*Article* **GABA-Alleviated Oxidative Injury Induced by Salinity, Osmotic Stress and their Combination by Regulating Cellular and Molecular Signals in Rice**

**Mohamed S. Sheteiwy 1,2, Hongbo Shao 1,3,4,\*, Weicong Qi 1, Yousef Alhaj Hamoud 1, Hiba Shaghaleh 1, Nasr Ullah Khan 1, Ruiping Yang <sup>3</sup> and Boping Tang <sup>3</sup>**


Received: 20 September 2019; Accepted: 11 November 2019; Published: 14 November 2019

**Abstract:** This study was conducted in order to determine the effect of priming with γ-aminobutyric acid (GABA) at 0.5 mM on rice (*Oryza sativa* L.) seed germination under osmotic stress (OS) induced by polyethylene glycol (30 g/L PEG 6000); and salinity stress (S, 150 mM NaCl) and their combination (OS+S). Priming with GABA significantly alleviated the detrimental effects of OS, S and OS+S on seed germination and seedling growth. The photosynthetic system and water relation parameters were improved by GABA under stress. Priming treatment significantly increased the GABA content, sugars, protein, starch and glutathione reductase. GABA priming significantly reduced Na<sup>+</sup> concentrations, proline, free radical and malonaldehyde and also significantly increased K<sup>+</sup> concentration under the stress condition. Additionally, the activities of antioxidant enzymes, phenolic metabolism-related enzymes, detoxification-related enzymes and their transcription levels were improved by GABA priming under stress. In the GABA primed-plants, salinity stress alone resulted in an obvious increase in the expression level of Calcineurin B-like Protein-interacting protein Kinases (*CIPKs*) genes such as *OsCIPK01*, *OsCIPK03*, *OsCIPK08* and *OsCIPK15*, and osmotic stress alone resulted in obvious increase in the expression of *OsCIPK02*, *OsCIPK07* and *OsCIPK09*; and OS+S resulted in a significant up-regulation of *OsCIPK12* and *OsCIPK17*. The results showed that salinity, osmotic stresses and their combination induced changes in cell ultra-morphology and cell cycle progression resulting in prolonged cell cycle development duration and inhibitory effects on rice seedlings growth. Hence, our findings suggested that the high tolerance to OS+S is closely associated with the capability of GABA priming to control the reactive oxygen species (ROS) level by inducing antioxidant enzymes, secondary metabolism and their transcription level. This knowledge provides new evidence for better understanding molecular mechanisms of GABA-regulating salinity and osmotic-combined stress tolerance during rice seed germination and development.

**Keywords:** rice; salinity; osmotic stress; combined stress; GABA; phenolic metabolism; *CIPKs* genes

#### **1. Introduction**

Rice is one of the most important cereal crops that serves as the staple food for almost half of the world's population. Rice is not a salt-tolerant crop, but is suited for cultivation in affected saline soils due to its highly consumption of fresh water for most of the growing season, which could dilute the salts and increase the availability of essential nutrients such as Fe, Mn, N, and P, which contribute to improving rice growth and yield. The mechanism by which rice can tolerate salinity stress is mainly related to the maintenance of ion homeostasis, predominantly low Na+/K<sup>+</sup> or high K+/Na<sup>+</sup> ratios, through exclusion, compartmentation, and partitioning of Na<sup>+</sup> [1]. In addition, rice plants can tolerate salt by ion exclusion which mainly involves Na<sup>+</sup> and Cl<sup>−</sup> transport processes in roots and prevention of the excess accumulation of Na<sup>+</sup> and Cl<sup>−</sup> in leaves [2], as well as osmotic stress tolerance which maintains leaf expansion and stomatal conductance [3]. However, rice productivity is affected by salinity stress, which originates from the accumulation of underground salt and is exacerbated by salt mining, deforestation and irrigation [4]. The tolerance limit of rice to saline conditions may vary among the different growth and developmental stages. In this regards, Zhu et al. [5] reported that rice is more tolerance to salinity during the germination and tillering stages, whereas it seems to be more sensitive during early vegetative and reproductive stages.

