**3. Discussion**

Enhancing plant tolerance to abiotic stresses by inducing a stress-responsive gene pathway in transgenic plants is a promising approach [28]. Combined stress is a new kind of abiotic stress in plants that requires integration defense or cross-talk response, and the plants should be tested for their tolerance to a combination of different stresses prior to cultivation under field conditions. The present study reported that priming with 0.5 mM GABA improved the physiological parameters under salinity and osmotic stress and their combination as compared with unprimed plants (Table 1). During seed germination and the stress condition, GABA could improve starch catabolism and mobilization of sugar and amino acids which are necessary for seedling growth [29]. GABA can also improve the antioxidant system for mitigation of oxidative damage, and increase Na+/K<sup>+</sup> transportation for osmotic regulation under salt stress [29]. Furthermore, GABA could maintain the hormones and mineral nutrients and reduce lipid peroxidation under different environmental stresses [30]. During the early stage of seed imbibition, the up-regulation of some germination-related genes contributed to the metabolic process prior to seed germination which could improve the germination and seedling growth under the stress

condition [31]. The reduction in rice growth from seed germination to the maturity stage under salinity stress may be due to the increase in osmotic pressure of the root medium and ion effects [32]. Several studies have reported that osmotic stress significantly decreased fresh and dry weights of shoots [33] and roots [34]. It has been found that exogenous GABA could significantly improve the shoot length, root length, and fresh weight of maize seedlings [35] and white clover [29] under abiotic stresses. In the present study, GABA induced starch catabolism, which is of primary importance for providing available carbohydrates for seed germination and growth under osmotic stress, salinity and the combined stress. These findings are consistent with previous studies, which found that environmental stresses such as osmotic stress, salt and heat stress decreased seed germination due to the inhibition of starch catabolism under these conditions [29,36,37]. It has been found that the metabolite mobilization of starch and soluble sugars is critical for the maintenance of cell turgor and energy sources when seeds are subjected to salinity and osmotic stress [36]. Additionally, GABA could increase starch catabolism and provide available carbohydrates for seed germination and growth of white clover under salt stress [29]. Moreover, starch catabolism could be accelerated by activating α- and β-amylase activities in GABA-pretreated seeds [29]. It is well-known that both organic and inorganic osmolytes such as sugars and starch are important osmotic regulators for plant adaption to environmental stresses [38–40]. These osmolytes were decreased in the present study under salinity, osmotic stress and OS+S, and were enhanced by priming with 0.5 mM GABA (Table 3). It has been found that the accumulation of osmolytes such as free starch, soluble sugars and protein regulated the osmotic pressure of the plants under abiotic stress including salinity and osmotic stress [41]. Furthermore, plants under stress conditions may accumulate small molecular weight proteins that could be used as a source of storage nitrogen and could be rapidly mobilized when required for the alleviation of stress [42]. These proteins could also have a role in osmotic adjustment [43]. Additionally, proline can also accumulate in plants to act as a solute for adaptation of plants in response to different environmental stresses [44].

The photosynthetic response to abiotic stress, especially salinity and osmotic stress, is highly complex. It involves the interaction of stress signaling with different plant cells which can promote plant growth and development. In the present study, rice plants grown under salinity, osmotic stress and OS+S treatments showed a marked reduction in Pn, gs and Tr (Table 2). The remarkable reduction in gs may be a consequence of stomatal closure caused by the higher osmotic pressure in guard cells under OS+S [45]. Another study observed a decrease of the chlorophyll content in rice under water stress, which may be related to the inhibition of the photosynthetic system of the plant under the water stress [46], or might be due to increases in ROS and lipid peroxidation levels leading to chlorophyll damage and a change in the leaf color from green to yellow [47]. In the present study, RWC was decreased by the salinity and osmotic stress and OS+S, and enhanced by GABA priming as compared with the control condition (Table 2). A decrease in the RWC under PEG-induced osmotic stress was also reported in rice leaves [46] and in tomato [48]; this might be due to the decreased water potential under salinity and osmotic stresses [49]. Similarly, the cellular accumulation of GABA could achieve a balance in the reduction of water potential that occurs during cellular dehydration under the stress condition [50].

