*2.10. E*ff*ects of GABA Priming on the Nuclear DNA Content and Ultramorphology of the Cell under Salinity, Osmotic Stress and OS*+*S*

In order to investigate whether the osmotic stress, salinity and their combination could inhibit cell cycle progression, the nuclear DNA content was analyzed using the flow cytometry technique. The results showed that seedlings without GABA priming under osmotic stress and salinity and their combination underwent changes in cell cycle progression (Figure 6A–D). Under the osmotic stress condition (Figure 6B), the cells were blocked in at the G2/M phase, but the cells showed pronounced nuclear accumulation at the G0/G1 phase. However, with GABA priming and under the osmotic stress (Figure 6F), the nuclear accumulation was more obvious in both phases of the cell cycle. The salinity and the OS+S stresses induced a pronounced cell accumulation in G2/M and a sharp inhibition in the cell progression through the G0/G1 phase irrespective of the priming treatment (Figure 6C,D,G,H). The flow cytometry analysis indicated that the G0/G1 phases were more sensitive under the salinity alone or the combined stress as compared to the osmotic stress. These findings suggested that the

osmotic stress, salinity and their combination result in a longer time for cells to progress through the cell cycle. However, each treatment affects cell cycle progression in a different way. The cell ultrastructure was affected due to the priming, salinity and the osmotic stress treatments (Figure 7). Under the control condition (Figure 7A) and priming treatment (Figure 7B), the transmission electron microcopy (TEM) analysis showed clear cell walls and developed chloroplasts (Chl) with uniform thylakoids (Thy). Under the salinity and the combined stress (OS+S), in the unprimed plants (Figure 7C,E), an unclear cell wall, a lot of vacuoles and raptured chloroplasts were observed. However, under the salinity and the combined stress (Figure 7D,F), the priming treatment somewhat improved the cell structure, which was represented by a clear cell wall, developed starch grain and the absence of the vacuoles. These results suggested that the priming with 0.5 mM GABA led to changes in the cell cycle progression and cell ultramorphology as a kind of cell signaling under salinity, osmotic stress and their combination.

**Figure 6.** Flow cytometric analysis of GABA-unprimed rice (**A**–**D**) showing the nuclear DNA content of the root cell under control (**A**); osmotic stress (**B**); salinity (**C**) and their combined stress (**D**) and GABA-primed rice (**E**–**H**) under control (**E**); osmotic stress (**F**); salinity (**G**) and their combined stress (**H**).

**Figure 7.** Transmission electron microscopic images of the leaf mesophyll of rice seedlings primed with 0.5 mM of GABA and grown under the control, salinity and the combination stress (OS+S) conditions. Control condition (**A**); primed with 0.5 mM GABA (**B**); salinity stress (**C**); salinity stress and primed with 0.5 mM GABA (**D**); combined stress (OS+S) (**E**); Combined stress (OS+S) and primed with 0.5 mM of GABA (**F**).
