NADPH oxidase AtrbohD and AtrbohF function in ROS-dependent regulation of Na+/K+ homeostasis in Arabidopsis under salt stress

Maintaining cellular Na+/K+ homeostasis is pivotal for plant survival in saline environments. However, knowledge about the molecular regulatory mechanisms of Na+/K+ homeostasis in plants under salt stress is largely lacking. In this report, the Arabidopsis double mutants atrbohD1/F1 and atrbohD2/F2, in which the AtrbohD and AtrbohF genes are disrupted and generation of reactive oxygen species (ROS) is pronouncedly inhibited, were found to be much more sensitive to NaCl treatments than wild-type (WT) and the single null mutant atrbohD1 and atrbohF1 plants. Furthermore, the two double mutant seedlings had significantly higher Na+ contents, lower K+ contents, and resultant greater Na+/K+ ratios than the WT, atrbohD1, and atrbohF1 under salt stress. Exogenous H2O2 can partially reverse the increased effects of NaCl on Na+/K+ ratios in the double mutant plants. Pre-treatments with diphenylene iodonium chloride, a widely used inhibitor of NADPH oxidase, clearly enhanced the Na+/K+ ratios in WT seedlings under salt stress. Moreover, NaCl-inhibited inward K+ currents were arrested, and NaCl-promoted increases in cytosolic Ca2+ and plasma membrane Ca2+ influx currents were markedly attenuated in atrbohD1/F1 plants. No significant differences in the sensitivity to osmotic or oxidative stress among the WT, atrbohD1, atrbohF1, atrbohD1/F1, and atrbohD2/F2 were observed. Taken together, these results strongly suggest that ROS produced by both AtrbohD and AtrbohF function as signal molecules to regulate Na+/K+ homeostasis, thus improving the salt tolerance of Arabidopsis.