NtPP2C42 negatively regulates NtCLE3-NtBAM3-3 mediated drought tolerance in tobacco

Members of the BAM family, as plant leucine-rich repeat receptor-like kinases (LRR-RLKs), play essential roles in the recognition and signaling of CLE peptides. Previous studies have demonstrated that NtCLE3 enhances drought resistance in Solanaceae crops. In this study, we showed that NtCLE3 induces stomatal closure in Arabidopsis, with the Atbam3 mutant failing to respond, underscoring the critical role of AtBAM3 in perceiving this peptide. The PP2C phosphatase family member AtPP2C15 negatively regulates this process by interacting with AtBAM3, thereby inhibiting its receptor activity and the expression of the ABA biosynthesis-related gene AtNCED3. In tobacco, silencing of AtBAM3 homologs using virus-induced gene silencing revealed that NtBAM3-3 mediates NtCLE3-induced stomatal closure and drought tolerance. Further analysis identified NtPP2C42 instead of NtPP2C15 as the negative regulator of NtCLE3 signaling in tobacco, where its silencing enhanced the stomatal response to NtCLE3. Luciferase complementation assays, and co-immunoprecipitation assays confirmed that the PP2C phosphatase NtPP2C42 directly interacts with NtBAM3-3 to negatively regulate the NtCLE3 signaling pathway in tobacco. These findings highlight both conserved and divergent nature of BAM3-mediated CLE peptide signaling across species and provide new insights into the potential application of CLE peptides for enhancing drought tolerance in crops.