Exogenous GSH protects tomatoes against salt stress by modulating photosystem ll efficiency, absorbed light allocation and H2O2-scavenging system in chloroplasts

ZHOU Yan , DlAO Ming , CUl Jin-xia , CHEN Xian-jun , WEN Ze-lin , ZHANG Jian-wei , LlU Huiying

1 Department of Horticulture, Agricultural College, Shihezi University, Shihezi 832003, P.R.China

2 Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization of Xinjiang Production and Construction Crops, Shihezi 832003, P.R.China

Abstract

Keywords: tomato, reduced glutathione, NaCl stress, chloroplast, photosynthesis, antioxidant enzyme

1. Introduction

Salinity is one of the most harmful environmental factors and it severely limits crop productivity. High salinity causes osmotic stress, ion imbalance, and oxidativestress, inhibiting plant growth through the destruction of physiological processes, such as photosynthesis (Horváth et al. 2015). Photosynthetic efficiency factors affected by salt stress in plants are both stomatal and non-stomatal factors, including stomatal closure, destruction of chlorophyll pigments, inhibition of photochemical efficiency of PSII and CO2assimilation, and degradation of membrane proteins in the photosynthetic apparatus (Athar et al. 2015). The chloroplast, which is the main site of photosynthesis, is plantspecific and is the most environmentally sensitive organelle.It is also the main organelle where reactive oxygen species(ROS) are generated. In general, under abiotic stress,excessive photonic energy can cause photoinhibition and promote the accumulation of ROS (including superoxidehydrogen peroxideand singlet oxygen(1O2)) in chloroplasts in the situation of lower antioxidant capacity to detoxify ROS. The accumulated ROS enhance membrane lipid peroxidation and severely damage the chloroplast membrane structure, disrupt the use of light energy, and alter the activities of antioxidative enzymes and antioxidant content, consequently leading to cell and plant death (Amjad et al. 2016). To eliminate ROS and protect against oxidative stress, chloroplasts in plants have formed an efficient antioxidant defense system pathway to maintain ROS equilibrium and participate in stress resistance and adaptation, while fulfilling other roles associated with redox signaling (Nazar et al. 2011; Fatma et al. 2014). In chloroplasts, superoxide dismutase (SOD) is the first line of defense to disproportionate superoxide radicals to H2O2and O2. Afterwards, H2O2is scavenged by the conversion of catalase (CAT), peroxidase (POD), the AsA-GSH cycle,Trx, and Grx systems to water (Gill and Tuteja 2010).

Reduced glutathione (γ-glutamyl-cysteinyl-glycine, GSH)is the most abundant low-molecular-weight thiol in cells,and it constitutes a redox buffer that keeps the intracellular environment reduced. GSH is involved in scavenging mostROS either directly or indirectly via the AsA-GSH cycle,Trx and Grx systems, sulfur metabolism, and regulation of intracellular environment homeostasis, as an antioxidant(Nahar et al. 2015a). In plants, the AsA-GSH cycle is indispensable in eliminating H2O2. In this cycle, GSH acts as a reducer that transforms ascorbate from its oxidized form (dehydroascorbate, DHA) to its reduced form (AsA)through the activity of dehydroascorbate reductase (DHAR).Subsequently, GR reduces oxidized glutathione (GSSG).GSH is also a substrate for glutathione peroxidase (GPx)and glutathione-S-transferase (GST). The GSH pool was used by both GPX and GST to detoxify H2O2and xenobiotics by catalyzing their conjugation with GSH (Anjum et al.2012; Noctor et al. 2012). Furthermore, GSH is involved in signal transduction through glutathionylation or thiol bridge redox reactions of proteins. Glutathione is also involved in anthocyanin accumulation (Sugiyama and Sekiya 2005),programmed cell death (PCD), and pathogen resistance(Mou et al. 2003; Foyer and Noctor 2005). Many studies have suggested that cellular endogenous GSH biosynthesis and accumulation also can improve resistance and adaptation of crops to biotic and abiotic stresses (Rausch et al. 2007; Foyer and Noctor 2011; Zhou et al. 2017).

In recent studies, application of exogenous GSH has been shown to be beneficial for crops under various abioticstresses as it improves crop physiological properties,the antioxidant defense system, glyoxalase system, and phenotypic appearance. It has been shown that exogenous GSH alleviates isoproturon and heavy metal toxicity (lead,chromium, cadmium and copper) in crops (Qiu et al. 2013;Cao et al. 2014; Mostofa et al. 2014; Yuan et al. 2014). For example, GSH enhanced drought tolerance in Arabidopsis(Chen et al. 2011), high temperature tolerance in mung beans (Nahar et al. 2015a), and low temperature tolerance in loquats (Wu et al. 2011). Exogenous GSH also enhances salt stress tolerance in canola, mung beans, Oryza sativa L. and in soybeans (Kattab 2007; Wang et al. 2014; Nahar et al. 2015b; Akram et al. 2017). Our previous work on tomatoes suggested that application of exogenous GSH increases salinity tolerance by improving the antioxidant system and regulating endogenous GSH synthesis and metabolism to maintain intracellular redox homeostasis in leaves of salt-stressed tomato seedlings (Zhou et al. 2016,2017). In another study, we observed that spraying tomato leaves with GSH and GSSG induces different redox states in leaves of salt-stressed tomato seedlings, and we supposed that exogenous GSH increases the net photosynthetic rate of salt-stressed tomato seedlings by mediating the redox state at tissue levels to up-regulate PSII photochemical efficiency and Rubisco activities (Liu et al. 2014).

