The Pepper E3 Ligase CaGIR1 Acts as a Negative Regulator of Drought Response via Controlling CaGRAS1 Stability

The ubiquitin-proteasome pathway modulates protein stability, which impacts plant responses to abiotic stresses, such as drought. Our previous study reported that the pepper GRAS-type transcription factor CaGRAS1 plays a positive role in drought resistance. However, the mechanism by which drought stress affects CaGRAS1 protein stability remains unknown. Here, we identified Capsicum annuum CaGRAS1-Interacting RING-type E3 ligase 1 (CaGIR1) through yeast two-hybrid analysis. The interaction between these two proteins was confirmed by both in vitro and in vivo assays, and interaction occurred in both the nucleus and cytoplasm, consistent with their subcellular localisation. In ubiquitination assays, CaGIR1 was shown to have ubiquitin E3 ligase activity, which is dependent on its RING domain. CaGIR1 also directly ubiquitinated CaGRAS1 in vitro and in vivo, and CaGRAS1 protein stability negatively correlated with CaGIR1 expression levels. In contrast to CaGRAS1, CaGIR1 was found to play a negative role in drought resistance. Phenotypic assays revealed that the silencing of CaGIR1 in pepper resulted in enhanced drought resistance through the modulation of stomatal responses and drought-responsive marker gene expression, whereas CaGIR1 overexpression led to the opposite results in Arabidopsis. Overall, our findings suggest that CaGIR1 negatively modulates ABA and drought responses by triggering CaGRAS1 protein degradation.