Molecular Mechanism Of Arabidopsis Transcription Factor RAV1in Negativly Regulating Abscisic Acid Signaling

The phytohormone abscisic acid (ABA) regulates many important plant growth and developmental events such as seed maturation, seed germination, seedling growth, stomatal movement and flowering, and responses to environmental stresses.The transcription factor RAV1is a member of the RAV (Related to ABI3/VP1) subfamily, which belongs to the APETALA2/Ethylene Responsive Factor (AP2/ERF) family. Previous studies showed that RAVI was involved in plant development and leaf senescence. In this study, molecular mechanism of RAV1involving in ABA signaling during seed germination and early seedling development was investigated. During the seed germination, RAVI expression increased markedly and exogenous ABA could repress its expression. The RAV1-underexpressing line (RAV1-U) was more sensitive to ABA than wild type plants during seed germination and early seedling development, whereas the RAV1-overexpressing lines showed strong ABA-insensitive phenotypes. The qRT-PCR result showed that expressions of several ABA-responseive genes (include ABI3, ABI4, ABI5, Eml and Em6) were up-regulated in RAVl-U, and down-regulated in RAV1OE2and RAV1OE3. These results demonstrate that RAV1was a negative regulator of ABA signaling during seed germination and early seedling development.Transient expression assay in tobacco (Nicotiana benthamiana) showed that RAV1negative regulated the ABI5expression. Sequence analysis revealed that there were three5’-CAACA-3’motifs within the ABI5promoter, which could be bound by RAV1. EMSA and ChIP assays indicated that RAV1could bind to the ABI5promoter, demonstrating that RAV1directly down-regulates ABI5expression. The interruption of ABI5function in RAV1-U abi5plants abolished the ABA-hypersensitive phenotype of,RAV1-U plants, demonstrating that ABI5is epistatic to RAV1.Previouse studies showed that three SNF1-RELATED PROTEIN KINASE2s (SnRK2s), SnRK2.2, SnKR2.3, and SnRK2.6, are important positive regulators of ABA signaling. RAV1interacted with SnRK2.2, SnRK2.3and SnRK2.6in the nucleus. In vitro kinase assays showed that SnRK2.2, SnRK2.3and SnRK2.6phosphorylated RAV1. Transient expression assays revealed that SnRK2.2, SnRK2.3and SnRK2.6reduced the RAV1-dependent repression of ABI5, and the ABA-insensitive phenotype of the RAV1-overexpressing line was impaired by overexpression of SnRK2.3in the RAV1OE3/SnRK2.3OE plants. Together, these results demonstrate that the Arabidopsis transcription factor RAV1plays an important role in ABA signaling through modulating ABI5expression, and that its activity is negatively regulated by SnRK2.2, SnRK2.3and SnRK2.6。...