Cinnamyl alcohol acyltransferase PmCAAT1 specifically catalyzes the formation of the characteristic volatile cinnamyl acetate of Prunus mume.

Abstract

Key message: Three cinnamyl alcohol acyltransferases (PmCAAT1-3) were identified in Prunus mume, with PmCAAT1 showing high substrate preference for cinnamyl alcohol and PmCAAT2 exhibiting broader substrate specificity including linalool. Prunus mume is renowned for its distinctive fragrance, with cinnamyl acetate serving as one of its key aroma components. However, the genes responsible for cinnamyl acetate biosynthesis have remained unidentified. In this study, a total of 116 acyltransferase family members were identified from the whole-genome data of P. mume var. tortuosa. Among these, three cinnamyl alcohol acyltransferase genes (PmCAAT1-3) were selected through transcriptome data of different cultivars and RT-qPCR analysis of petals at four developmental flowering stages. Then PmCAAT1-3 were cloned. Sequence alignment revealed that their amino acid sequences contain the conserved HXXXD catalytic motif and DFGWG substrate-binding motif of the BAHD family. Phylogenetic analysis showed that PmCAAT1 is evolutionarily closer to the CFAT clade, whereas PmCAAT2 and PmCAAT3 clustered together on a separate branch. Subcellular localization indicated that PmCAAT1-3 are localized in the nucleus and cytoplasm. Transient expression in P. mume petals demonstrated that all three genes could regulate cinnamyl acetate biosynthesis. Prokaryotic expression and In vitro enzyme activity assays confirmed that PmCAAT1 and PmCAAT2 could catalyze the conversion of cinnamyl alcohol to cinnamyl acetate. Kinetic analysis revealed that although both PmCAAT1 and PmCAAT2 display broad substrate selectivity, PmCAAT1 exhibits a strong preference for cinnamyl alcohol. This study elucidates the enzymatic basis for cinnamyl acetate biosynthesis in P. mume and offers a theoretical foundation for molecular breeding of floral fragrance in this species.