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Benzyl nitrile from tea plants attacked by various pests displays a diurnal pattern, which may be closely regulated by the endogenous circadian clock. However, the molecular mechanism by the circadian clock of tea plants that regulates the biosynthesis and release of volatiles remains unclear. In this study, the circadian clock gene CsPCL1 can activate both the expression of the benzyl nitrile biosynthesis-related gene CsCYP79 and the jasmonic acid signaling-related transcription factor CsMYC2 involved in upregulating CsCYP79 gene, thereby resulting in the accumulation and release of benzyl nitrile. Therefore, the anti-insect function of benzyl nitrile was explored in the laboratory. The application of slow-release beads of benzyl nitrile in tea plantations significantly reduced the number of tea geometrids and had positive effects on the yield of fresh tea leaves. These findings reveal the potential utility of herbivore-induced plant volatiles for the green control of pests in tea plantations.

Volatiles are important for plant root stress resistance. The diseases in tea root are serious, causing major losses. The volatile composition in tea root and whether it can resist diseases remain unclear. In this study, the volatile composition in different tea tissues was revealed. The vanillin content was higher in the root (mainly in root cortex) than in aerial parts. The antifungal effects of vanillin on pathogenic fungi in tea root were equal to or greater than those of other metabolites. O -methyltransferase (CsOMT), a key enzyme in one of two biosynthetic pathways of vanillin, converted protocatechualdehyde to vanillin in vitro . Furthermore, its characteristics and kinetic parameters were studied. In Arabidopsis thaliana protoplasts, the transiently expressed CsOMT was localized in the cytoplasm and nucleus. These findings have clarified the formation and bioactivities of volatiles in tea roots and provided a theoretical basis for understanding how tea plants resist root diseases.

Linalool, which is one of the most representative aroma substances in tea, is transformed into other aroma-related compounds, including linalool 3,6-oxides and linalool 3,7-oxides. The objective of this study was to elucidate the linalool oxide synthesis pathway and its response to stress in tea. By feeding experiment, chemical synthesis, and compound analysis, it was found that linalool can be transformed to linalool oxides via 6,7-epoxylinalool. The conversion rate from 6,7-epoxylinalool to linalool oxides was relatively high under acidic conditions. Four linalool oxide glucosides obtained from tea were structurally characterized. Additionally, tea green leafhopper infestation was observed to activate the whole metabolic flow from linalool into linalool oxides and their glucosides (p < 0.01). Moreover, light treatments further increased the accumulation of linalool oxides and their glucosides (p < 0.05). These results will be useful for elucidating the mechanism mediating linalool oxides content changes in response to stress in tea., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Polyamine oxidase (PAO) is a key enzyme maintaining polyamine homeostasis, which affects plant physiological activities. Until now, the gene members and function of PAOs in tea ( Camellia sinenesis ) have not been fully identified. Here, through the expression in Escherichia coli and Nicotiana benthamiana , we identified six genes annotated as CsPAO in tea genome and transcriptome and determined their enzyme reaction modes and gene expression profiles in tea cultivar 'Yinghong 9'. We found that CsPAO1,2,3 could catalyze spermine, thermospermine, and norspermidine, and CsPAO2,3 could catalyze spermidine in the back-conversion mode, which indicated that the precursor of γ-aminobutyric acid might originate from the oxidation of putrescin but not spermidine. We further investigated the changes of CsPAO activity with temperature and pH and their stability. Kinetic parameters suggested that CsPAO2 was the major PAO modifying polyamine composition in tea, and it could be inactivated by β-hydroxyethylhydrazine and aminoguanidine. Putrescine content and CsPAO2 expression were high in tea flowers. CsPAO2 responded to wound, drought, and salt stress; CsPAO1 might be the main member responding to cold stress; anoxia induced CsPAO3 . We conclude that in terms of phylogenetic tree, enzyme characteristics, and expression profile, CsPAO2 might be the dominant CsPAO in the polyamine degradation pathway.

Tea ( Camellia sinensis ) contains bioactive metabolites such as catechins, amino acids, caffeine, and aroma compounds that contribute to characteristic tea function and flavor. Therefore, studies on biochemical formation pathways and occurrences of these characteristic specialized metabolites in tea plants are important, providing essential information for the regulation and improvement of tea quality and function. Owing to the lack of a stable genetic transformation system, obtaining direct in vivo evidence of the formation of characteristic tea specialized metabolites is difficult. Herein, we review potential strategies for studying in vivo biochemical formation pathways and multilevel distributions of specialized metabolites in tea. At the individual plant level, stable isotope-labeled precursor tracing is an approach to discovering the pathways of some specialized metabolites specifically occurring in tea and elucidating the formation of tea specialized metabolites in response to stresses. At the within-tissue level, imaging mass spectrometry can be used to investigate the in situ localization of characteristic specialized metabolites within tea tissue without sample destruction. At the cellular or subcellular level, nonaqueous fractionation is a feasible method for characterizing the distributions of nonvolatile metabolites in subcellular organs. These approaches will help explain the characteristic scientific problems in tea secondary metabolism and provide more precise information to improve tea quality or function. HighlightsMultilevel distributions of metabolites in tea are important for tea quality improvement.Stable isotope-labeled precursor tracing method can be used to study formations of tea metabolites at individual plant level.Imaging mass spectrometry can be used to investigate the in situ localization of metabolites within tea tissue.Nonaqueous fractionation is a feasible method for characterizing the distributions of metabolites in subcellular organs.

