- Email: [email protected]
Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants
Accepted Manuscript Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants Maryam Seifi Kalhor, Aliniaeifard Sasan, Mehdi Seif, Elahe Javadi Asayesh, Françoise Bernard, Batool Hassani PII:
S0981-9428(18)30303-6
DOI:
10.1016/j.plaphy.2018.07.003
Reference:
PLAPHY 5326
To appear in:
Plant Physiology and Biochemistry
Received Date: 23 April 2018 Revised Date:
27 June 2018
Accepted Date: 2 July 2018
Please cite this article as: M.S. Kalhor, A. Sasan, M. Seif, E.J. Asayesh, Franç. Bernard, B. Hassani, Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2018.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Enhanced salt tolerance and photosynthetic performance: implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants
RI PT
Authors: Maryam Seifi Kalhora, Aliniaeifard Sasanb*, Mehdi Seifb, Elahe Javadi Asayeshb, Françoise Bernarda, Batool Hassania a
Faculty of Life Sciences and Biotechnology. Department of Plant Sciences, Shahid Beheshti
SC
University, Tehran, Iran b
EP
TE D
Corresponding email: [email protected]
AC C
*
M AN U
Department of Horticulture, Aburaihan campus, University of Tehran, Tehran, Iran
ACCEPTED MANUSCRIPT
1
Enhanced salt tolerance and photosynthetic performance: implication of ɤ-amino butyric
2
acid application in salt-exposed lettuce (Lactuca sativa L.) plants
RI PT
3
ABSTRACT
5
Gamma-Amino Butyric Acid (GABA) is a substantial component of the free amino acid pool
6
with low concentration in plant tissues. Enhanced GABA content occurs during plant growth and
7
developmental processes like seed germination. GABA level, basically, alters in response to
8
many endogenous and exogenous stimuli. In the current study, GABA effects were studied on
9
germination, photosynthetic performance and oxidative damages in salt-exposed lettuce plants.
10
Three NaCl (0, 40 and 80 mM) and two GABA (0 and 25 µM) concentrations were applied on
11
lettuce during two different developmental (seed germination and seedlings growth) stages.
12
Negative effects of salinity on germination and plant growth were removed by GABA
13
application. GABA significantly reduced mean germination time (MGT) in salt-exposed lettuce
14
seeds. Although, salinity caused a significant decline in maximum quantum yield of photosystem
15
II (Fv/Fm) during distinct steps of plant growth, GABA application improved Fv/Fm particularly
16
on high salinity level. GABA decreased specific energy fluxes per reaction center (RC) for
17
energy absorption and dissipation, while enhanced-electron transport flux in photosynthetic
18
apparatus of lettuce plants was observed in GABA-supplemented plants. Moreover, decline in
19
non-photochemical quenching (NPQ) and quenching coefficients (qP, qL, qN) by salt stress were
20
recovered by GABA application. Elevated electrolyte leakage considerably decreased by GABA
21
exposure on salt-treated plants. Although, proline level increased by NaCl treatments in a
22
concentration dependent manner, combined application of salt with GABA caused a significant
23
reduction in proline content. Catalase; EC 1.11.1.6 (CAT), L-ascorbate peroxidase; EC
AC C
EP
TE D
M AN U
SC
4
ACCEPTED MANUSCRIPT
1.11.1.11 (APX), and superoxide dismutase; EC 1.15.1.1 (SOD) activities were increased by
25
GABA exposure in salt-supplemented plants that resulted in regulated hydrogen peroxide level.
26
In conclusion, a multifaceted role for GABA is suggested for minimizing detrimental effects of
27
salinity on lettuce through improvement of photosynthetic functionality and regulation of
28
oxidative stress.
RI PT
24
29
Keywords: GABA, Germination, Growth, Lettuce, Oxidative stress, Photosynthesis, Salinity
SC
30
M AN U
31
1. Introduction
33
Various environmental stresses negatively influence agricultural productivity. Among them, soil
34
and water salinity progressively affect the agricultural lands by causing detrimental effects on
35
seed germination, plant growth and development (Li et al., 2017a). Ionic and osmotic stresses are
36
consequently induced by high salt concentrations in irrigation water (Zhang et al., 2009; Ahuja et
37
al., 2010; Aliniaeifard et al., 2016b). Salinity has adverse effects on seed germination by
38
affecting either germination percentage or germination longevity (Meot-Duros and Magné ,
39
2008). Under salinity stress, due to the disturbance in plant cell turgor, nutrient transport inside
40
of the plants would be also negatively affected (Chartzoulakis, 2005). Salinity influences the
41
rhizosphere osmotic potential which ultimately results in delayed and hampered seed
42
germination (Sosa et al., 2005). Decreases in leaf expansion, shoot and root development have
43
been reported when plants are exposed to saline conditions (Maggio et al., 2011; Giuffrida et al.,
44
2012; Machado and Serralheiro, 2017). Furthermore, various processes including enzymatic
45
activities, photosynthesis performance and respiration rate can also be negatively influenced by
46
salinity stress (Chartzoulakis, 2005; Aliniaeifard et al., 2016a; Aliniaeifard et al., 2016b).
AC C
EP
TE D
32
ACCEPTED MANUSCRIPT
Accumulation of Reactive Oxygen Species (ROS) in different plant organs has been frequently
48
reported following plant exposure to saline condition. ROS result in oxidative damages which
49
ultimately leading to the programmed cell death (Filomeni et al., 2015; You and Chan, 2015).
50
Negative effects of salt stress on photosynthesis can potentially occur through affecting stomata
51
functioning (Munns and Tester, 2008) or by ROS-induced oxidative damages in related enzymes
52
which incorporate in photosynthesis functionality (Chaves et al., 2009; Aliniaeifard et al.,
53
2016a). Detrimental effects of salt stress are associated with damages on distinct parts of the
54
photosynthetic apparatus (Mehta et al., 2010). To cope with these damages and to reduce the
55
stress related susceptibility, plants have co-opted numerous physiological, molecular, and
56
cellular adaptations during evolution (Shao et al., 2008). As an example, organic compounds
57
accumulate in the cells and through specific processes protect plants against oxidative damages
58
(Jutsz and Gnida, 2015; Slama et al., 2015). Accumulation of GABA, an endogenous organic
59
compound, under stressful conditions has been frequently reported (Kaplan et al., 2004; Xing et
60
al., 2007; Allan et al., 2008; Bor et al., 2009; Renault et al., 2011; Li et al., 2016b; Scholz et al.,
61
2017b). GABA is a four-carbon non-protein amino acid involving in plant responses to the
62
variety of environmental stresses (Ramesh et al., 2016; Vijayakumari and Puthur, 2016; Stuart,
63
2017). For the first time GABA identification was reported in 1949, when GABA accumulation
64
was detected in potato tuber tissues (Steward et al., 1949). Later, wide ranges of GABA effects
65
on plant growth and development were revealed with independent studies on various plant
66
species (Fait et al., 2008; Suo-ling et al., 2012; Ramesh et al., 2015). An increase in GABA level
67
in root and nodule tissues following exposure to salt stress has been reported in alfalfa
68
(Medicago sativa L.) plants (Fougere et al., 1991). Salt tolerance in Nicotiana sylvestris has been
69
shown by induction of GABA-related genes expression (e.g. GAD) which resulted in GABA
AC C
EP
TE D
M AN U
SC
RI PT
47
ACCEPTED MANUSCRIPT
accumulation under NaCl treatment (Akçay et al., 2012). Interestingly, plant deficient in GABA
71
transaminase enzyme (GABA-T), an enzyme involved in GABA metabolism, represented salt
72
sensitive responses when exposed to saline stress condition (Renault et al., 2013). These
73
observations indicate that salt tolerance in plant is associated with GABA metabolism. Positive
74
effect of GABA on photosynthetic functionality in plants imposed to salinity has also been
75
revealed (Li et al., 2017b). Recently it was shown that exogenous GABA can improve mask
76
melon (Cucumis melo L.) photosynthesis under saline conditions (Xiang et al., 2016); however,
77
GABA effects on seed germination and early stages of plant growth is not comprehensively
78
studied. In the current study we have used lettuce plant since it is an important edible crop,
79
widely consumed in the world and sensitive to the salt stress (Ünlükara et al., 2008). Negative
80
effects of salinity on different aspects of lettuce growth and cellular mechanisms have been
81
frequently reported (Andriolo et al., 2005; Mohammadi and Khoshgoftarmanesh, 2014; Bartha et
82
al., 2015; Xu and Mou, 2015). Although plant's responses to the salinity have been intensively
83
studied, few researches have investigated GABA effects on salt tolerance and its related
84
mechanisms. Therefore, the objectives of this study were to i) investigate whether GABA can
85
improve salinity tolerance during seed germination and early plant development, ii) reveal
86
GABA effects on photosynthetic functionality by analyzing distinct stages of electron flow in
87
photosynthetic electron transport chain, iii) study GABA effect on oxidative damages induced by
88
salinity stress. The result will be relevant to introduce a natural compound capable of reducing
89
negative consequence of salt stress on growth and physiology of lettuce plants.
SC
M AN U
TE D
EP
AC C
90
RI PT
70
91
2. Material and methods
92
2.1. Seed germination
ACCEPTED MANUSCRIPT
To analyze the seed germination under salt stress, the seeds of Lactuca sativa cv. Partavousi (45
94
seeds) were placed in 10 cm diameter petri dishes with three layers of filter paper and 3 ml of
95
double-distilled sterile water containing different concentrations of NaCl (0, 40 and 80 mM) and
96
GABA (0 and 25 µM). A pre-test experiment showed no significant difference between 25 and
97
50 µM GABA. Therefore, 25 µM GABA was used for this experiment. Plates were placed in a
98
growth chamber with 22±1°C and lighting period of 12 h light /12 h dark for 58 h. Seeds were
99
considered as germinated when the radicle emerged for 2 mm length. Three petri dishes were
100
used for each treatment and the experiment repeated for three times. MGT was calculated based
101
on the following equation:
102
MGT= ∑n.H / ∑n
103
Where n= number of newly germinated seeds at time H; H= hours from the onset of the
104
germination test, ∑n= final germination
TE D
105
M AN U
SC
RI PT
93
2.2. Plant growth conditions
107
In order to investigate lettuce plant growth under salt stress, the seeds were sown in a
108
transplanting tray with 80 sowing wells filled with fine perlite. After germination, in the stage of
109
cotyledon leaves emergence, homogeneous plants were selected and transplanted into the pots
110
(l×w×h=7 cm×7 cm×10 cm) filled with perlite (one plant per pot). Plants were kept inside a
111
growth chamber at 22±1°C with a 12 h light/12 h dark period, 50±5% relative humidity (RH),
112
400 ppm CO2 concentration (determined using Trotec BZ30 CO2 air quality data logger,
113
Heinsberg, Germany) and 250 µmol m-2 s-1 light intensity (measured with an sekonic
114
spectromaster c-7000, Japan) produced by white LED lights (400-700 nm). Daily irrigation was
115
applied with half strength of Hoagland solutions containing 0, 40 and 80 mM of NaCl together
AC C
EP
106
ACCEPTED MANUSCRIPT
116
with 0 and 25 µM of GABA. Medium substrates were fully washed with distilled water every 5
117
days to remove the effects of GABA and salt accumulation.
118
2.3. Growth measurements
120
Two weeks following germination, shoot and root fresh and dry weights were determined. Shoot
121
fresh weights were measured immediately after harvest, for root fresh weight, perlite's particles
122
were carefully removed using a sieve to minimize root loss and later biomass was measured
123
using a digital balance. Dry weights were measured after desiccation in an oven at 60ºC for 48 h
124
for all samples.
M AN U
SC
RI PT
119
125
2.4. Transient and slow induction of chlorophyll fluorescence
127
Outermost leaves following 4, 8 and 12 days application of treatments were used for measuring
128
slow chlorophyll fluorescence parameters. After dark adaptation for at least 20 minutes intact
129
attached leaves to the plants were immediately used to measure chlorophyll fluorescence
130
parameters using a fluorometer system (Handy FluorCam FC 1000-H Photon Systems
131
Instruments, PSI, Czech Republic). Images were recorded during short measuring flashes in
132
darkness. At the end of the short flashes, the samples were exposed to a saturating light pulse
133
(3900 µmol m-2 s-1) that resulted in a transitory saturation of photochemistry and reduction of
134
primary quinone acceptor of PSII (Genty et al., 1989; Aliniaeifard et al., 2014; Aliniaeifard and
135
van Meeteren, 2014). After reaching steady state fluorescence, two successive series of
136
fluorescence data were digitized and averaged, one during short measuring flashes in darkness
137
(F0), and the other during the saturating light flash (Fm). From these two images, variable
138
fluorescence (Fv) was calculated according to the ratio between Fm and F0. The Fv/Fm was
AC C
EP
TE D
126
ACCEPTED MANUSCRIPT
calculated using the ratio between Fv and Fm. Maximum fluorescence in light adapted steady
140
state (Fm′) was determined and was used for calculation of NPQ based on the following equation:
141
NPQ = (Fm/Fm')-1
142
Fraction of dark-adapted variable fluorescence lost upon adaptation to light (qN; NPQ
143
coefficient), photochemical quenching coefficient (qP); fraction of PSII open centers (qL) were
144
calculated as defined previously (Kramer et al., 2004). Following exposure to light, primary
145
charge separation in photosystem reaction centers occurs. In this step the fluorescence signal
146
rises due to reduction of plastoquinone pool by the generated editions from photosystem II
147
activity (FP). Later, the steady-state fluorescence levels attained in continuous light declines to
148
the fluorescence level called Ft. Rate of fluorescence decline (Rfd), an empirical parameter
149
proposed by (Lichtenthaler et al., 2005) to quantify plant fitness and vitality under stress
150
conditions, was calculated according to the following equation:
151
Rfd =(FP - Ft) / FP
152
The average values, and standard deviation per image were calculated by using of FluorCam
153
software 7.0. The polyphasic Chl a fluorescence (OJIP) transients were measured by a FluorPen
154
FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) on the outermost leaves
155
after 20 min dark adaptation. OJIP was used to study different biophysical and
156
phenomenological related to PSII status (Strasser, 1995). The OJIP transients measurement was
157
done according to the JIP test (Strasser et al., 2000). The following data from the original
158
measurements were used after extraction by Fluorpen software: fluorescence intensities at 50 µs
159
(F50 µs, considered as the minimum fluorescence F0), 2 ms (J-step, FJ), 60 ms (I-step, FI), and
160
maximum fluorescence (Fm). Performance index was calculated on the absorption basis (PIABS)
AC C
EP
TE D
M AN U
SC
RI PT
139
ACCEPTED MANUSCRIPT
and densities of QA- reducing PSII reaction centers at time 0 and time on maximum fluorescence
162
state. Furthermore, the yield ratios including: the probability that a trapped excision moves an
163
electron in electron transport chain beyond QA- (ψo), quantum yield of electron transport (φEo),
164
quantum yield of energy dissipation (φDo) and maximum quantum yield of primary
165
photochemistry (φPo) were also calculated based on the following equations:
RI PT
161
F0 FM
M AN U
φPo=1-
SC
Vj=(Fj-F0 )/(FM-F0 )
ψo=1-Vj
φDo=1-φPo- (
F0
F0 ) FM
. ψo
TE D
φEo= 1 −
From these data, the following parameters were calculated: the specific energy fluxes per
167
reaction center (RC) for energy absorption (ABS/RC= M0.(1/VJ).(1/φPo)), (M0= TR0/RC-
168
ET0/RC), trapped energy flux (TR0/RC= M0.(1/VJ)), electron transport flux (ET0/RC=
169
M0.(1/VJ). ψo) and dissipated energy flux (DI0/RC= (ABS/RC)–(TR0/RC)).
AC C
170
EP
166
171
2.5. Determination of electrolyte leakage percentage
172
For measuring electrolyte leakage, 10 leaf discs (0.5 cm diameter) were prepared using a cork
173
borer and washed with deionized water to remove surface-adhered electrolytes, placed in closed
174
vials containing 10 ml of deionized water and incubated at 25°C on a rotary shaker for 24h;
ACCEPTED MANUSCRIPT
subsequently, electrical conductivity of the solution (C1) was determined. Samples were
176
autoclaved at 120°C for 20 minutes and the resulting electrical conductivities (C2) were recorded
177
after equilibration at 25°C. The electrolyte leakage was calculated by the ratio between C1 and
178
C2 (Tuna et al., 2007).
179
RI PT
175
2.6. Determination of hydrogen peroxide level
181
Hydrogen peroxide (H2O2) was spectrophotometrically measured after reaction with potassium
182
iodide (KI). The reaction mixture contained 0.5 ml of 0.1% trichloroacetic acid (TCA), leaf
183
extract supernatant, 0.5 ml of 100 mM K-phosphate buffer and 2 ml reagent (1 M KI w/v in fresh
184
double-distilled water). The blank probe was contained 0.1% TCA in the absence of leaf extract.
185
The reaction was developed for 1 h in darkness and the absorbance was measured at 390 nm. The
186
amount of H2O2 was calculated using a standard curve prepared with known concentrations of
187
H2O2 levels according to the method described by (Patterson et al., 1984).
M AN U
TE D
188
SC
180
2.7. Determination of proline content
190
Determination of free proline content was performed according to (Bates et al., 1973). Fresh leaf
191
samples (0.5 g) were homogenized in 3% (w/v) sulphosalicylic acid. The homogenate was filtered by
192
using of filter paper. After addition of acid-ninhydrin and glacial acetic acid, the resulting mixture
193
was heated at 100°C for 1 h in a water bath. Reaction was stopped using an ice bath. The mixture
194
was extracted with toluene and the absorbance of fraction with toluene aspired from the liquid phase
195
was read at 520 nm. Proline concentration was determined using a calibration curve and expressed as
196
µmol proline g–1 Fresh weight.
197
AC C
EP
189
ACCEPTED MANUSCRIPT
2.8. Determination of antioxidant enzymes activity
199
APX activity was determined by oxidation of ascorbic acid (AA) at 265 nm ( = 13.7 mM-1 cm-1)
200
by slight modification from method described by (Nakano and Asada, 1981). The reaction
201
mixture contained 50 mM potassium phosphate buffer (pH 7.0), 5 mM AA, 0.5 mM H2O2 and
202
the enzyme extract. The reaction was started by adding H2O2. The rates were corrected for the
203
non-enzymatic oxidation of AA by the inclusion of a reaction mixture without the enzyme
204
extract (blind sample). The enzyme activity was expressed in µkat per mg-1 protein (Nakano and
205
Asada, 1981).
206
For determination of CAT activity, the decomposition of H2O2 was recorded by a decrease in
207
absorbance at 240 nm. Reaction mixture contained 1.5 ml of 50 mM sodium phosphate buffer
208
(pH 7.8), 0.3 ml of 100 mM H2O2, and 0.2 ml of enzyme extract. CAT unit was defined as the
209
amount of enzyme necessary to decompose 1 mM min-1 H2O2. Therefore the CAT activity was
210
expressed as µkat per mg-1 protein (Díaz-Vivancos et al., 2008).
211
SOD activity was measured according to the method described by Dhindsa et al. (1981). This
212
method is based on the ability of SOD to inhibit the photochemical reduction of nitro blue
213
tetrazolium (NBT). Reaction mixture contained 50 mM phosphate buffer (pH 7-8), 13 mM
214
methionine, 75 µM NBT, 0.15 mM riboflavin, 0.1 mM EDTA, and 0.50 ml enzyme extract.
215
Riboflavin was added as the last agent. The reaction mixture were shaken and placed under two
216
15 W fluorescent lamps. The reaction was started by switching on the light and was allowed to
217
run for 10 min. The reaction was stopped by turning off the light and the tubes were covered
218
using a black cloth. The absorbance by the reaction mixture was spectrophotometrically recorded
219
at 560 nm. A reaction mixture without exposure to the light was used as control. Controls were
AC C
EP
TE D
M AN U
SC
RI PT
198
ACCEPTED MANUSCRIPT
220
color free. Under assay conditions, one unit of SOD activity is expressed as the enzyme led to
221
50% inhibition of NBT reduction (Dhindsa et al., 1981).
