*4.8. Measurement of Na*<sup>+</sup> *and K*<sup>+</sup> *Ions in the Leaf and Root Tissues*

The concentrations of Na<sup>+</sup> and K<sup>+</sup> in leaf and root were measured according to the methods of Zhao et al. [98]. Briefly, the samples were washed with distilled water before the determination to remove any traces of the Na<sup>+</sup> from the leaf and root tissues, which were then dried at 50 ◦C for 4 d. Thereafter, the dried leaf and root tissues were ground into a fine powder in liquid nitrogen. The powder was then digested in 5 mL nitric acid overnight. Thereafter, the digested solution was diluted to 25 mL with double-distilled water. The concentration of Na<sup>+</sup> and K<sup>+</sup> in the acid-digested tissues was measured using a flame photometer according to the method of Zhao et al. [98].

#### *4.9. Analysis of Gene Expression*

In order to further study the mechanism by which GABA can alleviate the effects of both salinity and osmotic stresses alone or in combination on rice germination and seedling growth, the antioxidant enzymes, detoxification-related enzymes, phenolic metabolism-related enzymes and *OsCIPK* responses genes were investigated at the molecular levels. For this purpose, frozen leaf tissues (100 mg each) were ground thoroughly in liquid nitrogen using a pestle and mortar. Thereafter, the total RNA was isolated from the leaf and the concentration of the RNA was determined by a NanoDrop 2000/2000c (Thermo Scientific, Wilmington, Delaware, USA). The RNA purity was also checked by the spectrophotometer using the 260/280 nm ratio before quantitative real-time PCR. The primers of the *OsCIPK* genes presented in Supplementary Table S1 are the same as those used previously by Xiang et al. [75]. Quantitative real-time RT-PCR was performed using SYBR premix EX Taq (Takara, Japan). The PCR program used in this study is the same as that used recently by Sheteiwy et al. [88].