Tea plants ( Camellia sinensis ) specifically produce l-theanine, which contributes to tea function and taste. Ethylamine is a limiting factor differentiating l-theanine accumulation between tea and other plants. Ethylamine has long been assumed to be derived from l-alanine in tea. In this study, the l-alanine content in tea root cells was mainly located in vacuoles and mitochondria using a nonaqueous fractionation technique, while alanine decarboxylase in tea (CsADC) was located in the cytoplasm. Although CsADC was able to catalyze l-alanine decarboxylation to produce ethylamine in vitro , it may not provide the same enzyme activity in tea plants. Stable isotope-labeled precursor tracing in tea plants discovered that l-alanine is not a direct precursor of ethylamine but a precursor of l-glutamate, which is involved in l-theanine biosynthesis in tea. Cortex with epidermis from root tissue was the main location of ethylamine. In summary, l-alanine is converted to l-theanine via l-glutamate not ethylamine in tea plants in vivo .

Tea ( Camellia sinensis ) is the most popular nonalcoholic beverage worldwide. During cultivation, tea plants are susceptible to herbivores and pathogens, which can seriously affect tea yield and quality. A previous report showed that ( Z )-3-hexenol is a potentially efficient defensive substance. However, the molecular mechanism mediating ( Z )-3-hexenol signaling in tea plants and the resulting effects on plant defenses remain uncharacterized. To clarify the signaling mechanisms in which ( Z )-3-hexenol and light are involved, the gene transcription and metabolite levels were assessed, respectively. This study demonstrated that tea plants rapidly and continuously release ( Z )-3-hexen-1-ol in response to an insect infestation. ( Z )-3-Hexen-1-ol absorbed by adjacent healthy plants would be converted into three insect defensive compounds: ( Z )-3-hexenyl-glucoside, ( Z )-3-hexenyl-primeveroside, and ( Z )-3-hexenyl-vicianoside identified with laboratory-synthesized standards. Moreover, ( Z )-3-hexen-1-ol also activates the synthesis of jasmonic acid to enhance the insect resistance of tea plants. Additionally, a continuous light treatment induces the accumulation of ( Z )-3-hexenyl-glycosides. Hence, ( Z )-3-hexenol serves as a light-regulated signaling molecule that activates the systemic defenses of adjacent plants. Our study reveals the molecular mechanisms by which biotic and abiotic factors synergistically regulate the signaling functions of herbivore-induced plant volatiles in plants, providing valuable information for future comprehensive analyses of the systemic defense mechanisms in plants.

It is generally proposed that tea cultivars with larger leaves contain more linalool, an important tea aroma contributor, than ones with smaller leaves. The objective of this study was to confirm the trait and explore the involved reason. Investigation on ten tea cultivars with different leaf areas demonstrated a significant positive correlation between linalool content and leaf area (R 2  = 0.739, p = 0.010). Analysis of metabolite and gene expression level showed that the transform ability of linalool into linalool oxides was the key factor. Feeding experiments that supplied tea leaves of different leaf areas with [ 2 H 3 ]linalool under different light conditions revealed that the larger tea leaves receive more light and are less capable of transformation of linalool to linalool oxides, thus leading to linalool accumulation. This information will advance understanding of the variation of linalool content in tea varieties and will provide assistance in breeding and screening of high-linalool tea cultivars., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The manufacturing process of tea (Camellia sinensis), especially oolong tea, involves multiple postharvest stresses. These stresses can induce the formation and accumulation of many important aroma compounds, such as indole-a key floral aroma contributor of oolong tea. However, little is known about the regulation mechanisms of aroma compound formation, especially epigenetic regulation. DNA methylation is an important epigenetic modification. Changes in the DNA methylation levels of promoter sequences can regulate gene expression under stress conditions. In this study, the differences in DNA methylation levels and histone 3 lysine 9 dimethylation levels of indole key biosynthetic gene (tryptophan synthase β-subunit 2, CsTSB2) were detected between untreated and continuous wounding treatment tea leaves. The results show that the DNA methylation levels affect the ability of the basic helix-loop-helix family transcription factor CsMYC2a to bind to the promoter of CsTSB2. Analyses of the transcript levels of DNA methyltransferases during oolong tea processing screened out candidate genes involved in the regulation of secondary metabolite product biosynthesis/accumulation. The results suggest that the domains rearranged methyltransferase 3, a DNA methyltransferase, is involved in the DNA methylation regulation of indole formation during the oolong tea manufacturing process. This is the first report on the involvement of DNA methylation in the regulation of aroma compound formation in tea leaves exposed to postharvest stresses., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The plant hormone abscisic acid (ABA) accumulates in tea leaves under dehydration stress during the withering process. However, the mechanism underlying ABA biosynthesis regulation remains largely unclear. In the present study, we found increased expression of ABA biosynthesis genes under dehydration stress during postharvest processing of tea. Furthermore, dehydration stress promoted ABA accumulation by increasing histone acetylation of ABA anabolism genes but by decreasing the levels of histone H3 lysine 9 dimethylation and DNA methylation of ABA biosynthesis genes. We screened candidate regulators of histone deacetylation and DNA methylation under dehydration stress. Taken together, our results indicate a role for epigenetic modifications during postharvest processing of tea.