However, research is lacking on the role of GSH in balancing the excess excitation energy, redox state, and antioxidant capacity at the organelle level of plants under saltstress. Thus, in the present study, we investigated the effect of exogenous GSH on leaf photosynthetic performance,photosystem II (PSII) efficiency, absorbed light energy allocation, and the antioxidant system of chloroplasts in salt-stressed tomatoes. Meanwhile, to further determine the role of glutathione in balancing the excess excitation energy and modulating the ROS-scavenging system in chloroplasts of tomatoes under salt stress, we treated the salt-stressed plants with BSO (L-buthionine-sulfoximine, an inhibitor of key GSH synthesis enzyme γ-glutamylcysteine synthetase) prior to GSH treatment and compared them to plants that were treated with BSO. The purpose of using BSO is to reduce endogenous GSH content. We found that modulating photosynthetic activity at tissue levels and ROS metabolism at organelle levels by adding exogenous GSH was an important mechanism for mitigating the inhibition of plant growth and photosynthesis caused by salt stress.

2. Materials and methods

2.1. Plant materials, growth conditions and treatments

Tomato plants (Solanum lycopersicum L. cv. Zhongshu 4)were used for hydroponic experiments in a solar greenhouse at Shihezi University, Xinjiang Uygur Autonomous Region,China. The germinated seeds were sown in plug trays filled with 2:1 peat:vermiculite (v/v) before the seeds were incubated on damp filter paper in darkness at 28°C for 2 days. The seedlings were transplanted into 12-L plastic containers (with five seedlings per container) containing 10 L of oxygenated full-strength Hoagland nutrient solution(5 mmol L–1KNO3, 2 mmol L–1MgSO4, 5 mmol L–1Ca(NO3)2,2.5 mmol L–1KH2PO4, 50 μmol L–1Fe-EDTA, 29.6 μmol L–1H3BO3, 10 μmol L–1MnSO4, 1.0 μmol L–1ZnSO4, 0.05 μmol L–1H2MoO4, and 0.95 μmol L–1CuSO4at pH=6.2), when the second true leaves of seedlings were fully expanded.

After pre-culturing for 7 days, five treatments were conducted on tomato seedlings. NaCl was added to nutrient solutions and GSH and BSO were sprayed on the leaves resulting in five treatments: (1) no added NaCl, no sprayed GSH and BSO (Control); (2) added 100 mmol L–1NaCl(NaCl); (3) added 100 mmol L–1NaCl and sprayed 5 mmol L–1GSH (NG); (4) added 100 mmol L–1NaCl and sprayed 1 mmol L–1BSO (NB); and (5) added 100 mmol L–1NaCl and sprayed 1 mmol L–1BSO and 5 mmol L–1GSH (NBG).GSH and BSO were purchased from Roche (China) and Sigma (USA). For the NG, NB, and NBG treatments, the concentrations, volumes, and methods of GSH and BSO application were based on our previous study (Zhou et al.2017). The containers of five treatments for each replication were arranged in a randomized complete block with three replications. There were three containers per treatment.The light period of seedlings was 14 h and seedlings were held at 24–30°C during daytime and at 17–20°C during nighttime; nutrient solutions were replaced every 3 days.Tomato seedling leaves were sampled on the 10th day.

2.2. Growth measurements

After 10 days of treatments, each plant was divided into the aerial part and underground part, and both parts were dried at 75°C for 24 h. Dry weights were measured, and the relative growth rates (RGRs) of the aerial part and underground part were calculated according to equations as previously described (Van et al. 2016).

2.3. Determination of ion contents

Tomato leaves were ground after being dried at 75°C. A total of 0.2 g of samples was dry-ashed and digested in a mixture of HNO3and H2O2(2/1, v/v). The Na+and K+contents were analyzed using an inductively coupled plasma optical emission spectrometer (ICP-OES, USA), and then the ratio of Na+/K+was calculated. The content of Cl-was measured using a previously described method (Nazar et al. 2011).

2.4. Determination of photosynthetic parameters

The photosynthetic parameters including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2concentration (Ci), and transpiration rate (Tr) were measured using a portable photosynthetic system Li-6400 (LI-COR,USA) according to a method previously described by Diao et al. (2014).