RI PT
222
2.9. Statistical Analysis
224
For germination test, three petri dishes were used for each treatment with 45 seeds in each petri
225
dish and the experiment repeated for three times. The experiment was designed in a two-factorial
226
complete randomized experiment with six replications per treatment. Salt solutions were applied
227
to each plant per salt treatment to ensure that each plant received similar salt stress in all
228
treatments. Two-way analyses of variances (ANOVA) were used for significance tests of the
229
treatment effects. Tukey's multiple comparisons test were done for finding significant
230
differences. All statistical analyses were done using GraphPad Prism version 7.01 software
231
(GraphPad software, Inc. San Diego, CA).
232
TE D
M AN U
SC
223
3. Results
234
3.1. GABA improves seed germination and growth in salt-exposed lettuce seeds and seedlings
235
Monitoring seed germination after 12, 24, 36 and 48 h revealed that during first 12 h,
236
germination of seeds subjected to 40 and 80 mM NaCl were null except for those plants grown
237
under 40 mM NaCl treated with GABA (Fig. 1). Maximum germination percentage was
238
observed in control plants and those plants treated by GABA which represented 11.8% and 6.6%
239
seedlings respectively. After 24 h highest germination was recorded in control plants either
240
treated or non-treated with GABA. Lowest number of germinated seeds was observed in 80 mM
241
NaCl concentration. However, co-treatment with GABA remarkably increased the seedling
242
numbers in both NaCl concentrations, so that the seed germination (46%) in 40 mM salt
AC C
EP
233
ACCEPTED MANUSCRIPT
concentration by GABA treatment was equal with the control condition (without NaCl and
244
GABA treatment). Interestingly, 38% of seeds were germinated when GABA was added to 80
245
mM saline water, which was remarkably higher than seed germination ratio (29%) under 40 mM
246
NaCl irrigation regime. Changes in seedling numbers during third 12 h were more significant for
247
salt-imposed seeds. Triple increase in germination percentage was occurred for seeds co-treated
248
by 80 mM NaCl and GABA, however their germination percentage was lower than the control.
249
Significant changes were not observed in seedling numbers during fourth and fifth 12 h except
250
for those seeds exposed to high salt levels and moderate salinity without GABA treatment (Fig.
251
1). These results indicate positive regulatory role for GABA on germination of salt-exposed
252
lettuce seeds.
M AN U
SC
RI PT
243
100 90
TE D
80
60
NaCl 40
EP
50
C
40
NaCl 80 G G+NaCl 40
AC C
Germination, %
70
30
G+NaCl 80
20 10 0
0
253 254
10
20
30
40
Time (h)
50
60
70
ACCEPTED MANUSCRIPT
Fig. 1. Germination rate during 60 h exposure to three concentrations (0, 40 and 80 mM) of NaCl and two
256
levels (0 and 25 ppm) of GABA in lettuce seeds. Each value represents a mean ± standard error from
257
three independent experiments. C: Control condition without NaCl and G: GABA with GABA and
258
without NaCl.
RI PT
255
259
MGT was increased in a NaCl concentration-dependent manner (Fig. 2). Application of GABA
261
caused a decrease in MGT in both NaCl-treated plants; however; no remarkable difference was
262
found in MGT of plant seeds which treated by GABA.
EP
263
TE D
M AN U
SC
260
Fig. 2. Effects of salinity (0, 40 and 80 mM NaCl) and GABA [0 (black bars) and 25 µM GABA (white
265
bars)] on MGT of lettuce seeds. Each value represents a mean ± standard error from three independent
266
experiments. Bars with different letters are significantly different (ANOVA, P < 0.05).
267
AC C
264
268
Plant vegetative growth was measured following 14 days of NaCl and GABA applications. There
269
was significant difference between growth of control plants and those exposed to salt treatment.
270
Salt stress caused leaf chlorosis while, GABA application inhibited occurrence of NaCl-induced
271
chlorosis in lettuce leaves (Fig. 3)
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
272
273
Fig. 3. Lettuce plants exposed to different salt (0, 40 and 80 mM NaCl) and GABA [0 (control) and 25
275
µM GABA (G)] concentrations. Photographs were taken following 14 days of treatments.
276
TE D
274
Significant reductions of nearly 75% and 90% were observed in shoot fresh weight by 40 mM
278
and 80 mM NaCl respectively when compared to the shoot fresh weight of control plants. In
279
comparison with control plants, 50% and 75% decrease in shoot dry weights were observed in
280
seedlings exposed to 40 and 80 mM NaCl respectively. There was two-fold increase in shoot
281
fresh weight of GABA treated seedling compared with the shoot fresh weight of the control
282
plants. GABA exposure significantly increased shoot dry weights in both NaCl levels (Fig. 4B).
283
The highest increase in shoot dry weight was detected in plants treated with 80 mM NaCl.
284
GABA did not affect the shoot fresh and dry weights in non-stressed plants (Fig.4). In the term
285
of root growth, NaCl treatment reduced both fresh and dry weights in a NaCl dependent manner.
286
In comparison with the control plants, there were 50% and 85% reductions in root fresh weight
AC C
EP
277
ACCEPTED MANUSCRIPT
of 40 and 80 mM NaCl-treated plants, respectively (Fig. 4C). Similar results were obtained for
288
root dry weights (Fig. 4D). Interestingly, GABA increased root fresh and dry weights in control
289
plants. Significant increases in both root fresh and dry weights were detected in plants co-treated
290
with NaCl and GABA. Rate of root fresh and dry weights recovery by GABA was higher in
291
plants exposed to high NaCl concentration when compared to the medium and control NaCl
292
level.
SC
RI PT
287
GABA
Control 10
0.6
M AN U
8 0.4
6
4
0.2
2
0
0 N
aC
l8
0
0 aC N
N
aC
l4
l8
0
0 l8 N
aC
l4
aC
N
l
0.00
0
l
tr o
on
l4
0.05
0.0
C
aC
0.10
tr o
AC C
0.5
0.15
on
1.0
N
on
C
EP
1.5
0.20
C
2.0
tr ol
0 aC N
TE D
N
C
on
aC
l8
l4
0
tr ol
0.0
293 294
Fig. 4. Effects of salinity [0, 40 and 80 mM NaCl] and GABA [0 (black bars) and 25 µM GABA (white
295
bars)] on root and shoot fresh (A, C) and dry weights (B, D) of lettuce plants. Values are the means of six
296
replicates and bars indicate means ± SEM. Bars with different letters are significantly different (ANOVA,
297
P < 0.05).
ACCEPTED MANUSCRIPT
298
3.2. GABA improves photosynthetic electron flows in salt-exposed lettuce seedlings
300
Dark-adapted leaves of control and GABA-treated plants exposed to different NaCl
301
concentrations were used to measure the slow and fast inductions of Chl-a fluorescence. At the
302
beginning of the experiment (day 4), maximum Fv/Fm was observed in control plants treated with
303
GABA and minimum Fv/Fm was detected in plants exposed to 80 mM NaCl (Fig. 5A). Fv/Fm was
304
doubled by applying GABA on plants exposed to 80 mM NaCl. Fv/Fm value was increased after
305
8 days in all plants when compared with day 4. Notably, plants treated with 80 mM NaCl
306
showed the highest Fv/Fm recovery due to the GABA application (Fig. 5B). Fv/Fm value in plants
307
exposed to 80 mM NaCl was reached to the control plants through GABA treatment. In day 12,
308
Fv/Fm was not changed in control plants by GABA application. However, plants that were grown
309
under moderate and high saline conditions were positively responded to GABA and showed
310
significant increases in Fv/Fm values particularly in 80 mM NaCl treatment (Fig. 5C). These
311
results indicated the positive role of GABA on efficiency of photosystem II in salt-exposed
312
plants during early growth of lettuce plants.
SC
M AN U
TE D
EP AC C
313
RI PT
299
RI PT
1.0
a
a
a
a
a b
0.6
0.4
0.2
0 l8
0
N aC
l4
314
0 N aC l8
0
N aC l4
C on
tr ol
AC C
EP
TE D
N aC
C on
tr ol
0.0
M AN U
0.8
SC
0 N aC l8
N aC l4
C on tr o
0
l
ACCEPTED MANUSCRIPT
315
Fig. 5. Maximum quantum yield of photosystem II (Fv/Fm) in lettuce leaves following 4 (A), 8 (B) and 12
316
(C) days exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations.
317
Each value represents a mean ± standard error for six repetitions. Bars with different letters are
318
significantly different (ANOVA, P < 0.05).
ACCEPTED MANUSCRIPT
Specific energy fluxes per reaction center (RC) were measured based on the output of fast
320
inductions of Chl-a fluorescence. Parameters such as ABS/RC (Fig. 6A) and DI0/RC (Fig. 6B)
321
were decreased in plants subjected to both NaCl and GABA. In the case of TR0/RC, remarkable
322
differences were not detected among treatments (Fig. 6D). GABA caused increase in ET0/RC in
323
both salinity treatments; however, no significant effect was not observed in control plants (Fig.
324
6C), implying that under saline stress condition, GABA is able to recover electron transport in
325
PSII apparatus.
SC
RI PT
319
B
A
M AN U
GABA
Control
1.0
4.0
a
a
0.9
a
a
a
b
0.8
b
b
3.0
DI0/RC
3.5
b
b
0.7
c
c
0.6 0.4
326
0 N aC l8
0 N aC l4
on t C
D 3.0
2.5
2.0
1.5 1
N
aC
l8 0
l4 0 aC N
tr ol
l8 N aC
C on
0
0
0
l4
N aC
C on
tr ol
AC C
ET0/RC
EP
C
0.0
N aC l8 0
TE D
N aC l4 0
C on tr ol
1
ro l
2
327
Fig. 6. Specific energy fluxes per reaction center (RC) for energy absorption [A; (ABS/RC)], dissipated
328
energy flux [B; (DI0/RC)], electron transport flux [C; (ET0/RC)] and trapped energy flux [D; (TR0/RC)]
329
from the fluorescence transient exhibited by leaves of lettuce plants grown at different salt concentrations
ACCEPTED MANUSCRIPT
(black bars) and treated with GABA (white bars). Bars represent means of six repetitions ± SD. Bars with
331
different letters are significantly different (ANOVA, P < 0.05).
332
High NaCl concentration led to a significant reduction in performance index on absorption base
333
(PIABS) compared with the PIABS of control plants (Fig. 7). However, this reduction was not
334
significant for 40 mM NaCl-treated plants. Substantial increase in PIABS was occurred when
335
plants co-treated with GABA and NaCl. This increase was 30% and 50% for 40 mM and 80 mM
336
NaCl-treated plants respectively. Since PIABS represents validity index of PSII, these results can
337
imply that GABA affects photosynthesis functionality in a stress dependent manner.
M AN U
SC
RI PT
330
0
TE D aC l8
N
0 aC l4
EP
339
N
C on
tr ol
338
Fig. 7. Performance index on absorption basis (PIABS) in lettuce leaves following exposure to different salt
341
(0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value represents a mean ±
342
standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P <
343
0.05).
344
There was a gradual decrease in Performance index on absorption basis (PIABS) on a salt
345
concentration dependent manner, while under saline condition GABA caused remarkable
346
increases in the PIABS compared with the PIABS of control plants. Salinity stress led to a
AC C
340
ACCEPTED MANUSCRIPT
considerable decrease in NPQ; however, GABA application resulted in a full recovery of NPQ to
348
the value of control (Fig. 8A). GABA treatment caused an increase in qP of control plants and
349
plants grown under 40 mM NaCl (Fig. 8B). Significant change was not observed in 80 mM salt–
350
treated plants. GABA exposure led to the recovery of qL in high salt–treated plants to the value
351
of qL in control plants. Highest qN was recorded for non-treated GABA control plants (Fig. 8C).
352
Although, GABA had no effect on qN value in control plants, those plants exposed to moderate
353
and high saline condition showed a significant increase after GABA application. Monitoring the
354
state of active reaction centers (qL) showed a significant decrease in qL of plants subjected to
355
high level of salt stress (Fig. 8D). This indicates that 80 mM NaCl potentially force plants to
356
close reaction centers and consequently diminish qp during actual monitoring.
M AN U
SC
RI PT
347
AC C
EP
TE D
357
ACCEPTED MANUSCRIPT
Control
GABA
A
B
a
a
0.6
a
a ab
C
N aC l8 0
M AN U
D
0 N aC l8
0 N aC l4
tr ol C on
N aC l8 0
TE D
N aC l4 0
C
on t
ro
l
qL
qN
358
SC
C on tr ol
N aC l8 0
Na Cl 40
C
on tr ol
0.0
N aC l4 0
qP
NPQ 0.2
RI PT
b
0.4
Fig. 8. Values for NPQ and chlorophyll fluorescence quenching coefficients (qP, qL, qN) in lettuce leaves
360
following exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations.
361
Each value represents a mean ± standard error for six repetitions. Bars with different letters are
362
significantly different (ANOVA, P < 0.05).
AC C
363
EP
359
364
Chlorophyll fluorescence decrease ratio (Rfd) as plant vitality indicator was significantly
365
reduced in plants exposed to salinity stress in a salt concentration-dependent manner (Fig. 9).
366
However, application of GABA resulted in a remarkable increase in Rfd of moderate and high
367
salinity stresses in a salt concentration-dependent manner.
ACCEPTED MANUSCRIPT
SC
l8 0
l4 0
aC
aC
N
N
C on tr ol
RI PT
368
M AN U
369
Fig. 9. Chlorophyll fluorescence decrease ratio (Rfd) in lettuce leaves following exposure to different salt
371
(0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value represents a mean ±
372
standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P <
373
0.05).
374
TE D
370
3.3. GABA reduces oxidative stress in salt-exposed lettuce seedlings
376
Proline content was significantly increased in a NaCl concentration-dependent manner (Fig.
377
10A). However, with GABA application, the proline contents were decreased in both salt
378
exposed plants. These results indicated that under saline stress conditions, GABA possibly either
379
reduces the biosynthesis of proline or increases its conversion during certain metabolic pathway.
380
As shown in figure 8C the highest H2O2 level was detected in 80 mM NaCl-treated plants. H2O2
381
levels were significantly decreased in the leaves of plants treated with GABA (Fig. 10C).
382
Electrolyte leakage, which reflects damage to cell membrane, was considerably increased
383
following plant exposure to NaCl concentrations (Fig. 10B). GABA applications led to the
384
decline in the electrolyte leakages percentage of both salt-exposed plants.
AC C
EP
375
ACCEPTED MANUSCRIPT
385
387
SC 0
N aC l8
0
M AN U
N aC l8
0
0 N aC l4 N aC l4
N aC l8 0
N aC l4 0
ro l C on t
AC C
EP
TE D
C
C on tr ol
RI PT
386
388
Fig. 10. Proline content (A), electrolyte leakage (B) and H2O2 (C) content in lettuce leaves following
389
exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value
ACCEPTED MANUSCRIPT
390
represents a mean ± standard error for six repetitions. Bars with different letters are significantly different
391
(ANOVA, P < 0.05).
RI PT
392
As shown in figure 11, CAT enzyme amount was reduced in high NaCl concentration. Notably,
394
GABA treatments increased the enzyme level in all treatments so that similar amount of CAT
395
was observed after GABA application (Fig. 11A). APX level was reduced in a NaCl dependent
396
manner and after exposure to GABA an increase was detected in all treatments (Fig. 11B). SOD
397
level was increased by elevating the NaCl level; however, any increase in enzyme amount was
398
not observed after GABA application except for control plants (Fig. 11C).
M AN U
SC
393
AC C
EP
TE D
399
ol tr on C
l4 aC N
0 l8 aC N
0
0
N
aC
l8
0
EP
400
N
l4 aC
TE D
C
ol tr on
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 11. Salinity effect on antioxidant enzymes activity in lettuce plant under different salt (0, 40 and 80
402
mM NaCl) and GABA (0 and 25 µM) concentrations. A) CAT, B) APX, C) SOD. Each value represents a
403
mean ± standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P < 0.05).
404
AC C
401
405
4. Discussion
406
Soil salinity is a major abiotic stress which normally reduces plant growth and development
407
(Allakhverdiev et al., 2000) by hampering of water potential and nutrient deficiency
ACCEPTED MANUSCRIPT
(Chinnusamy et al., 2005; Genc et al., 2007). Inhibitory effect of salinity on seed germination has
409
been reported by different authors (Li, 2008; Meot-Duros and Magné , 2008; Panuccio et al.,
410
2014; Orlovsky et al., 2016). This effect can be due to the reduced available water required for
411
seed germination which results in a osmotic disruption and eventually hampered cell division
412
(Bonilla et al., 2004). Salinity tolerance during seed germination (in different plant species) can
413
be induced by exogenous applications such as boron (B+), Ca2+ (Bonilla et al., 2004), sodium
414
(Na+), magnesium (Mg2+) (Tobe et al., 2002) and salicylic acid (SA) (Al-Whaibi et al., 2012).
415
However, plants are able to produce endogenous growth regulators capable of modulating
416
developmental stages under certain stress condition. GABA as an endogenous signaling
417
molecule (Michaeli and Fromm, 2015; Žárský, 2015; Bown and Shelp, 2016; Gilliham and
418
Tyerman, 2016; Ramesh et al., 2016; Shelp et al., 2017) contributes in various stress related
419
responses (Akçay et al., 2012; Hu et al., 2015; Signorelli et al., 2015; Vijayakumari and Puthur,
420
2016; Scholz et al., 2017a). GABA role as a signaling metabolite was found when POP2 (Pollen-
421
Pistil incompatibility2) gene was identified as a regulator of pollen tube growth and germination
422
in Arabidopsis plants (Palanivelu et al., 2003). In current study, significant salt-induced-
423
reduction in seed germination of lettuce was improved by GABA application. This can be
424
explained based on its signaling role as endogenous modulator, which contributes in salinity
425
tolerance by protecting cell membrane against oxidative damages. Moreover, GABA exerts its
426
effects on plant cell through regulation of anion transporter that can alter plasma membrane
427
potential differences and osmotic adjustment (Ramesh et al., 2015) which is indispensible for
428
seed germination. In accordance with this, MGT was reduced in salt-exposed seeds that co-
429
treated with GABA. The probable reason for this could be explained by osmoprotecting role of
430
GABA (Nayyar et al., 2014), which potentially promotes early seed germination under salt-
AC C
EP
TE D
M AN U
SC
RI PT
408
ACCEPTED MANUSCRIPT
induced osmotic stress. Decreased MGT has been also reported in various osmopriming
432
conditions (Özbingöl et al., 1999). Analyzing of both root and shoot fresh and dry weighs
433
indicated that plant growth and/or development were stunted in response to the salinity. Similar
434
effects have been frequently reported in various plants, for example in tomato plant subjected to
435
120 mM salinity, both root and shoot fresh and dry weights have been negatively affected
436
(Rivero et al., 2014). Likewise in lettuce plant, shoot dry mass have been reduced when exposed
437
to salt stress (Cantrell and Linderman, 2001). The finding that fresh and dry shoot masses of
438
GABA-treated lettuce plants were less negatively affected by saline water than non-stresses
439
plants supports the hypothesis that, GABA favors lettuce plants to cope with the salinity stress in
440
rhizosphere. In fact, GABA role under salinity stress was more related to the stress signal
441
regulation rather than growth induction. By this result, it could be propose that plants engage
442
GABA as a stress mediator capable of regulating developmental stages influenced by ionic
443
(NaCl) stress in lettuce plants. This finding is consistent with defined role for GABA
444
transaminase (GABA-T), the enzyme involved in GABA catabolism, which has been reported as
445
''most responsive enzyme to NaCl'' in Arabidopsis thaliana plant (Renault et al., 2010).
