimilation
rate, was significantly reduced in plants stressed with NaCl
for 72 h to about 64.2 and 79.6% of the control in LcWT07
and LcJS0107, respectively (Fig. 4a). However, with the
addition of exogenous citric acid or proline, the 72 h saline
stress did not cause a significant decrease in the CO2
assimilation rate in either genotype, but the levels returned
to the control level. Treatment with Na2CO3 for 72 h also
induced a decrease in the CO2 assimilation rate, with
decrements of 46.6 and 52.6% of the control obtained for
LcWT07 and LcJS0107, respectively (Fig. 4b). The addi-
tion of exogenous citric acid or proline to plants under
alkaline stress also did not cause an increase in the CO2
assimilation rate of either LcWT07 or LcJS0107, with the
levels remaining comparable to the control.
The biomasses, as evaluated from the height of the
plants and their relative growth rates, were investigated in
this study. We did not observe any remarkable differences
in the height of plants because of the short experimental
Plant Growth Regul (2011) 64:129–139
Fig. 3 Relative water content (%) in the leaves of LcWT07 and
LcJS0107 plants stressed with saline stress (200 mM NaCl, a) and
alkaline stress (100 mM Na2CO3, b) 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 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
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
of the plants increased slightly compared to that without
exogenous application. For the relative growth rates, saline
stress for 72 h caused a significant decrease in LcWT07
and LcJS0107, with a 90.95 and 76.72% decline relative to
the controls, respectively (Fig. 5c). The addition of exog-
enous proline to plants under saline stress significantly
enhanced the relative growth rates in LcWT07 and
LcJS0107 over those without exogenous proline; the
addition of exogenous citric acid to plants under saline
stress also significantly enhanced the relative growth rates,
but the levels were comparable to those of the controls.
Alkaline stress significantly inhibited plant growth, and the
relative growth rates were reduced significantly almost to
zero (Fig. 5d). However, the level of alkaline stress in the
presence of exogenous proline did not decrease, but
the relative growth rates were increased compared to the
control, especially in LcWT07 plants. With the addition of
135
Fig. 4 Photosynthesis evaluated by the CO2 assimilation rate in
LcWT07 and LcJS0107 plants stressed with saline stress (200 mM
NaCl, a) and alkaline stress (100 mM Na2CO3, b) 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 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
exogenous citric acid to plants under alkaline stress, the
relative growth rates of both genotypes significantly
increased compared to plants without exogenous applica-
tion, but the levels observed were comparable to the
control.
Changes in lipid peroxidation and antioxidant
enzyme activities
Membrane damage was caused by the membrane lipid
peroxidation, as estimated by the contents of MDA in this
study (Fig. 6). For the control plants, the levels of MDA
were higher in LcWT07 than in LcJS0107, but under saline
stress or alkaline stress, both in the presence or absence of
either citric acid or proline, LcJS0107 showed higher
concentrations of MDA than did LcWT07. Although the
saline and alkaline stresses used in this study were very
effective in inhibiting plant growth as expressed by the
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136 Plant Growth Regul (2011) 64:129–139

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

relative growth rate, this effect was not able to promote leaf
lipid peroxidation in either genotype. Neither saline stress
nor alkaline stress used in this study caused any changes in
the MDA contents (Fig. 6).
Leaf CAT activity increased slightly in both genotypes
stressed with either saline stress or alkaline stress during
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-
mental day later than LcWT07, data not shown). We
therefore chose to use 72 h, a time point at which both
genotypes had strong vitality, as the experimental period in
this work. Under these stresses, plant growth was signifi-
cantly inhibited, with relative growth rates declining
rapidly in 6-week-old LcWT07 and LcJS0107 L. chinensis

123
Plant Growth Regul (2011) 64:129–139
Fig. 6 Lipid peroxidation evaluated by MDA content in the leaves of
LcWT07 and LcJS0107 plants under saline stress (200 mM NaCl)
and alkaline stress (100 mM Na2CO3) in the presence or absence of
either exogenous citric acid or proline for 72 h. The untreated plants
served as experimental control. Equal amounts of fresh leaf tissues
(0.5 g) for each of the experimental sets were sampled from more
than three untreated or treated plants. The amount of MDA was
determined as described in ‘‘Materials and methods’’. Values
represented graphically in the form of a histogram are the
mean ± standard error (SE, n = 10). The MDA levels in the same
series are analyzed according to two-way ANOVA using Duncan’s
multiple-range test at P \ 0.05, and no letter written means insignif-
icant difference
plants. The photosynthesis was also declined significantly
and cooperatively with the inhibition of plant growth,
although the relative water contents were not significantly
decreased by 72 h saline or alkaline stress. Lipid peroxi-
dation is an effective indicator of cellular oxidative damage
and was estimated here by measuring the levels of MDA.
