Functional characterizations of ß-glucosidases involved in aroma compound formation in tea (Camellia sinensis).

Tea (Camellia sinensis) aroma is an important factor affecting tea quality. Many tea aroma compounds are present as glycosidically conjugated forms in tea leaves, and can be hydrolyzed by ß-glucosidase (ß-Glu) and ß-primeverosidase to release free tea aromas. ß-Primeverosidase has been identified and functionally characterized, while ß-Glu has not been identified in tea leaves. In the present study, we established a yeast expression system to recombine CsGH1BG1, CsGH3BG1, and CsGH5BG1, which belonged to GH1, GH3, and GH5 families in plants, respectively. These three recombinant Csß-Glus hydrolyzed the ß-glucopyranosidically conjugated aromas to form free aromas, suggesting that there was no specific Csß-Glus for the hydrolysis of ß-glucopyranosidically conjugated aromas in vitro. Furthermore, subcellular localization of the Csß-Glus indicated that CsGH1BG1 and CsGH3BG1 were located in the cytosol and vacuole, respectively, while CsGH5BG1 was located in the cell wall. This suggested that CsGH1BG1 and CsGH3BG1 might be responsible for the hydrolysis of ß-glucopyranosidically conjugated aromas in tea leaves during the tea manufacturing process. This study provides the first evidence of Csß-Glus in tea leaves, and will advance understanding of tea aroma formation.

Formation of (E)-nerolidol in tea (Camellia sinensis) leaves exposed to multiple stresses during tea manufacturing.

(E)-Nerolidol is a volatile sesquiterpene that contributes to the floral aroma of teas (Camellia sinensis). The unique manufacturing process for oolong tea involves multiple stresses, resulting in a high content of (E)-nerolidol, which is not known to form in tea leaves. This study aimed to determine the formation mechanism of (E)-nerolidol in tea exposed to multiple stresses during tea manufacture. C. sinensis (E)-nerolidol synthase (CsNES) recombinant protein, found in the cytosol, was found to transform farnesyl diphosphate into (E)-nerolidol. CsNES was highly expressed during the oolong tea turn over process, resulting in (E)-nerolidol accumulation. Continuous mechanical damage, simulating the turn over process, significantly enhanced CsNES expression level and (E)-nerolidol content. The combination of low temperature stress and mechanical damage had a synergistic effect on (E)-nerolidol formation. This is the first evidence of (E)-nerolidol formation mechanism in tea leaves and a characteristic example of plant volatile formation in response to dual stresses.

Does oolong tea (Camellia sinensis) made from a combination of leaf and stem smell more aromatic than leaf-only tea? Contribution of the stem to oolong tea aroma.

The raw materials used to make oolong tea (Camellia sinensis) are a combination of leaf and stem. Oolong tea made from leaf and stem is thought to have a more aromatic smell than leaf-only tea. However, there is no available evidence to support the viewpoint. In this study, sensory evaluation and detailed characterization of emitted and internal volatiles (not readily emitted, but stored in samples) of dry oolong teas and infusions indicated that the presence of stem did not significantly improve the total aroma characteristics. During the enzyme-active processes, volatile monoterpenes and theanine were accumulated more abundantly in stem than in leaf, while jasmine lactone, indole, and trans-nerolidol were lower in stem than in leaf. Tissue-specific aroma-related gene expression and availability of precursors of aroma compounds resulted in different aroma distributions in leaf and stem. This study presents the first determination of the contribution of stem to oolong tea aroma.

Formation of Volatile Tea Constituent Indole During the Oolong Tea Manufacturing Process.

Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase ß-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time.

Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process?

It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. ß-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of ß-primeverosidase provided evidence that ß-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBV content was reduced. These findings reveal that, during the enzyme-active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and ß-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥4(4) were identified as relatively potent odorants in the oolong tea. These results suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process.

Diversity and evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae) whiteflies.

Wolbachia is the most prevalent symbiont described in arthropods to date. Wolbachia can manipulate host reproduction, provide nutrition to insect hosts and protect insect hosts from pathogenic viruses. So far, 13 supergroups of Wolbachia have been identified. The whitefly Bemisia tabaci is a complex containing more than 28 morphologically indistinguishable cryptic species. Some cryptic species of this complex are invasive. In this study, we report a comprehensive survey of Wolbachia in B. tabaci and its relative B. afer from 1658 insects representing 54 populations across 13 provinces of China and one state of Australia. Based on the results of PCR or sequencing of the 16S rRNA gene, the overall rates of Wolbachia infection were 79.6% and 0.96% in the indigenous and invasive Bemisia whiteflies, respectively. We detected a new Wolbachia supergroup by sequencing five molecular marker genes including 16S rRNA, groEL, gltA, hcpA, and fbpA genes. Data showed that many protein-coding genes have limitations in detecting and classifying newly identified Wolbachia supergroups and thus raise a challenge to the known Wolbachia MLST standard analysis system. Besides, the other Wolbachia strains detected from whiteflies were clustered into supergroup B. Phylogenetic trees of whitefly mitochondrial cytochrome oxidase subunit I and Wolbachia multiple sequencing typing genes were not congruent. In addition, Wolbachia was also detected outside the special bacteriocytes in two cryptic species by fluorescence in situ hybridization, indicating the horizontal transmission of Wolbachia. Our results indicate that members of Wolbachia are far from well explored.