Functional characterization of E3-ubiquitin ligases for improved drought tolerance – the case study of a rice ABA-responsive gene

Rice is one of the most important crops worldwide, being the primary source of calories for more than half of the world population and a very sensitive species to water deficit. The identification of important molecular mechanisms modulating drought response, for enhanced water and nutrient capture, will play a key role towards a more sustainable rice production. The Ubiquitin-Proteasome system (UPS) is a key regulator of the proteome pool and has been established as a potential candidate for crop improvement towards abiotic stress tolerance (Melo et al., 2021). Nevertheless, several of its components remain uncharacterized. In our group, we are following a candidate gene approach to identify and functionally characterize rice E3-ubiquitin ligases in stress response. After in silico analysis, we selected (and validated by RT-qPCR) 16 genes showing differential gene expression under stress. Among those, the most promising 4 were selected for a deeper functional characterization using both a knock-out (CRISPR/cas9 gene edition) and an overexpression approach. One of those genes, RiP4g, is up-regulated by ABA. To characterize its role in ABA signaling, we have performed germination and post-germination assays, using the KO and OX plants. These assays indicated that OX-RiP4g confers hypersensitivity and KO-RiP4g hyposensitivity to ABA. Furthermore, using a whole plant water loss assay, we observed a differential behavior that can involve RiP4g in the regulation of stomatal aperture through ABA sensitivity. Taken together, our results will enhance our understanding of the molecular mechanisms underlying the rice response to drought, particularly regarding the function of RiP4g