446
Arabidopsis plant deficient in GABA-T showed a supersensitive phenotype to the salinity stress.
447
GABA-T role in nitrogen and carbon metabolism in Arabidopsis root has also been revealed
448
(Renault et al., 2013). In corroborate with this report, in our experiment, exogenous GABA
449
increased root biomass in both stressed and non-stressed lettuce plants, indicative of higher
450
activity of GABA in the roots relative to the shoot tissues. Although these results are indicative
451
of positive effect of GABA on plant growth under saline condition, the mechanism underling
452
GABA-modulated tolerance to salinity still is the matter of debate.
AC C
EP
TE D
M AN U
SC
RI PT
431
ACCEPTED MANUSCRIPT
In the current study, salinity caused oxidative stress in lettuce plants by elevating H2O2 level,
454
while GABA reduced the magnitude of this oxidative stress. Under saline stress conditions,
455
overproduction of ROS results in wide cellular components damages (Kawano et al., 2002;
456
Demidchik et al., 2003; Demidchik et al., 2010). ROS like H2O2 has been suggested to activate
457
signal transduction pathways in plant cells (Gechev and Hille, 2005; Tripathy and Oelmüller,
458
2012) and modify function of genes involved in the early stress-induced signaling (Shu-Hsien et
459
al., 2005). For example in wheat cell, H2O2 reduction by NADPH in nucleus provides a redox
460
regulation status that is prerequisite for certain gene expressions and consequently led to the cells
461
protection during seed development (Pulido et al., 2009). Seedling treatment with H2O2 has
462
resulted in a chilling tolerance induction in mung bean (Vigna radiata L.) plants (Yu et al.,
463
2003). H2O2 interact with Calcium (Ca2+) in response to the abiotic stresses (Niu and Liao,
464
2016). The role of Ca2+ on seed germination (Kong et al., 2015), root growth (Han et al., 2015)
465
and salt tolerance (Tepe and Aydemir, 2015) have been reported. Under stress condition, Ca2+
466
level is elevated in the cell and induces the Calmodulin (CAM) gene expression. CAM protein
467
and Ca2+ interaction can make a complex capable of binding to the certain proteins and
468
regulating their functions. Ca2+/CAM complex also involves in GABA biosynthesis by
469
regulating glutamate decarboxylase (GAD) enzyme activity (Allan et al., 2009). GAD is
470
responsible for decarboxylation of glutamate during GABA metabolism. Therefore,
471
hypothetically, GABA/Ca2+ interactions have modulating roles over processes such as H2O2
472
signaling. GABA has been proposed to regulate the flux of anions through a plasma membrane
473
located, aluminum-activated malate transporter (ALMT) (Ramesh et al., 2017); however efflux
474
of other ions has also been reported by GABA transporter. For example, combined application of
475
both GABA and H2O2 in barely roots exhibited Ca2+ and K+ efflux induction as well as cell death
AC C
EP
TE D
M AN U
SC
RI PT
453
ACCEPTED MANUSCRIPT
reduction (Shabala et al., 2014; Gilliham and Tyerman, 2016). This indicating the indirect effect
477
of GABA on ion trafficking by changing the cell membrane potential (Gilliham and Tyerman,
478
2016). Our finding showed that GABA has significant subtractive effect on electrolyte leakage.
479
This observation could be attributed to the regulatory role of GABA on ALMT function which
480
consequently affects electrical potential across the cell membrane (Ramesh et al., 2015).
481
Alternatively, during saline stress condition, salt accumulation in root zone causes an osmotic
482
stress capable of inhibiting the essential minerals uptake such as Ca2+, K+ and NO3- and
483
accumulation of Na+ and Cl- which eventually result in the cell membrane damages induced by
484
disruption in cell homeostasis (Zhu, 2001; Chartzoulakis, 2005). Recent data also strongly
485
suggest that ROS production is tightly related to cell membrane damages and electrolyte
486
leakages (Demidchik et al., 2014).
487
The antioxidant system, comprising SOD, CAT and APX enzymes, plays pivotal role in ROS
488
metabolism by retaining of the balance between ROS generating and quenching in plant cells
489
(Qiu et al., 2013). GABA metabolism and accumulation have also been suggested to reduce the
490
oxidative damage caused by ROS, leading to improvement of tolerance to oxidative stresses
491
(AL-Quraan, 2015). For instance in musk melon (Cucumis melo) plant, exogenous GABA
492
application increase the saline-alkalin resistance which is caused through enhancement of SOD
493
and ascorbic acid-glutathione cycling in chloroplasts (Xiang et al., 2015). Moreover, in corn
494
plants GABA caused a profound effect on the antioxidant enzyme activity under salinity
495
condition (Wang et al., 2017). Furthermore, increase in wheat resistance to salt stress through
496
enhancement of antioxidant enzymes activities has been also reported following GABA
497
application (Li et al., 2016a). These data are consistent with our finding about enzymatic activity
498
under co-application of salt and GABA, in which, SOD, CAT and APX activities enhanced by
AC C
EP
TE D
M AN U
SC
RI PT
476
ACCEPTED MANUSCRIPT
GABA treatment as the salt dosage increased. This indirectly implies the involvement of GABA
500
in regulation of the lipid peroxidation and cell membrane protection by modulating ROS
501
regulatory mechanism. SOD enzyme mainly involved in dismutation of superoxide radical
502
which produce ordinary molecular oxygen or H2O2 (Hayyan et al., 2016). Last byproduct is
503
harmful, therefore should be removed from the cell Removing H2O2 is organized by enzymes
504
such as CAT and APX. According to our data, CAT and APX reduced after salt exposure
505
whereas SOD activity increased. Zhu et al. (2004) and Li et al. (2016) reported the same results
506
(Zhu et al., 2004; Li et al., 2016a). In our investigation, GABA had more effect on CAT and
507
APX enzyme rather than SOD. This is consistent with our data related to H2O2 content, which
508
showed significant reduction after GABA exposure. These are also supported by Renault and
509
coworkers (2011) findings which suggested that GABA regulates water channel proteins and
510
affect the cell elongation by osmotic adjustment and maintenance of the cell turgor (Renault et
511
al., 2011). This biological crosstalk could introduce how H2O2 content, GABA function and
512
electrolyte leakage are regulated in a retro-controlled mechanism.
513
High salt concentration may slightly reduce CAT and APX gene expression, leading to the
514
reduced enzymatic activity, but GABA either directly re-induced or indirectly hindered NaCl
515
negative effects on antioxidant gene expressions. In different scenario, SOD activity has been
516
elevated in plants received NaCl, indicative of salt concentration-dependent potential of SOD
517
to alleviate oxidative damages. Our finding showed an effective inter-linkage between GABA
518
and oxidative enzymes in plant cells. Although this will open the questions that i) how these
519
interactions are modulated during salt stress and ii) whether GABA intermediates this linkage
520
or its function is formulated by the component of this regulatory mechanism?
521
AC C
EP
TE D
M AN U
SC
RI PT
499
ACCEPTED MANUSCRIPT
Under salt stress, proline content increases in plants (Yoshiba et al., 1995). Similarly, in our
523
study proline level increased in leaves following plant exposure to salinity stress; however
524
GABA application reduced proline content in both saline conditions. Upon salt treatment, ROS
525
overproduced in the plant cells (You and Chan, 2015) and as a result organic osmolytes like
526
proline is produced to save the steady state of plant cells (Liang et al., 2013). During proline
527
biosynthesis, NADPH is produced and consumed in reductive biosynthesis pathways through
528
antioxidant defense system (Liang et al., 2013). NADPH is also produced during GABA
529
metabolism (Ludewig et al., 2008). In stress condition, tricarboxylic acid (TCA) cycle activity is
530
affected and results in a reduced pool of NADP+ which causes maintaining of ROS due to the
531
defect in antioxidant machinery. Hypothetically, under such situations, GABA and proline
532
biosynthesis provide adequate level of NADP+ and maintain antioxidant functionality. For many
533
years proline has been considered as an antioxidant, however new evidence demonstrates that
534
except for ·OH, it cannot react with most ROS (Signorelli et al., 2013; Signorelli et al., 2016).
535
Proline reaction with ·OH has been proposed as a natural pathway to produce precursor of
536
GABA under oxidative stress reaction (Signorelli et al., 2015). In our study proline content was
537
reduced by GABA application. One possible scenario could be that promoted antioxidant
538
machinery through GABA function can partially bypasses proline involvement in ROS
539
scavenging processes and result in the reduction of proline biosynthesis. In high NaCl
540
concentrations, proline reduction ratio was decreased. Apparently, by increasing salt level,
541
proline production is induced in the cell to provide proper activity of antioxidant machinery
542
along with GABA function. Our results suggested that GABA could be considered as plant-
543
boosting antioxidant component with ability to reduce oxidative damages by activation of
544
enzymatic/non-enzymatic metabolism under salt stress. In fact, positive effect of GABA on
AC C
EP
TE D
M AN U
SC
RI PT
522
ACCEPTED MANUSCRIPT
antioxidant enzyme such as CAT, SOD and APX activities under saline condition is indicative of
546
important role for GABA in maintaining of redox state and preventing of ROS accumulation in
547
plants during stress condition. Although further studies are required to determine how GABA
548
regulate enzymatic balances during stress conditions.
549
Photosynthetic capacity is negatively affected under saline condition (Chaves et al., 2009; Ashraf
550
and Harris, 2013). This reduction can be caused by i) stomata malfunctioning induced by salt
551
stress (Janagoudar Sr, 2007) or ii) non-stomata related defects which is attributed to disruption in
552
photosystem II function (He et al., 2009). In confirmation of the last scenario, we found that
553
maximum quantum yield of photosystem II (Fv/Fm) of dark-adapted leaves were reduced under
554
high saline condition in different stages of plant growth. GABA effectively removed the salt-
555
induced damages to photosystem II functioning in lettuce. Our data showed that high NaCl
556
concentration could affect photosynthesis yield from the early stage. In our study, NaCl
557
treatments significantly influenced the parameters related to the fast chlorophyll fluorescence
558
implying that salt has the potential of repressing the electron flows in photosynthesis machinery.
559
Improvement of electron transport capacity in plants exposed to NaCl stress indicates that
560
GABA is capable of removing defects in photosystem II induced by salinity stresses.
561
In the present study, parameters related to the fast and slow inductions of chlorophyll
562
fluorescence were regulated by GABA application in salt-treated plants. The absorbed photon
563
fluxes by photosystem lead to the excitation of chlorophylls. The energy of excitation can have
564
few fates: i) it will be partially converted to redox energy through electron transport which will
565
be consumed for CO2 fixation and ii) alternatively can be released as heat and iii) fluorescence
566
(Toth et al., 2007; Kalaji et al., 2017). Lower photosystem II photochemical performance (lower
567
PIABS, Rfd and Fv/Fm) in salt-treated plants can be due to the higher light energy absorption
AC C
EP
TE D
M AN U
SC
RI PT
545
ACCEPTED MANUSCRIPT
(ABS/RC) and dissipated energy flux (DI0/RC) and lower electron transport (ET0/ABS) per
569
reaction centers which consequently results in decreased quantum yield of absorbed photons.
570
Increase of ABS/RC in salt-exposed plants could be due to the inactivation of reaction centers
571
and a decrease in active QA reducing centers (Strasser and Stirbet, 1998). In stress conditions,
572
low proportion of absorbed energy passed on the electron transport chain (Sarkar and Ray,
573
2016).
574
It has been confirmed that plants protect themselves from excess light exposure through
575
dissipation of light energy as heat, known as NPQ.This process protects photosynthesis when
576
absorbed light energy exceeds the photosynthetic capacity to utilize it (Müller et al., 2001). In
577
our study, although NPQ was negatively affected by salinity stress, GABA kept the NPQ of salt-
578
treated plants on the same level of non-stressed plants. Increase in NPQ following exposure to
579
stress conditions depends on the xanthophyll cycle. When this cycle moves towards production
580
of zeaxanthin (usually under high light intensities), it would result in a higher NPQ. Production
581
of zeaxanthin is mediated with violaxanthin de-epoxidase (VDE) and APX activities (Sui et al.,
582
2007; Jahns and Holzwarth, 2012). In this study, APX activity was decreased in a salt-
583
concentration dependent manner in control plants while GABA caused considerable increase in
584
the APX activity. Confirming this finding, vde mutant plants showed decreased NPQ and Fv/Fm
585
under stress condition (Azzabi et al., 2012).
586
Positive effect of GABA on these parameters indicates the involvement of GABA in
587
photosynthesis functionality either directly by affecting the activity of reaction centers involved
588
in transferring of exited electron from chlorophyll to produce energy or indirectly by modulating
589
processes that crosstalk with photosynthesis performance. However the mechanism/s explain
590
how GABA interferes in photosynthesis enzymatic activities is not clear, GABA has been
AC C
EP
TE D
M AN U
SC
RI PT
568
ACCEPTED MANUSCRIPT
suggested to interplay with C and N metabolism in plants (Michaeli and Fromm, 2015) which
592
imply indirect effect of GABA on photosynthesis output. Alternatively, glutamine has long been
593
suggested as one the main components that intermediate carbon: nitrogen ratio in plants which
594
make it capable of affecting the photosynthesis functionality (Fait et al., 2008). Interestingly,
595
glutamine is involved in proline and chlorophyll synthesis when leaves are developing (Forde
596
and Lea, 2007) and also for GABA biosynthesis in whole plant growth stages (Signorelli et al.,
597
2015). This can introduce the indirect effect of GABA on photosynthesis performance. Although
598
the mechanism introduces how GABA affects photosynthesis function is remained to be
599
investigated.
M AN U
SC
RI PT
591
600
5. Conclusion
602
Based on finding of this study, GABA decreased the negative effects of NaCl on growth and
603
physiological parameters in early growth stage of lettuce plant. NaCl-treated seeds and seedlings
604
were able to germinate, show proper root and shoot growth, induce tolerance mechanism and
605
photosynthesis functionality when exposed to exogenous GABA. GABA effects on both
606
biophysical (chlorophyll fluorescence parameters) and cellular (parameters related to oxidative
607
stress) levels represent a multifunctional role for GABA as a biological component which can
608
exogenously adjust salt tolerance in lettuce plants. According to the salt dependent manner effect
609
of GABA on lettuce plant, it could be suggested that GABA role under salinity stress is more
610
related to stress signal regulation rather than protection. Moreover, GABA application in some
611
non-stressed plants imposed null effect implying relatively stress-responsive role for GABA. By
612
these results, we could propose that plants use GABA as a stress mediator capable of regulating
613
various biological processes influenced by salinity stress. However, the mechanism by which
AC C
EP
TE D
601
ACCEPTED MANUSCRIPT
614
GABA intermediates plant and saline stress interaction is an interesting question remained to be
615
answered.
RI PT
616
Author contribution statement
618
Maryam Seifi Kalhor and Sasan Aliniaiefard made substantial contributions to conception and
619
design, also performed statistical analysis, drafted the manuscript and critically revised the final
620
version. Mahdi Seif, Elahe Javadi and Batool Hassani carried out the experiments and helped in
621
the writing of manuscript. Françoise Bernard and Tao Li contributed to design of experiment and
622
critical revision of the final manuscript. All authors have read and approved the final manuscript.
M AN U
SC
617
623
Acknowledgments
625
We like to thank the Iran's National Elites Foundation for financial support of this project.
626
TE D
624
References
628 629 630 631 632 633 634 635 636 637 638 639 640
Ahuja, I., de Vos, R.C., Bones, A.M., Hall, R.D., 2010. Plant molecular stress responses face climate change. Trends Plant Sci. 15, 664-674. Akçay, N., Bor, M., Karabudak, T., Özdemir, F., Türkan, İ., 2012. Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants. J Plant Physiol. 169, 452-458. AL-Quraan, N.A., 2015. GABA shunt deficiencies and accumulation of reactive oxygen species under UV treatments: insight from Arabidopsis thaliana calmodulin mutants. Acta Physiol Plant. 37, 1-11. Al-Whaibi, M.H., Siddiqui, M.H., Basalah, M.O., 2012. Salicylic acid and calcium-induced protection of wheat against salinity. Protoplasma. 249, 769-778. Aliniaeifard, S., Hajilou, J., Tabatabaei, S.J., 2016a. Photosynthetic and Growth Responses of Olive to Proline and Salicylic Acid under Salinity Condition. Not. Bot. Horti Agrobot. Clujna. 44, 579-585. Aliniaeifard, S., Hajilou, J., Tabatabaei, S.J., Sifi-Kalhor, M., 2016b. Effects of Ascorbic Acid and Reduced Glutathione on the Alleviation of Salinity Stress in Olive Plants. Int. J. Fruit Sci. 16, 395-409.
AC C
EP
627
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Aliniaeifard, S., Malcolm Matamoros, P., van Meeteren, U., 2014. Stomatal malfunctioning under low Vapor Pressure Deficit (VPD) conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling. Physiol Plant. 152, 688-699. Aliniaeifard, S., van Meeteren, U., 2014. Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning. J. Exp. Bot. 65, 6529-6542. Allakhverdiev, S.I., Sakamoto, A., Nishiyama, Y., Inaba, M., Murata, N., 2000. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp. Plant Physiol. 123, 10471056. Allan, W.L., Clark, S.M., Hoover, G.J., Shelp, B.J., 2009. Role of plant glyoxylate reductases during stress: a hypothesis. Biochem J. 423, 15-22. Allan, W.L., Simpson, J.P., Clark, S.M., Shelp, B.J., 2008. γ-Hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms. J. Exp. Bot. 59, 2555-2564. Andriolo, J.L., Luz, G.L.d., Witter, M.H., Godoi, R.d.S., Barros, G.T., Bortolotto, O.C., 2005. Growth and yield of lettuce plants under salinity. Hort. Brasil. 23, 931-934. Ashraf, M., Harris, P., 2013. Photosynthesis under stressful environments: an overview. Photosynthetica. 51, 163-190. Azzabi, G., Pinnola, A., Betterle, N., Bassi, R., Alboresi, A., 2012. Enhancement of non-photochemical quenching in the Bryophyte Physcomitrella patens during acclimation to salt and osmotic stress. Plant Cell Physiol. 53, 1815-1825. Bartha, C., Fodorpataki, L., del Carmen Martinez-Ballesta, M., Popescu, O., Carvajal, M., 2015. Sodium accumulation contributes to salt stress tolerance in lettuce cultivars. J. App. Bot. Food Qual. 88. Bates, L., Waldren, R., Teare, I., 1973. Rapid determination of free proline for water-stress studies. Plant Soil. 39, 205-207. Bonilla, I., El-Hamdaoui, A., Bolaños, L., 2004. Boron and calcium increase Pisum sativum seed germination and seedling development under salt stress. Plant Soil. 267, 97-107. Bor, M., Seckin, B., Ozgur, R., Yılmaz, O., Ozdemir, F., Turkan, I., 2009. Comparative effects of drought, salt, heavy metal and heat stresses on gamma-aminobutryric acid levels of sesame (Sesamum indicum L.). Acta Physiol. Plant. 31, 655-659. Bown, A.W., Shelp, B.J., 2016. Plant GABA: not just a metabolite. Trends Plant Sci 21, 811-813. Cantrell, I.C., Linderman, R.G., 2001. Preinoculation of lettuce and onion with VA mycorrhizal fungi reduces deleterious effects of soil salinity. Plant Soil. 233, 269-281. Chartzoulakis, K., 2005. Salinity and olive: growth, salt tolerance, photosynthesis and yield. Agric. Water Manag. 78, 108-121. Chaves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann. Bot. 103, 551-560. Chinnusamy, V., Jagendorf, A., Zhu, J.-K., 2005. Understanding and improving salt tolerance in plants. Crop Sci. 45, 437-448. Demidchik, V., Cuin, T.A., Svistunenko, D., Smith, S.J., Miller, A.J., Shabala, S., Sokolik, A., Yurin, V., 2010. Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. J Cell Sci. 123, 1468-1479. Demidchik, V., Shabala, S.N., Coutts, K.B., Tester, M.A., Davies, J.M., 2003. Free oxygen radicals regulate plasma membrane Ca2+-and K+-permeable channels in plant root cells. J Cell Sci. 116, 81-88. Demidchik, V., Straltsova, D., Medvedev, S.S., Pozhvanov, G.A., Sokolik, A., Yurin, V., 2014. Stressinduced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. J. Exp. Bot. 65, 1259-1270. Dhindsa, R., Plumb-Dhindsa, P., Thorpe, T., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93-101.
