A comprehensive study of carboxylesterases involved in the degradation of volatile esters in horticultural crops

Volatile esters play a crucial role in shaping the aroma profiles and sensory attributes of horticultural crops. In this study, we comprehensively investigated the volatile esters, alcohols, and acids profiles of different tea plant varieties and tissues to identify the key metabolites responsible for tea flavors. Subsequently, two homologous genes, CXE2 and CXE3 , which encode a carboxylesterase and are involved in the hydrolysis of esters, were screened from Camellia sinensis , Vaccinium darrowii , Myrica rubra , and Actinidia chinensis via multiomics analysis. In vitro , the purified recombinant enzyme CXE2 and CXE3 catalyze the hydrolysis of acetate esters into alcohols and acids. Molecular docking, site-directed mutagenesis, and enzyme activity analysis revealed that Tyr-15 and Gly-81 were the key amino acids that imparted hydrolytic activity to CsCXE2 , whereas Gly-81 and Gly-82 were the key amino acids that imparted hydrolytic activity to CsCXE3 . In vivo , the low expression of CsCXE2 and CsCXE3 led to decreased normal pollen and an increased presence of triangular pollen. In tender tea shoots, the results of gene silencing and withering treatments revealed that CsCXE2 and CsCXE3 disrupted acetate metabolism and altered the dynamic balance between green grassy and fruity aroma-related compounds by hydrolyzing acetate esters. Overall, this study highlights the significant potential of targeting CXE2 and CXE3 for precise flavor modulation in horticultural crop cultivation and processing, offering novel strategies to optimize the aroma profiles of horticultural crops in industrial applications. • A method for excavating functional genes via multi-omics association analysis has been provided. • The functions of CXE2 and CXE3 were conserved in horticultural crops and catalyzed the hydrolysis of hexyl acetate. • Elucidated catalytic mechanisms of CXE2 and CXE3 via molecular docking, site-directed mutagenesis, and biochemical assays. • CXE2 and CXE3 alter the dynamic balance between green and fruity aroma by catalyzing the hydrolysis of hexyl acetate.