l-Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l-theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l-theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l-theanine synthetase and also to determine the subcellular localization of fluorescent-tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l-theanine biosynthesis, and cytosol-located CsTSI was the key l-theanine synthase. In tea shoot tissue, l-theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l-theanine synthases. In addition, l-theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Tea is the second most popular beverage globally after water and contains abundant specialized metabolites. These metabolites give tea unique quality and are beneficial to human health. Some secondary metabolites are produced to help plants, including tea plants ( Camellia sinensis ), adapt to variable environment and grow normally. Therefore, whether abundant specialized metabolites have biological functions and play roles in the environmental adaptability of tea plants is of interest., Aim of Review: Research progress regarding the biological functions of specialized metabolites (including catechins, l-theanine, caffeine, and volatile compounds) in tea plants is summarized. Furthermore, the main and characteristic scientific questions regarding tea plant growth in contrast to other economic crops are proposed, including (i) how tea plants adapt to acid soils, (ii) why tea plants have fewer diseases, and (iii) why tea plants and tea green leafhoppers have a symbiotic relationship. Accordingly, the potential adaptive mechanism is summarized, which is related to the function of specialized metabolites in tea plants., Key Scientific Concepts of Review: This is the most in-depth investigation of biological functions of volatile compounds in tea plants. Direct in vivo evidence in tea plants shows that volatile compounds help defend against insects through plant-to-plant signaling. Furthermore, abundant specialized metabolites are speculated to contribute to the environmental adaptability of tea plants. However, further in vivo evidence and exploration of relevant mechanisms are required for all aspects discussed. This review provides an important reference for basic biological research on the tea plant as a specialized metabolite studying model., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

l-Theanine has a significant role in the taste of tea ( Camellia sinensis ) infusions. Our previous research indicated that the lower l-theanine metabolism in ethylamine and l-glutamate is a key factor that explains the higher content of l-theanine in albino tea with yellow or white leaves, compared with that of normal tea with green leaves. However, the specific genes encoding l-theanine hydrolase in tea remains unknown. In this study, CsPDX2.1 was cloned together with the homologous Arabidopsis PDX2 gene and the recombinant protein was shown to catalyze l-theanine hydrolysis into ethylamine and l-glutamate in vitro . There were higher CsPDX2.1 transcript levels in leaf tissue and lower transcripts in the types of albino (yellow leaf) teas compared with green controls. The subcellular location of ethylamine in tea leaves was shown to be in the mitochondria and peroxisome using a nonaqueous fractionation method. This study identified the l-theanine hydrolase gene and subcellular distribution of ethylamine in tea leaves, which improves our understanding of the l-theanine metabolism and the mechanism of differential accumulation of l-theanine among tea varieties.

Linalool is abundant in tea leaves and contributes greatly to tea aroma. The two isomers of linalool, ( R )-linalool and ( S )-linalool, exist in tea leaves. Our study found that ( R )-linalool was the minor isomer in nine of Camellia sinensis var. sinensis cultivars. The ( R )-linalool synthase of tea plant CsRLIS was identified subsequently. It is a chloroplast-located protein and specifically catalyzes the formation of ( R )-linalool in vitro and in vivo . CsRLIS was observed to be a stress-responsive gene and caused the accumulation of internal ( R )-linalool during oolong tea manufacture, mechanical wounding, and insect attack. Further study demonstrated that the catalytic efficiency of CsRLIS was much lower than that of ( S )-linalool synthase CsSLIS, which might explain the lower ( R )-linalool proportion in C. sinensis var. sinensis cultivars. The relative expression levels of CsRLIS and CsSLIS may also affect the ( R )-linalool proportions among C. sinensis var. sinensis cultivars. This information will help us understand differential distributions of chiral aroma compounds in tea.

In tea ( Camellia sinensis ) plants, polyphenols are the representative metabolites and play important roles during their growth. Among tea polyphenols, catechins are extensively studied, while very little attention has been paid to other polyphenols such as gallic acid (GA) that occur in tea leaves with relatively high content. In this study, GA was able to be transformed into methyl gallate (MG), suggesting that GA is not only a precursor of catechins, but also can be transformed into other metabolites in tea plants. GA content in tea leaves was higher than MG content-regardless of the cultivar, plucking month or leaf position. These two metabolites occurred with higher amounts in tender leaves. Using nonaqueous fractionation techniques, it was found that GA and MG were abundantly accumulated in peroxisome. In addition, GA and MG were found to have strong antifungal activity against two main tea plant diseases, Colletotrichum camelliae and Pseudopestalotiopsis camelliae-sinensis . The information will advance our understanding on formation and biologic functions of polyphenols in tea plants and also provide a good reference for studying in vivo occurrence of specialized metabolites in economic plants.