AC C
641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Díaz-Vivancos, P., Clemente-Moreno, M.J., Rubio, M., Olmos, E., García, J.A., Martínez-Gómez, P., Hernández, J.A., 2008. Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus. J. Exp. Bot. 59, 2147-2160. Fait, A., Fromm, H., Walter, D., Galili, G., Fernie, A.R., 2008. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci. 13, 14-19. Filomeni, G., De Zio, D., Cecconi, F., 2015. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ. 22, 377. Forde, B.G., Lea, P.J., 2007. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339-2358. Fougere, F., Le Rudulier, D., Streeter, J.G., 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. Gechev, T.S., Hille, J., 2005. Hydrogen peroxide as a signal controlling plant programmed cell death. The J. Cell Biol. 168, 17-20. Genc, Y., Mcdonald, G.K., Tester, M., 2007. Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant Cell Environ. 30, 1486-1498. Genty, B., Briantais, J.-M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA)-General Subjects. 990, 87-92. Gilliham, M., Tyerman, S.D., 2016. Linking metabolism to membrane signaling: the GABA–malate connection. Trends Plant Sci. 21, 295-301. Giuffrida, F., Scuderi, D., Giurato, R., Leonardi, C., 2012. Physiological response of broccoli and cauliflower as affected by NaCl salinity, VI International Symposium on Brassicas and XVIII Crucifer Genetics Workshop. 1005, pp. 435-441. Han, S., Fang, L., Ren, X., Wang, W., Jiang, J., 2015. MPK6 controls H2O2‐induced root elongation by mediating Ca2+ influx across the plasma membrane of root cells in Arabidopsis seedlings. New Phytol. 205, 695-706. Hayyan, M., Hashim, M.A., AlNashef, I.M., 2016. Superoxide ion: generation and chemical implications. Chemical reviews. 116, 3029-3085. He, Y., Zhu, Z., Yang, J., Ni, X., Zhu, B., 2009. Grafting increases the salt tolerance of tomato by improvement of photosynthesis and enhancement of antioxidant enzymes activity. Environ. Exp. Bot. 66, 270-278. Hu, X., Xu, Z., Xu, W., Li, J., Zhao, N., Zhou, Y., 2015. Application of γ-aminobutyric acid demonstrates a protective role of polyamine and GABA metabolism in muskmelon seedlings under Ca(NO3)2 stress. Plant Physiol. Biochem. 92, 1-10. Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 1817, 182-193. Janagoudar Sr, B.S., 2007. Salinity induced changes on stomatal response, biophysical parameters, solute accumulation and growth in cotton (Gossypium spp.), The World Cotton Research Conference, pp. 10-14. Jutsz, A.M., Gnida, A., 2015. Mechanisms of stress avoidance and tolerance by plants used in phytoremediation of heavy metals. Arch. Environ. Protect. 41, 104-114. Kalaji, M.H., Goltsev, V.N., Zivcak, M., Brestic, M., 2017. Chlorophyll Fluorescence: Understanding Crop Performance—Basics and Applications. CRC Press. Kaplan, F., Kopka, J., Haskell, D.W., Zhao, W., Schiller, K.C., Gatzke, N., Sung, D.Y., Guy, C.L., 2004. Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol. 136, 4159-4168. Kawano, T., Kawano, N., Muto, S., Lapeyrie, F., 2002. Retardation and inhibition of the cation‐induced superoxide generation in BY‐2 tobacco cell suspension culture by Zn2+ and Mn2+. Physiol Plant. 114, 395404. Kong, D., Ju, C., Parihar, A., Kim, S., Cho, D., Kwak, J.M., 2015. Arabidopsis glutamate receptor homolog3. 5 modulates cytosolic Ca2+ level to counteract effect of abscisic acid in seed germination. Plant Physiol. 167, 1630-1642.
AC C
691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth Res. 79, 209-218. Li, H., Chang, J., Chen, H., Wang, Z., Gu, X., Wei, C., Zhang, Y., Ma, J., Yang, J., Zhang, X., 2017a. Exogenous Melatonin Confers Salt Stress Tolerance to Watermelon by Improving Photosynthesis and Redox Homeostasis. Front.n Plant Sci. 8. Li, M., Guo, S., Yang, X., Meng, Q., Wei, X., 2016a. Exogenous gamma-aminobutyric acid increases salt tolerance of wheat by improving photosynthesis and enhancing activities of antioxidant enzymes. Biol. Plant. 60, 123-131. Li, Y., 2008. Effect of salt stress on seed germination and seedling growth of three salinity plants. Pakistan journal of biological sciences: PJBS 11, 1268-1272. Li, Y., Fan, Y., Ma, Y., Zhang, Z., Yue, H., Wang, L., Li, J., Jiao, Y., 2017b. Effects of exogenous γaminobutyric acid (GABA) on photosynthesis and antioxidant system in pepper (Capsicum annuum L.) seedlings under low light stress. J. Plant Growth Regul. 36, 436-449. Li, Z., Yu, J., Peng, Y., Huang, B., 2016b. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera). Scientific Reports. 6. Liang, X., Zhang, L., Natarajan, S.K., Becker, D.F., 2013. Proline mechanisms of stress survival. Antioxidants & redox signaling. 19, 998-1011. Lichtenthaler, H., Langsdorf, G., Lenk, S., Buschmann, C., 2005. Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system. Photosynthetica. 43, 355-369. Ludewig, F., Hüser, A., Fromm, H., Beauclair, L., Bouché, N., 2008. Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis. PLoS One 3, e3383. Machado, R.M.A., Serralheiro, R.P., 2017. Soil salinity: Effect on vegetable crop growth. Management practices to prevent and mitigate soil salinization. Horticulturae. 3, 30. Maggio, A., De Pascale, S., Fagnano, M., Barbieri, G., 2011. Saline agriculture in Mediterranean environments. Italian j. Agron. 6, 7. Mehta, P., Jajoo, A., Mathur, S., Bharti, S., 2010. Chlorophyll a fluorescence study revealing effects of high salt stress on photosystem II in wheat leaves. Plant Physiol. Biochem. 48, 16-20. Meot-Duros, L., Magné , C., 2008. Effect of salinity and chemical factors on seed germination in the halophyte Crithmum maritimum L. Plant Soil 313, 83-87. Michaeli, S., Fromm, H., 2015. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?. Front. Plant Sci. 6, 419. Mohammadi, P., Khoshgoftarmanesh, A.H., 2014. The effectiveness of synthetic zinc (Zn)-amino chelates in supplying Zn and alleviating salt-induced damages on hydroponically grown lettuce. Sci. Hortic. 172, 117-123. Müller, P., Li, X.-P., Niyogi, K.K., 2001. Non-Photochemical Quenching. A response to excess light energy. Plant Physiol. 125, 1558-1566. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651-681. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867-880. Nayyar, H,. Kaur, R., Kaur, S., Singh, R. 2014. γ-Aminobutyric Acid (GABA) Imparts partial protection from heat stress injury to rice seedlings by improving leaf turgor and upregulating osmoprotectants and antioxidants. J. Plant Growth Regulation. 33, 408-419.
AC C
742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792
Niu, L., Liao, W., 2016. Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Frontiers in plant science. 7. Orlovsky, N., Japakova, U., Zhang, H., Volis, S., 2016. Effect of salinity on seed germination, growth and ion content in dimorphic seeds of Salicornia europaea L.(Chenopodiaceae). Plant Diversity 38, 183-189. Özbingöl, N., Corbineau, F., Groot, S., Bino, R., Côme, D., 1999. Activation of the cell cycle in tomato (Lycopersicon esculentum Mill.) seeds during osmoconditioning as related to temperature and oxygen. Ann. Bot. 84, 245-251.
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Palanivelu, R., Brass, L., Edlund, A.F., Preuss, D., 2003. Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 114, 47-59. Panuccio, M.R., Jacobsen, S.E., Akhtar, S.S., Muscolo, A., 2014. Effect of saline water on seed germination and early seedling growth of the halophyte quinoa. AoB PLANTS 6. Patterson, B.D., MacRae, E.A., Ferguson, I.B., 1984. Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical biochemistry. 139, 487-492. Pulido, P., Domínguez, F., Cejudo, F.J., 2009. A hydrogen peroxide detoxification system in the nucleus of wheat seed cells: protection or signaling role? Plant Signal. Behav. 4, 23-25. Qiu, Z., Li, J., Zhang, M., Bi, Z., Li, Z., 2013. He–Ne laser pretreatment protects wheat seedlings against cadmium-induced oxidative stress. Ecotoxicol Environ Saf. 88, 135-141. Ramesh, S.A., Kamran, M., Sullivan, W., Chirkova, L., Okamoto, M., Degryse, F., McLaughlin, M., Gilliham, M., Tyerman, S.D., 2017. Split personality of aluminum activated malate transporter family proteins: facilitation of both GABA and malate transport. bioRxiv. 215335. Ramesh, S.A., Tyerman, S.D., Gilliham, M., Xu, B., 2016. γ-Aminobutyric acid (GABA) signalling in plants. Cell. Mol. Life Sci. 1-27. Ramesh, S.A., Tyerman, S.D., Xu, B., Bose, J., Kaur, S., Conn, V., Domingos, P., Ullah, S., Wege, S., Shabala, S., 2015. GABA signalling modulates plant growth by directly regulating the activity of plantspecific anion transporters. Nature communications. 6. Renault, H., El Amrani, A., Berger, A., Mouille, G., Soubigou-Taconnat, L., Bouchereau, A., Deleu, C., 2013. γ‐Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots. Plant Cell Environ. 36, 1009-1018. Renault, H., El Amrani, A., Palanivelu, R., Updegraff, E.P., Yu, A., Renou, J.-P., Preuss, D., Bouchereau, A., Deleu, C., 2011. GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana. Plant Cell Physiol. 52, 894-908. Renault, H., Roussel, V., El Amrani, A., Arzel, M., Renault, D., Bouchereau, A., Deleu, C., 2010. The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance. BMC Plant Biol. 10, 20. Rivero, R.M., Mestre, T.C., Mittler, R., Rubio, F., GARCIA‐SANCHEZ, F., Martinez, V., 2014. The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell Environ. 37, 1059-1073. Sarkar, R., Ray, A., 2016. Submergence-tolerant rice withstands complete submergence even in saline water: Probing through chlorophyll a fluorescence induction OJIP transients. Photosynthetica. 54, 275287. Scholz, S.S., Malabarba, J., Reichelt, M., Heyer, M., Ludewig, F., Mithöfer, A., 2017a. Evidence for GABA-induced systemic GABA accumulation in arabidopsis upon wounding. Front. Plant Sci. 8. Scholz, S.S., Malabarba, J., Reichelt, M., Heyer, M., Ludewig, F., Mithöfer, A., 2017b. Evidence for GABA-induced systemic GABA accumulation in Arabidopsis upon wounding. Front. Plant Sci. 8, 388. Shabala, S., Shabala, L., Barcelo, J., Poschenrieder, C., 2014. Membrane transporters mediating root signalling and adaptive responses to oxygen deprivation and soil flooding. Plant Cell Environ. 37, 22162233. Shao, H.-B., Chu, L.-Y., Jaleel, C.A., Zhao, C.-X., 2008. Water-deficit stress-induced anatomical changes in higher plants. C R Biol. 331, 215-225. Shelp, B.J., Bown, A.W., Zarei, A., 2017. 4-Aminobutyrate (GABA): a metabolite and signal with practical significance. Botany. 95, 1015-1032. Shu-Hsien, H., Chih-Wen, Y., Lin, C.H., 2005. Hydrogen peroxide functions as a stress signal in plants. Botanical Bulletin of Academia Sinica. 46. Signorelli, S., Arellano, J.B., Melø, T.B., Borsani, O., Monza, J., 2013. Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants. Plant Physiology Biochem. 64, 80-83. Signorelli, S., Dans, P.D., Coitiño, E.L., Borsani, O., Monza, J., 2015. Connecting proline and γaminobutyric acid in stressed plants through non-enzymatic reactions. Plos One. 10, e0115349.
AC C
793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Signorelli, S., Imparatta, C., Rodríguez-Ruiz, M., Borsani, O., Corpas, F.J., Monza, J., 2016. In vivo and in vitro approaches demonstrate proline is not directly involved in the protection against superoxide, nitric oxide, nitrogen dioxide and peroxynitrite. Funct Plant Biol. 43, 870-879. Slama, I., Abdelly, C., Bouchereau, A., Flowers, T., Savouré, A., 2015. Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Ann. Bot. 115, 433-447. Sosa, L., Llanes, A., Reinoso, H., Reginato, M., Luna, V., 2005. Osmotic and specific ion effects on the germination of Prosopis strombulifera. Ann. Bot. 96, 261-267. Steward, F., Thompson, J., Dent, C., 1949. γ-Aminobutyric acid: a constituent of the potato tuber. Science 110, 439-440. Strasser, B.J., 1995. Measuring fast fluorescence transients to address environmental questions: the JIP test. Photosynthesis: from light to biosphere. 977-980. Strasser, R.J., Srivastava, A., zTsimilli-Michael, M.g., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. Probing photosynthesis: mechanisms, regulation and adaptation. 445-483. Strasser, R.J., Stirbet, A.D., 1998. Heterogeneity of photosystem II probed by the numerically simulated chlorophyll a fluorescence rise (O–J–I–P). Mathematics and Computers in Simulation. 48, 3-9. Stuart, K., 2017. Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an overview. J Sci Food Agric. Sui, N., Li, M., Liu, X.-Y., Wang, N., Fang, W., Meng, Q.-W., 2007. Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene. Photosynthetica. 45, 447-454. Suo-ling, S., Jing-rui, L., Hong-bo, G., Qing-yun, L., Li-wen, Y., Rui-juan, G., 2012. Effects of exogenous γ-aminobutyric acid on inorganic nitrogen metabolism and mineral elements contents of melon seedling under hypoxia stress. Acta Hortic. Sinica. 4, 012. Tepe, H.D., Aydemir, T., 2015. Protective effects of Ca2+ against NaCl induced salt stress in two lentil (Lens culinaris) cultivars. Afric. J. Agr. Res. 10, 2389-2398. Tobe, K., Li, X., Omasa, K., 2002. Effects of sodium, magnesium and calcium salts on seed germination and radicle survival of a halophyte, Kalidium caspicum (Chenopodiaceae). Aust J Bot 50, 163-169. Toth, S.Z., Schansker, G., Strasser, R.J., 2007. A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient. Photosynth Res. 93, 193-203. Tripathy, B.C., Oelmüller, R., 2012. Reactive oxygen species generation and signaling in plants. Plant Signal. Behav. 7, 1621-1633. Tuna, A.L., Kaya, C., Ashraf, M., Altunlu, H., Yokas, I., Yagmur, B., 2007. The effects of calcium sulphate on growth, membrane stability and nutrient uptake of tomato plants grown under salt stress. Environ Exp. Bot. 59, 173-178. Ünlükara, A., Cemek, B., Karaman, S., Erşahin, S., 2008. Response of lettuce (Lactuca sativa var. crispa) to salinity of irrigation water. N Z J Crop Hortic Sci. 36, 265-273. Vijayakumari, K., Puthur, J.T., 2016. γ-Aminobutyric acid (GABA) priming enhances the osmotic stress tolerance in Piper nigrum Linn. plants subjected to PEG-induced stress. Plant Growth Regul. 78, 57-67. Wang, Y., Gu, W., Meng, Y., Xie, T., Li, L., Li, J., Wei, S., 2017. γ-aminobutyric acid Imparts partial protection from salt stress injury to maize seedlings by improving photosynthesis and upregulating osmoprotectants and antioxidants. Scientific Reports. 7, 43609. Xiang, L., Hu, L., Hu, X., Pan, X., Ren, W., 2015. Response of reactive oxygen metabolism in melon chloroplasts to short-term salinity-alkalinity stress regulated by exogenous γ-aminobutyric acid. J. App. Ecol. 26, 3746-3752. Xiang, L., Hu, L., Xu, W., Zhen, A., Zhang, L., Hu, X., 2016. Exogenous γ-aminobutyric acid improves the structure and function of photosystem II in muskmelon seedlings exposed to salinity-alkalinity stress. PloS one. 11, e0164847. Xing, S.G., Jun, Y.B., Hau, Z.W., Liang, L.Y., 2007. Higher accumulation of γ-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots. Plant Physiol. Biochem. 45, 560-566.
AC C
844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894
ACCEPTED MANUSCRIPT
RI PT
Xu, C., Mou, B., 2015. Evaluation of lettuce genotypes for salinity tolerance. Hortscience 50, 1441-1446. Yoshiba, Y., Kiyosue, T., Katagiri, T., Ueda, H., Mizoguchi, T., Yamaguchi‐Shinozaki, K., Wada, K., Harada, Y., Shinozaki, K., 1995. Correlation between the induction of a gene for ∆1‐pyrroline‐5‐ carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J. 7, 751-760. You, J., Chan, Z., 2015a. ROS regulation during abiotic stress responses in crop plants. Front. Plant sci. 6, 1092. Yu, C.-W., Murphy, T.M., Lin, C.-H., 2003. Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Funct. Plant Biol. 30, 955-963. Žárský, V., 2015. Signal transduction: GABA receptor found in plants. Nature plants. 1, 15115. Zhang, L., Tian, L.-H., Zhao, J.-F., Song, Y., Zhang, C.-J., Guo, Y., 2009. Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis. Plant Physiol. 149, 916-928. Zhu, J.-K., 2001. Plant salt tolerance. Trends Plant Sci. 6, 66-71. Zhu, Z., Wei, G., Li, J., Qian, Q., Yu, J., 2004. Silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of salt-stressed cucumber (Cucumis sativus L.). Plant Sci. 167, 527-533.
SC
895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910
AC C
EP
TE D
M AN U
911
ACCEPTED MANUSCRIPT Highlights •
GABA improves seed germination and plant growth in saline conditions.
•
GABA reduces negative effects of salt stress on photosynthetic performance of lettuce plants. GABA decreases the levels of salt stress-induced oxidative stress in lettuce plants.
•
Salt-induced accumulation of proline is decreased by GABA application
AC C
EP
TE D
M AN U
SC
RI PT
•
ACCEPTED MANUSCRIPT Author contribution statement Maryam Seifi Kalhor and Sasan Aliniaiefard made substantial contributions to conception and design, also performed statistical analysis, drafted the manuscript and critically revised
RI PT
the final version. Mahdi Seif, Elahe Javadi and Batool Hassani carried out the experiments and helped in the writing of manuscript. Françoise Bernard and Tao Li contributed to design of experiment and critical revision of the final manuscript. All authors have read and
AC C
EP
TE D
M AN U
SC
approved the final manuscript.