No marked increase in the MDA contents of stressed plants
was obtained, suggesting that at least, no extensive lipid
peroxidation of cell-membrane components was caused by
salt-induced oxidative stress in this study. Antioxidant
enzymes including SOD, CAT, and APX, which promote
the scavenging of reactive oxygen species, also did not
show significant differences over 72 h experimental period,
suggesting that no oxidative damage was caused under
stressed conditions in this study.
To counteract environmental stresses, some plants,
especially halophytic plants induce intrinsic stress defense
mechanisms, of which the exudation of organic acids is
137
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
direct exogenous citric acid absorption. In addition,
exogenous proline also increased the accumulation of
citric acid in the leaves of saline stressed plants in vivo,
irrespective of genotype. Therewith, pronounced increases
in antioxidant enzymes caused by increased internal citric
acid were observed, suggesting exogenous citric acid
activated defense mechanisms to avoid stress damage.
The activities of CAT and APX, which plays a role in
scavenging and neutralizing hydrogen peroxide, were
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
or proline in both genotypes.
Taken together, these results demonstrate saline stress
(200 mM NaCl) and alkaline stress (100 mM Na2CO3)
used in this study induce stress defense mechanisms in
6-week-old L. chinensis plants, and exudation of citric acid
is one of stress-induced responses. Our results also suggest
that exogenous citric acid increases internal citric acid
concentrations in stressed plants, and this action might
enhance stress tolerance by improving photosynthesis, and
relative growth rate and increasing activities of antioxidant
enzymes. Further studies should focus on citric acid
metabolism-related gene expression and the relevant gene
expression profiles of the halophyte L. chinensis in com-
bination with the exogenous application of citric acid. The
results might provide a more comprehensive understanding
on the effect of exogenous citric acid on stress tolerance in
L. chinensis and a new strategy for researching adaptive
mechanisms by examining halophyte plants as models.
123
138
Fig. 7 Changes of the activities of antioxidant enzymes in the leaves
stressed with saline stress (200 mM NaCl, a, c, e) and alkaline stress
(100 mM Na2CO3, b, d, f) in the presence or absence of either
exogenous citric acid or proline for 72 h. The untreated plants served
as experimental control. a, b CAT; c, d SOD; e, f APX. The activities
of antioxidant enzymes were determined as described in ‘‘Materials
123
Plant Growth Regul (2011) 64:129–139
and methods’’. Values represented graphically in the form of a
histogram are the mean ± standard error (SE, n = 10). The levels in
the same series are analyzed according to two-way ANOVA using
Duncan’s multiple-range test at P \ 0.05, and no letter written means
insignificant difference
Plant Growth Regul (2011) 64:129–139
Acknowledgments This work was supported by Nutraceutical Bio
Brain Korea 21 Project Group.
References
Anamthawat Jónsson K, Schwarzacher T, Leitch AR, Bennett MD,
Heslop Harrison JS (1990) Discrimination between closely
related Triticeae species using genomic DNA as a probe. Theor
Appl Genet 79:721–728. doi:10.1007/BF00224236
Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved
assays and an assay applicable to acrylamide gels. Anal Biochem
44:276–287
Beers RF, Sizer IW (1952) A spectrophotometric method for
measuring the breakdown of hydrogen peroxide by catalase.
J Biol Chem 195:133–140
Boyer JS (1982) Plant productivity and environment. Science
218:443–448. doi:10.1126/science.218.4571.443
Bradford M (1976) A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248–254
De La Fuente JM, Ramı́rez Rodrı́guez V, Cabrera Ponce JL, Herrera
Estrella L (1997) Aluminum tolerance in transgenic plants by
alteration of citrate synthesis. Science 276:1566–1568. doi:
10.1126/science.276.5318.1566
Duncan DB (1955) Multiple range and multiple F tests. Biometrics
11:1–42
Flowers TJ, Troke PF, Yeo AR (1977) The mechanism of salt
tolerance in halophytes. Annu Rev Plant Physiol 28:89–121. doi:
10.1146/annurev.pp.28.060177.000513
Fougère F, Le Rudulier D, Streeter JG (1991) Effects of salt stress on
amino acid, organic acid, and carbohydrate composition of roots,
bacteroids and cytosol of alfalfa (Medicago sativa L.). Plant
Physiol 96:1228–1236
Godbold DL, Horst WJ, Collins JC, Thurman DA, Marschner H
(1984) Accumulation of zinc and organic acids in roots of zinc
tolerant and non-tolerant ecotypes of Deschampsia caespitosa.