Plants have to cope with various environmental stress factors which significantly impact plant physiology and secondary metabolism. Individual stresses, such as low temperature, are known to activate plant volatile compounds as a defense. However, less is known about the effect of multiple stresses on plant volatile formation. Here, the effect of dual stresses (wounding and low temperature) on volatile compounds in tea (Camellia sinensis) plants and the underlying signalling mechanisms were investigated. Indole, an insect resistance volatile, was maintained at a higher content and for a longer time under dual stresses compared with wounding alone. CsMYC2a, a jasmonate (JA)-responsive transcription factor, was the major regulator of CsTSB2, a gene encoding a tryptophan synthase β-subunit essential for indole synthesis. During the recovery phase after tea wounding, low temperature helped to maintain a higher JA level. Further study showed that CsICE2 interacted directly with CsJAZ2 to relieve inhibition of CsMYC2a, thereby promoting JA biosynthesis and downstream expression of the responsive gene CsTSB2 ultimately enhancing indole biosynthesis. These findings shed light on the role of low temperature in promoting plant damage responses and advance knowledge of the molecular mechanisms by which multiple stresses coordinately regulate plant responses to the biotic and abiotic environment., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

trans -Cinnamic acid (CA) is a precursor of many phenylpropanoid compounds, including catechins and aroma compounds, in tea ( Camellia sinensi s) leaves and is derived from l-phenylalanine (l-Phe) deamination. We have discovered an alternative CA formation pathway from l-Phe via phenylpyruvic acid (PPA) and phenyllactic acid (PAA) in tea leaves through stable isotope-labeled precursor tracing and enzyme reaction evidence. Both PPA reductase genes ( CsPPARs ) involved in the PPA-to-PAA pathway were isolated from tea leaves and functionally characterized in vitro and in vivo . CsPPAR1 and CsPPAR2 transformed PPA into PAA and were both localized in the leaf cell cytoplasm. Rosa hybrida flowers (economic crop flower), Lycopersicon esculentum Mill. fruits (economic crop fruit), and Arabidopsis thaliana leaves (leaf model plant) also contained this alternative CA formation pathway, suggesting that it occurred in most plants, regardless of different tissues and species. These results improve our understanding of CA biosynthesis in tea plants and other plants.

Catechins and theaflavins are important metabolites contributing to tea function and quality. Catechins are known to transform into theaflavins during the tea manufacturing process, but the same transformation in preharvest tea leaves is unknown. Herein, we determined that shade treatment (dark), an agronomic practise widely used in tea cultivation, reduced the contents of most catechins, but increased the theaflavin contents, in preharvest tea leaves (cv. Yinghong No.9). This was attributed to the activation of polyphenoloxidase (PPO) activity in darkness. Furthermore, CsPPO3 was highly expressed under darkness, and thus CsPPO3 had been cloned, sequenced, and characterization. The CsPPO3 recombinant protein exhibited PPO function. Furthermore, shade treatment also reduced the catechin contents and increased the theaflavin contents in Yabukita and Hoshinomidori, suggesting that this phenomenon might not be specific to certain tea cultivars. This information will aid in understanding of theaflavin formation and its response to environmental factors at the preharvest tea stage., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Volatiles affect tea ( Camellia sinensis ) aroma quality and have roles in tea plant defense against stresses. Some volatiles defend against stresses through their toxicity, which might affect tea safety. Benzyl nitrile is a defense-related toxic volatile compound that accumulates in tea under stresses, but its formation mechanism in tea remains unknown. In this study, l-[ 2 H 8 ]phenylalanine feeding experiments and enzyme reactions showed that benzyl nitrile was generated from l-phenylalanine via phenylacetaldoxime in tea. CsCYP79D73 showed activity for converting l-phenylalanine into phenylacetaldoxime, while CsCYP71AT96s showed activity for converting phenylacetaldoxime into benzyl nitrile. Continuous wounding in the oolong tea process significantly enhanced the CsCYP79D73 expression level and phenylacetaldoxime and benzyl nitrile contents. Benzyl nitrile accumulation under continuous wounding stress was attributed to an increase in jasmonic acid, which activated CsCYP79D73 expression. This represents the first elucidation of the formation mechanism of benzyl nitrile in tea.