S0981-9428(18)30303-6
DOI:
10.1016/j.plaphy.2018.07.003
Reference:
PLAPHY 5326
To appear in:
Plant Physiology and Biochemistry
Received Date: 23 April 2018 Revised Date:
27 June 2018
Accepted Date: 2 July 2018
Please cite this article as: M.S. Kalhor, A. Sasan, M. Seif, E.J. Asayesh, Franç. Bernard, B. Hassani, Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2018.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Enhanced salt tolerance and photosynthetic performance: implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants
RI PT
Authors: Maryam Seifi Kalhora, Aliniaeifard Sasanb*, Mehdi Seifb, Elahe Javadi Asayeshb, Françoise Bernarda, Batool Hassania a
Faculty of Life Sciences and Biotechnology. Department of Plant Sciences, Shahid Beheshti
SC
University, Tehran, Iran b
EP
TE D
Corresponding email: [email protected]
AC C
*
M AN U
Department of Horticulture, Aburaihan campus, University of Tehran, Tehran, Iran
ACCEPTED MANUSCRIPT
1
Enhanced salt tolerance and photosynthetic performance: implication of ɤ-amino butyric
2
acid application in salt-exposed lettuce (Lactuca sativa L.) plants
RI PT
3
ABSTRACT
5
Gamma-Amino Butyric Acid (GABA) is a substantial component of the free amino acid pool
6
with low concentration in plant tissues. Enhanced GABA content occurs during plant growth and
7
developmental processes like seed germination. GABA level, basically, alters in response to
8
many endogenous and exogenous stimuli. In the current study, GABA effects were studied on
9
germination, photosynthetic performance and oxidative damages in salt-exposed lettuce plants.
10
Three NaCl (0, 40 and 80 mM) and two GABA (0 and 25 µM) concentrations were applied on
11
lettuce during two different developmental (seed germination and seedlings growth) stages.
12
Negative effects of salinity on germination and plant growth were removed by GABA
13
application. GABA significantly reduced mean germination time (MGT) in salt-exposed lettuce
14
seeds. Although, salinity caused a significant decline in maximum quantum yield of photosystem
15
II (Fv/Fm) during distinct steps of plant growth, GABA application improved Fv/Fm particularly
16
on high salinity level. GABA decreased specific energy fluxes per reaction center (RC) for
17
energy absorption and dissipation, while enhanced-electron transport flux in photosynthetic
18
apparatus of lettuce plants was observed in GABA-supplemented plants. Moreover, decline in
19
non-photochemical quenching (NPQ) and quenching coefficients (qP, qL, qN) by salt stress were
20
recovered by GABA application. Elevated electrolyte leakage considerably decreased by GABA
21
exposure on salt-treated plants. Although, proline level increased by NaCl treatments in a
22
concentration dependent manner, combined application of salt with GABA caused a significant
23
reduction in proline content. Catalase; EC 1.11.1.6 (CAT), L-ascorbate peroxidase; EC
AC C
EP
TE D
M AN U
SC
4
ACCEPTED MANUSCRIPT
1.11.1.11 (APX), and superoxide dismutase; EC 1.15.1.1 (SOD) activities were increased by
25
GABA exposure in salt-supplemented plants that resulted in regulated hydrogen peroxide level.
26
In conclusion, a multifaceted role for GABA is suggested for minimizing detrimental effects of
27
salinity on lettuce through improvement of photosynthetic functionality and regulation of
28
oxidative stress.
RI PT
24
29
Keywords: GABA, Germination, Growth, Lettuce, Oxidative stress, Photosynthesis, Salinity
SC
30
M AN U
31
1. Introduction
33
Various environmental stresses negatively influence agricultural productivity. Among them, soil
34
and water salinity progressively affect the agricultural lands by causing detrimental effects on
35
seed germination, plant growth and development (Li et al., 2017a). Ionic and osmotic stresses are
36
consequently induced by high salt concentrations in irrigation water (Zhang et al., 2009; Ahuja et
37
al., 2010; Aliniaeifard et al., 2016b). Salinity has adverse effects on seed germination by
38
affecting either germination percentage or germination longevity (Meot-Duros and Magné ,
39
2008). Under salinity stress, due to the disturbance in plant cell turgor, nutrient transport inside
40
of the plants would be also negatively affected (Chartzoulakis, 2005). Salinity influences the
41
rhizosphere osmotic potential which ultimately results in delayed and hampered seed
42
germination (Sosa et al., 2005). Decreases in leaf expansion, shoot and root development have
43
been reported when plants are exposed to saline conditions (Maggio et al., 2011; Giuffrida et al.,
44
2012; Machado and Serralheiro, 2017). Furthermore, various processes including enzymatic
45
activities, photosynthesis performance and respiration rate can also be negatively influenced by
46
salinity stress (Chartzoulakis, 2005; Aliniaeifard et al., 2016a; Aliniaeifard et al., 2016b).
AC C
EP
TE D
32
ACCEPTED MANUSCRIPT
Accumulation of Reactive Oxygen Species (ROS) in different plant organs has been frequently
48
reported following plant exposure to saline condition. ROS result in oxidative damages which
49
ultimately leading to the programmed cell death (Filomeni et al., 2015; You and Chan, 2015).
50
Negative effects of salt stress on photosynthesis can potentially occur through affecting stomata
51
functioning (Munns and Tester, 2008) or by ROS-induced oxidative damages in related enzymes
52
which incorporate in photosynthesis functionality (Chaves et al., 2009; Aliniaeifard et al.,
53
2016a). Detrimental effects of salt stress are associated with damages on distinct parts of the
54
photosynthetic apparatus (Mehta et al., 2010). To cope with these damages and to reduce the
55
stress related susceptibility, plants have co-opted numerous physiological, molecular, and
56
cellular adaptations during evolution (Shao et al., 2008). As an example, organic compounds
57
accumulate in the cells and through specific processes protect plants against oxidative damages
58
(Jutsz and Gnida, 2015; Slama et al., 2015). Accumulation of GABA, an endogenous organic
59
compound, under stressful conditions has been frequently reported (Kaplan et al., 2004; Xing et
60
al., 2007; Allan et al., 2008; Bor et al., 2009; Renault et al., 2011; Li et al., 2016b; Scholz et al.,
61
2017b). GABA is a four-carbon non-protein amino acid involving in plant responses to the
62
variety of environmental stresses (Ramesh et al., 2016; Vijayakumari and Puthur, 2016; Stuart,
63
2017). For the first time GABA identification was reported in 1949, when GABA accumulation
64
was detected in potato tuber tissues (Steward et al., 1949). Later, wide ranges of GABA effects
65
on plant growth and development were revealed with independent studies on various plant
66
species (Fait et al., 2008; Suo-ling et al., 2012; Ramesh et al., 2015). An increase in GABA level
67
in root and nodule tissues following exposure to salt stress has been reported in alfalfa
68
(Medicago sativa L.) plants (Fougere et al., 1991). Salt tolerance in Nicotiana sylvestris has been
69
shown by induction of GABA-related genes expression (e.g. GAD) which resulted in GABA
AC C
EP
TE D
M AN U
SC
RI PT
47
ACCEPTED MANUSCRIPT
accumulation under NaCl treatment (Akçay et al., 2012). Interestingly, plant deficient in GABA
71
transaminase enzyme (GABA-T), an enzyme involved in GABA metabolism, represented salt
72
sensitive responses when exposed to saline stress condition (Renault et al., 2013). These
73
observations indicate that salt tolerance in plant is associated with GABA metabolism. Positive
74
effect of GABA on photosynthetic functionality in plants imposed to salinity has also been
75
revealed (Li et al., 2017b). Recently it was shown that exogenous GABA can improve mask
76
melon (Cucumis melo L.) photosynthesis under saline conditions (Xiang et al., 2016); however,
77
GABA effects on seed germination and early stages of plant growth is not comprehensively
78
studied. In the current study we have used lettuce plant since it is an important edible crop,
79
widely consumed in the world and sensitive to the salt stress (Ünlükara et al., 2008). Negative
80
effects of salinity on different aspects of lettuce growth and cellular mechanisms have been
81
frequently reported (Andriolo et al., 2005; Mohammadi and Khoshgoftarmanesh, 2014; Bartha et
82
al., 2015; Xu and Mou, 2015). Although plant's responses to the salinity have been intensively
83
studied, few researches have investigated GABA effects on salt tolerance and its related
84
mechanisms. Therefore, the objectives of this study were to i) investigate whether GABA can
85
improve salinity tolerance during seed germination and early plant development, ii) reveal
86
GABA effects on photosynthetic functionality by analyzing distinct stages of electron flow in
87
photosynthetic electron transport chain, iii) study GABA effect on oxidative damages induced by
88
salinity stress. The result will be relevant to introduce a natural compound capable of reducing
89
negative consequence of salt stress on growth and physiology of lettuce plants.
SC
M AN U
TE D
EP
AC C
90
RI PT
70
91
2. Material and methods
92
2.1. Seed germination
ACCEPTED MANUSCRIPT
To analyze the seed germination under salt stress, the seeds of Lactuca sativa cv. Partavousi (45
94
seeds) were placed in 10 cm diameter petri dishes with three layers of filter paper and 3 ml of
95
double-distilled sterile water containing different concentrations of NaCl (0, 40 and 80 mM) and
96
GABA (0 and 25 µM). A pre-test experiment showed no significant difference between 25 and
97
50 µM GABA. Therefore, 25 µM GABA was used for this experiment. Plates were placed in a
98
growth chamber with 22±1°C and lighting period of 12 h light /12 h dark for 58 h. Seeds were
99
considered as germinated when the radicle emerged for 2 mm length. Three petri dishes were
100
used for each treatment and the experiment repeated for three times. MGT was calculated based
101
on the following equation:
102
MGT= ∑n.H / ∑n
103
Where n= number of newly germinated seeds at time H; H= hours from the onset of the
104
germination test, ∑n= final germination
TE D
105
M AN U
SC
RI PT
93
2.2. Plant growth conditions
107
In order to investigate lettuce plant growth under salt stress, the seeds were sown in a
108
transplanting tray with 80 sowing wells filled with fine perlite. After germination, in the stage of
109
cotyledon leaves emergence, homogeneous plants were selected and transplanted into the pots
110
(l×w×h=7 cm×7 cm×10 cm) filled with perlite (one plant per pot). Plants were kept inside a
111
growth chamber at 22±1°C with a 12 h light/12 h dark period, 50±5% relative humidity (RH),
112
400 ppm CO2 concentration (determined using Trotec BZ30 CO2 air quality data logger,
113
Heinsberg, Germany) and 250 µmol m-2 s-1 light intensity (measured with an sekonic
114
spectromaster c-7000, Japan) produced by white LED lights (400-700 nm). Daily irrigation was
115
applied with half strength of Hoagland solutions containing 0, 40 and 80 mM of NaCl together
AC C
EP
106
ACCEPTED MANUSCRIPT
116
with 0 and 25 µM of GABA. Medium substrates were fully washed with distilled water every 5
117
days to remove the effects of GABA and salt accumulation.
118
2.3. Growth measurements
120
Two weeks following germination, shoot and root fresh and dry weights were determined. Shoot
121
fresh weights were measured immediately after harvest, for root fresh weight, perlite's particles
122
were carefully removed using a sieve to minimize root loss and later biomass was measured
123
using a digital balance. Dry weights were measured after desiccation in an oven at 60ºC for 48 h
124
for all samples.
M AN U
SC
RI PT
119
125
2.4. Transient and slow induction of chlorophyll fluorescence
127
Outermost leaves following 4, 8 and 12 days application of treatments were used for measuring
128
slow chlorophyll fluorescence parameters. After dark adaptation for at least 20 minutes intact
129
attached leaves to the plants were immediately used to measure chlorophyll fluorescence
130
parameters using a fluorometer system (Handy FluorCam FC 1000-H Photon Systems
131
Instruments, PSI, Czech Republic). Images were recorded during short measuring flashes in
132
darkness. At the end of the short flashes, the samples were exposed to a saturating light pulse
133
(3900 µmol m-2 s-1) that resulted in a transitory saturation of photochemistry and reduction of
134
primary quinone acceptor of PSII (Genty et al., 1989; Aliniaeifard et al., 2014; Aliniaeifard and
135
van Meeteren, 2014). After reaching steady state fluorescence, two successive series of
136
fluorescence data were digitized and averaged, one during short measuring flashes in darkness
137
(F0), and the other during the saturating light flash (Fm). From these two images, variable
138
fluorescence (Fv) was calculated according to the ratio between Fm and F0. The Fv/Fm was
AC C
EP
TE D
126
ACCEPTED MANUSCRIPT
calculated using the ratio between Fv and Fm. Maximum fluorescence in light adapted steady
140
state (Fm′) was determined and was used for calculation of NPQ based on the following equation:
141
NPQ = (Fm/Fm')-1
142
Fraction of dark-adapted variable fluorescence lost upon adaptation to light (qN; NPQ
143
coefficient), photochemical quenching coefficient (qP); fraction of PSII open centers (qL) were
144
calculated as defined previously (Kramer et al., 2004). Following exposure to light, primary
145
charge separation in photosystem reaction centers occurs. In this step the fluorescence signal
146
rises due to reduction of plastoquinone pool by the generated editions from photosystem II
147
activity (FP). Later, the steady-state fluorescence levels attained in continuous light declines to
148
the fluorescence level called Ft. Rate of fluorescence decline (Rfd), an empirical parameter
149
proposed by (Lichtenthaler et al., 2005) to quantify plant fitness and vitality under stress
150
conditions, was calculated according to the following equation:
151
Rfd =(FP - Ft) / FP
152
The average values, and standard deviation per image were calculated by using of FluorCam
153
software 7.0. The polyphasic Chl a fluorescence (OJIP) transients were measured by a FluorPen
154
FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) on the outermost leaves
155
after 20 min dark adaptation. OJIP was used to study different biophysical and
156
phenomenological related to PSII status (Strasser, 1995). The OJIP transients measurement was
157
done according to the JIP test (Strasser et al., 2000). The following data from the original
158
measurements were used after extraction by Fluorpen software: fluorescence intensities at 50 µs
159
(F50 µs, considered as the minimum fluorescence F0), 2 ms (J-step, FJ), 60 ms (I-step, FI), and
160
maximum fluorescence (Fm). Performance index was calculated on the absorption basis (PIABS)
AC C
EP
TE D
M AN U
SC
RI PT
139
ACCEPTED MANUSCRIPT
and densities of QA- reducing PSII reaction centers at time 0 and time on maximum fluorescence
162
state. Furthermore, the yield ratios including: the probability that a trapped excision moves an
163
electron in electron transport chain beyond QA- (ψo), quantum yield of electron transport (φEo),
164
quantum yield of energy dissipation (φDo) and maximum quantum yield of primary
165
photochemistry (φPo) were also calculated based on the following equations:
RI PT
161
F0 FM
M AN U
φPo=1-
SC
Vj=(Fj-F0 )/(FM-F0 )
ψo=1-Vj
φDo=1-φPo- (
F0
F0 ) FM
. ψo
TE D
φEo= 1 −
From these data, the following parameters were calculated: the specific energy fluxes per
167
reaction center (RC) for energy absorption (ABS/RC= M0.(1/VJ).(1/φPo)), (M0= TR0/RC-
168
ET0/RC), trapped energy flux (TR0/RC= M0.(1/VJ)), electron transport flux (ET0/RC=
169
M0.(1/VJ). ψo) and dissipated energy flux (DI0/RC= (ABS/RC)–(TR0/RC)).
AC C
170
EP
166
171
2.5. Determination of electrolyte leakage percentage
172
For measuring electrolyte leakage, 10 leaf discs (0.5 cm diameter) were prepared using a cork
173
borer and washed with deionized water to remove surface-adhered electrolytes, placed in closed
174
vials containing 10 ml of deionized water and incubated at 25°C on a rotary shaker for 24h;
ACCEPTED MANUSCRIPT
subsequently, electrical conductivity of the solution (C1) was determined. Samples were
176
autoclaved at 120°C for 20 minutes and the resulting electrical conductivities (C2) were recorded
177
after equilibration at 25°C. The electrolyte leakage was calculated by the ratio between C1 and
178
C2 (Tuna et al., 2007).
179
RI PT
175
2.6. Determination of hydrogen peroxide level
181
Hydrogen peroxide (H2O2) was spectrophotometrically measured after reaction with potassium
182
iodide (KI). The reaction mixture contained 0.5 ml of 0.1% trichloroacetic acid (TCA), leaf
183
extract supernatant, 0.5 ml of 100 mM K-phosphate buffer and 2 ml reagent (1 M KI w/v in fresh
184
double-distilled water). The blank probe was contained 0.1% TCA in the absence of leaf extract.
185
The reaction was developed for 1 h in darkness and the absorbance was measured at 390 nm. The
186
amount of H2O2 was calculated using a standard curve prepared with known concentrations of
187
H2O2 levels according to the method described by (Patterson et al., 1984).
M AN U
TE D
188
SC
180
2.7. Determination of proline content
190
Determination of free proline content was performed according to (Bates et al., 1973). Fresh leaf
191
samples (0.5 g) were homogenized in 3% (w/v) sulphosalicylic acid. The homogenate was filtered by
192
using of filter paper. After addition of acid-ninhydrin and glacial acetic acid, the resulting mixture
193
was heated at 100°C for 1 h in a water bath. Reaction was stopped using an ice bath. The mixture
194
was extracted with toluene and the absorbance of fraction with toluene aspired from the liquid phase
195
was read at 520 nm. Proline concentration was determined using a calibration curve and expressed as
196
µmol proline g–1 Fresh weight.
197
AC C
EP
189
ACCEPTED MANUSCRIPT
2.8. Determination of antioxidant enzymes activity
199
APX activity was determined by oxidation of ascorbic acid (AA) at 265 nm ( = 13.7 mM-1 cm-1)
200
by slight modification from method described by (Nakano and Asada, 1981). The reaction
201
mixture contained 50 mM potassium phosphate buffer (pH 7.0), 5 mM AA, 0.5 mM H2O2 and
202
the enzyme extract. The reaction was started by adding H2O2. The rates were corrected for the
203
non-enzymatic oxidation of AA by the inclusion of a reaction mixture without the enzyme
204
extract (blind sample). The enzyme activity was expressed in µkat per mg-1 protein (Nakano and
205
Asada, 1981).
206
For determination of CAT activity, the decomposition of H2O2 was recorded by a decrease in
207
absorbance at 240 nm. Reaction mixture contained 1.5 ml of 50 mM sodium phosphate buffer
208
(pH 7.8), 0.3 ml of 100 mM H2O2, and 0.2 ml of enzyme extract. CAT unit was defined as the
209
amount of enzyme necessary to decompose 1 mM min-1 H2O2. Therefore the CAT activity was
210
expressed as µkat per mg-1 protein (Díaz-Vivancos et al., 2008).
211
SOD activity was measured according to the method described by Dhindsa et al. (1981). This
212
method is based on the ability of SOD to inhibit the photochemical reduction of nitro blue
213
tetrazolium (NBT). Reaction mixture contained 50 mM phosphate buffer (pH 7-8), 13 mM
214
methionine, 75 µM NBT, 0.15 mM riboflavin, 0.1 mM EDTA, and 0.50 ml enzyme extract.
215
Riboflavin was added as the last agent. The reaction mixture were shaken and placed under two
216
15 W fluorescent lamps. The reaction was started by switching on the light and was allowed to
217
run for 10 min. The reaction was stopped by turning off the light and the tubes were covered
218
using a black cloth. The absorbance by the reaction mixture was spectrophotometrically recorded
219
at 560 nm. A reaction mixture without exposure to the light was used as control. Controls were
AC C
EP
TE D
M AN U
SC
RI PT
198
ACCEPTED MANUSCRIPT
220
color free. Under assay conditions, one unit of SOD activity is expressed as the enzyme led to
221
50% inhibition of NBT reduction (Dhindsa et al., 1981).
RI PT
222
2.9. Statistical Analysis
224
For germination test, three petri dishes were used for each treatment with 45 seeds in each petri
225
dish and the experiment repeated for three times. The experiment was designed in a two-factorial
226
complete randomized experiment with six replications per treatment. Salt solutions were applied
227
to each plant per salt treatment to ensure that each plant received similar salt stress in all
228
treatments. Two-way analyses of variances (ANOVA) were used for significance tests of the
229
treatment effects. Tukey's multiple comparisons test were done for finding significant
230
differences. All statistical analyses were done using GraphPad Prism version 7.01 software
231
(GraphPad software, Inc. San Diego, CA).
