Exogenous γ-Aminobutyric Acid Improves the Structure and Function of Photosystem II in Muskmelon Seedlings Exposed to Salinity-Alkalinity Stress

Gamma-aminobutyric acid (GABA) is important in plant responses to environmental stresses. We wished to clarify the role of GABA in maintenance of photosynthesis in muskmelon seedlings (Cucumis melo L., cv. Yipintianxia) during saline-alkaline stress. To this end, we assessed the effect of GABA on the structure and function of the photosynthetic apparatus in muskmelon seedlings grown under saline-alkaline stress. These stresses in combination reduced net photosynthetic rate, gas-exchange, and inhibited photosystem II (PSII) electron transport as measured by the JIP-test. They also reduced the activity of chloroplast ATPases and disrupted the internal lamellar system of the thylakoids. Exogenous GABA alleviated the stress-induced reduction of net photosynthesis, the activity of chloroplast ATPases, and overcame some of the damaging effects of stress on the chloroplast structure. Based on interpretation of the JIP-test, we conclude that exogenous GABA alleviated stress-related damage on the acceptor side of PSII. It also restored energy distribution, the reaction center status, and enhanced the ability of PSII to repair reaction centers in stressed seedlings. GABA may play a crucial role in protecting the chloroplast structure and function of PSII against the deleterious effects of salinity-alkalinity stress.


Introduction
Both saline and alkaline soil conditions are deleterious to many plant species. The effects of salinity, an abiotic stress factor of global importance, are well known. Alkaline conditions have also been shown to inhibit plant growth, to inhibit photosynthesis, to alter metabolism of reactive oxygen species, and to cause cell death [1,2]. Numerous studies have shown that, when both salt and alkaline stresses are present, these deleterious effects are compounded and are more severe than those that result from salinity alone [3,4,5,6]. In tomato seedlings subjected to this combination of stresses, photochemical quenching parameters were reduced, including media for a final concentration of 50 mM. The pH of the final nutrient solution is 8.6. GABA was applied by spraying leaves with 50 mM GABA in water daily; this concentration was chosen based on previous results [17]. There were four treatments: untreated control plants (Control), plants treated with GABA only (CG), plants treated with the complex salts only (S), and plants treated with complex salts and GABA (SG). Seedlings were treated with same amount of GABA and H 2 O at 9:00am every day and continued to be handled for five days. Five days after the plants had been exposed to 50 mM complex salts, the following measurements were made, using the third fully expanded leaf from the shoot apex: net photosynthetic rate, the chl a fluorescence transient, and chemical analyses. Same melon lamina with different plants was used to measure chlorophyll fluorescence and gas exchange and analyses ultrastructure. Four times to measure gas exchange and chlorophyll fluorescence for per plant and total used 3 plants for per treatment. We also assessed the structure of the chloroplasts. Samples used for chemical analyses were stored at -80°C until analysis.

Measurement of photosynthesis
The net photosynthetic rate (P n ) and gas-exchange parameters were measured with a portable photosynthesis system LI-6400 (Li-COR Inc., USA). Measurements were made at a CO 2 concentration of 380 ± 10 μmolÁmol -1 , 25°C, and with 800 μmol of photons m -2 s -1 at the surface of the leaf. Stomatal limitation (L s ) was calculated as L s = 1-Ci /C a , where C i and C a represent the intercellular and ambient CO 2 concentration, respectively [7].

Measurement of Chl a fluorescence transient and JIP-test parameters
Chl a fluorescence transients were recorded with a Plant Efficiency Analyzer fluorometer (PEA; Hansatech, Ltd., UK) at room temperature. Measurements were carried out at 9:30 am, and leaves for dark adaptation at least 30 min. After that, the leaves exposed to red light of 650 nm from three high intensity light-emitting diodes, which is readily absorbed by the chloroplasts. Light intensity reaching the leaf was 3000 μmol photonsÁm -2 Ás -1 , which was sufficient to generate maximal fluorescence for all treatments. Use the program PEA plus 1.0.01 to collecting the data and the Biolyzer 3.0 to calculation of the OJIP test parameters. Data were transferred into Excel (Microsoft, Redmond, USA) for further expound, including the parameters introduced by Strasser et al. [10]. A summary of OJIP test parameters used in this study is shown in Table 1. The fraction of the oxygen evolving complex was compared to the control by calculating according to Appenroth et al. [23] as

