OsRAV9 positively regulates seed germination and negatively modulates the drought tolerance

In plants, transcription factors play vital roles in abiotic stress responses. Related to ABI3/VP1 (RAV) is a special subgroup of AP2 transcription factors. However, the functions of rice RAV members in the response to abiotic stresses remain largely unknown. In the present study, we characterized the functions of OsRAV9 by generating knockout and over-expression lines. Compared with wild-type seeds, OsRAV9 mutant seeds germinated more slowly, indicating that OsRAV9 positively regulates seed germination. In addition, OsRAV9 mutant seedlings were more resistant to drought. After drought stress, the survival rates of two OsRAV9 mutant lines, osrav9-1 and osrav9-2 seedlings, were 86.47 ± 2.30 % and 48.17 ± 4.86 %, respectively, which were much higher than that of the control 9.51 ± 2.98 %. In contrast, transgenic plants over-expressing OsRAV9 were more sensitive to PEG treatment, implying that OsRAV9 negatively regulates drought tolerance. To elucidate the mechanism underlying this modulation of drought tolerance, OsRAV9 mutant and wild-type seedlings treated by drought were collected for RNA-sequencing. A total of 1261 differentially expressed genes were identified, and several of these genes are involved in abscisic acid (ABA) signaling. In particular, the expression levels of osbZIP23 and NCED4 in osrav9-1 seedlings increased about one fold and three fold respectively, compared to the expresssion levels in wild-type seedlings. Furthermore, the results of electrophoretic mobility shift assay (EMSA) and yeast one hybrid (Y1H) assays showed that OsRAV9 directly repressed the expression of OsbZIP23, which is a positive regulator of drought tolerance. Further analysis showed that mutations in osbZIP23 weakened the drought tolerance of osrav9 seedlings, suggesting that OsbZIP23 is a key downstream gene of OsRAV9 in response to drought stress. In addition, we found that the expression levels of six TIFY genes were up-regulated in OsRAV9 mutant seedlings. These findings extend our understanding of rice RAV transcription factors.