232
TE D
M AN U
SC
223
3. Results
234
3.1. GABA improves seed germination and growth in salt-exposed lettuce seeds and seedlings
235
Monitoring seed germination after 12, 24, 36 and 48 h revealed that during first 12 h,
236
germination of seeds subjected to 40 and 80 mM NaCl were null except for those plants grown
237
under 40 mM NaCl treated with GABA (Fig. 1). Maximum germination percentage was
238
observed in control plants and those plants treated by GABA which represented 11.8% and 6.6%
239
seedlings respectively. After 24 h highest germination was recorded in control plants either
240
treated or non-treated with GABA. Lowest number of germinated seeds was observed in 80 mM
241
NaCl concentration. However, co-treatment with GABA remarkably increased the seedling
242
numbers in both NaCl concentrations, so that the seed germination (46%) in 40 mM salt
AC C
EP
233
ACCEPTED MANUSCRIPT
concentration by GABA treatment was equal with the control condition (without NaCl and
244
GABA treatment). Interestingly, 38% of seeds were germinated when GABA was added to 80
245
mM saline water, which was remarkably higher than seed germination ratio (29%) under 40 mM
246
NaCl irrigation regime. Changes in seedling numbers during third 12 h were more significant for
247
salt-imposed seeds. Triple increase in germination percentage was occurred for seeds co-treated
248
by 80 mM NaCl and GABA, however their germination percentage was lower than the control.
249
Significant changes were not observed in seedling numbers during fourth and fifth 12 h except
250
for those seeds exposed to high salt levels and moderate salinity without GABA treatment (Fig.
251
1). These results indicate positive regulatory role for GABA on germination of salt-exposed
252
lettuce seeds.
M AN U
SC
RI PT
243
100 90
TE D
80
60
NaCl 40
EP
50
C
40
NaCl 80 G G+NaCl 40
AC C
Germination, %
70
30
G+NaCl 80
20 10 0
0
253 254
10
20
30
40
Time (h)
50
60
70
ACCEPTED MANUSCRIPT
Fig. 1. Germination rate during 60 h exposure to three concentrations (0, 40 and 80 mM) of NaCl and two
256
levels (0 and 25 ppm) of GABA in lettuce seeds. Each value represents a mean ± standard error from
257
three independent experiments. C: Control condition without NaCl and G: GABA with GABA and
258
without NaCl.
RI PT
255
259
MGT was increased in a NaCl concentration-dependent manner (Fig. 2). Application of GABA
261
caused a decrease in MGT in both NaCl-treated plants; however; no remarkable difference was
262
found in MGT of plant seeds which treated by GABA.
EP
263
TE D
M AN U
SC
260
Fig. 2. Effects of salinity (0, 40 and 80 mM NaCl) and GABA [0 (black bars) and 25 µM GABA (white
265
bars)] on MGT of lettuce seeds. Each value represents a mean ± standard error from three independent
266
experiments. Bars with different letters are significantly different (ANOVA, P < 0.05).
267
AC C
264
268
Plant vegetative growth was measured following 14 days of NaCl and GABA applications. There
269
was significant difference between growth of control plants and those exposed to salt treatment.
270
Salt stress caused leaf chlorosis while, GABA application inhibited occurrence of NaCl-induced
271
chlorosis in lettuce leaves (Fig. 3)
ACCEPTED MANUSCRIPT
M AN U
SC
RI PT
272
273
Fig. 3. Lettuce plants exposed to different salt (0, 40 and 80 mM NaCl) and GABA [0 (control) and 25
275
µM GABA (G)] concentrations. Photographs were taken following 14 days of treatments.
276
TE D
274
Significant reductions of nearly 75% and 90% were observed in shoot fresh weight by 40 mM
278
and 80 mM NaCl respectively when compared to the shoot fresh weight of control plants. In
279
comparison with control plants, 50% and 75% decrease in shoot dry weights were observed in
280
seedlings exposed to 40 and 80 mM NaCl respectively. There was two-fold increase in shoot
281
fresh weight of GABA treated seedling compared with the shoot fresh weight of the control
282
plants. GABA exposure significantly increased shoot dry weights in both NaCl levels (Fig. 4B).
283
The highest increase in shoot dry weight was detected in plants treated with 80 mM NaCl.
284
GABA did not affect the shoot fresh and dry weights in non-stressed plants (Fig.4). In the term
285
of root growth, NaCl treatment reduced both fresh and dry weights in a NaCl dependent manner.
286
In comparison with the control plants, there were 50% and 85% reductions in root fresh weight
AC C
EP
277
ACCEPTED MANUSCRIPT
of 40 and 80 mM NaCl-treated plants, respectively (Fig. 4C). Similar results were obtained for
288
root dry weights (Fig. 4D). Interestingly, GABA increased root fresh and dry weights in control
289
plants. Significant increases in both root fresh and dry weights were detected in plants co-treated
290
with NaCl and GABA. Rate of root fresh and dry weights recovery by GABA was higher in
291
plants exposed to high NaCl concentration when compared to the medium and control NaCl
292
level.
SC
RI PT
287
GABA
Control 10
0.6
M AN U
8 0.4
6
4
0.2
2
0
0 N
aC
l8
0
0 aC N
N
aC
l4
l8
0
0 l8 N
aC
l4
aC
N
l
0.00
0
l
tr o
on
l4
0.05
0.0
C
aC
0.10
tr o
AC C
0.5
0.15
on
1.0
N
on
C
EP
1.5
0.20
C
2.0
tr ol
0 aC N
TE D
N
C
on
aC
l8
l4
0
tr ol
0.0
293 294
Fig. 4. Effects of salinity [0, 40 and 80 mM NaCl] and GABA [0 (black bars) and 25 µM GABA (white
295
bars)] on root and shoot fresh (A, C) and dry weights (B, D) of lettuce plants. Values are the means of six
296
replicates and bars indicate means ± SEM. Bars with different letters are significantly different (ANOVA,
297
P < 0.05).
ACCEPTED MANUSCRIPT
298
3.2. GABA improves photosynthetic electron flows in salt-exposed lettuce seedlings
300
Dark-adapted leaves of control and GABA-treated plants exposed to different NaCl
301
concentrations were used to measure the slow and fast inductions of Chl-a fluorescence. At the
302
beginning of the experiment (day 4), maximum Fv/Fm was observed in control plants treated with
303
GABA and minimum Fv/Fm was detected in plants exposed to 80 mM NaCl (Fig. 5A). Fv/Fm was
304
doubled by applying GABA on plants exposed to 80 mM NaCl. Fv/Fm value was increased after
305
8 days in all plants when compared with day 4. Notably, plants treated with 80 mM NaCl
306
showed the highest Fv/Fm recovery due to the GABA application (Fig. 5B). Fv/Fm value in plants
307
exposed to 80 mM NaCl was reached to the control plants through GABA treatment. In day 12,
308
Fv/Fm was not changed in control plants by GABA application. However, plants that were grown
309
under moderate and high saline conditions were positively responded to GABA and showed
310
significant increases in Fv/Fm values particularly in 80 mM NaCl treatment (Fig. 5C). These
311
results indicated the positive role of GABA on efficiency of photosystem II in salt-exposed
312
plants during early growth of lettuce plants.
SC
M AN U
TE D
EP AC C
313
RI PT
299
RI PT
1.0
a
a
a
a
a b
0.6
0.4
0.2
0 l8
0
N aC
l4
314
0 N aC l8
0
N aC l4
C on
tr ol
AC C
EP
TE D
N aC
C on
tr ol
0.0
M AN U
0.8
SC
0 N aC l8
N aC l4
C on tr o
0
l
ACCEPTED MANUSCRIPT
315
Fig. 5. Maximum quantum yield of photosystem II (Fv/Fm) in lettuce leaves following 4 (A), 8 (B) and 12
316
(C) days exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations.
317
Each value represents a mean ± standard error for six repetitions. Bars with different letters are
318
significantly different (ANOVA, P < 0.05).
ACCEPTED MANUSCRIPT
Specific energy fluxes per reaction center (RC) were measured based on the output of fast
320
inductions of Chl-a fluorescence. Parameters such as ABS/RC (Fig. 6A) and DI0/RC (Fig. 6B)
321
were decreased in plants subjected to both NaCl and GABA. In the case of TR0/RC, remarkable
322
differences were not detected among treatments (Fig. 6D). GABA caused increase in ET0/RC in
323
both salinity treatments; however, no significant effect was not observed in control plants (Fig.
324
6C), implying that under saline stress condition, GABA is able to recover electron transport in
325
PSII apparatus.
SC
RI PT
319
B
A
M AN U
GABA
Control
1.0
4.0
a
a
0.9
a
a
a
b
0.8
b
b
3.0
DI0/RC
3.5
b
b
0.7
c
c
0.6 0.4
326
0 N aC l8
0 N aC l4
on t C
D 3.0
2.5
2.0
1.5 1
N
aC
l8 0
l4 0 aC N
tr ol
l8 N aC
C on
0
0
0
l4
N aC
C on
tr ol
AC C
ET0/RC
EP
C
0.0
N aC l8 0
TE D
N aC l4 0
C on tr ol
1
ro l
2
327
Fig. 6. Specific energy fluxes per reaction center (RC) for energy absorption [A; (ABS/RC)], dissipated
328
energy flux [B; (DI0/RC)], electron transport flux [C; (ET0/RC)] and trapped energy flux [D; (TR0/RC)]
329
from the fluorescence transient exhibited by leaves of lettuce plants grown at different salt concentrations
ACCEPTED MANUSCRIPT
(black bars) and treated with GABA (white bars). Bars represent means of six repetitions ± SD. Bars with
331
different letters are significantly different (ANOVA, P < 0.05).
332
High NaCl concentration led to a significant reduction in performance index on absorption base
333
(PIABS) compared with the PIABS of control plants (Fig. 7). However, this reduction was not
334
significant for 40 mM NaCl-treated plants. Substantial increase in PIABS was occurred when
335
plants co-treated with GABA and NaCl. This increase was 30% and 50% for 40 mM and 80 mM
336
NaCl-treated plants respectively. Since PIABS represents validity index of PSII, these results can
337
imply that GABA affects photosynthesis functionality in a stress dependent manner.
M AN U
SC
RI PT
330
0
TE D aC l8
N
0 aC l4
EP
339
N
C on
tr ol
338
Fig. 7. Performance index on absorption basis (PIABS) in lettuce leaves following exposure to different salt
341
(0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value represents a mean ±
342
standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P <
343
0.05).
344
There was a gradual decrease in Performance index on absorption basis (PIABS) on a salt
345
concentration dependent manner, while under saline condition GABA caused remarkable
346
increases in the PIABS compared with the PIABS of control plants. Salinity stress led to a
AC C
340
ACCEPTED MANUSCRIPT
considerable decrease in NPQ; however, GABA application resulted in a full recovery of NPQ to
348
the value of control (Fig. 8A). GABA treatment caused an increase in qP of control plants and
349
plants grown under 40 mM NaCl (Fig. 8B). Significant change was not observed in 80 mM salt–
350
treated plants. GABA exposure led to the recovery of qL in high salt–treated plants to the value
351
of qL in control plants. Highest qN was recorded for non-treated GABA control plants (Fig. 8C).
352
Although, GABA had no effect on qN value in control plants, those plants exposed to moderate
353
and high saline condition showed a significant increase after GABA application. Monitoring the
354
state of active reaction centers (qL) showed a significant decrease in qL of plants subjected to
355
high level of salt stress (Fig. 8D). This indicates that 80 mM NaCl potentially force plants to
356
close reaction centers and consequently diminish qp during actual monitoring.
M AN U
SC
RI PT
347
AC C
EP
TE D
357
ACCEPTED MANUSCRIPT
Control
GABA
A
B
a
a
0.6
a
a ab
C
N aC l8 0
M AN U
D
0 N aC l8
0 N aC l4
tr ol C on
N aC l8 0
TE D
N aC l4 0
C
on t
ro
l
qL
qN
358
SC
C on tr ol
N aC l8 0
Na Cl 40
C
on tr ol
0.0
N aC l4 0
qP
NPQ 0.2
RI PT
b
0.4
Fig. 8. Values for NPQ and chlorophyll fluorescence quenching coefficients (qP, qL, qN) in lettuce leaves
360
following exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations.
361
Each value represents a mean ± standard error for six repetitions. Bars with different letters are
362
significantly different (ANOVA, P < 0.05).
AC C
363
EP
359
364
Chlorophyll fluorescence decrease ratio (Rfd) as plant vitality indicator was significantly
365
reduced in plants exposed to salinity stress in a salt concentration-dependent manner (Fig. 9).
366
However, application of GABA resulted in a remarkable increase in Rfd of moderate and high
367
salinity stresses in a salt concentration-dependent manner.
ACCEPTED MANUSCRIPT
SC
l8 0
l4 0
aC
aC
N
N
C on tr ol
RI PT
368
M AN U
369
Fig. 9. Chlorophyll fluorescence decrease ratio (Rfd) in lettuce leaves following exposure to different salt
371
(0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value represents a mean ±
372
standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P <
373
0.05).
374
TE D
370
3.3. GABA reduces oxidative stress in salt-exposed lettuce seedlings
376
Proline content was significantly increased in a NaCl concentration-dependent manner (Fig.
377
10A). However, with GABA application, the proline contents were decreased in both salt
378
exposed plants. These results indicated that under saline stress conditions, GABA possibly either
379
reduces the biosynthesis of proline or increases its conversion during certain metabolic pathway.
380
As shown in figure 8C the highest H2O2 level was detected in 80 mM NaCl-treated plants. H2O2
381
levels were significantly decreased in the leaves of plants treated with GABA (Fig. 10C).
382
Electrolyte leakage, which reflects damage to cell membrane, was considerably increased
383
following plant exposure to NaCl concentrations (Fig. 10B). GABA applications led to the
384
decline in the electrolyte leakages percentage of both salt-exposed plants.
AC C
EP
375
ACCEPTED MANUSCRIPT
385
387
SC 0
N aC l8
0
M AN U
N aC l8
0
0 N aC l4 N aC l4
N aC l8 0
N aC l4 0
ro l C on t
AC C
EP
TE D
C
C on tr ol
RI PT
386
388
Fig. 10. Proline content (A), electrolyte leakage (B) and H2O2 (C) content in lettuce leaves following
389
exposure to different salt (0, 40 and 80 mM NaCl) and GABA (0 and 25 µM) concentrations. Each value
ACCEPTED MANUSCRIPT
390
represents a mean ± standard error for six repetitions. Bars with different letters are significantly different
391
(ANOVA, P < 0.05).
RI PT
392
As shown in figure 11, CAT enzyme amount was reduced in high NaCl concentration. Notably,
394
GABA treatments increased the enzyme level in all treatments so that similar amount of CAT
395
was observed after GABA application (Fig. 11A). APX level was reduced in a NaCl dependent
396
manner and after exposure to GABA an increase was detected in all treatments (Fig. 11B). SOD
397
level was increased by elevating the NaCl level; however, any increase in enzyme amount was
398
not observed after GABA application except for control plants (Fig. 11C).
M AN U
SC
393
AC C
EP
TE D
399
ol tr on C
l4 aC N
0 l8 aC N
0
0
N
aC
l8
0
EP
400
N
l4 aC
TE D
C
ol tr on
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig. 11. Salinity effect on antioxidant enzymes activity in lettuce plant under different salt (0, 40 and 80
402
mM NaCl) and GABA (0 and 25 µM) concentrations. A) CAT, B) APX, C) SOD. Each value represents a
403
mean ± standard error for six repetitions. Bars with different letters are significantly different (ANOVA, P < 0.05).
404
AC C
401
405
4. Discussion
406
Soil salinity is a major abiotic stress which normally reduces plant growth and development
407
(Allakhverdiev et al., 2000) by hampering of water potential and nutrient deficiency
ACCEPTED MANUSCRIPT
(Chinnusamy et al., 2005; Genc et al., 2007). Inhibitory effect of salinity on seed germination has
409
been reported by different authors (Li, 2008; Meot-Duros and Magné , 2008; Panuccio et al.,
410
2014; Orlovsky et al., 2016). This effect can be due to the reduced available water required for
411
seed germination which results in a osmotic disruption and eventually hampered cell division
412
(Bonilla et al., 2004). Salinity tolerance during seed germination (in different plant species) can
413
be induced by exogenous applications such as boron (B+), Ca2+ (Bonilla et al., 2004), sodium
414
(Na+), magnesium (Mg2+) (Tobe et al., 2002) and salicylic acid (SA) (Al-Whaibi et al., 2012).
415
However, plants are able to produce endogenous growth regulators capable of modulating
416
developmental stages under certain stress condition. GABA as an endogenous signaling
417
molecule (Michaeli and Fromm, 2015; Žárský, 2015; Bown and Shelp, 2016; Gilliham and
418
Tyerman, 2016; Ramesh et al., 2016; Shelp et al., 2017) contributes in various stress related
419
responses (Akçay et al., 2012; Hu et al., 2015; Signorelli et al., 2015; Vijayakumari and Puthur,
420
2016; Scholz et al., 2017a). GABA role as a signaling metabolite was found when POP2 (Pollen-
421
Pistil incompatibility2) gene was identified as a regulator of pollen tube growth and germination
422
in Arabidopsis plants (Palanivelu et al., 2003). In current study, significant salt-induced-
423
reduction in seed germination of lettuce was improved by GABA application. This can be
424
explained based on its signaling role as endogenous modulator, which contributes in salinity
425
tolerance by protecting cell membrane against oxidative damages. Moreover, GABA exerts its
426
effects on plant cell through regulation of anion transporter that can alter plasma membrane
427
potential differences and osmotic adjustment (Ramesh et al., 2015) which is indispensible for
428
seed germination. In accordance with this, MGT was reduced in salt-exposed seeds that co-
429
treated with GABA. The probable reason for this could be explained by osmoprotecting role of
430
GABA (Nayyar et al., 2014), which potentially promotes early seed germination under salt-
AC C
EP
TE D
M AN U
SC
RI PT
408
ACCEPTED MANUSCRIPT
induced osmotic stress. Decreased MGT has been also reported in various osmopriming
432
conditions (Özbingöl et al., 1999). Analyzing of both root and shoot fresh and dry weighs
433
indicated that plant growth and/or development were stunted in response to the salinity. Similar
434
effects have been frequently reported in various plants, for example in tomato plant subjected to
435
120 mM salinity, both root and shoot fresh and dry weights have been negatively affected
436
(Rivero et al., 2014). Likewise in lettuce plant, shoot dry mass have been reduced when exposed
437
to salt stress (Cantrell and Linderman, 2001). The finding that fresh and dry shoot masses of
438
GABA-treated lettuce plants were less negatively affected by saline water than non-stresses
439
plants supports the hypothesis that, GABA favors lettuce plants to cope with the salinity stress in
440
rhizosphere. In fact, GABA role under salinity stress was more related to the stress signal
441
regulation rather than growth induction. By this result, it could be propose that plants engage
442
GABA as a stress mediator capable of regulating developmental stages influenced by ionic
443
(NaCl) stress in lettuce plants. This finding is consistent with defined role for GABA
444
transaminase (GABA-T), the enzyme involved in GABA catabolism, which has been reported as
445
''most responsive enzyme to NaCl'' in Arabidopsis thaliana plant (Renault et al., 2010).
446
Arabidopsis plant deficient in GABA-T showed a supersensitive phenotype to the salinity stress.
447
GABA-T role in nitrogen and carbon metabolism in Arabidopsis root has also been revealed
448
(Renault et al., 2013). In corroborate with this report, in our experiment, exogenous GABA
449
increased root biomass in both stressed and non-stressed lettuce plants, indicative of higher
450
activity of GABA in the roots relative to the shoot tissues. Although these results are indicative
451
of positive effect of GABA on plant growth under saline condition, the mechanism underling
452
GABA-modulated tolerance to salinity still is the matter of debate.
