Whole-genome characterization of CKX genes in Prunus persica and their role in bud dormancy and regrowth

Bud dormancy is a complex physiological process of perennial woody plants living in temperate regions,and it can be affected by various phytohormones.Cytokinin oxidase/dehydrogenases(CKXs)are a group of enzymes essential for maintaining cytokinin homeostasis,yet a comprehensive analysis of these enzymes in peach remains lacking.Here,a total of 51 CKX members from different species,including six from peach,eleven from apple,nine from poplar,seven from Arabidopsis,eight from strawberry,and ten from rice,were identified using the Simple HMM Search tool of TBtools and a BLASTP program and classified into four groups using phylogenetic analysis.Conserved motif and gene structure analysis of these 51 CKX members showed that 10 conserved motifs were identified,and each CKX gene contained at least two introns.Cis-element analysis of PpCKXs showed that all PpCKX genes have light-responsive elements and at least one hormone-responsive element.The changed relative expression levels of six PpCKX genes in peach buds from endodormancy to bud-break were observed by qRT-PCR.Among them,the expression trend of PpCKX6 was almost opposite that of PpEBB1,a positive bud-break regulator in woody plants,around the bud-break stage.Y1H,EMSA,and dual-luciferase assays indicated that PpEBB1negatively regulated PpCKX6 through direct binding to a GCC box-like element located in the promoter region of PpCKX6.In addition,a transient assay showed that overexpression of PpCKX6 delayed the bud-break of peach.These results indicate that the PpCKX genes play an essential role in the dormancy-regrowth process,and Pp CKX6may act downstream of PpEBB1 directly to regulate the bud-break process,which further improves the hormoneregulatory network of dormancy-regrowth of woody plants,and provides new insights for molecular breeding and genetic engineering of peach.