Pepper RING-Type E3 Ligase, CaAIAR1, Negatively Modulates ABA Signaling and Drought Stress Response by Controlling...

The ubiquitin-proteasome pathway plays a crucial role in plant abiotic stress responses by regulating protein stability. Our previous work demonstrated that the pepper bZIP transcription factor CaADBZ1 regulates abscisic acid (ABA) signaling and drought stress responses. However, the regulatory factors controlling CaADBZ1 protein stability remain unknown. Here, we performed a yeast two-hybrid assay to identify the E3 ubiquitin ligase involved in regulating CaADBZ1 stability. The pepper RING-type E3 ligase CaAIAR1 (Capsicum annuum ABA-Induced Ankyrin repeat RING-type E3 ligase 1) interacts with CaADBZ1 both in vitro and in vivo. CaAIAR1 ubiquitinates CaADBZ1 and exhibits E3 ligase activity that depends on its RING domain. A cell-free degradation assay revealed that CaADBZ1 was unstable under non-stress conditions, and CaAIAR1 accelerated its degradation via the 26S proteasome pathway. CaAIAR1 expression was significantly induced by various abiotic stresses and was associated with altered phenotypic responses to drought stress and exogenous ABA treatment. Moreover, CaAIAR1-silenced pepper plants exhibited enhanced drought tolerance compared to control plants, as evidenced by reduced transpiration rates and increased ABA sensitivity in leaves. Conversely, CaAIAR1-overexpressing Arabidopsis plants showed decreased drought tolerance compared to control plants and exhibited reduced ABA sensitivity at the seedling stage. In addition, co-silencing CaAIAR1 and CaADBZ1 increased drought sensitivity, producing a phenotype opposite to that of CaAIAR1 silencing alone, indicating that CaAIAR1 functions upstream of CaADBZ1 in drought stress. Collectively, our findings suggest that CaAIAR1 negatively regulates the drought response and ABA signaling by modulating CaADBZ1 protein stability.