The Circadian Clock Gene PHYTOCLOCK1 Mediates the Diurnal Emission of the Anti-Insect Volatile Benzyl Nitrile from Damaged Tea (Camellia sinensis) Plants.

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.

Tea green leafhopper infestations affect tea plant growth by altering the synthesis of brassinolide.

Tea green leafhoppers are insects widely distributed in major tea-growing areas. At present, less attention has been paid to the study on effect of tea green leafhopper infestation on tea growth phenotype. In this study, tea green leafhoppers were used to treat tea branches in laboratory and co-treated with brassinolide (BL), the highest bioactivity of brassinosteroids (BRs), in tea garden. The results showed that the expression of genes related to BRs synthesis was inhibited and BL content was reduced in tea shoots after infestation by tea green leafhoppers. In addition, area of each leaf position, length and diameter of internodes, and the biomass of the tender shoots of tea plant were decreased after infestation by tea green leafhoppers. The number of trichomes, leaf thickness, palisade tissue thickness and cuticle thickness of tea shoots were increased after tea green leafhoppers infestation. BL spraying could partially recover the phenotypic changes of tea branches caused by tea green leafhoppers infestation. Further studies showed that tea green leafhoppers infestation may regulate the expression of CsDWF4 (a key gene for BL synthesis) through transcription factors CsFP1 and CsTCP1a, which finally affect the BL content. Moreover, BL was applied to inhibit the tea green leafhoppers infestation on tea shoots. In conclusion, our study revealed the effect of plant hormone BL-mediated tea green leafhoppers infestation on the growth phenotype of tea plants.

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.

Natural allelic variation confers high resistance to sweet potato weevils in sweet potato.

Sweet potato (Ipomoea batatas L.) is a major root crop worldwide. Sweet potato weevils (SPWs) pose one of the most significant challenges to sweet potato production in tropical and subtropical regions, causing deleterious economic and environmental effects. Characterizing the mechanisms underlying natural resistance to SPWs is therefore crucial; however, the genetic basis of host SPW resistance (SPWR) remains unclear. Here we obtained two sweet potato germplasm with high SPWR and, by map-based cloning, revealed two major SPW-resistant genes-SPWR1 and SPWR2-that are important regulators of natural defence against SPWs. The SPW-induced WRKY transcriptional factor SPWR1 directly activates the expression of SPWR2, and SPWR2, the conserved dehydroquinate synthase, promotes the accumulation of quinate derivative metabolites that confer SPWR in sweet potato. Generally, our results provide new insights into the molecular mechanism underlying sweet potato-SPW interactions and will aid future efforts to achieve eco-friendly SPW management.

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.

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.

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.

Feasible strategies for studying the involvement of DNA methylation and histone acetylation in the stress-induced formation of quality-related metabolites in tea (Camellia sinensis).

Tea plants are subjected to multiple stresses during growth, development, and postharvest processing, which affects levels of secondary metabolites in leaves and influences tea functional properties and quality. Most studies on secondary metabolism in tea have focused on gene, protein, and metabolite levels, whereas upstream regulatory mechanisms remain unclear. In this review, we exemplify DNA methylation and histone acetylation, summarize the important regulatory effects that epigenetic modifications have on plant secondary metabolism, and discuss feasible research strategies to elucidate the underlying specific epigenetic mechanisms of secondary metabolism regulation in tea. This information will help researchers investigate the epigenetic regulation of secondary metabolism in tea, providing key epigenetic data that can be used for future tea genetic breeding.

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.

The Morphological and Dynamic Changes of Ultrasound in the Evaluation of Effects of Oral Steroids Treatment for Patients with Carpal Tunnel Syndrome.

