Lanting Zeng

Proteolysis of chloroplast proteins is responsible for accumulation of free amino acids in dark-treated tea (Camellia sinensis) leaves.

Shade management (dark treatment) on tea (Camellia sinensis) plants is a common approach to improve free amino acids in raw materials of tea leaves. However, the reason for amino acid accumulation in dark-treated tea leaves is still unknown. In the present study, dark treatment significantly increased content of free amino acids and reduced content of soluble proteins in tea leaves. Quantitative proteomics analysis showed that most enzymes involved in biosyntheses of amino acids were down-accumulated by dark treatment. Chloroplast numbers reduced in dark-treated leaves and the content of soluble proteins reduced in the chloroplasts isolated from dark-treated leaves compared to control. These suggest that proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves. Two chloroplasts proteases, ATP-dependent Clp protease proteolytic subunit 3 and protease Do-like 2, were up-accumulated in dark-treated leaves. This study firstly elucidated the mechanism of accumulation of amino acids in dark-treated tea leaves. BIOLOGICAL SIGNIFICANCE Effect of dark on crop growth has been widely studied, while less attention has been paid to effect of dark on quality-related metabolites in crops. Shade management (dark treatment) on tea plants is a common approach to improve free amino acids in tea leaves. However, the reason for accumulation of free amino acids in dark-treated tea leaves is still unknown. In the present study, an iTRAQ-based quantitative proteomic analysis was performed and the results revealed the accumulation of free amino acids in dark-treated tea leaves was not due to activation of biosyntheses of amino acids, but resulted from proteolysis of chloroplast proteins. The information will advance our understanding of formation of quality or function-related metabolites in agricultural crops exposed to dark stress/shade management.

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 β-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.

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.

Functional characterizations of β-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 β-glucosidase (β-Glu) and β-primeverosidase to release free tea aromas. β-Primeverosidase has been identified and functionally characterized, while β-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 Csβ-Glus hydrolyzed the β-glucopyranosidically conjugated aromas to form free aromas, suggesting that there was no specific Csβ-Glus for the hydrolysis of β-glucopyranosidically conjugated aromas in vitro. Furthermore, subcellular localization of the Csβ-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 β-glucopyranosidically conjugated aromas in tea leaves during the tea manufacturing process. This study provides the first evidence of Csβ-Glus in tea leaves, and will advance understanding of tea aroma formation.

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 and emission of linalool in tea (Camellia sinensis) leaves infested by tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda)

Famous oolong tea (Oriental Beauty), which is manufactured by tea leaves (Camellia sinensis) infected with tea green leafhoppers, contains characteristic volatile monoterpenes derived from linalool. This study aimed to determine the formation mechanism of linalool in tea exposed to tea green leafhopper attack. The tea green leafhopper responsible for inducing the production of characteristic volatiles was identified as Empoasca (Matsumurasca) onukii Matsuda. E. (M.) onukii attack significantly induced the emission of linalool from tea leaves (p<0.05) as a result of the up-regulation of the linalool synthases (CsLIS1 and CsLIS2) (p<0.05). Continuous mechanical damage significantly enhanced CsLIS1 and CsLIS2 expression levels and linalool emission (p<0.05). Therefore, continuous wounding was a key factor causing the formation and emission of linalool from tea leaves exposed to E. (M.) onukii attack. This information should prove helpful for the future use of stress responses of plant secondary metabolism to improve quality components of agricultural products.

Studies on the Biochemical Formation Pathway of the Amino Acid l-Theanine in Tea (Camellia sinensis) and Other Plants

Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [2H5]ethylamine, all the plants produce [2H5]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

Elucidation of Differential Accumulation of 1-Phenylethanol in Flowers and Leaves of Tea (Camellia sinensis) Plants

1-Phenylethanol (1PE) is a major aromatic volatile in tea (Camellia sinensis) flowers, whereas it occurs in a much smaller amounts in leaves. Enzymes involved in the formation of 1PE in plants and the reason why 1PE differentially accumulates in plants is unknown. In the present study, enzymes in the last step leading from acetophenone to 1PE were isolated from tea flowers by traditional biochemical chromatography. The two types of partially purified enzymes were proposed to be responsible for formations of (R)-1PE and (S)-1PE, respectively. Tea leaves also contained such enzymes having equivalent activities with flowers. Stable isotope labeling experiments indicated that weak transformation from l-phenylalanine to acetophenone in leaves mainly resulted in little occurrence of 1PE in leaves. This study provided an example that differential distribution of some metabolites in plant tissues was not only determined by enzyme(s) in the last step of metabolite formation, but also can be due to substrate availability.

An alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid in tea (Camellia sinensis (L.) O. Kuntze) leaves

Aromatic aroma compounds contribute to flavor of tea (Camellia sinensis (L.) O. Kuntze) and they are mostly derived from l-phenylalanine via trans-cinnamic acid or directly from l-phenylalanine. The objective of this study was to investigate whether an alternative pathway derived from l-phenylalanine via phenylpyruvic acid is involved in formation of aroma compounds in tea. Enzyme reaction with phenylpyruvic acid showed that benzaldehyde, benzyl alcohol, and methyl benzoate were derived from phenylpyruvic acid in tea leaves. Feeding experiments using [2H8]l-phenylalanine indicated that phenylpyruvic acid was derived from l-phenylalanine in a reaction catalyzed by aromatic amino acid aminotransferases (AAATs). CsAAAT1 showed higher catalytic efficiency towards l-phenylalanine (p ≤ 0.001) while CsAAAT2 showed higher catalytic efficiency towards l-tyrosine (p ≤ 0.001). Both CsAAATs were localized in the cytoplasm of leaf cells. In conclusion, an alternative pathway for the formation of aromatic aroma compounds derived from l-phenylalanine via phenylpyruvic acid occurred in tea leaves.

Biosynthesis of Jasmine Lactone in Tea (Camellia sinensis) Leaves and Its Formation in Response to Multiple Stresses

Jasmine lactone has a potent odor that contributes to the fruity, sweet floral aroma of tea ( Camellia sinensis). Our previous study demonstrated that jasmine lactone was mostly accumulated at the turnover stage of the oolong tea manufacturing process. This study investigates the previously unknown mechanism of formation of jasmine lactone in tea leaves exposed to multiple stresses occurring during the growth and manufacturing processes. Both continuous mechanical damage and the dual stress of low temperature and mechanical damage enhanced jasmine lactone accumulation in tea leaves. In addition, only one pathway, via hydroperoxy fatty acids from unsaturated fatty acid, including linoleic acid and α-linolenic acid, under the action of lipoxygenases (LOXs), especially CsLOX1, was significantly affected by these stresses. This is the first evidence of the mechanism of jasmine lactone formation in tea leaves and is a characteristic example of plant volatile formation in response to dual stress.