Tea (Camellia sinensis) contains two active glutamate decarboxylases (CsGADs), whose unclear properties were examined here. CsGAD1 was 4-fold higher than CsGAD2 in activity. Their K m values for L-glutamate were around 5 mM. CsGAD1 and CsGAD2 performed best at 55 and 40 °C, respectively, and were both stimulated by calcium/calmodulin (Ca 2+ /CaM). Over 40 °C, their calmodulin-binding domains degraded. CsGADs were most active at pH 5.6, and were stimulated by Ca 2+ /CaM at pH 5.6-6.6, but inactivated at pH 3.6. Ca 2+ /CaM restored the CsGAD1 activity suppressed by inhibitors. CsGADs and CsCaM were localized to the cytosol. CsGAD1 was more highly expressed in most tissues, while CsGAD2 expression was more induced under stresses. The characteristics we first elucidated here revealed that CsGAD1 is the predominant isoform in tea plant, with CsGAD2 exhibiting a supplementary role under certain conditions. The information will contribute to regulation of GABA tea quality., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Amino acids are the main contributors to tea ( Camellia sinensis ) flavor and function. Tea leaves contain not only proteinaceous amino acids but also specialized non-proteinaceous amino acids such as L-theanine and γ-aminobutyric acid (GABA). Here, we review different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea. The key findings were: (1) High accumulations of proteinaceous amino acids mainly result from protein degradation, which occurs in each tea stage, including preharvest, postharvest, manufacturing, and deep processing; (2) L-Theanine is the most represented non-proteinaceous amino acid that contributes to tea taste and function. Its accumulation is influenced more by the variety than by exogenous factors; and (3) GABA is the second most represented non-proteinaceous amino acid that contributes to tea function. Its formation, and resulting accumulation, are responses to stress. The combination of anoxic stress and mechanical damage are essential for a high GABA accumulation. An understanding of the biosynthesis, metabolism, and regulatory mechanisms of the proteinaceous and non-proteinaceous amino acids during the whole process from raw materials to tea products is necessary to safely and effectively alter tea flavor and function.

When insects attack plants, insect-derived elicitors and mechanical damage induce the formation and emission of plant volatiles that have important ecological functions and flavor properties. These events have mainly been studied in model plants, rather than crop plants. Our study showed that tea green leafhopper ( Empoasca ( Matsumurasca ) onukii Matsuda), a major pest infesting tea attack significantly induced the emission of geraniol from tea leaves, but did not affect the crude enzyme activity of geraniol synthase in tea leaves. An enzyme extract of E. (M.) onukii specifically produced geraniol from geraniol diphosphate. Furthermore, a terpene synthase (EoTPS) was isolated from E. (M.) onukii . This terpene synthase was able to convert geraniol diphosphate to geraniol in vitro. In addition, geraniol had in vitro ability to inhibit the growth of Acinetobacter johnsonii that is endobacterial isolated from E. (M.) onukii . This information illustrates that elicitors from piercing-sucking insects can induce the formation of volatiles from crop plants and advances our understanding of the roles of plant volatiles in the interaction among crops-insects-microorganisms.

Although specialized metabolite distributions in different tea (Camellia sinensis) tissues has been studied extensively, little is known about their within-tissue distribution owing to the lack of nondestructive methodology. In this study, desorption electrospray ionization imaging mass spectrometry was used to investigate the within-tissue spatial distributions of specialized metabolites in tea. To overcome the negative effects of the large amount of wax on tea leaves, several sample preparation methods were compared, with a Teflon-imprint method established for tea leaves. Polyphenols are characteristic metabolites in tea leaves. Epicatechin gallate/catechin gallate, epigallocatechin gallate/gallocatechin gallate, and gallic acid were evenly distributed on both sides of the leaves, while epicatechin/catechin, epigallocatechin/gallocatechin, and assamicain A were distributed near the leaf vein. L-Theanine was mainly accumulated in tea roots. L-Theanine and valinol were distributed around the outer root cross-section. The results will advance our understanding of the precise localizations and in-vivo biosyntheses of specialized metabolites in tea., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

2-Phenylethanol (2PE) is a representative aromatic aroma compound in tea ( Camellia sinensis ) leaves. However, its formation in tea remains unexplored. In our study, feeding experiments of [ 2 H 8 ]L-phenylalanine (Phe), [ 2 H 5 ]phenylpyruvic acid (PPA), or ( E / Z )-phenylacetaldoxime (PAOx) showed that three biosynthesis pathways for 2PE derived from L-Phe occurred in tea leaves, namely, pathway I (via phenylacetaldehyde (PAld)), pathway II (via PPA and PAld), and pathway III (via ( E / Z )-PAOx and PAld). Furthermore, increasing temperature resulted in increased flux into the pathway for 2PE from L-Phe via PPA and PAld. In addition, tomato fruits and petunia flowers also contained the 2PE biosynthetic pathway from L-Phe via PPA and PAld and increasing temperatures led to increased flux into this pathway, suggesting that such a phenomenon might be common among most plants containing 2PE. This represents a characteristic example of changes in flux into the biosynthesis pathways of volatile compounds in plants in response to stresses.

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

The enzymatic hydrolysis of glycosidically bound volatiles (GBVs) plays an important role in tea aroma formation during the tea manufacturing process. However, during the enzyme-active manufacturing process of oolong tea, most GBVs showed no reduction, while ( Z)-3-hexenyl-β-glucopyranoside significantly enhanced at the turnover stage. This study aimed to determine the reason for this increase in ( Z)-3-hexenyl-β-glucopyranoside. Continuous wounding stress in the turnover stage did not enhance the expression level of glycosyltransferase 1 ( CsGT1), while it induced a significant increase in the ( Z)-3-hexenol content ( p ≤ 0.05). Furthermore, observing the cell structures of tea leaves exposed to continuous wounding and subcellular localizations of CsGTs suggested that the interaction of ( Z)-3-hexenol (substrate) and CsGT1 (enzyme) was available. In conclusion, both continuous wounding and subcellular localizations led to a ( Z)-3-hexenyl-β-glucopyranoside enhancement mechanism during the oolong tea's turnover stage. These results advance our understanding of GBV formation during the tea manufacturing process and their relationship with the stress from the tea manufacturing process. In addition, the information will help us further evaluate contribution of GBVs to enzymatic formation of oolong tea aroma compounds.

