Arabidopsis RAV1, RAV1L and RAV2/TEM2 are Related to ABI3/VP1 (RAV) transcription factors that contain both plant-specific B3 and AP2 domains. RAV1 was known to be a negative regulator of growth and its transcript level was repressed by brassinolide (BL). In this study, we found that the expressions of RAV1, and its closest homologs RAV1L and RAV2 were also regulated by other plant hormones, and especially repressed significantly by BL and abscisic acid (ABA), which mediate various abiotic stress responses in plants. Therefore, to further investigate the physiological functions of RAV1, RAV1L and RAV2 in abiotic stress responses, we isolated T-DNA insertional knockout mutants of each gene and produced transgenic plants overexpressing the RAVs. Under normal conditions, each single mutant showed slightly promoted growth patterns only at an early stage of development. In comparison, the RAV1-overexpressing plants exhibited strong growth retardation with semi-dwarfed stature. In drought conditions, RAVs-overexpressing transgenic plants exhibited higher transpirational water loss than the wild type. In salt conditions, seed germination of the RAVs-overexpressing transgenic plants was more inhibited than that of the wild type, while ravs mutants showed promoted seed germination. We also found that RAVs expressions were reduced by dryness and salt. RAV1-overexpressing plants showed the same patterns of increased expression as stress-inducible genes such as RD29A, RD29B and the genes encoding ABA biosynthetic enzymes, as did the wild type and rav1 mutant. However, the RAV1-overexpressing transgenic plants were insensitive to ABA, regardless of the higher accumulation of ABA even in normal conditions. Taken together, these results suggest that RAVs are versatile negative regulators for growth and abiotic stresses, drought and salt, and that negative regulatory effects of RAVs on abiotic stresses are likely to be operated independently of ABA.

Stomatal movement in response to water availability is an important physiological process in the survival of land plants. The plant hormone abscisic acid (ABA) and brassinosteroids (BRs) regulate stomatal closure. The physiological functions of ABA and BRs, including germination, cell elongation and stomatal movement, are generally known to be antagonistic. Here, we investigated how BRs affect stomatal movement alone and in combination with ABA. We demonstrate that brassinoslide (BL), the most active BR, promotes stomatal closure in an ABA-independent manner. Interestingly, BL also inhibited ABA-induced stomatal closure when a high concentration of BL was added to ABA. Furthermore, we found that the induction of some genes for reactive oxygen species (ROS) generation by ABA (AtrbohD, NIA1 and NIA2) and subsequent ROS production were repressed by BL treatment. The BR signaling mutant bri1-301 failed to inhibit ABA-induced stomatal closure upon BL treatment. However, BRI1-overexpressing transgenic plants were hypersensitive to ABA during stomatal closure, and BL reversed ABA-induced stomatal closure more completely than in wild type plants. Taken together, these results suggest that BRs can positively and negatively modulate ABA-induced stomatal closure. Therefore, interactions between ABA and BR signaling are important for the regulation of stomatal closure.

We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.