2.5. Chlorophyll fluorescence parameters

Chlorophyll fluorescence parameters were measured using a portable fluorescence monitoring system (FMS2,Han-satech, England), a modulated fluorescence imaging system, and the ImagingWin software application (Imaging-PAM, WALZ, Germany) from the same leaves that were previously used for photosynthetic measurements. After 30 min of dark adaption, the leaves were illuminated under a high saturating light pulse with a frequency of 0.05 Hz for 260 s. Foand Fmwere determined (Table 1). Fv/Fmwas calculated as previously described (Genty et al. 1989).False-color images of Fv/Fmwere recorded, stored, and compared using ImagingWin (Yu et al. 2002). Images of Fv/Fmwere shown with a false color code ranging from 0.000(black) to 1.000 (purple). 1–qP, qP, NPQ, ETR, Y(NPQ) and Y(NO) were measured on light-adapted leaves using the equations described previously (Maxwell and Johnson 2000;Kramer et al. 2004) (Table 1). The allocation of the fraction of excitation energy was analyzed, including D, P, Ex, and(β/α–1), according to the equations previously described (Li et al. 2003) (Table 1).

2.6. lsolation of chloroplasts

Chloroplasts were isolated from leaves according to a previously described method (Diao et al. 2014) with some modifications. Ten grams of fresh tomato leaves were pulverized with 20 mL buffer A (50 mmol L–1MES, 0.3 mmol L–1sorbitol, 10 mmol L–1NaCl, 2 mmol L–1MgCl2, 2 mmolL–1EDTA, 0.5 mmol L–1KH2PO4, and 2 mmol L–1sodium ascorbic acid, pH 6.1) in a mortar. The leaves were filtered with four layers of gauze filter homogenate, and then the filtrate was centrifuged at 2 000×g for 30 s. The pellets were resuspended in 2 mL buffer B (50 mmol L–1HEPES,0.3 mmol L–1sorbitol, 10 mmol L–1NaCl, 2 mmol L–1MgCl2,2 mmol L–1EDTA, 0.5 mmol L–1KH2PO4, and 2 mmol L–1sodium ascorbic acid, pH 7.6), and then centrifuged at 2 000×g for 1 min. Pellets were resuspended in 5 mL of buffer A, and then put into a tube containing 15 mL of resuspension medium plus 40–80% (v/v) Percoll and centrifuged at 2 000×g for 3 min. The interlayer between 40 and 80%Percoll containing intact chloroplasts was collected, and then mixed with 3 mL of ice-cold HEPES buffer (25 mmol L–1,pH 7.8) containing 0.2 mmol L–1EDTA and 2% (w/v) PVP. The mixed solution was centrifuged at 12 000×g for 20 min at 4°C,and the supernatants were used for analysis of antioxidant enzyme activity, and determination of antioxidant, hydrogen peroxide (H2O2), and malondialdehyde (MDA) contents. All procedures were carried out at 4°C.

Table 1 Parameters of chlorophyll fluorescence

2.7. Determination of the redox state in the chloroplasts

The determination of GSH and GSSG were done using the supernatant from the chloroplast isolation. Total glutathione(GSH+GSSG) and oxidized glutathione (GSSG) contents in the chloroplast were determined based on previous methods (Yu et al. 2002). The GSH content was calculated by subtracting GSSG content from GSH+GSSG, and then the ratio of GSH/GSSH was calculated.

2.8. Determination of H2O2 and MDA contents in the chloroplasts

H2O2and MDA contents of chloroplasts were determined using a previously described method (Diao et al. 2014).

2.9. Determination of antioxidant enzyme activity in the chloroplasts

Superoxidase dismutase (SOD; EC:1.15.1.1) activity was assayed using a xanthine-xanthine oxidase system (El-Shabrawi et al. 2010). The SOD activity was expressed as the amount of enzyme needed to inhibit NTB reduction by 50%. The change in absorbance was read at 560 nm.

Peroxidase (POD; EC:1.11.1.7) activity was determined using a previously described method (Shannon et al. 1966).The change in absorbance was read at 470 nm for 4 min.One enzyme unit was defined as the change in 1 unit of absorbance min–1.

Catalase (CAT; EC:1.11.1.6) activity was assayed as described previously (Hasanuzzaman et al. 2011). The reaction was initiated with the enzyme extract. The decrease in absorbance (due to decomposition of H2O2) at 240 nm was recorded for 1 min. The activity was calculated using an extinction coefficient of 39.4 mol L–1cm–1.

Ascorbate peroxidase (APX; EC:1.11.1.11) activity was measured as previously described (Nakano and Asada 1981). The absorbance was measured at 290 nm for 1 min. The APX activity was determined using an extinction coefficient of 2.8 mmol L–1cm–1.

Monodehydroascorbate reductase (MDHAR; EC: 1.6.5.4)activity was assayed as described previously (Hossain et al. 1984). The absorbance was measured at 340 nm.The activity was calculated from the change in absorbance after 1 min (extinction coefficient was 6.2 mmol L–1cm–1).