1-Phenylethanol is a chiral flavor compound that has enantiomers, (R)- and (S)-1-phenylethanol, with different flavor properties. Given that isolating these enantiomers from plants is low yielding and costly, enzymatic synthesis presents an alternative approach. However, the genes/enzymes that specifically produce (R)- and (S)-1-phenylethanol in plants are unknown. To identify these enzymes in tea (Camellia sinensis) flowers, 21 short chain dehydrogenase (SDR) genes were isolated from tea flowers, cloned, and functionally characterized. Several recombinant SDRs in Escherichia coli exhibited activity for converting acetophenone to (S)-1-phenylethanol (CsSPESs, >99.0%), while only one SDR produced (R)-1-phenylethanol (CsRPES, 98.6%). A pair of homologue enzymes (CsSPES and CsRPES) showed a strong preference for NADPH cofactor, with optimal enzymatic reaction conditions of 45-55 °C and pH 8.0. Identification of the tea flower-derived gene responsible for specific synthesis of (R)- and (S)-1-phenylethanolsuggests enzymatic synthesis of enantiopure 1-phenylethanol is possible using a plant-derived gene., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

l-Theanine is a specialized metabolite in tea (Camellia sinensis) leaves that contributes to tea function and quality. Yellow tea leaves (albino) generally have higher l-theanine contents than green tea leaves (normal), but the reason is unknown. The objective of this study was to investigate why l-theanine is accumulated in yellow tea leaves. We compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. The l-theanine content was significantly higher in yellow leaves than in green leaves (p ≤ 0.01). After supplementation with [ 2 H 5 ]-l-theanine, yellow leaves catabolized less [ 2 H 5 ]-l-theanine than green leaves (p ≤ 0.05). Furthermore, most plants contained the enzyme catalyzing l-theanine conversion to ethylamine and l-glutamic acid. In conclusion, l-theanine accumulation in albino-induced yellow tea leaves was due to weak l-theanine catabolism. The differential accumulation mechanism differed from the l-theanine accumulation mechanism in tea and other plants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Characteristic aroma formation in tea ( Camellia sinensis ) leaves during the oolong tea manufacturing process might result from the defense responses of tea leaves against these various stresses, which involves upregulation of the upstream signal phytohormones related to leaf chloroplasts, such as jasmonic acid (JA). Whether chloroplast changes affect the formation of JA and characteristic aroma compounds in tea leaves exposed to stresses is unknown. In tea germplasms, albino-induced yellow tea leaves have defects in chloroplast ultrastructure and composition. Herein, we have compared the differential responses of phytohormone and characteristic aroma compound formation in normal green and albino-induced yellow tea leaves exposed to continuous wounding stress, which is the main stress in oolong tea manufacture. In contrast to single wounding stress (from picking, as a control), continuous wounding stress can upregulate the expression of CsMYC2 , a key transcription factor of JA signaling, and activate the synthesis of JA and characteristic aroma compounds in both normal tea leaves (normal chloroplasts) and albino tea leaves (chloroplast defects). Chloroplast defects had no significant effect on the expression levels of CsMYC2 and JA synthesis-related genes in response to continuous wounding stress, but reduced the increase in JA content in response to continuous wounding stress. Furthermore, chloroplast defects reduced the increase in volatile fatty acid derivatives, including jasmine lactone and green leaf volatile contents, in response to continuous wounding stress. Overall, the formation of metabolites derived from fatty acids, such as JA, jasmine lactone, and green leaf volatiles in tea leaves, in response to continuous wounding stress, was affected by chloroplast defects. This information will improve understanding of the relationship of the stress responses of JA and aroma compound formation with chloroplast changes in tea.

Metabolite formation is a biochemical and physiological feature of plants developed as an environmental response during the evolutionary process. These metabolites help defend plants against environmental stresses, but are also important quality components in crops. Utilizing the stress response to improve natural quality components in plants has attracted increasing research interest. Tea, which is processed by the tender shoots or leaves of tea plant ( Camellia sinensis (L.) O. Kuntze), is the second most popular beverage worldwide after water. Aroma is an important factor affecting tea character and quality. The defense responses of tea leaves against various stresses during preharvest (tea growth process) and postharvest (tea manufacturing) processing can result in aroma formation. Herein, we summarize recent investigations into the biosyntheses of several characteristic aroma compounds prevalent in teas and derived from volatile fatty acid derivatives, terpenes, and phenylpropanoids/benzenoids. Several key aroma synthetic genes from tea leaves have been isolated, cloned, sequenced, and functionally characterized. Biotic stress (such as tea green leafhopper attack) and abiotic stress (such as light, temperature, and wounding) could enhance the expression of aroma synthetic genes, resulting in the abundant accumulation of characteristic aroma compounds in tea leaves. Understanding the specific relationships between characteristic aroma compounds and stresses is key to improving tea quality safely and effectively.

