Identification and functional analysis of CLE genes associated with drought tolerance in pepper

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides constitute one of the largest families of small signaling peptides, playing crucial roles in plant development and stress responses. Despite extensive research on CLE genes in various species, our understanding of these genes in pepper (Capsicum annuum) remains limited. In this study, we identified and characterized 10 CLE-like peptide genes (CaCLEs) from the pepper genome. All CaCLEs possess a conserved C-terminal CLE motif, and most contain an N-terminal signal peptide. Several CaCLE genes displayed high expression levels in roots and shoot apices. Furthermore, CaCLE1, CaCLE2, and CaCLE3 were strongly upregulated in leaves and/or roots by drought stress, with CaCLE3 showing the most rapid and pronounced induction in roots, whereas other CaCLE genes displayed varied expression patterns under drought, osmotic, and salt stress conditions. Subcellular localization assays revealed that CaCLE proteins localize to the plasma membrane or nucleus; notably, CaCLE1 was detected in both compartments. Virus-mediated overexpression (VOX) of CaCLE1, CaCLE2, and CaCLE3 in tobacco and pepper plants resulted in significantly enhanced drought tolerance, as evidenced by the reduced wilting and improved survival following drought imposition and re-watering; however, CaCLE1 and CaCLE3 overexpression also led to growth inhibition. Among these, CaCLE3-overexpressing plants exhibited the highest drought tolerance. Collectively, these findings suggest that CaCLE1, CaCLE2, and CaCLE3 contribute positively to drought stress tolerance, indicating their potential application in enhancing drought stress resilience in pepper.