Xinjiang Province is one of the main producing regions of processing tomatoes in China. However, because of excessive use of fertilizers, unreasonable methods of irrigation and drainage, and the special climatic conditions (such as dryness due to less rain and strong evaporation in Xinjiang), the area is threatened by soil salinization, and soil secondary salinization has exceeded more than half of the total cultivated land dedicated to processing tomatoes. In recent years, this salinization problem became more and more serious, as it results in declines in photosynthetic rates, environmental resistance, and yield and quality of processing tomatoes. Increasing levels of salt in soil have become a primary abiotic stress that limits the productivity of processing tomatoes in Xinjiang Province (Pang et al., 2005). High salinity inhibits crop growth by disrupting a range of physiological processes involved in cell metabolism, especially photosynthesis (Thagela et al., 2017; Yang et al., 2020). Photosynthesis is the primary metabolic mechanism in plants (Singh et al., 2014) and is particularly sensitive to salinity (Ashraf and Harris, 2013). Salinity causes stomatal closure, destruction of chlorophyll pigments, inhibition of photochemical efficiency of photosystem II (PSII), and CO₂ assimilation (Dietz and Pfannschmidt, 2011). The presence of salt increases the buildup of reactive oxygen species (ROS) (Oukarroum et al., 2015; Hossain et al., 2017). The accumulated ROS severely damage the ultrastructure of photosynthetic apparatus, disrupting the use of light energy and causing the photoinhibition of PSII, consequently leading to plant growth retardation, and even cell and plant death (Jiang et al., 2017). Therefore, it is very important for plants to increase their salt tolerance and maintain their economic yield to counteract the adverse effects of salt stress on photosynthesis.

Reduced ascorbic acid (AsA) is by far the most abundant low-molecular weight antioxidant in plants, and AsA plays a key role in protecting plants from ROS generated during normal metabolic processes and under stress conditions including wounding, pathogen attack, salt, heat, light, and ozone exposure (Conklin, 2010). As an efficient electron donor and important non-enzymatic antioxidant, AsA constitutes a redox buffer that keeps the intracellular environment reduced and maintains the cellular redox homeostasis by directly or indirectly scavenging ROS (including H₂O₂, O2-, ·OH⁻, and ¹O₂). AsA can act as a signaling agent participating in the interaction with the environment (Linster and Clarke, 2008). AsA is also associated with several biosynthetic pathways, such as the xanthophyll cycle, which guard against the negative effects of surplus energy from excitation. Furthermore, many authors have demonstrated in the past years that AsA still exerts other significant roles in a wide range of processes, such as plant growth control, α-tocopherol regeneration, cell division and expansion regulation, resistance to insect feeding (Foyer and Graham, 2011), carbon–nitrogen metabolism, fruit quality and seed germination, as well as the expression of genes related to hormone synthesis and signal transduction. The cellular AsA pool size in plants can be regulated by the coordinated action of many related enzymes, and an enhanced AsA level has been confirmed to increase the tolerance and adaptation of crops to abiotic stresses such as high light intensity, cold, drought, salinity, UV-B, and ozone stresses (Xie et al., 2009). Similarly, exogenous application of AsA to plants has been shown to confer tolerance to several abiotic stresses in many crop plants (Xie et al., 2009; Wang et al., 2014; Alhasnawi et al., 2016; Akram et al., 2017; Penella et al., 2017). Our previous research demonstrated that optimal concentrations of exogenous AsA could relieve the salt-induced inhibition on the seedling growth of processing tomatoes, increasing its adaptability to salt stress (Supplementary Materials). It also showed that tomato salt tolerance improvement induced by exogenous selenium, glutathione (GSH), and nitric oxide (NO) is accompanied by the increase of endogenous AsA content and AsA/dehydroascorbate (DHA) ratio (He et al., 2015; Wen et al., 2018; Zhou et al., 2018).