Tea ( Camellia sinensis ) cultivars with green leaves are the most widely used for making tea. Recently, tea mutants with white or yellow young shoots have attracted increasing interest as raw materials for making "high-quality" tea products. Albino teas are generallycharacterized as having metabolites of relatively high amino acid content and lower catechin content. However, little is known about aroma compounds in albino tea leaves. Herein, we compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. GC-MS was employed to analyze endogenous tea aroma compounds and related precursors. Quantitative real time PCR was used to measure expression levels of genes involved in biosyntheses of tea aromas.The total contents of most endogenous free tea aromas, including aroma fatty acid derivatives, aroma terpenes, and aroma phenylpropanoids/benzenoids, and their glycosidically bound aroma compounds, were lower in yellow leaves than in green leaves. The content of the key precursor geranyl diphosphate (GDP) and expression levels of key synthetic genes involved in the formation of linalool, a major aroma compound in cv. Yinghong No. 9, were investigated. Linalool content was lower in albino-induced yellow leaves, which was due to the lower GDP content compared with normal green leaves.

After tea leaves, tea (Camellia sinensis) flowers are becoming a second tea plant resource because they contain not only functional metabolites similar to those found in tea leaves, but also predominant amounts of functional metabolites that only occur in tea leaves in small amounts. 1-Phenylethanol (1PE) is a predominant aroma compound found in tea flowers. A 1PE synthase in tea flowers was isolated, functionally characterized, and shown to have the highest catalytic efficiency for the conversion of acetophenone (AP). To determine why 1PE accumulates more in tea flowers than other plants, we compared their 1PE contents and used a stable isotope labeling method to elucidate the 1PE biosynthetic route. Supplementation with [ 2 H 8 ]l-phenylalanine and [ 2 H 5 ]AP suggested that most plants containing the enzyme/gene catalyzed the conversion of AP to 1PE. Furthermore, the availability of AP derived from l-phenylalanine was responsible for the difference in 1PE accumulation between tea flowers and other plants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Jasmonic acid (JA) is reportedly involved in the interaction between insects and the vegetative parts of horticultural crops; less attention has, however, been paid to its involvement in the interaction between insects and the floral parts of horticultural crops. Previously, we investigated the allene oxide synthase 2 ( AOS2 ) gene that was found to be the only JA synthesis gene upregulated in tea ( Camellia sinensis ) flowers exposed to insect ( Thrips hawaiiensis (Morgan)) attacks. In our present study, transient expression analysis in Nicotiana benthamiana plants confirmed that CsAOS2 functioned in JA synthesis and was located in the chloroplast membrane. In contrast to tea leaves, the metabolite profiles of tea flowers were not significantly affected by 10 h JA (2.5 mM) treatment as determined using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, and gas chromatography-mass spectrometry. Moreover, JA treatment did not significantly influence ethylene formation in tea flowers. These results suggest that JA in tea flowers may have different functions from JA in tea leaves and other flowers.

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.

Tea ( Camellia sinensis ) is an important crop, and its leaves are used to make the most widely consumed beverage, aside from water. People have been using leaves from tea plants to make teas for a long time. However, less attention has been paid to the flowers of tea plants, which is a waste of an abundant resource. In the past 15 years, researchers have attempted to discover, identify, and evaluate functional molecules from tea flowers, and have made insightful and useful discoveries. Here, we summarize the recent investigations into these functional molecules in tea flowers, including functional molecules similar to those in tea leaves, as well as the preponderant functional molecules in tea flowers. Tea flowers contain representative metabolites similar to those of tea leaves, such as catechins, flavonols, caffeine, and amino acids. The preponderant functional molecules in tea flowers include saponins, polysaccharides, aromatic compounds, spermidine derivatives, and functional proteins. We also review the safety and biological functions of tea flowers. Tea flower extracts are proposed to be of no toxicological concern based on evidence from the evaluation of mutagenicity, and acute and subchronic toxicity in rats. The presence of many functional metabolites in tea flowers indicates that tea flowers possess diverse biological functions, which are mostly related to catechins, polysaccharides, and saponins. Finally, we discuss the potential for, and challenges facing, future applications of tea flowers as a second resource from tea plants., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Herbivore-induced plant volatiles (HIPVs) act as direct defenses against herbivores and as indirect defenses by attracting herbivore enemies. However, the involvement of HIPVs in within-plant or plant-to-plant signaling is not fully clarified. Furthermore, in contrast to model plants, HIPV signaling roles in crops have hardly been reported. Here, we investigated HIPVs emitted from tea (Camellia sinensis) plants, an important crop used for beverages, and their involvement in tea plant-to-plant signaling. To ensure uniform and sufficient exposure to HIPVs, jasmonic acid combined with mechanical damage (JAMD) was used to simulate herbivore attacks. Metabonomics techniques based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and gas chromatography-mass spectrometry were employed to determine metabolite changes in undamaged tea plants exposed to JAMD-stimulated volatiles. JAMD-stimulated volatiles mainly enhanced the amounts of 1-O-galloyl-6-O-luteoyl-α-d-glucose, assamicain C, 2,3,4,5-tetrahydroxy-6-oxohexyl gallate, quercetagitrin, 2-(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-8-yl)-4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-3-yl, 3,4-dimethoxybenzoate, 1,3,4,5,6,7-hexahydroxyheptan-2-one, and methyl gallate in neighboring undamaged tea leaves. Furthermore, α-farnesene and β-ocimene, which were produced after JAMD treatments, were identified as two main JAMD-stimulated volatiles altering metabolite profiles of the neighboring undamaged tea leaves. This research advances our understanding of the ecological functions of HIPVs and can be used to develop crop biological control agents against pest insects in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 [ 2 H 5 ]ethylamine, all the plants produce [ 2 H 5 ]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea ( Camellia sinensis ) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.