Ultrastructure of the chloroplast
Leaf samples were cut into discs with an area of approximately 1 mm 2 and incubated in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4; this buffer was used for the entire preparation of samples for microscopy) overnight. The samples were washed three times, for 15 min each time, with buffer; immersed in 1% osmic acid in buffer for 2 h; and washed again in buffer following the same protocol. The samples were dehydrated in a graded ethanol series (50%, 70%, 90%, and 100%), and then in absolute acetone for 15 min. After dehydration, the samples were embedded in Durcupan ACM epoxy resin (Sigma Aldrich, USA). Ultra-thin sections were cut from the samples and stained with uranium acetate followed by lead citrate. The sections were mounted on copper grids and examined with a JEM-1230 transmission electron microscope (JEOL, Peabody, MA, USA) at an accelerating voltage of 80 kV.

Isolation of intact chloroplasts
Intact chloroplasts were isolated as described by Shu et al. [24] with slight modification. Ten grams of leaves were homogenized for 5 s in 30 mL of 330 mM sorbitol, 30 mM Mes, 2 mM ascorbate, 0.1% bovine serum albumin (BSA) in 50 mM Tris-HCl adjusted to pH 7.6. The homogenate was filtered through four layers of cheesecloth and centrifuged at 800×g for 3 min. The resultant supernatant was centrifuged at 3000×g for 5 min and the pellet was resuspended in 3 mL of 330 mM sorbitol, 30 mM Hepes, and 0.2% BSA in Tris, adjusted to pH 7.6. This suspension was mixed with 40-80% (v/v) Percoll density centrifugation medium and centrifuged for 3 min at 3000×g. The region between 40 and 80% Percoll contained the intact chloroplasts.
All procedures were carried out at 4°C.

Statistical analysis
All data presented are the mean values. All experiments were conducted in triplicate and data were analyzed with statistical software SPSS (version 20.0, SPSS Institute, Chicago, USA) using Duncan's multiple range test at P< 0.05 level of significance.

Photosynthesis and photosynthetic efficiency of PSII
The net photosynthetic (P n ), stomatal conductance (G s ), intercellular CO 2 concentration (C i ), and stomatal limitation (L s ) values in leaves of control plants and plants treated with GABA only were not significantly different (p < 0.05) after 5 days of 50 mM complex salts stress treatment. However, in plants exposed to saline-alkaline stress P n declined by 61.6%, G s declined by 73.4%, and L s increased 44.9% compared to the control plants (Fig 1). Foliar application of GABA to stressed plants significantly increased P n , G s and C i , and significantly reduced (p < 0.05) L s as compared to the salinity-alkalinity treatment, partly restoring the effects of stress on these parameters.

Fast chlorophyll a fluorescence transient
The Chl a transient curves for all treatments are presented in Fig 2. Muskmelon seedling leaves would be trapped a saturating light pulse after 30 min dark adaptation, the chlorophyll a fluorescence is form OJIP curve rapidly. After maximum fluorescence, it began to decline. We observed no significant difference in the transient curves of the control and GABA-treated plants. The curve of only complex salts treated plants had 8.4% more fluorescence at the J phase than the control and less fluorescence at the I and P phase. The J phase of stressed plants treated with GABA was no different than the J phase of the control, but the I phase had even less fluorescence (it was further reduced by 2.6%) than the stressed plant (Fig 2).

The donor side of PSII
An analysis of the fluorescence emission data from the JIP-test yielded values for the ratio of fluorescence intensity of the K phase to that of the J phase (W k values) and the fractions of oxygen evolving complex for all treatments (Fig 3). These values were statistically similar for the control plants and plants treated with GABA alone (p < 0.05). Plants exposed to saline-alkaline stress had W k values that were 11.1% greater than the control plants and fraction of oxygen  evolving complex values that were 19.0% less than the control; both values were significantly different than the control values (p < 0.05). However, stressed plants that had been treated with foliar application of GABA had W k values and fraction of O 2 evolving complex values that were not different from the control. We suggest that saline-alkaline stress had a negative effect on the donor of the photosynthetic electron transport chain and that GABA alleviated this effect.  13.1% and 38.8% greater, while the ψ o value was 11.5% less, in the stressed plants than in the control (significant differences at p < 0.05). These results are consistent with the interpretation that saline-alkaline stress inhibited photosynthetic electron transport. Stressed plants that were treated with GABA had lower value of V J , M o , and higher value of ψ o that were significantly different (p < 0.05) than the only stress values.