The mechanisms of salt tolerance are very complex. Recent studies aiming to understand the mechanisms of AsA effects on plant salt tolerance have shown that salt tolerance induced by endogenous AsA or application of exogenous AsA mainly ascribed to the improvement of antioxidant, ion balance, and osmotic balance regulation (Wang et al., 2019). Although the photosynthetic apparatus is the most sensitive component for evaluating the degree of salt stress damage, few studies concerning the protection mechanisms of exogenous AsA on its structure and function under salt stress were carried out and more detailed ones are needed. The fluorescence transient (OJIP) technique is a quick, precise, and non-invasive method for reliably detecting and analyzing the effects of abiotic and biotic stress on the composition and processes of PSII and photosystem I (PSI), such as the redox condition of both photosystems, alterations in the main photosynthetic processes, and the efficiency of the transfer of electrons via the cross-system bond to the electron acceptors on the PSI acceptor node (Brestic et al., 2014). The conditions observed in the JIP test as well as the composition of the OJIP transient are easily affected by various stressors and have also been successfully employed in assessing and screening the tolerance of plants to environmental stresses (Chen et al., 2016; Çiçek et al., 2017; Essemine et al., 2017; Banks, 2018; Maliba et al., 2019). As such, we created tomato plants with various levels of endogenous AsA by applying AsA, the leaves of tomato seedlings that have been exposed to salt. Hence, we constructed tomato plants with different endogenous AsA levels by spraying salt-stressed tomato seedling leaves with AsA, Lycorine (LYC, an inhibitor of key AsA synthesis enzyme l-galactono-γ-lactone dehydrogenase activity), or AsA and LYC. The purpose of this experiment was to use OJIP transient analyses to assess the processes by which AsA impacts the photosystem stability, the processes of the photosynthetic electron transport chain, and the relationship between energy use and expenditure in the photosynthetic system of processing tomatoes subjected to salt stress. Increasing the exogenous AsA to expand PSII stability and regulate the photosynthetic activity and efficiency of the distribution of energy significantly contributed to mitigating the negative effects of salt stress, namely, the inhibition of photosynthetic processes and plant growth.

Growth is the comprehensive embodiment of plant response to stress, and it is also a common index to determine stress tolerance (Liu et al., 2018). Salt stress is one of the primary abiotic factors affecting the quality and yield of crops (Landi et al., 2017). In this study, the results showed that AsA addition can alleviate the deleterious effects of salt stress (Figure 1; Table 2). This observation on the role of AsA in circumventing the adverse effects of salinity on plant biomass coincides with earlier reports (Wang et al., 2014; Terzi et al., 2015; Aliniaeifard et al., 2016). Meanwhile, we found that exogenous AsA application on salt-stress tomato seedlings is accompanied by an increase of endogenous AsA level and AsA/DHA ratio (Figure 2). Significant evidence demonstrates that the intracellular redox condition and the ROS scavenging capacity affect the resistance of abiotic stress in plants (Dietz, 2010). Keeping high redox pools and antioxidative level of GSH and AsA (or GSH/GSSG and AsA/DHA ratios) is necessary for plants to consume surplus ROS and maintain the sulfhydryl groups of membrane and soluble proteins in a lowered condition (Wang et al., 2020a). Our findings demonstrate that the exogenous application of AsA can induce tomato seedling growth under salt-stress conditions, and it was associated with increases in endogenous AsA levels. To further probe the role of AsA, we applied lycorine (LYC, an inhibitor of AsA synthesis key enzyme l-galactono-γ-lactone dehydrogenase activity) on the leaves of salt-stressed tomato seedlings prior or not to AsA treatment. LYC was used to reduce endogenous AsA content (Arrigoni et al., 1997). In this study, we observed that application of exogenous LYC under salt stress exacerbated growth inhibition and also significantly decreased the endogenous AsA level and the ratio of AsA/DHA (Figure 1; Table 2). However, the application of exogenous AsA can also effectively reverse the effects of LYC on the growth of tomato seedlings and the redox state of endogenous AsA (Figures 1, 2). These results give strong evidence that AsA can enhance the resistance of tomato seedlings to salt stress or salt stress with LYC application by mediating high reducing power and ROS scavenging capacity of endogenous AsA and redox state. This is consistent with the reports in barley by Agami (2014).