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.

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.

γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. It has multiple positive effects on mammalian physiology and is an important bioactive component of tea (Camellia sinensis). GABA generally occurs at a very low level in plants but GABA content increases substantially after exposure to a range of stresses, especially oxygen-deficiency. During processing of tea leaves, a combination of anoxic stress and mechanical damage are essential for the high accumulation of GABA. This is believed to be initiated by a change in glutamate decarboxylase activity, but the underlying mechanisms are unclear. In the present study we characterized factors regulating the expression and activity of three tea glutamate decarboxylase genes (CsGAD1, 2, and 3), and their encoded enzymes. The results suggests that, unlike the model plant Arabidopsis thaliana, there are dual mechanisms regulating the accumulation of GABA in tea leaves exposed to multiple stresses, including activation of CsGAD1 enzymatic activity by calmodulin upon the onset of the stress and accumulation of high levels of CsGAD2 mRNA induced by a combination of anoxic stress and mechanical damage.

A recently developed method enabled us to simultaneously characterize and quantitate glycosidically bound volatiles (GBVs) at picomole levels using liquid chromatography-mass spectrometry (LC-MS). On the basis of the analytical data it is possible to screen tea varieties most suitable for black tea processing, in which higher concentrations of primeverosides accumulate. The primeverosides decreased at the rolling step in black tea processing, whereas the glucopyranosides did not change much. The total contents of GBVs gradually increased at the withering steps and then remarkably increased after the fixing step at 230 °C, during oolong tea processing. The presence of 6'-O-malonyl ester type β-D-glucopyranosides in the tea samples suggested a contribution to the increment in glucopyranosides during oolong tea processing. The method was also used to analyze GBVs and their derivatives to understand their possible role in the metabolic pathway of tea.

Regulation of plant growth and development by light wavelength has been extensively studied. Less attention has been paid to effect of light wavelength on formation of plant metabolites. The objective of this study was to investigate whether formation of volatiles in preharvest and postharvest tea (Camellia sinensis) leaves can be regulated by light wavelength. In the present study, in contrast to the natural light or dark treatment, blue light (470 nm) and red light (660 nm) significantly increased most endogenous volatiles including volatile fatty acid derivatives (VFADs), volatile phenylpropanoids/benzenoids (VPBs), and volatile terpenes (VTs) in the preharvest tea leaves. Furthermore, blue and red lights significantly up-regulated the expression levels of 9/13-lipoxygenases involved in VFADs formation, phenylalanine ammonialyase involved in VPBs formation, and terpene synthases involved in VTs formation. Single light wavelength had less remarkable influences on formation of volatiles in the postharvest leaves compared with the preharvest leaves. These results suggest that blue and red lights can be promising technology for remodeling the aroma of preharvest tea leaves. Furthermore, our study provided evidence that light wavelength can activate the expression of key genes involved in formation of plant volatiles for the first time.

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. β-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided evidence that β-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 β-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.

A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE β-d-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE β-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase β-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, β-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time.

We produced low temperature (15 °C) processed green tea (LTPGT) with higher aroma contents than normal green tea (Sencha). Normal temperature processed green tea (NTPGT), involved storing at 25 °C, and Sencha had no storing process. Sensory evaluation showed LTPGT had higher levels of floral and sweet odorants than NTPGT and Sencha. Aroma extract dilution analysis and gas chromatography-mass spectrometry-olfactometry indicated LTPGT had 12 aroma compounds with high factor dilution values (FD). Amongst LTPGT's 12 compounds, indole, jasmine lactone, cis-jasmone, coumarin, and methyl epijasmonate contributed to floral, fruity and sweet characters. In particular, indole increased initially, peaking at 16 h, then gradually decreased. Feeding experiments suggested [(15)N]indole and [(15)N]oxygenated indoles (OX-indoles) were produced from [(15)N]anthranilic acid. We proposed the increase in indole was due to transformation of anthranilic acid during the 16 h storage and the subsequent decline in indole level was due to its conversion to OX-indoles., (Copyright © 2013 Elsevier Ltd. All rights reserved.)