Salinity, being an important environmental factor, severely causes a significant reduction in the seed germination, seedling growth and development of rice. It has been reported that more than 800 million hectares of the global cultivated area are severely affected by salt stress [2]. A previous study has showed that rice plants experience osmotic stress in saline soil as a result of reduced osmotic potential of the soil solution, and ultimately reduced water uptake by plants [6]. Under the salinity stress condition, the photosynthetic rate decreased due to stomatal closure and resulted in limited availability of CO2 and thus altered carbohydrate content of the leaf [7]. Plants can adapt to these conditions by accumulation of compatible solutes such as proline and starch, which function as osmoprotectants and have a vital role in plant adaptation to osmotic stress through stabilization of the tertiary structure of proteins [2]. Salinity reduces the ability of plants to take up water, which leads to increasing of osmotic substances, and causes inhibition of plant growth rate accompanied by metabolic changes similar to those induced by osmotic stress [8]. This action of salinity can induce osmotic stress, oxidative damage, stomatal closure, inhibition of photosynthesis, and damage of cellular structures, and decreased gas exchange rates [9]. Osmotic stress can cause a significant crop yield loss worldwide. It can reduce the plant productivity and seedling growth [10] by affecting the stomatal closure and photosynthesis process [9]. In the present study, PEG (6000) was used to induce osmotic stress in rice plants, being frequently used to induce osmotic stress in several plant species [11,12]. Moreover, the osmotic stress induced by PEG can reduce the photosynthetic rate and chlorophyll content by inhibiting the electron transport system.

The feedback regulation of plants to the combination of salinity and osmotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the two stresses applied individually [13]. The physiological response of barley was investigated under combinations of two different abiotic stresses [8]; however, the molecular mechanism of plant adaptation to a combination of two different stresses remains a matter of debate [14]; this adaptation might require conflicting or antagonistic responses [14,15]. As such, plants can be adapted to heat stress by increasing the transpiration rate through opening stomata to recover from the high temperature of their leaves. Nevertheless, when plants are exposed to the combination of heat and osmotic stresses, plants have to close their stomata to reduce water loss under the osmotic stress condition [14].

GABA accumulates rapidly in response to biotic and abiotic stresses [16]. Despite the rapid accumulation of GABA during stresses, the specificity of the response and the specific role of GABA under these conditions are still elusive [17]. The accumulation of GABA in different plant cells under the osmotic stress condition requires a specific response which can act as a signaling molecule by modulating the activity of H+-ATPase and regulating stomatal movement [17,18]. This action of GABA accumulation has been observed in plants under different environmental stresses such as osmotic

stress, oxygen deficiency, mechanical stimulation, low temperature and pathogen attack [16,19]. As a physiological response under abiotic stress, GABA plays vital roles in plants for maintaining the C/N balance, regulating cytosolic pH and scavenging reactive oxygen species (ROS) [20]. Under salinity stress, exogenous GABA application acts as a signaling molecule and functions in phenolic compound enrichment [21], scavenging of ROS [22] and modulating antioxidant enzyme activities in nitrogen metabolic pathways [23]. Recent studies reported that GABA has been implicated in signaling processes affecting the nitrate-uptake system [24] and guidance of the pollen tube [25].

Salinity and osmotic stresses have a significant devastating effect limiting worldwide crop production. Taking into consideration the expected increasing world population and food demand, finding ways to improve crop tolerance to abiotic stress constraints is an urgent issue for further improving agricultural production and enhancing global food security. GABA metabolism could be involved in regulation of plant development under abiotic stress through regulation of C and N metabolism [26]. Therefore, the present study hypothesized that GABA could be involved in rice tolerance to salinity and osmotic stresses and their combination. Moreover, the *CIPKs* genes were frequently expressed and participated in plant growth and development during abiotic stresses such as heat, drought, salinity, and chilling stresses [27]. For this reason, we have investigated the effects of GABA on rice *CIPK* genes in responses to the combination of salinity and osmotic stresses to evaluate the potential usefulness of the stress-responsive *CIPK* genes in genetic improvement of stress tolerance. In addition, the present study hypothesized that high accumulation of secondary metabolites during salinity and osmotic stress could facilitate osmotic adjustment and ultimately increase rice tolerance to the combined stresses. This study was undertaken to elucidate the mechanism by which exogenously supplied GABA is involved in the stresses tolerance, in the context of regulation of Na<sup>+</sup> and K<sup>+</sup> balance, the photosynthetic system, antioxidant system, cell cycle development, cellular regulation, and controlling stomatal conductance which ultimately might result in improving rice growth under the stress conditions. The current study could significantly contribute to further understanding the tolerance mechanism induced by priming rice seeds with GABA under salinity and osmotic stresses.

#### **2. Results**
