sitl1 mutant enhances salinity stress insensitivity
sitl1 mutant showed significantly decreased root biomass and leaf chlorophyll content when plants were grown in NS (Figure 4a-e). In contrast, when plants were treated with NS containing 50 and 100 mM NaCl, the sitl1 produced 1.22-fold, 1.19-fold, and 1.26-fold more biomass in root, leaf sheath, and leaf blade than WT, respectively (Figure 4b). Theses increased root and shoot biomass in the sitl1was also observed when plants were treated with DW containing 50 and 100 mM NaCl (Figure 4c). The sitl1 also had higher chlorophyll content than the WT plant when plants were subjected to the treatment with NS or DW containing 50 and 100 mM NaCl (Figure 4d,e). We further confirmed that the sitl1 showed improved salinity insensitivity when plants were irrigated with 50 mM NaCl solution under soil-grown conditions but the sitl1 did not show drought stress insensitivity (Figure S5). These results confirm that the sitl1have a higher ability to tolerate salinity stress but not drought stress.
In order to visualize and quantitate the H2O2 accumulations, we conducted histochemical DAB staining and H2O2quantification analysis at 7 days after treatment with 0, 50, 100 mM NaCl (Figure 4f-h). Under unstressed condition, no significant differences were observed in the amounts of H2O2 between the root tissues of thesitl1 and WT, whereas the sitl1 line showed a 1.47-fold increase in H2O2 amount in leaf tissues compared to the WT (Figure 4g-h). Under salinity stress condition, thesitl1 showed a 0.45- (50 mM NaCl) to 0.5-fold (100 mM NaCl) decrease in root tissues, and 0.53- (50 mM NaCl) to 0.58-fold (100 mM NaCl) decrease in leaf tissues, respectively, relative to the WT (Figure 4f-h).
Sodium ion concentration within the lateral roots was measured using the cell-permeant CoroNa Green AM dye, which is a green-fluorescent Na+-specific indicator that exhibits an increase in fluorescence emission intensity upon binding Na+levels (Cho, Park, Kim, & Jang, 2010). Seven-day-old seedlings were transferred to nutrient solution containing 50 mM NaCl for 3 h. The salinity-treated WT plants showed strong fluorescence signals both in the vacuoles and the cytoplasm of outer epidermis and inner cells, whereas the sitl1 showed very little fluorescence signals, which tightly sequestrated into the vacuoles in roots (Figure 4i).
In order to validate the ability of Na+ influx across the plant plasma membranes, we used freshly isolated protoplasts from leaf tissues because it was observed the difference in root development of the sitl1 could alter the apoplastic permeability of Na+ or the ability of root apopolastic barriers, which block Na+ transport to shoot tissues in rice (Meier, Kovalchuk, & Rose, 2006; Zhou et al., 2011). The salinity-treated WT protoplasts showed obvious CoroNa Green AM staining, whereas the protoplasts of the sitl1 showed very little staining (Figure 4j). Quantitation of the CoroNa-Green AM intensity showed that the WT displayed significantly higher fluorescence than the sitl1 in the vacuole and the cytoplasm. Under unstressed conditions, there was no significant differences in fluorescence intensity. However, under 50 mM NaCl, the WT protoplast showed 6.95-fold and 4.15-fold higher fluorescence intensity than the sitl1 , in the cytoplasm and in the vacuoles, respectively (Figure 4k). These results demonstrate that the improved salinity tolerance of the sitl1 caused major changes such as reduced Na+ influx rate across the plasma membrane in both root and leaf tissues.