Dehydroascorbate reductase (DHAR; EC:1.8.5.1) activity was measured as previously described (Nakano and Asada 1981). The activity was calculated from the change in absorbance at 265 nm after 1 min (extinction coefficient was 14 mmol L–1cm–1).

Glutathione reductase (GR; EC:1.6.4.2) activity was measured using a previously described method (Cakmak et al. 1993). The decrease in absorbance at 340 nm due to NADPH oxidation was recorded for 1 min. The activity was calculated using the extinction coefficient of 6.2 mmol L–1cm–1.

Glutathione peroxidase (GPX; EC: 1.11.1.9) activity was measured as previously described (Elia et al. 2003) using H2O2as a substrate. The oxidation of NADPH was recorded at 340 nm for 1 min and the activity was calculated using an extinction coefficient of 6.62 mmol L–1cm–1.

2.10. Statistical analysis

All treatments were replicated at least three times. Data were analyzed with SPSS version 19.0 (SPSS Inc., Chicago,IL, USA). Differences were considered to be significant when P＜0.05. The values in the figures are means±SE(n=3).

3. Results

3.1. Plant growth and leaf ion content

Compared to the control, NaCl treatment decreased the relative growth rate (RGR) in the underground part and aerial part of tomato plants (P＜0.05) (Fig. 1). Exogenous GSH application significantly increased the RGR in the underground and aerial parts by 37.79 and 9.38%,respectively, when plants were exposed to salt stress(P＜0.05). BSO application decreased the RGR in the underground part and aerial part of salt-stressed plants.NBG treatment significantly increased the RGR in the underground part and aerial part of plants when compared with NB-treated plants (P＜0.05).

Fig. 1 The relative growth rates in aerial part (A) and underground part (B) of salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG,added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB,added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG,added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3).Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

The Na+and Cl–contents, and the Na+/K+ratios were significantly increased by 1 842.54%, 286.11% and 3 078.59% (P＜0.05), respectively, and K+content was significantly decreased by 63.58% in NaCl-stressed plants, compared to control plants (P＜0.05) (Fig. 2). GSH application significantly decreased the Na+content by 38.52%, Cl–content by 32.22%, and Na+/K+ratio by 55.32%and increased the K+content by 12.20% in the leaves of NaCl-stressed seedlings, compared with seedlings that were only NaCl-stressed (P＜0.05). In contrast, compared with the NaCl-stressed seedlings, application of BSO significantly increased Na+, Cl–, and Na+/K+by 10.85, 8.63, and 28.29%,respectively, and decreased K+by 15.68% in the leaves of NaCl-stressed plants (P＜0.05). The Na+and Cl–contents and Na+/K+ratio in the NBG treatment were significantly lower, and the K+content was higher than in NB treatment.

3.2. Photosynthetic parameters

Fig. 2 The Na+ content (A), Cl– content (B), K+ content (C) and Na+/K+ (D) in leaves of salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO;NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3). Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

Fig. 3 Values of net photosynthetic rate (Pn, A), stomatal conductance (Gs, B), transpiration rate (Tr, C), and intercellular CO2 concentration (Ci, D) in salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthioninesulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3). Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

As shown in Fig. 3, NaCl treatment resulted in the reduction of Pnby 97.73%, Gsby 263.73%, Trby 60.39% and Ciby 9.44%, compared to the controls (P＜0.05). Application of GSH significantly increased Pnby 35.91%, Gsby 218.63%,Trby 29.70% and Ciby 8.34% when the plants were exposed to salinity stress. BSO application under salt-stressed conditions significantly decreased by 18.71% in Pnand 24.58% in Trbut had no significant influence on Gsand Cicompared with NaCl treatment. Compared with NB treatment, NBG treatment had no significant influence on Cibut significantly increased Pn, Gs, and Trby 96.76, 124.59,and 63.65%, respectively.

3.3. Chlorophyll fluorescence parameters

Compared to controls, Fv/Fm, qP, NPQ, ETR, Y(II), and Y(NPQ) values were significantly reduced under salt stress(Fig. 4). Salt stress significantly increased 1–qPand Y(NO)values in tomato leaves. Application of exogenous GSH significantly decreased the values of 1–qPand Y(NO),and significantly increased the values of Fv/Fm, qP, NPQ,ETR, and Y(II) in the leaves of the salt-stressed seedlings(P＜0.05). In contrast, the application of exogenous BSO significantly increased 1–qP, Y(NO), and Y(NPQ), and decreased Fv/Fm, qP, NPQ, ETR, and Y(II) values in the leaves of the salt-stressed seedlings (P＜0.05). Compared with NB treatment, application of GSH decreased 1-qPby 31.64%, and Y(NO) by 16.21%, and increased Fv/Fm, qP,NPQ, ETR, Y(II), and Y(NPQ) (P＜0.05) by 7.26, 4.86, 20.75,11.60, 7.82, and 16.24%, respectively (Fig. 4).

