Pepper RING-type E3 ligase, CaANKR1, negatively regulates drought tolerance by modulating CaMAPK1 stability.

Abstract

Drought stress severely restricts plant growth and productivity, and abscisic acid (ABA)-mediated signaling plays a central role in coordinating adaptive responses to water deficit. Mitogen-activated protein kinase (MAPK) cascades are widely involved in plant stress signaling; however, the ubiquitin-mediated regulation of MAPK stability during drought responses remains poorly understood. In this study, we conducted a yeast two-hybrid screening and identified CaMAPK1, a pepper (Capsicum annuum) MAPK, as an interacting partner of the RING-type E3 ubiquitin ligase CaANKR1, which was further confirmed via in vitro and in vivo assays. CaMAPK1 expression was induced by dehydration, osmotic stress, salinity stress, and ABA treatment. Furthermore, CaMAPK1 exhibited kinase activity and was predominantly localized to the nucleus. Ubiquitination assays revealed that CaANKR1 promoted K48-linked polyubiquitination of CaMAPK1 in a RING domain-dependent manner. Consistently, cell-free degradation assays showed that CaMAPK1 protein stability negatively correlated with CaANKR1 expression levels, indicating that CaANKR1 promotes the proteasome-dependent degradation of CaMAPK1. Phenotypic assays revealed that CaMAPK1 silencing in pepper reduced drought tolerance by altering ABA-mediated stomatal responses and water retention, whereas CaMAPK1 overexpression in Arabidopsis produced the opposite phenotype. Collectively, these findings suggest that CaANKR1 negatively modulates ABA-mediated drought tolerance through ubiquitin-dependent destabilization of CaMAPK1. Together, they reveal a mechanism underlying ABA-driven drought responses and highlight a promising target for enhancing crop drought resilience.