*2.9. E*ff*ects of GABA Priming on Gene Expression under Salinity, Osmotic Stress and OS*+*S*

In order to further understand the molecular mechanism by which GABA priming could alleviate the detrimental effects of osmotic stress, salinity and their combination, we investigated the transcript levels of *APXa*, *CATa*, *SOD1* (Figure 3D–F)*, PAL1*, *PPO*, *SKDH* (Figure 3J–L), *IbCAD1*, *SbGST*, *Chi2* (Figure 4D–F), *OsCIPK01*, *OsCIPK02*, *OsCIPK03*, *OsCIPK07*, *OsCIPK08*, *OsCIPK09*, *OsCIPK12*, *OsCIPK15*, *OsCIPK17* genes (Figure 5A–I). As shown in Figure 3D–F, *SOD1*, *CATa* and *APXa* were significantly up-regulated in the GABA-primed seedlings under osmotic stress, salinity and OS+S stress relative to their controls. In the primed seedlings, *SOD1* was significantly up-regulated under osmotic stress as compared with salinity and the combined stress, while *CATa* was highly significantly up-regulated under the combined stress, whereas the *APXa* was highly significantly up-regulated under the salinity stress. The transcript levels of these genes under different treatments are somewhat consistent with the activity of their corresponding enzymes activity (Figure 3A–C). Under the unstressed conditions, priming with 0.5 mM GABA up-regulated *SOD1*, *CATa* and *APXa* by 62.26, 17.00 and 19.17-fold, respectively as compared with the control condition. The transcription level of *SOD1* was up-regulated in the GABA-primed seeds under the osmotic stress, salinity and OS+S by 22.24, 24.82 and 32.33-fold, *CATa* by 70.04, 69.64, and 64.74-fold; and *APXa* by 32.13, 63.37 and 45.67-fold, respectively (Figure 3D–F). Similarly, priming seeds with 0.5 mM GABA resulted in up-regulation of the *PAL1*, *PPO* and *SKDH* genes under osmotic stress, salinity and OS+S stress relative to their controls (Figure 3J–L). The transcription level of *PAL1* and *SKDH* greatly increased under salinity and combined stress without significant differences between them. In contrast, *PPO* expression was greatly increased under the combined stress. In the absence of stress conditions, priming with 0.5 mM GABA resulted in up-regulation of the *PAL1*, *PPO* and *SKDH* genes by 3.06, 2.85 and 8.00-fold, respectively, as compared with the control condition. The relative expression level of *PAL1*, *PPO* and *SKDH* genes was up-regulated in the primed seedlings under the osmotic stress, by 50.07, 50.57 and 47.72-fold; salinity by 32.39, 38.15 and 32.81-fold, and OS+S by 28.38, 32.53 and 27.58-fold, respectively, as compared with their controls (Figure 3J–L). The transcription levels of *ibCAD1* (Figure 4D) and *Chi2* (Figure 4F) in the GABA-primed plants were highly significantly up-regulated when the seedlings were exposed to salinity stress as compared to osmotic stress and the combined stress, while the transcription level of *SbGST* was highly significantly up-regulated in the GABA-primed plants when exposed to osmotic stress as compared with salinity and combined stress (Figure 4E). Osmotic stress, salinity, and OS+S, priming with 0.5 mM GABA up-regulated *ibCAD1* by 36.55, 41.66 and 29.77-fold; *SbGST* by 64.19, 25.14, and 36.91-fold; and *Chi2* by 48.70, 64.02 and 60.46-fold, respectively, as compared with the controls (Figure 4D–F). The results reported that the *OsCIPK* genes in the GABA-primed seedlings were up-regulated under the osmotic, salinity and OS+S conditions as compared to the controls (Figure 5). Salinity stress resulted in significant increases in the expression level of the *OsCIPK01*, *OsCIPK03*, *OsCIPK08* and *OsCIPK15* genes as compared to osmotic stress and the combined stress (Figure 5A,C,E,H). Osmotic stress resulted in a greater increase in the expression levels of *OsCIPK02*, *OsCIPK07* and *OsCIPK09* genes as compared with salinity and the combined stress (Figure 5B,D,F), whereas the combined stress (OS+S) resulted in a significant up-regulation of *OsCIPK12* and *OsCIPK17* genes as compared with salinity and osmotic stress (Figure 5G,I).

**Figure 5.** Effects of GABA treatment on the transcription levels of *OsCIPK01* (**A**), *OsCIPK02* (**B**), *OsCIPK03* (**C**), *OsCIPK07* (**D**), *OsCIPK08* (**E**), *OsCIPK09* (**F**), *OsCIPK012* (**G**), *OsCIPK015* (**H**) and *OsCIPK017* (**I**) genes in rice seedlings exposed to salinity, osmotic stress, and their combined stress (OS+S). OS (Osmotic stress); S (Salinity).
