Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.

Biosynthetic Pathway and Bioactivity of Vanillin, a Highly Abundant Metabolite Distributed in the Root Cortex of Tea Plants (Camellia sinensis).

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.

Light induces an increasing release of benzyl nitrile against diurnal herbivore Ectropis grisescens Warren attack in tea (Camellia sinensis) plants.

Herbivore-induced plant volatiles (HIPVs) are critical compounds that directly or indirectly regulate the tritrophic interactions among herbivores, natural enemies and plants. The synthesis and release of HIPVs are regulated by many biotic and abiotic factors. However, the mechanism by which multiple factors synergistically affect HIPVs release remains unclear. Tea plant (Camellia sinensis) is the object of this study because of its rich and varied volatile metabolites. In this study, benzyl nitrile was released from herbivore-attacked tea plants more in the daytime than at night, which was consistent with the feeding behaviour of tea geometrid (Ectropis grisescens Warren) larvae. The Y-tube olfactometer assay and insect resistance analysis revealed that benzyl nitrile can repel tea geometrid larvae and inhibit their growth. On the basis of enzyme activities in transiently transformed Nicotiana benthamiana plants, CsCYP79 was identified as a crucial regulator in the benzyl nitrile biosynthetic pathway. Light signalling-related transcription factor CsPIF1-like and the jasmonic acid (JA) signalling-related transcription factor CsMYC2 serve as the activator of CsCYP79 under light and damage conditions. Our study revealed that light (abiotic factor) and herbivore-induced damage (biotic stress) synergistically regulate the synthesis and release of benzyl nitrile to protect plants from diurnal herbivorous tea geometrid larvae.

Effects of temperature and light on quality-related metabolites in tea [Camellia sinensis (L.) Kuntze] leaves.

In China, the environmental conditions in mountainous regions are suitable for producing high-quality tea, but the underlying mechanism is unknown. Analyses of published data revealed tea leaves harvested at high altitudes are associated with high-quality tea, possibly because of the effects of light and temperature. This was confirmed by simulating the changes induced by temperature and light at different altitudes. Catechin biosynthesis was inhibited by high altitudes, whereas free amino acid contents increased, leading to a decrease in polyphenol to amino acids ratio. Under simulated high-altitude conditions, increases in the abundance of l-theanine and other amino acids were due to the increase in precursor contents and chloroplast protein hydrolysis, respectively. In addition, some aroma compound contents increased in fresh or wounded leaves because of up-regulated expression of key structural genes. Overall, tea taste and aroma were enhanced by simulated high-altitude conditions. This study provides the basis for enhancing low-quality tea raw materials.

Characterizing the cultivar-specific mechanisms underlying the accumulation of quality-related metabolites in specific Chinese tea (Camellia sinensis) germplasms to diversify tea products.

The diverse tea (Camellia sinensis) germplasms in China include those that specifically accumulate metabolites, such as anthocyanin, catechin, amino acid, caffeine, aroma compound, and chlorophyll. There is interest in the derived products because of special flavor quality or high efficacy activity. This review describes the characteristics of specific tea germplasms and associated regulatory mechanisms. High expression levels of the corresponding biosynthetic genes lead to the substantial accumulation of anthocyanins. The increased metabolic flux from anthocyanins to galloylated catechins is responsible for the occurrence of high-catechin germplasms. The precursor ethylamine determines the differential abundance of l-theanine between tea and other plants. The high amino acid contents in albino germplasms are the result of decreased l-theanine hydrolysis. In low-caffeine tea germplasms, caffeine synthase genes are minimally expressed or mutated. High-aroma germplasms are associated with an increase in the precursors or strong stress-induced responses. Enhanced chloroplast and chlorophyll synthesis is a hallmark of the high-chlorophyll germplasms. Overall, biosynthetic metabolism might have contributed to the occurrence of specific tea germplasms. Furthermore, elucidation the deeper molecular mechanisms in specific tea germplasms are significant and urgent. The information will enhance our understanding of the metabolic activities in tea plants, with implications for tea breeding.

Light synergistically promotes the tea green leafhopper infestation-induced accumulation of linalool oxides and their glucosides in tea (Camellia sinensis).

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.