Fig. 4 Values of 1–qp (A), Fv/Fm (B), qp (C), NPQ (D), ETR (E), and Y(II), Y(NPQ), Y(NO) (F) in salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. The explanations for 1–qp, Fv/Fm, qp, NPQ, ETR, and Y(II), Y(NPQ), Y(NO) can be seen in Table 1. Error bars represent SD values(n=3). Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

In order to understand the changes of Fv/Fminduced by different treatments, a false-color image application was used (Fig. 5). Under saline conditions, leaf colors shifted from blue to green representing reductions in the Fv/Fmratio.Compared with NaCl treatment, leaf color was recovered to near the control level when tomato seedlings were grown in NaCl and GSH conditions, whereas NaCl+BSO treatment had an opposite effect. NB and NBG treatment had different colors of the Fv/Fmimages in the leaves of tomato seedlings. Smaller yellow leaf areas and less brown spots were observed in NBG than in NB (Fig. 5).

3.4. Absorbed light allocation

Fig. 5 False-color images of the maximal photochemical efficiency of PSII (Fv/Fm) in leaves of salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). A false color code from left to ranging from 0.000 (black) to 1.000 (purple). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH.

Fig. 6 Values of P (A), D (B), Ex (C) and β/α–1 (D) in salt-stressed tomato seedlings as affected by exogenous reduced glutathione(GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl;NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG,added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. The explanations for P, D, Ex and β/α–1 can be seen in Table 1. Error bars represent SD values (n=3). Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

NaCl treatment significantly decreased P and significantly increased D, Ex, and β/α–1 in the tomato seedling leaves(P＜0.05) (Fig. 6). Compared with the NaCl-stressed seedlings, NG treatment significantly increased P by 17.40%and significantly decreased D by 18.26%, Ex by 14.32%,and β/α–1 by 31.65% (P＜0.05). NB treatment significantly decreased P by 10.94% and significantly increased Ex by 17.08% and β/α–1 by 27.27% (P＜0.05) but had no significant effect on D. The P value in NB was significantly increased and Ex and β/α–1 in NB were significantly decreased by GSH application.

3.5. Redox status, oxidative damage and the activity of antioxidant enzymes in the chloroplast

Endogenous GSH content and redox state in tomatochloroplastsExposure of tomato seedlings to salt stress significantly decreased the chloroplast GSH contents by 39.73% and GSH/GSSG ratios by 34.68%. Application of exogenous GSH significantly increased the chloroplastGSH contents by 17.93% and GSH/GSSG ratios by 17.37%. Similarly, compared to NaCl-treated plants, GSH contents and GSH/GSSG ratios were significantly reduced by 13.07 and 16.27% in the chloroplasts of NB-treated tomato plants. NBG treatment significantly increased the chloroplast GSH contents and GSH/GSSG ratios by 17.45 and 17.67%, respectively, compared to NB treated plants(Fig. 7).

H2O2 and MDA contents in tomato chloroplastsThe plants suffered severely at 10 days of salt stress, as indicated by the increase in H2O2and MDA contents in tomato chloroplasts (Fig. 8-A and B). However, the H2O2and MDA contents in the chloroplasts of salt-stressed tomato seedlings were significantly decreased by application of GSH (P＜0.05). In contrast, NB treatment induced a significant increase in H2O2by 36.78% and MDA by 38.28%compared to plants that had been treated only with NaCl(P＜0.05). NBG treatment significantly decreased the MDA and H2O2contents in tomato chloroplasts compared to NB-treated seedlings. Differences between the NB and NBG treatments in terms of H2O2and MDA contents reached 15.11 and 22.16%, respectively (Fig. 8-A and B).

Fig. 7 The reduced glutathione (GSH) content (A) and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio (B) in leaf chloroplasts of salt-stressed tomato seedlings as affected by exogenous GSH and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3). Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

Fig. 8 The H2O2 content (A) and malondialdehyde (MDA) content (B) in leaf chloroplasts of salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3). Different letters indicate the significant differences at same sampling date among the treatments(P＜0.05).

Antioxidant enzyme activities in tomato chloroplastsSOD,POD, CAT, APX, DHAR, MDHAR, GR, and GPX activities were significantly decreased in the chloroplasts of tomatoes under salt stress compared with the control (P＜0.05)(Fig. 9-A–H). Application of GSH to NaCl-treated plants significantly increased POD, CAT, APX, DHAR, MDHAR,GR, and GPX activities in chloroplasts by 70.04, 787.04,72.91, 89.35, 47.45, 61.28, and 100.12%, respectively,compared with only NaCl treated plants. In contrast,application of BSO to NaCl-stressed seedlings significantly decreased activities of SOD, POD, CAT, APX, DHAR,MDHAR, GR, and GPX in chloroplasts by 52.58, 21.27,62.52, 71.51, 41.64, 71.04, 60.54 and 263.66%, respectively,compared with the NaCl-stressed seedlings. Compared with NB treatment, GSH application significantly increased SOD, CAT, APX, DHAR, MDHAR, GR, and GPX activities in the chloroplasts of NB-treated tomato seedlings (P＜0.05)(Fig. 9-A–H).

