Foliar application of glycinebetaine and Zn fertilizer improves both the apparent and functional qualities of albino tea [Camellia sinensis (L.) O. Kuntze].

With Zn deficiency increasing in the global population, functional plant food (including tea) can help to fill the nutrition gap that the main crops cannot meet. Glycinebetaine (GB), an important bioactive substance with a wide range of natural sources, has received limited attention towards its effects on Zn biofortification and the quality of tea. The Zn enrichment and metabolite responses of albino tea [cv. White leaf No. 1 (WL-1)] to the foliar application of GB, Zn, and their combination (Zn + GB) were investigated in a field experiment. The result indicated that the 100-buds weight, total N, Zn, Thea, and total amino acid content in the young leaves of WL-1 with Zn2 + GB2 treatment were significantly increased, whereas the Chla contents were decreased (p < 0.05). The total catechins and CAF contents of Zn2 + GB2 treatment were lower than those of other treatments, with significance (p < 0.05). Multivariate analysis and general quantitative analysis returned complementary results, revealing that Zn2 + GB2 treatment was better for the apparent and functional quality of WL-1. The more theanine and Zn, limited chlorophyll, catechin, and caffeine contributed to the quality improvement, as well as to maintaining the leaf albinistic characteristics, inhibiting astringency and bitterness, exerting flavor and umami, and improving the ultimate beneficial functions. The combined application of Zn and GB is a promising practice for Zn biofortification and for the quality improvement of tea, with spraying 750 L ha-1 of 2.0 g L-1 Zn fertilizer and 3.2 g L-1 GB mixture recommended.

Menghai Huangye, a novel albino tea germplasm with high theanine content and a high catechin index.

Yunnan Province has a very wide diversity of tea germplasm resources. A variety of special tea germplasms with outstanding traits have been discovered, including tea germplasms with high anthocyanin content and low caffeine content. Albino tea cultivars generally have higher contents of theanine that contribute to the umami taste, and the quality of tea brewed from it is higher. The catechin index (CI), the ratio of dihydroxylated catechins (DIC) to trihydroxylated catechins (TRIC), is a crucial index of suitability for processing tea. In this study, the albino tea plant Menghai Huangye (MHHY) with yellow leaves was identified. Analysis of the biochemical components revealed that MHHY was enriched in theanine and the total catechins (TC) were lower than Yunkang 10 (YK10). In addition, the CI value of MHHY was extremely significantly higher than that of YK10. Metabolic profile of catechins and the related gene expression profile analysis found that the coordinated expression of the key branch genes F3'H and F3'5'Ha for the synthesis of DIC and TRIC in tea plant was closely related to the high CI and low TC of MHHY. Further analysis of the F3'H promoter showed that a 284-bp deletion mutation was present in the F3'H promoter of MHHY, containing the binding sites of the transcriptional repressor MYB4 involved in flavonoid metabolism, which might be an important reason for the up-regulated expression of F3'H in MHHY. Overall, this study provides a theoretical basis for understanding the characteristics of albino tea germplasm resources and efficiently utilizing high-CI tea germplasm resources.

From Tea Leaves to Factories: A Review of Research Progress in l-Theanine Biosynthesis and Production.

l-Theanine is the most popular nonprotein amino acid contained in tea leaves. It is one of the umami components of green tea, contributing to the unique flavor of tea. Because of its various health functions, l-theanine has been commercially developed as a valuable ingredient easily used for various applications in food and pharmaceutical industries. Nowadays, l-theanine is mass-produced by plant extraction, chemical synthesis, or enzymatic transformation in factories. This review embodies the available up to date information on the l-theanine synthesis metabolism in the tea plant as well as approaches to produce it, placing emphasis on the biotransformation of l-theanine. It also gives insight into the challenges of l-theanine production on a large scale, as well as directions for future research. This review comprehensively summarizes information on l-theanine to provide an approach for an in-depth study of l-theanine production.

Characterization of L-theanine in tea extracts and synthetic products using stable isotope ratio analysis.