Strategies for studying in vivo biochemical formation pathways and multilevel distributions of quality or function-related specialized metabolites in tea (Camellia sinensis).

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.

How does tea (Camellia sinensis) produce specialized metabolites which determine its unique quality and function: a review.

Tea (Camellia sinensis) is both a plant and a foodstuff. Many bioactive compounds, which are present in the final tea product and related to its quality or functional properties, are produced during the tea manufacturing process. However, the characteristic secondary metabolites, which give tea its unique qualities and are beneficial to human health, are produced mainly in the leaves during the process of plant growth. Therefore, it is important to understand how tea leaves produce these specialized metabolites. In this review, we first compare the common metabolites and specialized metabolites in tea, coffee, cocoa, and grape and discuss the occurrence of characteristic secondary metabolites in tea. Progress in research into the formation of these characteristic secondary metabolites in tea is summarized, including establishing a biological database and genetic transformation system, and the biosynthesis of characteristic secondary metabolites. Finally, speculation on future research into the characteristic secondary metabolites of tea is provided from the viewpoints of the origin, resources, cultivation, and processing of tea. This review provides an important reference for future research on the specialized metabolites of tea in terms of its characteristics.

Stable Isotope-Labeled Precursor Tracing Reveals that l-Alanine is Converted to l-Theanine via l-Glutamate not Ethylamine in Tea Plants In Vivo.

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.

Herbivore-Induced (Z)-3-Hexen-1-ol is an Airborne Signal That Promotes Direct and Indirect Defenses in Tea (Camellia sinensis) under Light.

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.

Elucidation of the Regular Emission Mechanism of Volatile ß-Ocimene with Anti-insect Function from Tea Plants (Camellia sinensis) Exposed to Herbivore Attack.

Herbivore-induced plant volatiles (HIPVs) play an important role in insect resistance. As a common HIPV in tea plants (Camellia sinensis), ß-ocimene has shown anti-insect function in other plants. However, whether ß-ocimene in tea plants also provides insect resistance, and its mechanism of synthesis and emission are unknown. In this study, ß-ocimene was confirmed to interfere with tea geometrid growth via signaling. Light was identified as the key factor controlling regular emission of ß-ocimene induced by the wounding from tea geometrids. ß-Ocimene synthase (CsBOS1) was located in plastids and catalyzed ß-ocimene formation in overexpressed tobacco. CsBOS1 expression in tea leaves attacked by tea geometrids showed a day-low and night-high variation pattern, while CsABCG expression involved in volatile emission showed the opposite pattern. These two genes might regulate the regular ß-ocimene emission from tea plants induced by tea geometrid attack. This study advances the understanding on HIPV emission and signaling in tea plants.

Epigenetic Regulation of the Phytohormone Abscisic Acid Accumulation under Dehydration Stress during Postharvest Processing of Tea (Camellia sinensis).

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.

Nonaqueous fractionation and overexpression of fluorescent-tagged enzymes reveals the subcellular sites of L-theanine biosynthesis in 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.

Roles of specialized metabolites in biological function and environmental adaptability of tea plant (Camellia sinensis) as a metabolite studying model.

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.

Characterization of l-Theanine Hydrolase in Vitro and Subcellular Distribution of Its Specific Product Ethylamine in Tea (Camellia sinensis).

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.

Metabolism of Gallic Acid and Its Distributions in Tea (Camellia sinensis) Plants at the Tissue and Subcellular Levels.

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.

Alternative Pathway to the Formation of trans-Cinnamic Acid Derived from l-Phenylalanine in Tea (Camellia sinensis) Plants and Other Plants.

trans-Cinnamic acid (CA) is a precursor of many phenylpropanoid compounds, including catechins and aroma compounds, in tea (Camellia sinensis) 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.

Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment.

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.

Low temperature synergistically promotes wounding-induced indole accumulation by INDUCER OF CBF EXPRESSION-mediated alterations of jasmonic acid signaling in Camellia sinensis.

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 ß-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.

Biochemical Pathway of Benzyl Nitrile Derived from l-Phenylalanine in Tea (Camellia sinensis) and Its Formation in Response to Postharvest Stresses.

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-[2H8]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.