J Plant Physiol 116:59–69
Guo JX (1994) Decomposer Subsystem of Leymus chinensis Grass-
lands. Jilin Science and Technology Press, Jilin, China (in
Chinese)
Harinasut P, Tsutsui K, Takabe T, Nomura M, Takabe T, Kishitani S
(1996) Exogenous glycinebetaine accumulation and increased
salt-tolerance in rice seedlings. Biosci Biotech Bioch
60:366–368
Heath RL, Packer L (1968) Photoperoxidation in isolated chloro-
plasts. I. Kinetics and stoichiometry of fatty acid peroxidation.
Arch Biochem Biophys 125:189–198
Hernández JA, Almansa MS (2002) Short-term effects of salt stress
on antioxidant systems and leaf water relations of pea leaves.
Physiol Plant 115:251–257
Hoagland DR, Arnon DI (1950) The water-culture method for
growing plants without soil. Calif Agric Exp Stn Citc 347:1–32
Huang ZH, Zhu JM, Mu XJ, Lin JX (2004) Pollen dispersion, pollen
viability and pistil receptivity in Leymus chinensis. Ann Bot
93:295–301. doi:10.1093/aob/mch044
139
Jin H, Plaha P, Park JY, Hong CP, Lee IS, Yang ZH, Jiang GB, Kwak
SS, Liu SK, Lee JS, Kim YA, Lim YP (2006) Comparative EST
profiles of leaf and root of Leymus chinensis, a xerophilous grass
adapted to high pH sodic soil. Plant Sci 170:1081–1086. doi:
10.1016/j.plantsci.2006.01.002
Krotz RM, Evangelou BP, Wagner GJ (1989) Relationships between
cadmium, zinc, Cd-peptide, and organic acid in tobacco
suspension cells. Plant Physiol 91:780–787
Ma JF, Furukawa J (2003) Recent progress in the research of external
Al detoxification in higher plants: a minireview. J Inorg Biochem
97:46–51. doi:10.1016/S0162-0134(03)00245-9
Mailloux RJ, Lemire J, Kalyuzhnyi S, Appanna V (2008) A novel
metabolic network leads to enhanced citrate biogenesis in
Pseudomonas fluorescens exposed to aluminum toxicity.
Extremophiles 12:451–459
Melderis A (1980) Leymus. In: Tutin TG, Heywood VH, Burges NA,
Moore DM, Valentine DM, Walters SM, Webb DA (eds) Flora
Europeae, vol 5. Cambridge University Press, Cambridge,
pp 190–192
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell
Physiol 22:867–880
Okuma E, Murakami Y, Shimoishi Y, Tada M, Murata Y (2004)
Effects of exogenous application of proline and betaine on the
growth of tobacco cultured cells under saline conditions. Soil
Sci Plant Nutr 50:1301–1305
Pál M, Horváth E, Janda T, Páldi E, Gabriella S (2006) Physiological
changes and defense mechanisms induced by cadmium stress in
maize. J Plant Nutr Soi Sci 169:239–246. doi:10.1002/jpln.
200520573
Shlizerman L, Marsh K, Blumwald E, Sadka A (2007) Iron-shortage-
induced increase in citric acid content and reduction of cytosolic
aconitase activity in Citrus fruit vesicles and calli. Physiol Plant
131:72–79. doi:10.1111/j.1399-3054.2007.00935.x
Sun YL, Hong SK (2010a) Exogenous proline mitigates the
detrimental effects of saline and alkaline stresses in Leymus
chinensis (Trin.). J Plant Biotechnol (accepted)
Sun YL, Hong SK (2010b) Effects of plant growth regulators and
L-glutamic acid on shoot organogenesis in the halophyte Leymus
chinensis (Trin.). Plant Cell Tiss Organ Cult 100:317–328. doi:
10.1007/s11240-009-9653-4
Tesfaye M, Dufault NS, Dornbusch MR, Allan DL, Vance CP, Samac
DA (2003) Influence of enhanced malate dehydrogenase expres-
sion by alfalfa on diversity of rhizobacteria and soil nutrient
availability. Soil Biol Biochem 35:1103–1113. doi:10.1016/
S0038-0717(03)00162-7
Timpa JD, Burke JJ, Quisenberry JE, Wendt CW (1986) Effects of
water stress on the organic acid and carbohydrate compositions
of cotton plants. Plant Physiol 82:724–728
Wang KP, Lou X (1987) Studies on the species differentiation of
Leymus chinensis. III. Analysis on the genetic base of different
ecological type. Grassland China 4:42–45
Zeng FR, Mao Y, Cheng WD, Wu FB, Zhang GP (2008) Genotypic
and environmental variation in chromium, cadmium and lead
concentrations in rice. Environ Pollut 153:309–314. doi:
10.1016/j.envpol.2007.08.022
Zimmermann U (1978) Physics of turgor- and osmoregulation. Annu
Rev Plant Physiol 29:121–148
123