L-theanine involves a great number of health benefits and dietary supplements containing this molecule are becoming increasingly popular. There is, therefore, a growing need to find ways to discriminate between natural L-theanine extracted from tea leaves and the cheaper, synthetic one obtained using specific bacterial enzymes. A first attempt of stable isotope ratio analysis characterization of the possible synthetic adulterant L-theanine (δ13C of -14.3 ± 1.5‰), obtained from vegetable substrates with C4 photosynthetic cycle, and of the more expensive natural L-theanine (δ13C of -24.4 ± 1.3‰), extracted from Camellia sinensis plants with C3 photosynthetic cycle, is reported here.

The Biosynthesis of Main Taste Compounds Is Coordinately Regulated by miRNAs and Phytohormones in Tea Plant (Camellia sinensis).

Based on the abundance of taste compounds in leaves at different leaf positions on the same shoot, green tea made from one bud and one leaf, or even just one bud, has the best quality. To elucidate the mechanism underlying the regulation of the biosynthesis of these compounds, we profiled the metabolome, transcriptome, sRNA, degradome, and WGCNA using leaves from five leaf positions of shoots. Through this analysis, we found 139 miRNA-target pairs related to taste compound biosynthesis and 96 miRNA-target pairs involved in phytohormone synthesis or signal transduction. Moreover, miR166-HD-ZIP, miR169-NF-YA, IAA, ZA, ABA, and JA were positively related to the accumulation of gallated catechin, caffeine, and theanine. However, miR396-GRF, miR393-bHLH, miR156-SBP, and SA were negatively correlated with these compounds. Among these important pairs, the miR396-GRF and miR156-SBP pairs were further validated by using qRT-PCR, Northern blots, and cotransformation. This is the first report describing that miRNA-TF pairs and phytohormones might synergistically regulate the biosynthesis of taste compounds in the leaves of tea plants.

"Oral" tribology study on saliva-tea compound mixtures: Correlation between sweet aftertaste (Huigan) perception and friction coefficient.

Sweet aftertaste (Huigan) is a sensation perceived after drinking tea, and lasts in the mouth and throat, leading to salivation for an extended period of time. The study aimed to reveal the underpinning mechanisms of Huigan and the its influencing factors. Tribology approach was applied to this study in conjunction with sensory analysis and other physiology assessments. Tea compounds of commercial interest were selected for the study. Preliminary sensory tests of 24 subjects were carried out to evaluate the Huigan intensity of these tea compounds. Based on these tests, 12 subjects were selected and divided into two groups, each with 6 subjects: sensitive subjects and non-sensitive subjects. In vitro tribology measurements were made for samples prepared either prepared as 1:1 mixtures of tea compound solution and human whole saliva or expectorated tea compound solutions along with the saliva which were collected from the subjects after oral processing. The Huigan intensity perceived by the sensitive group was found to be highly correlated with the friction coefficient measured, especially at the sliding speeds lower than 0.5 mm/s.

Comparative Transcriptomic Analysis Reveals Regulatory Mechanisms of Theanine Synthesis in Tea (Camellia sinensis) and Oil Tea (Camellia oleifera) Plants.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (Camellia sinensis) and oil tea (Camellia oleifera) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.

Untargeted metabolomics coupled with chemometrics analysis reveals potential non-volatile markers during oolong tea shaking.

Shaking is a critical process in the formation of oolong tea quality, although the metabolites and their changes in this sensitive process have not yet been determined. In this study, untargeted analysis based on ultrahigh-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry was conducted to comprehensively profile metabolite changes in different cultivars. Theanine glucoside was identified for the first time in oolong tea. Hierarchical cluster analysis indicated that shaking caused major changes in metabolite levels in oolong tea. Seventy-one, 83 and 73 potential features showed significant differences between pre- and post-shaking samples for var. sinensiscv. "Zimudan", "Shuixian", and "Huangmeigui," respectively. Chemometrics analysis of the three cultivars led to the identification of 18 shared metabolites, including epigallocatechin gallate, phenylalanine, tryptophan, proline, and hydroxy-jasmonic acid, as potential markers. This study identified the metabolites that allow monitoring of tea quality formation during both processing and preservation, and it provides a novel strategy for data reduction in studies to discover key metabolites.