Under stressful environment, the decline in plant growth often relates to a decrease in photosynthetic capacity (Li et al., 2017; Zahoor et al., 2017). Reduced photosynthesis under salinity is not only attributed to stomatal closure factors but also attributed to non-stomatal limitation factors (Melesse and Caesar, 2010). Our findings demonstrate that a decrease in leaf P_n due to salt (Figure 3A) was mainly induced by non-stomatal factors, since salt-stress response elevated C_i (Figure 3D), even though G_s decreased (Figure 3B). In this experiment, exogenously applying AsA effectively combatted the negative effects of salt stress and NL treatment on C_i, G_s, and P_n in tomato seedling leaves (Figure 3). These results showed that AsA might help to relieve salt-induced photosynthetic inhibition mainly by the regulation of non-stomatal limitation factors, such as the decrease in photosynthetic pigments content, the chloroplast ultrastructure disorganization, the photochemical reaction activity decrease, or the decrease of enzyme activity involved in the process of carbon assimilation.

Chlorophyll molecules have high sensitivity to environmental stresses, and chlorophyll content, due to positive correlation with photosynthesis, is widely used as an index of abiotic tolerance in plants (An et al., 2018; Wang et al., 2020b). High salinity causes an increased ROS generation in chloroplasts, which can destroy chlorophyll. Also, reduction of chlorophyll content induced by salinity stress is due to the enhanced activity of chlorophyll enzymes and the instability of pigment–protein complex (Aghaleh et al., 2009; Hamid and Ashraf, 2010). In addition, salt stress reduces chlorophyll content by decreasing the uptake and accumulation of key nutrients such as Mg²⁺, Fe²⁺, and Cu²⁺, which are involved in the biosynthesis of chlorophyll (Evelin et al., 2012). The activities of Mg-chelatase and ferrochelatase are regulated by the redox state in chloroplast. Under the oxidized state in chloroplast, the activity of Mg-chelatase is inhibited and the activity of ferrochelatase is improved, finally resulting in the inhibition of chlorophyll biosynthesis (Walker and Willows, 1997; Adhikari et al., 2011). In this study, the reduced chlorophyll contents in salt-stressed plant leaves might be due to chlorophyll degradation and chlorophyll biosynthesis inhibition (Table 3). This is in line with what has been reported earlier in Phaseolus vulgaris L. (Taïbi et al., 2016), Gossypium spp (Saleh, 2012), and Brassica chinensis L. (Muhammad and Shafiq, 2014). AsA levels in plants increase when subjected to stress and serve important roles when defending against oxidative stress (Xu et al., 2015). Exogenous application of AsA has been reported to increase the chlorophyll contents in diverse plants (Saeidi-Sar et al., 2012; Ahmad et al., 2013; Kostopoulou et al., 2014). In this study, the exogenous application of AsA counteracted the adverse effects on photosynthetic pigments of salt stress with or without LYC (Figure 3; Table 3), and the AsA application to NaCl-treated or NL-treated tomato seedlings caused a considerable increase in the endogenous AsA content and AsA/DHA ratio (Figure 2), thereby promoting the biosynthesis of chlorophyll and protecting the chloroplasts from oxidative damage. Furthermore, higher carotenoids contents were also observed in the NA-treated and NLA-treated plants compared with the NaCl-treated and NL-treated plants (Table 3). Carotenoids can protect the photosynthetic apparatus from photooxidative damage by quenching the triplet state of chlorophyll and helping dissipate the excessive energy of excitation, thereby preventing the formation of ROS (Derks et al., 2015). Above all, these results suggest that alleviating chloroplasts oxidative stress damage and promoting chlorophyll synthesis, by enhancing the antioxidant capacity and modulating redox state in chloroplast of tomato seedlings under salt stress, is one of the mechanisms by which exogenous AsA could increase the photosynthetic capacity of salt-stressed tomato seedlings.