Rice controls the transport of salts initially by selective uptake by root cells and ions entering into the root along with water through symplastic and apoplastic routes [51]. The ratio of Na+/K<sup>+</sup> can be used as a physiological index for salt response in several crop plants such as tomato (*Solanum lycopersicum*) [52], chickpea (*Cicer arietinum*) [53], barley (*Hordeum vulgare*) [54] and white clover (*Trifolium repens*) [29]. Moreover, the concentration of Na<sup>+</sup> is key for the salinity tolerance mechanism, interacting with K<sup>+</sup> homeostasis, and especially given its involvement in numerous metabolic processes, maintaining a balanced cytosolic Na+/K<sup>+</sup> ratio [55]. In the present study, priming with 0.5 mM GABA under salinity, osmotic stress and OS+S caused a significant reduction in the Na<sup>+</sup> concentration in the leaf and root as compared with unprimed seeds (Figure 2). Under the salinity stress, plant cell loses the balance in the Na+/K<sup>+</sup> ratio due to a continuously increasing Na<sup>+</sup> ion concentration and decrease in K<sup>+</sup> ion uptake [56]. In the current study, a higher concentration of Na<sup>+</sup> was recorded in

roots as compared to the leaf under the salinity and osmotic stress and their combination (Figure 2). The high accumulation of Na<sup>+</sup> especially in the leaf resulted in a reduction in the photosynthesis due to stomatal and non-stomatal limitation [57]. Therefore, plants have to reduce the concentration of Na<sup>+</sup> in the plant leaves by either minimizing the entry from the root symplast to reduce loading, maximizing Na<sup>+</sup> retrieval from the xylem [58], or exporting Na<sup>+</sup> from the leaf to the phloem [59]. As such, the down-regulation of genes encoding Na<sup>+</sup> influx transporters (*OsCNGC1*) in rice root significantly contributed to the salinity tolerance, as it could avert toxic Na<sup>+</sup> influx [60].

In the present study, antioxidant enzymes were measured to investigate the capability of priming with 0.5 mM GABA to induce such enzymes for mitigating the oxidative stress by scavenging ROS in the cell and thus increase the rice tolerance under the salinity, osmotic stress and their combination. In the current work, salt and osmotic stress and their combination (OS+S) significantly decreased the activity of CAT, SOD and APX (Figure 3), which was consistent with that of barely exposed to salinity stress [21]. However, the decrease in these enzymes was inhibited when the seeds were primed with 0.5 mM of GABA under the stress conditions (Figure 3). Similar findings were observed in different plant species primed with GABA such as rice seedlings [61], black pepper seedlings [62], perennial ryegrass [63] and white clover [29]. The accumulation of ROS and oxidative stress signal are the main common mechanisms for the plant tolerance could be observed under the salinity and osmotic stresses. The intensive accumulation of ROS in the different plant cells resulted in significant pigment loss, a reduction in the photosynthetic system efficiency and decreased protein assimilation [64]. However, induction of the antioxidant defense system could protect plants from oxidative damage induced by ROS accumulation under stress conditions [65]. In the present study, salinity and osmotic stress and OS+S improved the ROS, i.e., H2O2, O2 − and OH− accumulation in the leaf tissue as compared with the control condition (Table 2). The present study reported that GABA-activated antioxidant enzymes could play vital roles in scavenging free radicals induced by H2O2 and O2 − and reduced lipid peroxidation by up-regulating the genes (*APXa*, *CATa* and *SOD1*) involved in antioxidant enzymes during rice seed germination and seedling growth under salinity and osmotic stress and their combination (Figure 3).