Fig. 9 Activities of superoxidase dismutase (SOD, A), peroxidase (POD, B), catalase (CAT, C), ascorbate peroxidase (APX,D), dehydroascorbate reductase (DHAR, E), monodehydroascorbate reductase (MDAHR, F), glutathione reductase (GR, G)and glutathione peroxidase (GPX, H) in leaf chloroplasts of salt-stressed tomato seedlings as affected by exogenous reduced glutathione (GSH) and L-buthionine-sulfoximine (BSO). Control, no added NaCl, no sprayed GSH and BSO; NaCl, added 100 mmol L–1 NaCl; NG, added 100 mmol L–1 NaCl and sprayed 5 mmol L–1 GSH; NB, added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO; NBG,added 100 mmol L–1 NaCl and sprayed 1 mmol L–1 BSO and 5 mmol L–1 GSH. Error bars represent SD values (n=3).Different letters indicate the significant differences at same sampling date among the treatments (P＜0.05).

4. Discussion

Decreased growth in plants subjected to a stressful environment, including salt stress, is often associated with a reduction in the photosynthetic capacity (Ali et al. 2008;Hu et al. 2013). Osmotic imbalance and iron stress induced by a high Na+tissue content and Na+/K+ratio cause water deficits and reduce leaf area expansions and stomatal closures, which ultimately lessen photosynthesis and growth(Roy et al. 2014). Because of stomata abnormality (such as stomatal closure, lower stoma density, and high stomatal resistance), Pnand gas change parameters (especially Ci, Gs, and Ex) were decreased under high Cd stress in P. divaricata (Ying et al. 2010). In the present study, salttreated plants exhibited disturbed homeostasis of Na+and Cl-ions by increasing their contents and the Na+/K+ratio,leading to a decrease in Pnand in the relative growth rate(RGR) in aerial parts (Figs. 1–3). At the same time, the decrease in Pnunder salt stress treatment was accompanied by a significant decrease in Gs, Ci, and Ex(Fig. 3-A, B and D). These results suggested that stomatal factors, such as stomatal closure and high stomatal resistance, were responsible for this reduction in photosynthesis becausestomatal closure and high stomatal resistance limited the supply of intercellular CO2for CO2fixation. A similar result was found in mustard (Fatma et al. 2014).

Many studies have shown that endogenous GSH is the main player in coordinating the activation of antioxidant defense and glyoxalase systems, and applications of exogenous GSH have been reported to promote plant growth and improve stress tolerance by detoxifying stressinduced ROS (Mostofa et al. 2014; Wang et al. 2014; Cao et al. 2015; Nahar et al. 2015a, b; Ding et al. 2016). The maintenance of a high K+concentration and low Na+/K+ratio in the cytosol is also critical for salt tolerance (Kiani et al. 2017). Our results also showed that application of exogenous GSH was more effective in reducing the contents of Na+and Cl–and the ratio of Na+/K+, and mitigating the effects of salt stress on the growth of plants and Pn, Gsand Cias was found in cotton (Ibrahim et al. 2017). In addition, the application of BSO significantly decreased Pnand Trbut had no influence on Gsand Ciin the leaves of salt-stressed plants compared with NaCl-treated plants. However, exogenous GSH application effectively relieved the growth inhibition and increased Pn, Gs, and Trbut had no influence on Ciin the leaves of NB-treated plants. These results indicated that exogenous GSH helps to relieve salt-induced photosynthetic inhibition by overcoming stomatal limitations and increasing photochemical efficiency and light use efficiency.