AC C
EP
TE D
M AN U
SC
RI PT
431
ACCEPTED MANUSCRIPT
In the current study, salinity caused oxidative stress in lettuce plants by elevating H2O2 level,
454
while GABA reduced the magnitude of this oxidative stress. Under saline stress conditions,
455
overproduction of ROS results in wide cellular components damages (Kawano et al., 2002;
456
Demidchik et al., 2003; Demidchik et al., 2010). ROS like H2O2 has been suggested to activate
457
signal transduction pathways in plant cells (Gechev and Hille, 2005; Tripathy and Oelmüller,
458
2012) and modify function of genes involved in the early stress-induced signaling (Shu-Hsien et
459
al., 2005). For example in wheat cell, H2O2 reduction by NADPH in nucleus provides a redox
460
regulation status that is prerequisite for certain gene expressions and consequently led to the cells
461
protection during seed development (Pulido et al., 2009). Seedling treatment with H2O2 has
462
resulted in a chilling tolerance induction in mung bean (Vigna radiata L.) plants (Yu et al.,
463
2003). H2O2 interact with Calcium (Ca2+) in response to the abiotic stresses (Niu and Liao,
464
2016). The role of Ca2+ on seed germination (Kong et al., 2015), root growth (Han et al., 2015)
465
and salt tolerance (Tepe and Aydemir, 2015) have been reported. Under stress condition, Ca2+
466
level is elevated in the cell and induces the Calmodulin (CAM) gene expression. CAM protein
467
and Ca2+ interaction can make a complex capable of binding to the certain proteins and
468
regulating their functions. Ca2+/CAM complex also involves in GABA biosynthesis by
469
regulating glutamate decarboxylase (GAD) enzyme activity (Allan et al., 2009). GAD is
470
responsible for decarboxylation of glutamate during GABA metabolism. Therefore,
471
hypothetically, GABA/Ca2+ interactions have modulating roles over processes such as H2O2
472
signaling. GABA has been proposed to regulate the flux of anions through a plasma membrane
473
located, aluminum-activated malate transporter (ALMT) (Ramesh et al., 2017); however efflux
474
of other ions has also been reported by GABA transporter. For example, combined application of
475
both GABA and H2O2 in barely roots exhibited Ca2+ and K+ efflux induction as well as cell death
AC C
EP
TE D
M AN U
SC
RI PT
453
ACCEPTED MANUSCRIPT
reduction (Shabala et al., 2014; Gilliham and Tyerman, 2016). This indicating the indirect effect
477
of GABA on ion trafficking by changing the cell membrane potential (Gilliham and Tyerman,
478
2016). Our finding showed that GABA has significant subtractive effect on electrolyte leakage.
479
This observation could be attributed to the regulatory role of GABA on ALMT function which
480
consequently affects electrical potential across the cell membrane (Ramesh et al., 2015).
481
Alternatively, during saline stress condition, salt accumulation in root zone causes an osmotic
482
stress capable of inhibiting the essential minerals uptake such as Ca2+, K+ and NO3- and
483
accumulation of Na+ and Cl- which eventually result in the cell membrane damages induced by
484
disruption in cell homeostasis (Zhu, 2001; Chartzoulakis, 2005). Recent data also strongly
485
suggest that ROS production is tightly related to cell membrane damages and electrolyte
486
leakages (Demidchik et al., 2014).
487
The antioxidant system, comprising SOD, CAT and APX enzymes, plays pivotal role in ROS
488
metabolism by retaining of the balance between ROS generating and quenching in plant cells
489
(Qiu et al., 2013). GABA metabolism and accumulation have also been suggested to reduce the
490
oxidative damage caused by ROS, leading to improvement of tolerance to oxidative stresses
491
(AL-Quraan, 2015). For instance in musk melon (Cucumis melo) plant, exogenous GABA
492
application increase the saline-alkalin resistance which is caused through enhancement of SOD
493
and ascorbic acid-glutathione cycling in chloroplasts (Xiang et al., 2015). Moreover, in corn
494
plants GABA caused a profound effect on the antioxidant enzyme activity under salinity
495
condition (Wang et al., 2017). Furthermore, increase in wheat resistance to salt stress through
496
enhancement of antioxidant enzymes activities has been also reported following GABA
497
application (Li et al., 2016a). These data are consistent with our finding about enzymatic activity
498
under co-application of salt and GABA, in which, SOD, CAT and APX activities enhanced by
AC C
EP
TE D
M AN U
SC
RI PT
476
ACCEPTED MANUSCRIPT
GABA treatment as the salt dosage increased. This indirectly implies the involvement of GABA
500
in regulation of the lipid peroxidation and cell membrane protection by modulating ROS
501
regulatory mechanism. SOD enzyme mainly involved in dismutation of superoxide radical
502
which produce ordinary molecular oxygen or H2O2 (Hayyan et al., 2016). Last byproduct is
503
harmful, therefore should be removed from the cell Removing H2O2 is organized by enzymes
504
such as CAT and APX. According to our data, CAT and APX reduced after salt exposure
505
whereas SOD activity increased. Zhu et al. (2004) and Li et al. (2016) reported the same results
506
(Zhu et al., 2004; Li et al., 2016a). In our investigation, GABA had more effect on CAT and
507
APX enzyme rather than SOD. This is consistent with our data related to H2O2 content, which
508
showed significant reduction after GABA exposure. These are also supported by Renault and
509
coworkers (2011) findings which suggested that GABA regulates water channel proteins and
510
affect the cell elongation by osmotic adjustment and maintenance of the cell turgor (Renault et
511
al., 2011). This biological crosstalk could introduce how H2O2 content, GABA function and
512
electrolyte leakage are regulated in a retro-controlled mechanism.
513
High salt concentration may slightly reduce CAT and APX gene expression, leading to the
514
reduced enzymatic activity, but GABA either directly re-induced or indirectly hindered NaCl
515
negative effects on antioxidant gene expressions. In different scenario, SOD activity has been
516
elevated in plants received NaCl, indicative of salt concentration-dependent potential of SOD
517
to alleviate oxidative damages. Our finding showed an effective inter-linkage between GABA
518
and oxidative enzymes in plant cells. Although this will open the questions that i) how these
519
interactions are modulated during salt stress and ii) whether GABA intermediates this linkage
520
or its function is formulated by the component of this regulatory mechanism?
521
AC C
EP
TE D
M AN U
SC
RI PT
499
ACCEPTED MANUSCRIPT
Under salt stress, proline content increases in plants (Yoshiba et al., 1995). Similarly, in our
523
study proline level increased in leaves following plant exposure to salinity stress; however
524
GABA application reduced proline content in both saline conditions. Upon salt treatment, ROS
525
overproduced in the plant cells (You and Chan, 2015) and as a result organic osmolytes like
526
proline is produced to save the steady state of plant cells (Liang et al., 2013). During proline
527
biosynthesis, NADPH is produced and consumed in reductive biosynthesis pathways through
528
antioxidant defense system (Liang et al., 2013). NADPH is also produced during GABA
529
metabolism (Ludewig et al., 2008). In stress condition, tricarboxylic acid (TCA) cycle activity is
530
affected and results in a reduced pool of NADP+ which causes maintaining of ROS due to the
531
defect in antioxidant machinery. Hypothetically, under such situations, GABA and proline
532
biosynthesis provide adequate level of NADP+ and maintain antioxidant functionality. For many
533
years proline has been considered as an antioxidant, however new evidence demonstrates that
534
except for ·OH, it cannot react with most ROS (Signorelli et al., 2013; Signorelli et al., 2016).
535
Proline reaction with ·OH has been proposed as a natural pathway to produce precursor of
536
GABA under oxidative stress reaction (Signorelli et al., 2015). In our study proline content was
537
reduced by GABA application. One possible scenario could be that promoted antioxidant
538
machinery through GABA function can partially bypasses proline involvement in ROS
539
scavenging processes and result in the reduction of proline biosynthesis. In high NaCl
540
concentrations, proline reduction ratio was decreased. Apparently, by increasing salt level,
541
proline production is induced in the cell to provide proper activity of antioxidant machinery
542
along with GABA function. Our results suggested that GABA could be considered as plant-
543
boosting antioxidant component with ability to reduce oxidative damages by activation of
544
enzymatic/non-enzymatic metabolism under salt stress. In fact, positive effect of GABA on
AC C
EP
TE D
M AN U
SC
RI PT
522
ACCEPTED MANUSCRIPT
antioxidant enzyme such as CAT, SOD and APX activities under saline condition is indicative of
546
important role for GABA in maintaining of redox state and preventing of ROS accumulation in
547
plants during stress condition. Although further studies are required to determine how GABA
548
regulate enzymatic balances during stress conditions.
549
Photosynthetic capacity is negatively affected under saline condition (Chaves et al., 2009; Ashraf
550
and Harris, 2013). This reduction can be caused by i) stomata malfunctioning induced by salt
551
stress (Janagoudar Sr, 2007) or ii) non-stomata related defects which is attributed to disruption in
552
photosystem II function (He et al., 2009). In confirmation of the last scenario, we found that
553
maximum quantum yield of photosystem II (Fv/Fm) of dark-adapted leaves were reduced under
554
high saline condition in different stages of plant growth. GABA effectively removed the salt-
555
induced damages to photosystem II functioning in lettuce. Our data showed that high NaCl
556
concentration could affect photosynthesis yield from the early stage. In our study, NaCl
557
treatments significantly influenced the parameters related to the fast chlorophyll fluorescence
558
implying that salt has the potential of repressing the electron flows in photosynthesis machinery.
559
Improvement of electron transport capacity in plants exposed to NaCl stress indicates that
560
GABA is capable of removing defects in photosystem II induced by salinity stresses.
561
In the present study, parameters related to the fast and slow inductions of chlorophyll
562
fluorescence were regulated by GABA application in salt-treated plants. The absorbed photon
563
fluxes by photosystem lead to the excitation of chlorophylls. The energy of excitation can have
564
few fates: i) it will be partially converted to redox energy through electron transport which will
565
be consumed for CO2 fixation and ii) alternatively can be released as heat and iii) fluorescence
566
(Toth et al., 2007; Kalaji et al., 2017). Lower photosystem II photochemical performance (lower
567
PIABS, Rfd and Fv/Fm) in salt-treated plants can be due to the higher light energy absorption
AC C
EP
TE D
M AN U
SC
RI PT
545
ACCEPTED MANUSCRIPT
(ABS/RC) and dissipated energy flux (DI0/RC) and lower electron transport (ET0/ABS) per
569
reaction centers which consequently results in decreased quantum yield of absorbed photons.
570
Increase of ABS/RC in salt-exposed plants could be due to the inactivation of reaction centers
571
and a decrease in active QA reducing centers (Strasser and Stirbet, 1998). In stress conditions,
572
low proportion of absorbed energy passed on the electron transport chain (Sarkar and Ray,
573
2016).
574
It has been confirmed that plants protect themselves from excess light exposure through
575
dissipation of light energy as heat, known as NPQ.This process protects photosynthesis when
576
absorbed light energy exceeds the photosynthetic capacity to utilize it (Müller et al., 2001). In
577
our study, although NPQ was negatively affected by salinity stress, GABA kept the NPQ of salt-
578
treated plants on the same level of non-stressed plants. Increase in NPQ following exposure to
579
stress conditions depends on the xanthophyll cycle. When this cycle moves towards production
580
of zeaxanthin (usually under high light intensities), it would result in a higher NPQ. Production
581
of zeaxanthin is mediated with violaxanthin de-epoxidase (VDE) and APX activities (Sui et al.,
582
2007; Jahns and Holzwarth, 2012). In this study, APX activity was decreased in a salt-
583
concentration dependent manner in control plants while GABA caused considerable increase in
584
the APX activity. Confirming this finding, vde mutant plants showed decreased NPQ and Fv/Fm
585
under stress condition (Azzabi et al., 2012).
586
Positive effect of GABA on these parameters indicates the involvement of GABA in
587
photosynthesis functionality either directly by affecting the activity of reaction centers involved
588
in transferring of exited electron from chlorophyll to produce energy or indirectly by modulating
589
processes that crosstalk with photosynthesis performance. However the mechanism/s explain
590
how GABA interferes in photosynthesis enzymatic activities is not clear, GABA has been
AC C
EP
TE D
M AN U
SC
RI PT
568
ACCEPTED MANUSCRIPT
suggested to interplay with C and N metabolism in plants (Michaeli and Fromm, 2015) which
592
imply indirect effect of GABA on photosynthesis output. Alternatively, glutamine has long been
593
suggested as one the main components that intermediate carbon: nitrogen ratio in plants which
594
make it capable of affecting the photosynthesis functionality (Fait et al., 2008). Interestingly,
595
glutamine is involved in proline and chlorophyll synthesis when leaves are developing (Forde
596
and Lea, 2007) and also for GABA biosynthesis in whole plant growth stages (Signorelli et al.,
597
2015). This can introduce the indirect effect of GABA on photosynthesis performance. Although
598
the mechanism introduces how GABA affects photosynthesis function is remained to be
599
investigated.
M AN U
SC
RI PT
591
600
5. Conclusion
602
Based on finding of this study, GABA decreased the negative effects of NaCl on growth and
603
physiological parameters in early growth stage of lettuce plant. NaCl-treated seeds and seedlings
604
were able to germinate, show proper root and shoot growth, induce tolerance mechanism and
605
photosynthesis functionality when exposed to exogenous GABA. GABA effects on both
606
biophysical (chlorophyll fluorescence parameters) and cellular (parameters related to oxidative
607
stress) levels represent a multifunctional role for GABA as a biological component which can
608
exogenously adjust salt tolerance in lettuce plants. According to the salt dependent manner effect
609
of GABA on lettuce plant, it could be suggested that GABA role under salinity stress is more
610
related to stress signal regulation rather than protection. Moreover, GABA application in some
611
non-stressed plants imposed null effect implying relatively stress-responsive role for GABA. By
612
these results, we could propose that plants use GABA as a stress mediator capable of regulating
613
various biological processes influenced by salinity stress. However, the mechanism by which
AC C
EP
TE D
601
ACCEPTED MANUSCRIPT
614
GABA intermediates plant and saline stress interaction is an interesting question remained to be
615
answered.
RI PT
616
Author contribution statement
618
Maryam Seifi Kalhor and Sasan Aliniaiefard made substantial contributions to conception and
619
design, also performed statistical analysis, drafted the manuscript and critically revised the final
620
version. Mahdi Seif, Elahe Javadi and Batool Hassani carried out the experiments and helped in
621
the writing of manuscript. Françoise Bernard and Tao Li contributed to design of experiment and
622
critical revision of the final manuscript. All authors have read and approved the final manuscript.
M AN U
SC
617
623
Acknowledgments
625
We like to thank the Iran's National Elites Foundation for financial support of this project.
626
TE D
624
References
628 629 630 631 632 633 634 635 636 637 638 639 640
Ahuja, I., de Vos, R.C., Bones, A.M., Hall, R.D., 2010. Plant molecular stress responses face climate change. Trends Plant Sci. 15, 664-674. Akçay, N., Bor, M., Karabudak, T., Özdemir, F., Türkan, İ., 2012. Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants. J Plant Physiol. 169, 452-458. AL-Quraan, N.A., 2015. GABA shunt deficiencies and accumulation of reactive oxygen species under UV treatments: insight from Arabidopsis thaliana calmodulin mutants. Acta Physiol Plant. 37, 1-11. Al-Whaibi, M.H., Siddiqui, M.H., Basalah, M.O., 2012. Salicylic acid and calcium-induced protection of wheat against salinity. Protoplasma. 249, 769-778. Aliniaeifard, S., Hajilou, J., Tabatabaei, S.J., 2016a. Photosynthetic and Growth Responses of Olive to Proline and Salicylic Acid under Salinity Condition. Not. Bot. Horti Agrobot. Clujna. 44, 579-585. Aliniaeifard, S., Hajilou, J., Tabatabaei, S.J., Sifi-Kalhor, M., 2016b. Effects of Ascorbic Acid and Reduced Glutathione on the Alleviation of Salinity Stress in Olive Plants. Int. J. Fruit Sci. 16, 395-409.
AC C
EP
627
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Aliniaeifard, S., Malcolm Matamoros, P., van Meeteren, U., 2014. Stomatal malfunctioning under low Vapor Pressure Deficit (VPD) conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling. Physiol Plant. 152, 688-699. Aliniaeifard, S., van Meeteren, U., 2014. Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning. J. Exp. Bot. 65, 6529-6542. Allakhverdiev, S.I., Sakamoto, A., Nishiyama, Y., Inaba, M., Murata, N., 2000. Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp. Plant Physiol. 123, 10471056. Allan, W.L., Clark, S.M., Hoover, G.J., Shelp, B.J., 2009. Role of plant glyoxylate reductases during stress: a hypothesis. Biochem J. 423, 15-22. Allan, W.L., Simpson, J.P., Clark, S.M., Shelp, B.J., 2008. γ-Hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms. J. Exp. Bot. 59, 2555-2564. Andriolo, J.L., Luz, G.L.d., Witter, M.H., Godoi, R.d.S., Barros, G.T., Bortolotto, O.C., 2005. Growth and yield of lettuce plants under salinity. Hort. Brasil. 23, 931-934. Ashraf, M., Harris, P., 2013. Photosynthesis under stressful environments: an overview. Photosynthetica. 51, 163-190. Azzabi, G., Pinnola, A., Betterle, N., Bassi, R., Alboresi, A., 2012. Enhancement of non-photochemical quenching in the Bryophyte Physcomitrella patens during acclimation to salt and osmotic stress. Plant Cell Physiol. 53, 1815-1825. Bartha, C., Fodorpataki, L., del Carmen Martinez-Ballesta, M., Popescu, O., Carvajal, M., 2015. Sodium accumulation contributes to salt stress tolerance in lettuce cultivars. J. App. Bot. Food Qual. 88. Bates, L., Waldren, R., Teare, I., 1973. Rapid determination of free proline for water-stress studies. Plant Soil. 39, 205-207. Bonilla, I., El-Hamdaoui, A., Bolaños, L., 2004. Boron and calcium increase Pisum sativum seed germination and seedling development under salt stress. Plant Soil. 267, 97-107. Bor, M., Seckin, B., Ozgur, R., Yılmaz, O., Ozdemir, F., Turkan, I., 2009. Comparative effects of drought, salt, heavy metal and heat stresses on gamma-aminobutryric acid levels of sesame (Sesamum indicum L.). Acta Physiol. Plant. 31, 655-659. Bown, A.W., Shelp, B.J., 2016. Plant GABA: not just a metabolite. Trends Plant Sci 21, 811-813. Cantrell, I.C., Linderman, R.G., 2001. Preinoculation of lettuce and onion with VA mycorrhizal fungi reduces deleterious effects of soil salinity. Plant Soil. 233, 269-281. Chartzoulakis, K., 2005. Salinity and olive: growth, salt tolerance, photosynthesis and yield. Agric. Water Manag. 78, 108-121. Chaves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann. Bot. 103, 551-560. Chinnusamy, V., Jagendorf, A., Zhu, J.-K., 2005. Understanding and improving salt tolerance in plants. Crop Sci. 45, 437-448. Demidchik, V., Cuin, T.A., Svistunenko, D., Smith, S.J., Miller, A.J., Shabala, S., Sokolik, A., Yurin, V., 2010. Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. J Cell Sci. 123, 1468-1479. Demidchik, V., Shabala, S.N., Coutts, K.B., Tester, M.A., Davies, J.M., 2003. Free oxygen radicals regulate plasma membrane Ca2+-and K+-permeable channels in plant root cells. J Cell Sci. 116, 81-88. Demidchik, V., Straltsova, D., Medvedev, S.S., Pozhvanov, G.A., Sokolik, A., Yurin, V., 2014. Stressinduced electrolyte leakage: the role of K+-permeable channels and involvement in programmed cell death and metabolic adjustment. J. Exp. Bot. 65, 1259-1270. Dhindsa, R., Plumb-Dhindsa, P., Thorpe, T., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93-101.
