Distribution of and Temporal Variation in Volatiles in Tea (Camellia sinensis) Flowers during the Opening Stages.

Tea (Camellia sinensis) flowers emit a large amount of volatiles that attract pollinators. However, few studies have characterized temporal and spatial variation in tea floral volatiles. To investigate the distribution of volatiles within tea flowers and their variation among opening stages, volatile components from different parts of tea flowers and different opening stages were collected by headspace solid-phase microextraction and analyzed by gas chromatography-mass spectrometry. A total of 51 volatile compounds of eight chemical classes were identified in the tea flowers. Volatile compounds were most abundant in tea flowers of the Shuchazao cultivar. Acetophenone, 1-phenylethanol, 2-phenylethanol, and benzyl alcohol were the most abundant volatiles. Terpenes were common in the sepals, and benzoids were common in the stamens. The fatty acid derivatives were mainly distributed in the pistils and receptacles and were less abundant in the petals, sepals, and stamens. During the opening phase of tea flowers, the volatile content increased 12-fold, which mainly stemmed from the increase in benzoids. These results enhance our understanding of the formation of volatiles in tea flowers.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition.

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 µM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 µM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Characterization of Key Odorants in Xinyang Maojian Green Tea and Their Changes During the Manufacturing Process.

Xinyang Maojian (XYMJ) green tea is a famous high-grade Chinese green tea, but the key odorants contributing to its aroma have been poorly understood. In this study, solid-phase microextraction and solvent-assisted flavor evaporation were used for sample preparation, and gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) were used for both qualitative and quantitative analysis. A total of 50 volatile compounds of five chemical classes were identified in XYMJ tea infusion. Among them, nine odorants including nonanal, ß-ionone, octanal, E-nerolidol, linalool, cis-3-hexenyl hexanoate, geraniol, decanal, and ß-cyclocitral were identified as key odorants of XYMJ based on GC-O, odor activity values, and aroma combination experiments. Changes in the content of these aroma-active compounds during the manufacturing process of XYMJ (fresh leaves, fixing, rolling, shaping, and drying) were also determined. Most aroma-active compounds decreased after the fixation process, with the exception of cis-3-hexenyl hexanoate. This is the first study to investigate the key odorants in XYMJ using the sensomics approach. The findings of this study provide novel information on the aroma quality of XYMJ.