PhCBP60b positively regulates drought tolerance in Petunia by transcriptionally activating PhSWEET11 and PhUGT74E2

Abstract

The calmodulin-binding protein 60-like protein family plays important roles in plant immunity and stress responses, but its functions in ornamental plants remain poorly understood. In this study, we characterized PhCBP60b from Petunia × hybrida ‘Mitchell Diploid’ and investigated its role in drought tolerance. PhCBP60b encoded a nuclear-localized protein containing a conserved calmodulin-binding domain and was phylogenetically closely related to Arabidopsis AtCBP60b. PhCBP60b was predominantly expressed in roots. Overexpression of PhCBP60b in Arabidopsis and petunia enhanced drought tolerance, as shown by reduced leaf water loss, higher relative water content, and increased survival rates after drought stress. Transgenic plants also exhibited enhanced drought-induced stomatal closure. Under mannitol-induced osmotic stress at 150 and 300 mM, PhCBP60b-overexpressing lines showed improved seedling growth compared with WT plants, including higher germination rates and longer primary roots. Transcriptome analysis identified 167 differentially expressed genes associated with PhCBP60b overexpression, among which several drought-responsive genes, including DDF1, LEA3, SWEET11, and UGT74E2, were highlighted. Integrated transcriptomic, DAP-seq, and dual-luciferase assays further demonstrated that PhCBP60b could bind to and activate the promoters of PhSWEET11 and PhUGT74E2, resulting in an approximately two-fold increase in transcriptional activity. These results indicate that PhCBP60b functions as a positive regulator of drought tolerance by modulating stomatal traits and activating downstream drought-responsive metabolic genes. This study provides new insights into the functional role of CBP60 family proteins in ornamental plants and highlights PhCBP60b as a promising candidate for improving drought resistance in petunia and other crops.