Analysis of Young Shoots of 'Anji Baicha' (Camellia sinensis) at Three Developmental Stages Using Nontargeted LC-MS-Based Metabolomics.

'Anji Baicha' (Camellia sinensis) is a low-temperature-sensitive tea variety. During the development of young shoots, the leaves of 'Anji Baicha' exhibit periodic albinism. The quality of 'Anji Baicha' is closely related to the color of the fresh leaves, with whiter leaves affording a higher amino acid content and superior sensory quality after processing. However, the metabolic mechanism of its quality formation is still unclear. In this study, we analyzed the metabolomic changes of young shoots of 'Anji Baicha' and screened for metabolic markers that may be involved in the periodic albinism. Positive- and negative-mode UPLC-QTOF-MS was applied to the metabolomic analysis of young leaves of 'Anji Baicha' during three developmental stages (i.e., the pre-albescent, albescent, and regreening stages). The results revealed significant differences in the metabolic profiles of the young leaves at the three stages. The differential metabolites were mainly related to the pathways of flavonoid, phenylpropanoid, and amino acid biosynthesis. The concentrations of several amino acids (primarily l-theanine, l-glutamate, N2 -acetyl-l-ornithine, l-aspartic acid, d-proline, l-glutamine, l-leucine, and pyroglutamic acid) and 12-OPDA were significantly higher in the albescent stage. In contrast, during the albescent stages, the concentrations of several carbohydrates (d-fructose, ß-d-galactopyranose, 3-O-fucopyranosyl-2-acetamido-2-deoxyglucopyranose, galactose-ß-1, 4-xylose acetyl-maltose, and 2-fucosyllactose) were significantly lower. Moreover, catechins (mainly epigallocatechin and catechin derivatives), dimeric catechins (primarily proanthocyanidins), and flavonol and flavonol/flavone glycosides (mainly kaempferol, myricetin, quercetin, cyanidin, and delphinidin glycosides) were detected at the highest levels in the regreening or pre-albescent stages. The obtained results enhance the current understanding of the metabolic mechanisms of periodic albinism and quality development formation in 'Anji Baicha'. PRACTICAL APPLICATION: The obtained results not only provide information regarding differential metabolites but also advance the understanding of the mechanism of periodic albinism in 'Anji Baicha' at the metabolite level and open up new possibilities for the genetic improvement of tea cultivars.

Effect of Major Tea Insect Attack on Formation of Quality-Related Nonvolatile Specialized Metabolites in Tea ( Camellia sinensis) Leaves.

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.

Visualized analysis of within-tissue spatial distribution of specialized metabolites in tea (Camellia sinensis) using desorption electrospray ionization imaging mass spectrometry.

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.

Aroma compositions of large-leaf yellow tea and potential effect of theanine on volatile formation in tea.

Large-leaf yellow tea (LYT) imparting unique toasty flavor is a traditional beverage in China. The volatile composition of LYT after full fire processing (FF-YT) was determined by different extraction methods. Steam distillation (SD), solid-phase microextraction (SPME) and headspace (HS) were applied for analysis of tea body notes, tea infusion and dry tea aroma, respectively. A total of 143 volatile compounds was identified by the three methods, of which SD-FF-YT, SPME-FF-YT, and HS-FF-YT extracts contained 100, 72, and 56 volatiles, respectively. Heterocyclics and aromatic compounds were the main volatile composition of LYT and might be contributors to its crispy-rice-like odor. l-Theanine was demonstrated to contribute to the formation of tea volatiles. N-Ethylacetamide, formed in the l-theanine only model thermal reaction, was probably involved in the LYT volatile formation. The current results provide new insight into l-theanine on aroma formation, although the detailed formation mechanism remains largely unknown.

Differential accumulation of specialized metabolite l-theanine in green and albino-induced yellow tea (Camellia sinensis) leaves.

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 [2H5]-l-theanine, yellow leaves catabolized less [2H5]-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.

Effect of shading intensity on morphological and color traits and on chemical components of new tea (Camellia sinensis L.) shoots under direct covering cultivation.