Chlorophyll fluorescence technology can reflect the process of light energy absorption and transmission during photosynthesis, so it is a fast, non-invasive probe for measuring photosynthetic function in leaves. The quantum efficiency of PSII and the extent of photoinhibition are continually used to ensure the energy distribution state in the thylakoid membrane (Hu et al. 2014; Zhang et al. 2015). The most vital chlorophyll fluorescence parameters are Fv/Fm,ETR, NPQ, qP, and 1–qPvalues, and they are broadly applied to plant stress physiology studies (Baker and Rosenqvist2004; Olvera-González et al. 2013; Thwe et al. 2014). Excess energy may cause photoinhibition and even severe damage to the photosynthetic apparatus (Sanda et al. 2011; Wilhelm and Selmar 2011; Wu et al. 2011; Wu and Bao 2011). In the present study, after a 10-day NaCl exposure, salt stress decreased Pn, and significantly decreased Fv/Fm, qP, NPQ,and ETR (Figs. 3 and 4). The decrease in Fv/Fmreflected the inhibition in the direct vicinity of PSII reaction centers or was due to secondary damage to the photosynthetic apparatus caused by photoinhibition (Murchie and Lawson 2013). A low NPQ implies a low ability of thermal energy dissipation through the xanthophylls cycle, which would disable the dissipation of excessive absorbed light energy(Maricle and Adler 2011). These results indicated that saltinduced photoinhibition is due to absorbed light energy in excess of the level required for photosynthesis, and excess energy induced by salt stress also causes severe damage to the photosynthetic apparatus. Another indication of such photoinhibition and damage is the decrease of Y(II) (Fig. 4).Decreases of Y(II) and Y(NPQ) are generally paralleled by an increase of Y(NO), which indicates the inhibition of photosynthesis (Bonfig et al. 2006). The data of Fig. 4 illustrate the decrease in Y(II) in the leaves of salt-stressed plants compared with the leaves of control plants, suggesting that the ability for photochemical energy conversion isstrongly compromised due to salt stress. Concomitantly,the decrease in Y(NPQ) coupled with the increase in Y(NO) suggests that the mechanisms of energy dissipation via the regulated photoprotective NPQ-mechanism (i.e.,dissipation of excessive excitation energy into innocuous heat) are inefficient. Therefore, our findings imply that the negative effect of salt stress on Pnis attributed to complex physiological disturbances, mainly including the inhibition of PSII reaction centers, and damage to the photosynthetic apparatus. Exogenous GSH could significantly alleviate the inhibition induced by salt stress (Fig. 4). The addition of GSH significantly increased Pn, Fv/Fm, qP, NPQ, ETR, and Y(II) of plants under salt stress but decreased 1–qPand Y(NO) (Fig. 4). The addition of BSO further aggravated the photosynthetic inhibition and damage to the photosynthetic apparatus by salt stress, while exogenous GSH application increased Fv/Fm, qP, NPQ, ETR, Y(II), and Y(NPQ) in plants under NB treatment. These results suggested that exogenous GSH effectively contributes to improving the photosynthesis rate of salt-stressed plants by enhancing photochemical conversion, photochemical activity, and the photosynthetic electron transport rate of PSII in the form of heat dissipation to protect the photosynthetic apparatus under salt stress from damage due to excess energy.

In the present study, salt stress caused an uneven distribution of light energy in chloroplasts. The photochemical process in chloroplasts of the salt-stressed tomato was weakened and more energy was consumed by nonphotochemical processes, which was reflected in the significant decrease in P and significant increase in the values of β/α–1, Ex, and D (Fig. 6-A–D). Application of exogenous GSH resulted in an increase in P and a decrease in D, Ex, and β/α–1 values of salt-stressed tomatoes. By contrast, BSO addition further increased the Ex and β/α–1 values and decreased the P-value of salt-stressed tomatoes,while exogenous GSH suppressed the reduction in P and the increase in Ex and β/α–1 values of the NB-treated tomatoes.These results suggested that exogenous GSH promotes photosynthetic electron transport and redistribution of energy in PSII reaction centers, and protects PSII from excess excitation energy damage induced by salt stress without BSO or with BSO by promoting photochemical,rather than non-photochemical, processes. It is likely that the improved carbon fixation capacity results in greater consumption of electrons, which is consistent with GSH-induced increases in Pnin salt-stressed tomatoes.

Chloroplasts are the major site of photosynthesis and are also the most important primary sites of ROS production in plants and are extremely sensitive to salt stress (Sairam and Tyagi 2004). The photoproduction of ROS is largely affected by physiological and environmental factors (Asada 2006). Under normal conditions, plants can maintain a balance between producing and regulating ROS. However,under stress conditions, accumulation of ROS may not be adequately regulated, resulting in oxidative stress and damaging the chloroplast structure and function (Chaves et al. 2009, 2011; Wang et al. 2009), which is closely related to excess light energy exposure (Edreva 2005; Zhang et al.2011). In the present study, salt-induced oxidative stress was visualized by the overproduction of H2O2and MDA in tomato chloroplasts over the salt-free control (Fig. 8-A and B), suggesting that chloroplast membrane functionality and integrity were severely affected. This is in agreement with the reports in processing tomatoes (Diao et al. 2014) and in Oryza sativa L. (Wang et al. 2014). The application of BSO further exacerbated the oxidative stress caused by NaCl and enhanced oxidation degradation of lipids in the chloroplasts. To protect from oxidative stress, enzymatic and non-enzymatic ROS scavenging systems evolved in plant chloroplasts. The above systems play an important role in maintaining the redox state of chloroplasts and protecting thestructure and function of the membrane system (Mittler et al.2004; Asada 2006; Diao et al. 2014). In chloroplasts, SOD,POD, CAT and the AsA-GSH cycle, Trx, and Grx systems mainly scavenge ROS and modulate the redox balance.Enhanced antioxidant defense is one of the mechanisms by which plants adapt to adverse environmental conditions including salt stress and this is important to prevent injury to photosynthetic apparatus (Foyer and Noctor 2009; Foyer and Noctor 2011).