The accumulation of phenolic compounds under environmental stresses can protect plants from damage caused by ROS-induced oxidative stress [66] by scavenging free radicals, breaking radical chain reactions, and decomposing peroxides [67]. In the present study, the enzymes involved in the phenolic metabolism such as PAL, PPO, SKDH and CAD were improved by priming with 0.5 mM GABA under the osmotic, salinity and OS+S stresses (Figure 3G–I and Figure 4A). This phenolic metabolism may provide an effective defense tool for plant tolerance under environmental stresses [68]. Recently, SKDH activity was increased in plants exposed to salinity stress for 3 days [69]. Moreover, the activity and the expression level of the *PAL* protein were increased in barley exposed to 200 μM of Al for 24 h [70]. The increased PAL and PPO levels might help plants to cope with oxidative stress by scavenging ROS [45,71]. In the present study, the accumulation of PPO in the leaf under salinity and osmotic stresses (Figure 3H), might be due to the induction of *PPO* genes in the leaf tissues under osmotic stress [72]. However, the PPO and CAD activities were not affected in *Matricaria chamomilla* plants exposed to salinity stress, but a higher activity of SKDH was observed in the root [68]. In the present study, the induction of the defense genes involved in secondary metabolites such as *PAL1*, *PPO*, *SKDH* and *IbCAD1* in the primed plants may increase the tolerance of plants to salinity, osmotic stress and OS+S (Figure 3J–L and Figure 4B), which is consistent with the finding of Ahammed et al. [71], who also found that plant growth regulators help in alleviating the oxidative stress in plant leaves through the induction of phenolic metabolism defense under the stress condition. The mechanism by which GABA treatment increases the tolerance to salt stress might be due to the ability of GABA to induce endogenous GABA, proline and the total phenolic content, thus enhancing the antioxidant capacity [67]. The present study revealed that the GST and chitinase activities and their transcript levels were increased by priming with 0.5 mM of GABA under salinity, osmotic stresses and their combination (Figure 4). Similarly, a previous study reported that the *Chi2* gene was up-regulated in pepper leaves under salinity and osmotic stresses which could protect plant tissues against osmotic stress via an

ABA-independent signal transduction pathway [73]. Another study reported that chitinase is involved in heavy metal stress tolerance and chilling tolerance [74]. Moreover, GST activity and expression level increased in salinity and osmotic -stressed barley plants [45].

In the present study, up-regulation of *OsCIPK* genes was observed in the GABA-primed seedlings under salinity and osmotic stress and OS+S as compared to their controls (Figure 5). The *OsCIPK* genes induced by different stresses may provide new signaling pathway to reveal the molecular mechanism of rice response to different stresses alone or in combination considering the nature of *CIPKs* as putative signaling components [75]. The signaling pathway of *OsCIPK* genes may be involved in the substantial common regulatory systems or cross talks triggered by different stresses [75]. Our findings indicated that expression patterns of *OsCIPK* genes were induced under the salinity, osmotic stress and OS+S, which is consistent with cross talk between salinity and osmotic stress as previously reported by Seki et al. [76]. The interaction between co-activated pathways is likely to be mediated at different levels under the combined stresses [13]. This pathway could include the interaction between different transcription factors and mitogen-activated protein kinase (MAPK) cascades [77], different stress hormones such as ethylene, jasmonic acid and abscisic acid [78], between calcium and/or ROS signaling [79] and between different receptors and signaling complexes [80].

The present study revealed that nuclear accumulation was inhibited under the stress condition especially under salinity and the combined stress in the G0/G1 as compared with the control or osmotic stress (Figure 6). A recent study showed that root growth was inhibited under the abiotic stress conditions, and the cell division and cell cycle regulation might be involved in this inhibition [80], or might be associated with the reduction of cell production [81]. The reduced cell production under the salinity and osmotic stress and their combination might be due to a smaller number of dividing cells such as a meristem size reduction, and the temporary inhibition of mitotic activity that allows the adaptation to the stress condition is most likely mediated by post-translational control of cyclin-dependent kinase activity (CDK) [80].
