Abstract:Prunus mume Siebold & Zucc. has many varieties and diverse floral scent types, among which cinnamyl alcohol and cinnamyl acetate are unique floral scent components in pink varieties of P. mume. In plants, cinnamaldehyde is reduced to cinnamyl alcohol under the catalysis of cinnamyl alcohol dehydrogenase (CAD), which in turn generates cinnamyl acetate under the catalysis of acetyltransferase. Therefore, cinnamyl alcohol is not only an important floral scent component in P. mume, but also an important precursor for the synthesis of cinnamyl acetate. This study identified 56 PmCADs homologous genes based on the second-generation genome of P. mume. It was found that the PmCADs family can be divided into five subgroups, and the protein conserved motif composition of subgroups I, II, and III is relatively similar, with significant differences from subgroups IV and V suggesting potential functional differentiation between them. Chromosome fragment replication and tandem replication were identified as two forms of PmCADs replication in genome evolution. It was found that the promoter regions of PmCADs genes extensively contained abscisic acid, methyl jasmonate, salicylic acid responsive elements, and meristem related elements suggesting that the PmCADs genes are mainly involved in the growth, development, and stress response of P. mume. Phylogenetic analysis of CAD from P. mume and other species suggested that the three PmCADs genes located in subgroup III may be involved in the floral scent synthesis in P. mume. Heat map analysis of PmCADs genes in different tissues and organs indicated that the three PmCADs were all expressed in flowers. Furthermore, analysis of relative gene expression revealed that the expression patterns of the 3 candidate PmCADs genes varied at different stages of flowering, with higher expression levels after the flowers bloom. The in vitro enzyme activity analysis confirmed that all three PmCADs have cinnamyl alcohol dehydrogenase activity, indicating that they jointly participate in the synthesis of cinnamyl alcohol, an important floral scent component of P. mume. Subcellular localization analysis revealed differences in their distribution within the cell, suggesting that they may play roles in different parts of the cell. This study revealed the evolution and functional differentiation of the PmCADs genes in P. mume, laying theoretical foundations for the synthesis of important floral scent components and providing theoretical bases for floral fragrance molecular breeding in P. mume.