Our previous work indicated that salt-induced H2O2and MDA accumulation at the tissue level of tomatoes is due to the inhibited activities of SOD and H2O2-scavenging enzymes (including POD, CAT, and APX) (Zhou et al. 2017).The present results also showed that salt stress significantly decreased H2O2-scavenging enzymes (including SOD, POD and CAT), four AsA-GSH cycle enzymes (including APX,MDHAR, DHAR and GR), and Grx systems (including GPX and GR) in chloroplasts. This disagrees with a report on processing tomatoes (Diao et al. 2014), which indicated that salt stress-induced oxidative stress in the chloroplasts of processing tomatoes is due to the inhibited activities of MDAR, DHAR, and GR, and enhanced activities of SOD and APX. This difference might be attributed to the different cultivated tomato types used. However, application of GSH significantly increased SOD, POD, CAT, and the key enzymes of the AsA-GSH cycle and Grx systems in the chloroplasts of salt-stressed seedlings, which is consistent with an increase in ROS detoxification. BSO sprayed on seedlings that were salt stressed further exacerbated the oxidative stress induced by salt stress, and therefore, all measured enzyme activities significantly decreased in the chloroplasts, whereas exogenous GSH sprayed on seedlings increased activities of all measured enzymes(except for POD) in the chloroplasts and stimulated the detoxification of oxidative degradation of lipids in the saltstressed seedlings sprayed with BSO. Our results further confirmed that exogenous GSH is able to induce a high ROS detoxification capacity in the chloroplasts of salt-stressed tomato seedlings and this might be an important mechanism for GSH to alleviate salt-induced oxidative stress and enhance tolerance to salinity.

An important factor for quenching excess ROS is maintaining a high reducing power level for plants. GSH has a low-molecular weight and acts directly as an antioxidant,maintaining homeostasis among different components of the antioxidant system, and plays an important role in scavenging and removing toxic products before membrane damage occurs in the chloroplasts (Foyer and Noctor 2011;András et al. 2012). Plants maintain an appropriate redox environment and reduce the oxidative stress caused by biotic and abiotic stress because of higher GSH content and GSH/GSSG ratio. Previous studies showed that a higher leaf GSH level reduces oxidative stress induced by salt stress (Babu and Rangaiah 2008; Fatma et al. 2014).Cell membrane properties improved following exogenous GSH application (Wu et al. 2011; Alla and Hassan 2014),and oxidative stress was reduced by high endogenous GSH levels under different abiotic stresses (Wu et al. 2011;Hasanuzzaman et al. 2014). Our previous studies show that exogenous GSH maintains leaf GSH redox homeostasis and helps to meet the increasing demand for GSH by regulating GSH synthesis and regeneration, thus increasing tomato resistance to the damaging oxidative effects of salt stress(Zhou et al. 2017). In the present study, exogenous GSH and BSO increased and decreased GSH contents and GSH/GSSG ratios in the chloroplasts of salt-stressed tomato seedlings, respectively. Furthermore, GSH application also significantly increased the contents of GSH and the ratios of GSH/GSSG in the chloroplasts of the NB treated plants. Meanwhile, lower H2O2and MDA accumulation in NB-stressed seedlings sprayed with GSH was observed,compared with NB-stressed seedlings. The results showed that exogenous GSH could maintain the cellular redox state in the chloroplast and keep the sulfhydryl groups of soluble and membrane protein reduced, thus alleviating the saltstress. Therefore, the alleviation of salt-induced chloroplastmembrane damage following exogenous GSH is associated with a higher level of reducing power, thus protecting the chloroplast from oxidative stress.

5. Conclusion

Saline stress disturbed homeostasis of Na+and Cl–ions, and inhibition of plant growth and reduction of photosynthesis were ameliorated by the exogenous application of GSH. GSH application promoted plant growth and photosynthesis of salt-stressed tomato seedlings mainly by overcoming stomatal limitations,enhancing the efficiency of light utilization and dissipation of excitation energy in the PSII, balancing the absorbed light allocation and regulating chloroplast redox homeostasis and the antioxidant defense system to protect chloroplasts from oxidative damage. Thus, GSH regulates photosynthetic activities at the tissue level and ROS metabolism at the organelle level. This is one of the important mechanisms to mitigate the inhibition of plant growth and the reduction of photosynthesis caused by salt stress. This study shows an interesting effect of GSH in the stress response that should be important not only for a basic understanding of the role of GSH but also for potential use of the chemical in agriculture. Further study using molecular genetic approaches can be undertaken to understand the detailed mechanism of GSH-induced salt stress amelioration and detoxification.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31360478), and the International Cooperation Project of Xinjiang Production and Construction Corps, China (2014BC002).