In order to further decipher the mechanism of action of salt stress on the electron transport chain and the mechanism of how exogenous AsA improve the performance of photosynthetic organs in salt-stressed tomato seedlings, the effects of AsA application on the structure and functioning of the photochemical apparatus of tomato seedlings under salt stress with or without LYC were investigated by rapid chlorophyll fluorescence technique. Our findings indicate that OJIP curve shapes are sensitive to salt stress. The OJIP curve was flatter in salt-stressed leaves than that in the control, because of decreases in F_m (P-step) and increases in F_o (O-step) (Figures 4A, 7). The normalized curve demonstrated relative J-step elevations, which can also be seen in the elevated V_J parameter, compared with that of control (Figure 7). These alterations in the values of F_o, F_m, and V_J suggest that the PSII donor end deteriorates as a result of salt stress and that the capacity of the PSII donor end to give electrons also decreases due to an increase in the closed PSII RCs (Strasser et al., 2010). The appearance of K- and L-bands in the OJIP transient curve of salt-treated leaves, as well as the increases in W_K and W_L values (Figures 5 and 6), also confirms the destruction of OEC and the integrity of the thylakoid membrane in the donor side of PSII, leading to the decrease in the electron donation capacity of the PSII donor side (Gomes et al., 2012; Li et al., 2014), as found for barley and passion fruit under drought stress (Oukarroum et al., 2007). The parameters, such as V_J, M_o, S_m, ϕE_o, and ψ_o, mainly reflect the changes of the acceptor side of PSII. According to the higher V_J in salt-treated leaves (Figure 7), we concluded that the acceptor side of PSII also suffered damage from salt stress. Photoinhibitory damage on the PSII acceptor end of leaves that have been treated with salt is confirmed by our observations that the stress of salt decreases S_m, ϕE_o and ψ_o and increases M_o (Figure 7) (Alaka Srivastava and Strasser, 2003; Oukarroum et al., 2004). Additionally, our results demonstrate that salt stress lowered the maximal redox threshold of PSI (I/I_o and ΔI/I_o), the quantum yield necessary to reduce the end electron acceptors on the PSI acceptor end (ϕR_o), and how efficiently an electron is moved from the intersystem electron carriers to reduce the end electron accepted on the PSI acceptor end (δR_o) (Figure 12). These results indicate that salt stress reduced the capability of the photosynthetic electron transport from the PSII donor side and reduced the PSI end acceptors, reducing the photosynthetic capacity of the leaves. These findings suggest that both donor and acceptor sides of PSII and PSI are the target sites under salinity stress in tomato seedlings. Those results are consistent with the conclusions suggested by Shamshiri and Fattahi (2016) in pistachio (Pistacia vera). However, the application of AsA on the leaves of the tomato seedlings salt-stressed with or without LYC effectively reversed these changes on the donor side and acceptor side of photosystem (including PSII and PSI). These findings confirm that exogenously applying AsA serves an important role during the movement of electrons from the PSII donor end to the PSI acceptor ends. AsA can promote the electron transport activity (ϕE_o and ψ_o), enhance the PQ pool size (S_m), and lower the rate of reduction of Q_A to QA- (Mo) on the PSII acceptor end. As such, there are increases in both the electron transport flux per reaction center (ET_o/RC) and the primary photochemical efficiency (ψ_o), improving the efficiency of photochemical processes in tomato seedlings under salt stress.