AC C
641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Díaz-Vivancos, P., Clemente-Moreno, M.J., Rubio, M., Olmos, E., García, J.A., Martínez-Gómez, P., Hernández, J.A., 2008. Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus. J. Exp. Bot. 59, 2147-2160. Fait, A., Fromm, H., Walter, D., Galili, G., Fernie, A.R., 2008. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci. 13, 14-19. Filomeni, G., De Zio, D., Cecconi, F., 2015. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ. 22, 377. Forde, B.G., Lea, P.J., 2007. Glutamate in plants: metabolism, regulation, and signalling. J. Exp. Bot. 58, 2339-2358. Fougere, F., Le Rudulier, D., Streeter, J.G., 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. Gechev, T.S., Hille, J., 2005. Hydrogen peroxide as a signal controlling plant programmed cell death. The J. Cell Biol. 168, 17-20. Genc, Y., Mcdonald, G.K., Tester, M., 2007. Reassessment of tissue Na+ concentration as a criterion for salinity tolerance in bread wheat. Plant Cell Environ. 30, 1486-1498. Genty, B., Briantais, J.-M., Baker, N.R., 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA)-General Subjects. 990, 87-92. Gilliham, M., Tyerman, S.D., 2016. Linking metabolism to membrane signaling: the GABA–malate connection. Trends Plant Sci. 21, 295-301. Giuffrida, F., Scuderi, D., Giurato, R., Leonardi, C., 2012. Physiological response of broccoli and cauliflower as affected by NaCl salinity, VI International Symposium on Brassicas and XVIII Crucifer Genetics Workshop. 1005, pp. 435-441. Han, S., Fang, L., Ren, X., Wang, W., Jiang, J., 2015. MPK6 controls H2O2‐induced root elongation by mediating Ca2+ influx across the plasma membrane of root cells in Arabidopsis seedlings. New Phytol. 205, 695-706. Hayyan, M., Hashim, M.A., AlNashef, I.M., 2016. Superoxide ion: generation and chemical implications. Chemical reviews. 116, 3029-3085. He, Y., Zhu, Z., Yang, J., Ni, X., Zhu, B., 2009. Grafting increases the salt tolerance of tomato by improvement of photosynthesis and enhancement of antioxidant enzymes activity. Environ. Exp. Bot. 66, 270-278. Hu, X., Xu, Z., Xu, W., Li, J., Zhao, N., Zhou, Y., 2015. Application of γ-aminobutyric acid demonstrates a protective role of polyamine and GABA metabolism in muskmelon seedlings under Ca(NO3)2 stress. Plant Physiol. Biochem. 92, 1-10. Jahns, P., Holzwarth, A.R., 2012. The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 1817, 182-193. Janagoudar Sr, B.S., 2007. Salinity induced changes on stomatal response, biophysical parameters, solute accumulation and growth in cotton (Gossypium spp.), The World Cotton Research Conference, pp. 10-14. Jutsz, A.M., Gnida, A., 2015. Mechanisms of stress avoidance and tolerance by plants used in phytoremediation of heavy metals. Arch. Environ. Protect. 41, 104-114. Kalaji, M.H., Goltsev, V.N., Zivcak, M., Brestic, M., 2017. Chlorophyll Fluorescence: Understanding Crop Performance—Basics and Applications. CRC Press. Kaplan, F., Kopka, J., Haskell, D.W., Zhao, W., Schiller, K.C., Gatzke, N., Sung, D.Y., Guy, C.L., 2004. Exploring the temperature-stress metabolome of Arabidopsis. Plant Physiol. 136, 4159-4168. Kawano, T., Kawano, N., Muto, S., Lapeyrie, F., 2002. Retardation and inhibition of the cation‐induced superoxide generation in BY‐2 tobacco cell suspension culture by Zn2+ and Mn2+. Physiol Plant. 114, 395404. Kong, D., Ju, C., Parihar, A., Kim, S., Cho, D., Kwak, J.M., 2015. Arabidopsis glutamate receptor homolog3. 5 modulates cytosolic Ca2+ level to counteract effect of abscisic acid in seed germination. Plant Physiol. 167, 1630-1642.
AC C
691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Kramer, D.M., Johnson, G., Kiirats, O., Edwards, G.E., 2004. New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth Res. 79, 209-218. Li, H., Chang, J., Chen, H., Wang, Z., Gu, X., Wei, C., Zhang, Y., Ma, J., Yang, J., Zhang, X., 2017a. Exogenous Melatonin Confers Salt Stress Tolerance to Watermelon by Improving Photosynthesis and Redox Homeostasis. Front.n Plant Sci. 8. Li, M., Guo, S., Yang, X., Meng, Q., Wei, X., 2016a. Exogenous gamma-aminobutyric acid increases salt tolerance of wheat by improving photosynthesis and enhancing activities of antioxidant enzymes. Biol. Plant. 60, 123-131. Li, Y., 2008. Effect of salt stress on seed germination and seedling growth of three salinity plants. Pakistan journal of biological sciences: PJBS 11, 1268-1272. Li, Y., Fan, Y., Ma, Y., Zhang, Z., Yue, H., Wang, L., Li, J., Jiao, Y., 2017b. Effects of exogenous γaminobutyric acid (GABA) on photosynthesis and antioxidant system in pepper (Capsicum annuum L.) seedlings under low light stress. J. Plant Growth Regul. 36, 436-449. Li, Z., Yu, J., Peng, Y., Huang, B., 2016b. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera). Scientific Reports. 6. Liang, X., Zhang, L., Natarajan, S.K., Becker, D.F., 2013. Proline mechanisms of stress survival. Antioxidants & redox signaling. 19, 998-1011. Lichtenthaler, H., Langsdorf, G., Lenk, S., Buschmann, C., 2005. Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system. Photosynthetica. 43, 355-369. Ludewig, F., Hüser, A., Fromm, H., Beauclair, L., Bouché, N., 2008. Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis. PLoS One 3, e3383. Machado, R.M.A., Serralheiro, R.P., 2017. Soil salinity: Effect on vegetable crop growth. Management practices to prevent and mitigate soil salinization. Horticulturae. 3, 30. Maggio, A., De Pascale, S., Fagnano, M., Barbieri, G., 2011. Saline agriculture in Mediterranean environments. Italian j. Agron. 6, 7. Mehta, P., Jajoo, A., Mathur, S., Bharti, S., 2010. Chlorophyll a fluorescence study revealing effects of high salt stress on photosystem II in wheat leaves. Plant Physiol. Biochem. 48, 16-20. Meot-Duros, L., Magné , C., 2008. Effect of salinity and chemical factors on seed germination in the halophyte Crithmum maritimum L. Plant Soil 313, 83-87. Michaeli, S., Fromm, H., 2015. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?. Front. Plant Sci. 6, 419. Mohammadi, P., Khoshgoftarmanesh, A.H., 2014. The effectiveness of synthetic zinc (Zn)-amino chelates in supplying Zn and alleviating salt-induced damages on hydroponically grown lettuce. Sci. Hortic. 172, 117-123. Müller, P., Li, X.-P., Niyogi, K.K., 2001. Non-Photochemical Quenching. A response to excess light energy. Plant Physiol. 125, 1558-1566. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651-681. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867-880. Nayyar, H,. Kaur, R., Kaur, S., Singh, R. 2014. γ-Aminobutyric Acid (GABA) Imparts partial protection from heat stress injury to rice seedlings by improving leaf turgor and upregulating osmoprotectants and antioxidants. J. Plant Growth Regulation. 33, 408-419.
AC C
742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792
Niu, L., Liao, W., 2016. Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Frontiers in plant science. 7. Orlovsky, N., Japakova, U., Zhang, H., Volis, S., 2016. Effect of salinity on seed germination, growth and ion content in dimorphic seeds of Salicornia europaea L.(Chenopodiaceae). Plant Diversity 38, 183-189. Özbingöl, N., Corbineau, F., Groot, S., Bino, R., Côme, D., 1999. Activation of the cell cycle in tomato (Lycopersicon esculentum Mill.) seeds during osmoconditioning as related to temperature and oxygen. Ann. Bot. 84, 245-251.
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Palanivelu, R., Brass, L., Edlund, A.F., Preuss, D., 2003. Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels. Cell 114, 47-59. Panuccio, M.R., Jacobsen, S.E., Akhtar, S.S., Muscolo, A., 2014. Effect of saline water on seed germination and early seedling growth of the halophyte quinoa. AoB PLANTS 6. Patterson, B.D., MacRae, E.A., Ferguson, I.B., 1984. Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical biochemistry. 139, 487-492. Pulido, P., Domínguez, F., Cejudo, F.J., 2009. A hydrogen peroxide detoxification system in the nucleus of wheat seed cells: protection or signaling role? Plant Signal. Behav. 4, 23-25. Qiu, Z., Li, J., Zhang, M., Bi, Z., Li, Z., 2013. He–Ne laser pretreatment protects wheat seedlings against cadmium-induced oxidative stress. Ecotoxicol Environ Saf. 88, 135-141. Ramesh, S.A., Kamran, M., Sullivan, W., Chirkova, L., Okamoto, M., Degryse, F., McLaughlin, M., Gilliham, M., Tyerman, S.D., 2017. Split personality of aluminum activated malate transporter family proteins: facilitation of both GABA and malate transport. bioRxiv. 215335. Ramesh, S.A., Tyerman, S.D., Gilliham, M., Xu, B., 2016. γ-Aminobutyric acid (GABA) signalling in plants. Cell. Mol. Life Sci. 1-27. Ramesh, S.A., Tyerman, S.D., Xu, B., Bose, J., Kaur, S., Conn, V., Domingos, P., Ullah, S., Wege, S., Shabala, S., 2015. GABA signalling modulates plant growth by directly regulating the activity of plantspecific anion transporters. Nature communications. 6. Renault, H., El Amrani, A., Berger, A., Mouille, G., Soubigou-Taconnat, L., Bouchereau, A., Deleu, C., 2013. γ‐Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots. Plant Cell Environ. 36, 1009-1018. Renault, H., El Amrani, A., Palanivelu, R., Updegraff, E.P., Yu, A., Renou, J.-P., Preuss, D., Bouchereau, A., Deleu, C., 2011. GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana. Plant Cell Physiol. 52, 894-908. Renault, H., Roussel, V., El Amrani, A., Arzel, M., Renault, D., Bouchereau, A., Deleu, C., 2010. The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance. BMC Plant Biol. 10, 20. Rivero, R.M., Mestre, T.C., Mittler, R., Rubio, F., GARCIA‐SANCHEZ, F., Martinez, V., 2014. The combined effect of salinity and heat reveals a specific physiological, biochemical and molecular response in tomato plants. Plant Cell Environ. 37, 1059-1073. Sarkar, R., Ray, A., 2016. Submergence-tolerant rice withstands complete submergence even in saline water: Probing through chlorophyll a fluorescence induction OJIP transients. Photosynthetica. 54, 275287. Scholz, S.S., Malabarba, J., Reichelt, M., Heyer, M., Ludewig, F., Mithöfer, A., 2017a. Evidence for GABA-induced systemic GABA accumulation in arabidopsis upon wounding. Front. Plant Sci. 8. Scholz, S.S., Malabarba, J., Reichelt, M., Heyer, M., Ludewig, F., Mithöfer, A., 2017b. Evidence for GABA-induced systemic GABA accumulation in Arabidopsis upon wounding. Front. Plant Sci. 8, 388. Shabala, S., Shabala, L., Barcelo, J., Poschenrieder, C., 2014. Membrane transporters mediating root signalling and adaptive responses to oxygen deprivation and soil flooding. Plant Cell Environ. 37, 22162233. Shao, H.-B., Chu, L.-Y., Jaleel, C.A., Zhao, C.-X., 2008. Water-deficit stress-induced anatomical changes in higher plants. C R Biol. 331, 215-225. Shelp, B.J., Bown, A.W., Zarei, A., 2017. 4-Aminobutyrate (GABA): a metabolite and signal with practical significance. Botany. 95, 1015-1032. Shu-Hsien, H., Chih-Wen, Y., Lin, C.H., 2005. Hydrogen peroxide functions as a stress signal in plants. Botanical Bulletin of Academia Sinica. 46. Signorelli, S., Arellano, J.B., Melø, T.B., Borsani, O., Monza, J., 2013. Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants. Plant Physiology Biochem. 64, 80-83. Signorelli, S., Dans, P.D., Coitiño, E.L., Borsani, O., Monza, J., 2015. Connecting proline and γaminobutyric acid in stressed plants through non-enzymatic reactions. Plos One. 10, e0115349.
AC C
793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
SC
RI PT
Signorelli, S., Imparatta, C., Rodríguez-Ruiz, M., Borsani, O., Corpas, F.J., Monza, J., 2016. In vivo and in vitro approaches demonstrate proline is not directly involved in the protection against superoxide, nitric oxide, nitrogen dioxide and peroxynitrite. Funct Plant Biol. 43, 870-879. Slama, I., Abdelly, C., Bouchereau, A., Flowers, T., Savouré, A., 2015. Diversity, distribution and roles of osmoprotective compounds accumulated in halophytes under abiotic stress. Ann. Bot. 115, 433-447. Sosa, L., Llanes, A., Reinoso, H., Reginato, M., Luna, V., 2005. Osmotic and specific ion effects on the germination of Prosopis strombulifera. Ann. Bot. 96, 261-267. Steward, F., Thompson, J., Dent, C., 1949. γ-Aminobutyric acid: a constituent of the potato tuber. Science 110, 439-440. Strasser, B.J., 1995. Measuring fast fluorescence transients to address environmental questions: the JIP test. Photosynthesis: from light to biosphere. 977-980. Strasser, R.J., Srivastava, A., zTsimilli-Michael, M.g., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. Probing photosynthesis: mechanisms, regulation and adaptation. 445-483. Strasser, R.J., Stirbet, A.D., 1998. Heterogeneity of photosystem II probed by the numerically simulated chlorophyll a fluorescence rise (O–J–I–P). Mathematics and Computers in Simulation. 48, 3-9. Stuart, K., 2017. Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an overview. J Sci Food Agric. Sui, N., Li, M., Liu, X.-Y., Wang, N., Fang, W., Meng, Q.-W., 2007. Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene. Photosynthetica. 45, 447-454. Suo-ling, S., Jing-rui, L., Hong-bo, G., Qing-yun, L., Li-wen, Y., Rui-juan, G., 2012. Effects of exogenous γ-aminobutyric acid on inorganic nitrogen metabolism and mineral elements contents of melon seedling under hypoxia stress. Acta Hortic. Sinica. 4, 012. Tepe, H.D., Aydemir, T., 2015. Protective effects of Ca2+ against NaCl induced salt stress in two lentil (Lens culinaris) cultivars. Afric. J. Agr. Res. 10, 2389-2398. Tobe, K., Li, X., Omasa, K., 2002. Effects of sodium, magnesium and calcium salts on seed germination and radicle survival of a halophyte, Kalidium caspicum (Chenopodiaceae). Aust J Bot 50, 163-169. Toth, S.Z., Schansker, G., Strasser, R.J., 2007. A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient. Photosynth Res. 93, 193-203. Tripathy, B.C., Oelmüller, R., 2012. Reactive oxygen species generation and signaling in plants. Plant Signal. Behav. 7, 1621-1633. Tuna, A.L., Kaya, C., Ashraf, M., Altunlu, H., Yokas, I., Yagmur, B., 2007. The effects of calcium sulphate on growth, membrane stability and nutrient uptake of tomato plants grown under salt stress. Environ Exp. Bot. 59, 173-178. Ünlükara, A., Cemek, B., Karaman, S., Erşahin, S., 2008. Response of lettuce (Lactuca sativa var. crispa) to salinity of irrigation water. N Z J Crop Hortic Sci. 36, 265-273. Vijayakumari, K., Puthur, J.T., 2016. γ-Aminobutyric acid (GABA) priming enhances the osmotic stress tolerance in Piper nigrum Linn. plants subjected to PEG-induced stress. Plant Growth Regul. 78, 57-67. Wang, Y., Gu, W., Meng, Y., Xie, T., Li, L., Li, J., Wei, S., 2017. γ-aminobutyric acid Imparts partial protection from salt stress injury to maize seedlings by improving photosynthesis and upregulating osmoprotectants and antioxidants. Scientific Reports. 7, 43609. Xiang, L., Hu, L., Hu, X., Pan, X., Ren, W., 2015. Response of reactive oxygen metabolism in melon chloroplasts to short-term salinity-alkalinity stress regulated by exogenous γ-aminobutyric acid. J. App. Ecol. 26, 3746-3752. Xiang, L., Hu, L., Xu, W., Zhen, A., Zhang, L., Hu, X., 2016. Exogenous γ-aminobutyric acid improves the structure and function of photosystem II in muskmelon seedlings exposed to salinity-alkalinity stress. PloS one. 11, e0164847. Xing, S.G., Jun, Y.B., Hau, Z.W., Liang, L.Y., 2007. Higher accumulation of γ-aminobutyric acid induced by salt stress through stimulating the activity of diamine oxidases in Glycine max (L.) Merr. roots. Plant Physiol. Biochem. 45, 560-566.
AC C
844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894
ACCEPTED MANUSCRIPT
RI PT
Xu, C., Mou, B., 2015. Evaluation of lettuce genotypes for salinity tolerance. Hortscience 50, 1441-1446. Yoshiba, Y., Kiyosue, T., Katagiri, T., Ueda, H., Mizoguchi, T., Yamaguchi‐Shinozaki, K., Wada, K., Harada, Y., Shinozaki, K., 1995. Correlation between the induction of a gene for ∆1‐pyrroline‐5‐ carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J. 7, 751-760. You, J., Chan, Z., 2015a. ROS regulation during abiotic stress responses in crop plants. Front. Plant sci. 6, 1092. Yu, C.-W., Murphy, T.M., Lin, C.-H., 2003. Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Funct. Plant Biol. 30, 955-963. Žárský, V., 2015. Signal transduction: GABA receptor found in plants. Nature plants. 1, 15115. Zhang, L., Tian, L.-H., Zhao, J.-F., Song, Y., Zhang, C.-J., Guo, Y., 2009. Identification of an apoplastic protein involved in the initial phase of salt stress response in rice root by two-dimensional electrophoresis. Plant Physiol. 149, 916-928. Zhu, J.-K., 2001. Plant salt tolerance. Trends Plant Sci. 6, 66-71. Zhu, Z., Wei, G., Li, J., Qian, Q., Yu, J., 2004. Silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of salt-stressed cucumber (Cucumis sativus L.). Plant Sci. 167, 527-533.
SC
895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910
AC C
EP
TE D
M AN U
911
ACCEPTED MANUSCRIPT Highlights •
GABA improves seed germination and plant growth in saline conditions.
•
GABA reduces negative effects of salt stress on photosynthetic performance of lettuce plants. GABA decreases the levels of salt stress-induced oxidative stress in lettuce plants.
•
Salt-induced accumulation of proline is decreased by GABA application
AC C
EP
TE D
M AN U
SC
RI PT
•
ACCEPTED MANUSCRIPT Author contribution statement Maryam Seifi Kalhor and Sasan Aliniaiefard made substantial contributions to conception and design, also performed statistical analysis, drafted the manuscript and critically revised
RI PT
the final version. Mahdi Seif, Elahe Javadi and Batool Hassani carried out the experiments and helped in the writing of manuscript. Françoise Bernard and Tao Li contributed to design of experiment and critical revision of the final manuscript. All authors have read and
AC C
EP
TE D
M AN U
SC
approved the final manuscript.