The role of oral steroids in carpal tunnel syndrome (CTS) remains elusive. This study aims to depict the ultrasound findings and conceivable mechanisms in relation to the efficacy of oral steroids for patients with CTS by measuring the morphological and motion changes in the median nerve. In this study, CTS patients were randomized to the oral steroid group (14 participants and 22 wrists) or nicergoline group (22 participants and 35 wrists) for 4 weeks. Both treatment arms were given global symptom score (GSS) measurements and completed an ultra-sound at baseline and at 2- and 4-weeks post-treatment. In the nerve conduction study (NCS), distal motor latency (DML) was used to assess the treatment response at baseline and 4 weeks post-treatment. The cross-sectional area (CSA) and amplitude (AMP) evaluated by the maximum lateral sliding displacement represented the morphological and dynamic changes in the median nerve, respectively. The results showed that AMP, CSA, GSS, and DML were significantly im-proved in the steroid group, as compared to the nicergoline group at weeks 2 and 4 (p < 0.05). The mean improvement in ultrasound parameters CSA (15.03% reduction) and AMP (466.09% increase) was better than the DML (7.88% reduction) parameter of NCS, and ultrasound changes were detectable as early as 2 weeks after oral steroid administration. Ultrasounds can serve as a tool for the quantitative measurement of treatment effects and can potentially elucidate the pathogenesis of CTS in a non-invasive and more effective manner.

Echotexture Analysis of L4 Supraspinous Enthesis in Ankylosing Spondylitis.

BACKGROUND: Enthesopathy is a main characteristic of ankylosing spondylitis (AS). However, ultrasonographic features of supraspinous enthesis in AS have not yet been reported. METHODS: Forty-seven AS patients and 22 healthy individuals were enrolled and completed the study. L4 supraspinous entheses were assessed through an ultrasound (US) unit with the participants in a lateral decubitus position. Entheseal echogenicity was interpreted upon inspection of the US image. An entheseal grayscale (GS) value determination, along with an echotexture analysis using a gray-level co-occurrence matrix algorithm, was performed. The thoracolumbar fascia just above the enthesis was also analyzed. An enthesis-to-fascia ratio (EFR) of each texture feature was used for the purpose of intergroup comparison. RESULTS: The prevalence of abnormal entheseal echogenicity in the AS and healthy groups was 19.1% and 13.6%, respectively (P = 0.42). The AS group experienced a higher GS EFR (0.56 [0.10-1.08] vs. 0.40 [0.12-0.89], P = 0.007), higher contrast EFR (0.62 [0.15-1.23] vs. 0.49 [0.23-1.33], P = 0.049), higher variance EFR (0.44 [0.06-1.21] vs. 0.35 [0.13-1.10], P = 0.023), and lower homogeneity EFR (1.07 [0.97-1.27] vs. 1.11 [1.04-1.19], P = 0.011) in comparison to the healthy group. CONCLUSION: Echotexture analysis identified the subtle structural changes in L4 supraspinous enthesis in AS patients. It proved to be superior to the inspection method and may possess the potential for providing early detection of supraspinous enthesopathy in AS.

Transformation of Salicylic Acid and Its Distribution in Tea Plants (Camellia sinensis) at the Tissue and Subcellular Levels.

Salicylic acid (SA) is a well-known immune-related hormone that has been well studied in model plants. However, less attention has been paid to the presence of SA and its derivatives in economic plants, such as tea plants (Camellia sinensis). This study showed that tea plants were rich in SA and responded differently to different pathogens. Feeding experiments in tea tissues further confirmed the transformation of SA into salicylic acid 2-O-ß-glucoside (SAG) and methyl salicylate. Nonaqueous fractionation techniques confirmed that SA and SAG were mostly distributed in the cytosol of tea leaves, consistent with distributions in other plant species. Furthermore, the stem epidermis contained more SA than the stem core both in C. sinensis cv. "Jinxuan" (small-leaf species) and "Yinghong No. 9" (large-leaf species). Compared with cv. "Yinghong No. 9", cv. "Jinxuan" contained more SAG in the stem epidermis, which might explain its lower incidence rate of wilt disease. This information will improve understanding of SA occurrence in tea plants and provide a basis for investigating the relationship between SA and disease resistance 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.