The energy distribution
The measurements for quantum yield for electron transport (φE o ), an expression for the probability that the energy of an absorbed photon is dissipated as heat (φD o ), and the maximum quantum yield for primary photochemistry (φP o ) are presented in

The reaction center status
The parameters relate to the status of the reaction centers of the chloroplast of the experimental plants are reported in S1 Table. GABA treatment by itself did not affect these parameters as

The performance index and driving force
The performance index and driving force are presented, expressed on an absorption basis (PI ABS and DF ABS , respectively), in Fig 6. There was no significant difference in these values in plants treated with GABA alone and the untreated control plants (p < 0.05). Plants under saline-alkaline stress had PI ABS and DF ABS that were 34.8% and 35.7% less than the control, respectively. When the stressed plants were treated with GABA, PI ABS and DF ABS values

Activity of ATPase
There was no significant difference in the activity of the different ATPases that were assessed in the chloroplasts of leaves from control or GABA-treated plants (p < 0.05), as shown in Fig  7. In leaves from saline-alkaline stressed plants, the activities of H + -ATPase, Mg 2+ -ATPase, and Ca 2+ ATPase were decreased by 17.2%, 21.3% and 28.4%, respectively, compared to the control. Fig 7 also shows that these activities were greater in stressed plants treated with GABA than in the only stressed plants, although the activities were still lower than in the control plants. We conclude that exogenous GABA maintained the activities of these three ATPase, in plants exposed to saline-alkaline stress.  (Fig 8A-8D). The chloroplasts from plants treated with saline-alkaline stress were markedly different in appearance: they were swollen compared to the control and the membranes of the granal and stromal thylakoids were indistinct under the microscope (Fig 8E and 8F). Chloroplasts from stressed plants that received exogenous GABA had more normal appearing chloroplasts that those from stressed plants (Fig 8G and 8H). Specifically, the images reveal that the internal lamellae of these thylakoids were better integrated than those from stressed plants not treated with GABA. Apparently, exogenous GABA maintained the stability of photosynthetic apparatus during saline-alkaline stress.

