Functional Characterization And Molecular Regulation Mechanism Of Wheat Ubiquitin E3 Ligase TaSAP5

Based on the previous work of homology-based cloning of wheat TaSAP5, we finished drought tolerance characterization and molecular regulation mechanism for TaSAP5. TaSAP5 was a functionl ubiquitin E3 ligase and could mediate degradation of DREB2A INTERACTING PROTEIN (DRIP), to increase expression of DREB2A protein. The main results are as follows:1. The expression levels of TaSAP5-A, TaSAP5-B, and TaSAP5-D in response to drought stress were measured by RT-qPCR analysis. TaSAP5 overexpression confers drought tolerance in both Arabidopsis and wheat.2. We further performed RNA sequencing on wheat treated with PEG and found that amongst the genes that are induced in TaSAP5 overexpression line, six genes were previously described as being DREB2A target genes. As the protein level of DREB2A increased without any significant alteration in transcription level, we build a working hypothesis in which TaSAP5 would regulate the stability of an intermediate protein, on its turn regulating the DREB2A protein.3. Cellular localization of TaSAP5 in wheat protoplasts indicated that TaSAP5 localized to both the cytosol and nucleus. In vitro ubiquitination assay was conducted to investigate the potential E3 ubiquitin ligase activity of TaSAP5.4. Co-IP assays were performed following Agrobacterium-mediated transient expression to prove that TaSAP5 interacts with DRIP and facilitates their ubiquitination in vivo. DRIP could be degraded by 26S proteasome under drought stress condition and the presence of TaSAP5 accelerated DRIP degradation. These results indicate that overexpression of TaSAP5 could accelerate degradation of DRIP in Arabidopsis and wheat and that this occurs at least in part through degradation via the 26S proteasome pathway.5. All together, we demonstrated that TaSAP5 overexpression enhances DRIP degradation and increases the levels of DREB2A protein and its downstream targets. These results suggest that TaSAP5 acts to link drought with DREB2A accumulation and illustrate the molecular mechanisms involved in this process. Alleles of TaSAP5 that confer improved drought tolerance can be identified and used as markers in marker-assisted selection in wheat breeding.