Biochemical characterization of CsCXEs: Carboxylesterase enhances the biosynthesis of green odor volatiles during tea processing

Tea flavor is a comprehensive representation of its aroma and other characteristics. The formation of volatile odor compounds during tea processing depends on a variety of enzymatic and non-enzymatic activities. (Z)-3-hexenol is considered the primary source of the green odor and is also the most important component in tea aroma, significantly affecting the overall aroma. However, the biosynthesis and accumulation of (Z)-3-hexenol during tea processing have not been fully analyzed. In this study, we found that withering treatment at different times and withering plus shaking treatment at different degrees promoted the accumulation of important volatile components of green tea odor, especially (Z)-3-hexenol by GC–MS. The RNA-seq and qRT-PCR results showed that withering and withering plus shaking treatments enhanced the expression of (Z)-3-hexenol-related genes in tea leaves, including synthetic pathway 1 genes (CsLOX3, CsHPL1, CsADH4, and CsAHD1), synthetic pathway 2 genes (CsGLU), and synthetic pathway 3 genes (CsCXEs). Correlation analysis of the key odorants and important genes in the three synthetic pathways revealed that some CsCXEs were positively correlated with green odor compounds. The in vitro enzyme activity results showed that rCsCXE3 (GWHTASIV011658), and rCsCXE6 (GWHTASIV031480) exhibited hydrolytic activity against three tea acetate compounds [hexyl acetate, (E)-2-hexyl acetate, and (Z)-3-hexyl acetate], resulting in the production of corresponding alcohol compounds. In summary, withering and shaking treatment during tea processing promoted the expression of CsCXE3 and CsCXE6, thereby enhancing the production of hexenol compounds. These compounds play a crucial role in increasing the green odor of tea.