Discussion
Saline stress often decreases the photochemical efficiency of plants, which has been ascribed to suppression of PSII activity [29]. The relationship between L s and non-stomatal factors has been evaluated by [30]. When both C i and G s decrease, P n is limited by stomatal conductance. When G s decreases and C i either does not change or increases, the reduced P n that is observed may be due to non-stomatal factors. In this study, saline-alkaline stress reduced P n and G s , and increased L s (Fig 1). This suggests that stress-induced limitation of photosynthesis in this study was mainly unrelated to effects on the stomata. Exogenous GABA supplied to saline-alkaline stressed plants alleviated stress-induced reductions in P n , G s and Ci, and increased L s . This suggests that exogenous GABA absorbed by plants could act as a temporary nitrogen source and osmotic substances which reduced reduce the injury of mesophyll cells under saline-alkaline stress. Meanwhile, GABA may be involved in regulation of ABA levels in plant and maintain higher P n levels by enhancing the level of stomatal openness. We interpret these results as evidence that saline-alkaline stress blocked electron transport, which in turn damaged the photosynthetic apparatus (Fig 8), and that exogenous GABA alleviated these effects.
Photosynthesis, the process of changing light energy to chemical energy, is critical in the growth and development of plants. In this study, saline-alkaline stress altered the chl a fluorescence transient (Fig 2) and reduced the maximum quantum yield for primary photochemistry (Fig 5). This is evidence that photoinhibition occurred, on both the acceptor and donor side of PSII [31]. The W k value assesses the activity of the oxygen evolution complexes on the donor side of PSII and an increase in W k indicates damage to the complexes [9]. In this study, we can observe that stress increased the W k value (Fig 3A), which indicative of damage to the donor side of PSII. Specifically, the decrease of fraction of O 2 evolving complex further more supported it (Fig 3B). V J represents the closed degree of the reaction center at 2ms, M o represents the maximum reduction rate of Q A , and ψ o represents the probability that a trapped exaction moves an electron into the electron transport chain beyond Q A - [10]. The values of V J and M o increased and the value of ψ o decreased under stress, which consistent with blockage of electron transfer from the primary acceptor Q A to the secondary acceptor plastoquinone B (Q B ) on the acceptor side of PSII (Fig 4). φE o , the probability that an absorbed photon leads to an electron transport farther than Q A , and φP o , the probability that an absorbed photon leads to a reduction of Q A and φD o , the probability that the energy of an absorbed photon was dissipated. Stress induced the reduction of φE o and φP o and increase of φD o , these variation illuminated stress can re-distribution of the quantum efficiency of PSII ( Fig 5). As S1 Table shows These indicated that exogenous GABA reduced the heat dissipation and distributed more energy for transfer electron, and promoted probability that an absorbed photon lead to an electron transport further than Q A . Hu et al. [17] research showed exogenous GABA with stressed plants could further more increase the endogenous GABA. The GABA accumulation has two pathways. One is converted glutamate to succinate via GABA that called the GABA shunt [19]. Another is come from polyamines (PAs) oxidation [16,32]. In our study, exogenous GABA alleviation effect of stressed plants may though the GABA shunt to promoting the tricarboxylic acid cycle (TCA) and ensuring the operation of photosynthetic electron transport chain. In addition, exogenous GABA may mediate the PAs metabolism to improving tolerance of salinity-alkalinity stress. The polyamines could enhance antioxidant, increase ATP synthase, Qb, psbA protein and the saturated fatty acid contents of thylakoid membranes, decrease the content of D2 protein and LHCII type III in NaCl-stressed thylakoid membranes to overcoming the damaging effects of stress on the structure and function of the photosynthetic apparatus and improving the photochemical efficiency of PSII of the salt stressed plants [24,33,34].
The generation of ATP is the last step in the light reactions of photosynthesis. In this study, saline-alkaline stress inhibited the activity of three ATPases: the H + -ATPase, the Mg 2+ -ATPase, and the Ca 2+ -ATPase (Fig 7) and this inhibition was partly reversed by GABA. It has been reported that GABA could regulate cytosolic pH by consuming a proton and it could also function as a nitrogen source to promote the tricarboxylic acid cycle [17]. This effect of GABA might improve ATPase function [17]. Another possiility is that exogenous GABA could affect the endogenous GABA levels that stimulated CaM-dependent GAD activity [17] and amplifyed the GABA accumulation/Ca 2+ release cycle. The observed effects of stress and GABA on the driving force (DF ABS value) support this suggestion (Fig 6).
In higher plants, the photosynthetic machinery is mainly localized in the thylakoids membranes of the chloroplasts. Intact thylakoids are essential to efficient photosynthesis and the structure of thylakoids is a major factor that affects functionality and efficiency of the photosynthetic apparatus. Saline stress reduces photosynthetic efficiency and electron transport; it may be that this inhibition is due to effects of stress on the structure of photosynthetic apparatus [35]. In this study, we detected degradation of thylakoid membranes accompanied by the accumulation of plastoglobuli under saline-alkaline stress (Fig 8E and 8F). Plants exposed to environmental stress accumulate large quantities of reactive oxygen species (ROS) in their chloroplasts [2]. In this study, we suggest that inactivation of the electron transport chain prevented the alternate oxidation and reduction of P680, leading to increased oxidation potential on the donor side of PSII (Fig 3). This could, in turn, cause peroxidation of the chloroplast membrane and other structural abnormalities (Fig 8). Additionally, the prevention of electron transfer from the primary acceptor Q A to the secondary acceptor Q B would form the triplet chlorophyll ( 3 P680) which, when combined with oxygen, would generate singlet oxygen (Fig  4). The singlet oxygen could cause D1 protein, important in repair, to be degraded and cause pigment decomposition, directly inhibiting photosynthesis [36,37]. GABA, which appears to stabilize the photosynthetic electron transport chain and the photosynthetic apparatus (Fig 8), may enhance the activity of superoxide dismutase, accelerate ascorbic acid-glutathione cycling, and increase the content of antioxidants that scavenge ROS, leading to stabilization of the structure and function of chloroplast [22,38]. The similar results with Malekzadeh [39], who indicated that exogenous GABA by enhancing some antioxidant enzymes activity and reducing MDA content to alleviate the damage of tomato seedlings under chilling stress.
Supporting Information S1 Table. Status of the reaction centers in chloroplasts of muskmelon seedlings. Control, plants grown in medium only; CG, medium with leaf spraying with GABA; S, nutrient medium with complex neutral and alkali salt; SG, medium with both complex neutral and alkali salt and leaf spraying with GABA. Data represent the mean ± SE of three independent experiments (n = 3). Different letters indicate significant differences between treatments (p < 0.05). (DOCX)