Regulating the activity of PSII reaction centers and improving the ability of chloroplasts to absorb and dissipate energy is the key to improving plant salt tolerance. In this study, the increase of ABS/CS_m, TR/CS_m, and ET_o/CS_m in tomato seedlings under salt stress (Figures 9, 10) may be a protective mechanism under salt stress (Sane et al., 2002). In contrast, the decreased ABS/RC and TR_o/RC under salt stress (Figures 9, 10) means that the effective average absorption of the antenna and trapping per active RC are both higher because of certain RC inactivation (Yusuf et al., 2010). The increased DI_o/RC indicates that the proportion of total dissipation to active RCs increases due to high dissipation of the inactive RCs (Gerosa et al., 2003), which is consistent with previous results in cucumber under salt stress (Yuan et al., 2014). However, the negative effects of salt stress (in the presence of LYC or not) on active RC density and the use and expenditure of absorbed energy in PSII RCs were counteracted by exogenous application of AsA. The majority of plants dissipate excitation energy surpluses thermally, along with decreased regulation of PSII activity. This defends the photosynthetic apparatus abiotic environmental stressors, which can damage them. Xu (2017) reported that exogenous AsA can effectively promote xanthophyll loops in wheat under Cr⁶⁺ stress to dissipate excessive excitation light energy, thus avoiding the occurrence of photoinhition. So, our results suggest that AsA may act as a cofactor of violaxanthin de-epoxidase in the xanthophyll cycle (Muller-Moule et al., 2002) which helps protects tomatoes against the harmful effects of excess excitation energy induced by salt stress. Additionally, the role of AsA in regulating the photosynthetic capacity has been extensively studied by assessing patterns in the fluorescence parameters F_v/F_m as well as the performance index PI_ABS. We determined that lower F_v/F_m levels in the leaves of tomatoes due to salt stress were associated with lower levels of PI_ABS (Figure 11), suggesting that salt-induced photoinhibition was due to damaged photosynthetic apparatus caused by excess light energy absorption compared with the level required for photosynthesis and limited component mechanism efficiency like the movement of electrons within a system and the trapping of energy (Song et al., 2003; Campos et al., 2014). The results in Figure 11 show that AsA played a key role in regulating the use and expenditure of energy absorbed by PSII RCs in seedlings subjected to salt stress, both in the presence of LYC and without LYC. This is supported by the increased F_v/F_m and PI_ABS values (Figure 11). Therefore, these findings indicate that photosynthetic defense due to AsA was a result of increased capacity not only to protect photosynthetic processes from the negative effects of salt stress but also to inactivate the RCs of PSII replacement and regulation of the light striking PSII RCs, which is where absorbed energy is captured and energy from the dissipation of heat is transferred to a photochemical reaction. Additional energy from excitation was converted to transport electrons, a process that could provide additional transport capacity for electrons on the PSII donor end. As such AsA serves an important role in protecting the photosynthetic capacity during salt-stress conditions.

In conclusion, this study clearly demonstrated that salt stress lowered the capacity of several parts of the photosynthetic electron transport path. The OEC was damaged, PSII RCs were inactivated, the connections among independent PSII units were decreased, there was little electron transport beyond Q_A, and electron transporters on the PSI acceptor end in the leaves of tomato seedlings were damaged. However, exogenously applying AsA decreased the photoinhibition and lessened the negative effects on photosynthesis. Thus, under salt-stress conditions AsA can enhance salt-stress tolerance by promoting chlorophyll synthesis and alleviate the damage of oxidative stress to chloroplasts by modulating the redox state in chloroplast and dissipating excitation energy in the PSII antennae. AsA can also stabilize the molecular structure of photosynthetic reaction center, keeping the electron transport chain opened and enhancing the electron transfer efficiency, which is helpful to promote the improvement of photochemical activity and photosynthetic performance and the efficient distribution of energy. Moreover, the several beneficial effects of AsA during salt stress make AsA a good candidate for potential use as a chemical in agriculture.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The work presented here was carried out in collaboration between all authors. H-yL, MD, and XC defined the research theme. XC designed methods and experiments, carried out the laboratory experiments, analyzed the data, interpreted the results, and wrote the manuscript. YZ co-designed the experiments, carried out the laboratory experiments, and discussed the analyses and interpretation. YC, PZ, JC, WX, and QS co-worked on the analysis of fast fluorescence induction kinetics curve and JIP test parameters and discussed analyses and interpretation. H-yL and MD conceived and coordinated the study. All authors have contributed to, seen, and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.