Genome-wide identification of CKX genes in petunia reveals PhCKX3 as a key regulator of flower and leaf development

• First comprehensive analysis of the CKX gene family was performed in petunia. • The expression of PhCKX3 and PhCKX7 are related to flower size and development stages. • Silencing of PhCKX3 resulted in enlarged size of flowers and leaves in petunia. Cytokinin oxidase/dehydrogenase (CKX) is a critical regulator of cytokinin homeostasis, influencing plant growth and developmental processes. Despite their importance, the evolutionary dynamics and functional roles of CKX genes in ornamental species like petunia remain poorly characterized. Here, we conducted a genome-wide identification of CKX genes from four wild Petunia species ( P. axillaris, P. exserta, P. secreta , and P. inflata ) and a garden petunia line ( P. hybrida Line A), totally identifying 29 CKX genes with five or six members in each genome. Phylogenetic analysis demonstrated that CKX genes were classified into five clades in angiosperms, with PhCKX s distributed across four of these clades. Promoter analysis identified cis -elements responsive to light, hormones, and stress, indicating that CKX expression in petunia is regulated through multiple pathways. Expression profiles of PhCKX s in various tissues and flower development stages were investigated, which revealed tissue- and stage-specific expression patterns of PhCKX s, moreover, the expression levels of PhCKX3 and PhCKX7 showed significant correlation with floral organ size. Functional inhibition of PhCKX3 through virus-induced gene silencing (VIGS) resulted in enlarged leaf and floral organs, confirming its role as a negative regulator of cell proliferation. This study provides the first comprehensive analysis of CKX genes in Petunia , offering insights into cytokinin-mediated floral development and strategies for molecular breeding of flower size.