BACKGROUND: Use of covering cultivation to shade tea (Camellia sinensis L.) trees to produce high-quality, high-priced green tea has recently increased in Japan. Knowledge of shading effects on morphological and color traits and on chemical components of new tea shoots is important for product quality and productivity. We assessed these traits of tea shoots and their relationships under covering cultivation of various radiation intensities. RESULTS: Leaf thickness, leaf mass per area, and leaf density of new tea leaves were smaller under covering culture than under open-field culture. SPAD values and chlorophyll contents were larger under covering culture than under open culture. The derived exponential equation for estimating chlorophyll contents from SPAD values was improved by considering leaf thickness. Covering culture decreased epicatechin and epigallocatechin contents, and increased theanine and caffeine contents. Principal component analysis on shoot and leaf traits indicated that leaf mass per area, chlorophyll, epicatechin, and epigallocatechin contents were strongly associated with shading effects. CONCLUSION: The morphological traits, color traits, and chemical components of new tea shoots and leaves varied depending on radiation intensity, shoot growth, and cropping season. These findings are useful for covering cultivation with high quality and high productivity in tea gardens. © 2018 Society of Chemical Industry.

Contribution of l-theanine to the formation of 2,5-dimethylpyrazine, a key roasted peanutty flavor in Oolong tea during manufacturing processes.

l-Theanine, the most abundant amino acid in tea, is widely believed to be associated with the tea taste, however, its contribution to the formation of tea aroma is still unknown. Volatiles were determined and nitrogen-containing compounds formed during manufacturing processes were quantified. Lower levels of total sugar and l-theanine were detected in the Oolong tea product undergoing full fire processing (FFOT) suggesting that l-theanine probably involved in the volatile formation during manufacturing processes. Methylpyrazine and 2,5-dimethylpyrazine, two newly formed compounds in FFOT, together with other volatiles were successfully detected in a model thermal reaction of d-glucose and l-theanine (GT-MTR) but not detectable in thermal reactions with single d-glucose (G-MTR) or l-theanine (T-MTR). The concentration of 2,5-dimethylpyrazine increased significantly by adding additional l-theanine to 2nd roasted tea. Our study demonstrated that l-theanine, at least partly, contributed to the formation of 2,5-dimethylpyrazine, a key roasted peanutty flavor in Oolong tea.

l-Theanine: An astounding sui generis integrant in tea.

l-theanine (l-Th), a non-protein amino acid present in tea, is a valuable nutraceutical product with unique health benefits and used as an additive in food industry. l-Th enhances the umami taste but its use is limited due to its inadequate production. Different extraction approaches from tea shoots, chemical synthesis to microbial transformation have been tried to meet its demand. In vitro, in vivo as well as clinical studies have shown its positive effect in regulating CNS disorders. l-Th has become choice ingredient in CNS active products due to its anti-stress and neuroprotective role in dementias particularly in retrogression of Alzheimer's. l-Th biochemically modulates various anti-neoplastic agents by increasing their bioavailability in tumour cells. The review, is an effort to condense the recent research on l-Th highlighting its biological resource, plausible role in tea plant, production approaches, its physiological role on human health and future prospects.

CsGOGAT Is Important in Dynamic Changes of Theanine Content in Postharvest Tea Plant Leaves under Different Temperature and Shading Spreadings.

We analyzed the changes of theanine content in postharvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using ultrahigh-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology. The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain reaction. The correlations between the dynamic changes of theanine content and expression profiles of related genes and DEPs were analyzed. This study preliminarily proved the importance of CsGOGAT in dynamic changes of theanine content in postharvest tea leaves during spreading.

The synergistic potential of various teas, herbs and therapeutic drugs in health improvement: a review.

Tea is one of the most widely consumed non-alcoholic beverages in the world next to water. It is classified as Camellia sinensis and non-Camellia sinensis (herbal teas). The common bioactive compounds found mainly in green teas are flavan-3-ols (catechins) (also called flavanols), proanthocyanidins (tannins) and flavonols. Black tea contains theaflavins and thearubigins and white tea contains l-theanine and gamma-aminobutyric acid (GABA), while herbal teas contain diverse polyphenols. Phytochemicals in tea exhibit antimicrobial, anti-diabetic and anti-cancer activities that are perceived to be helpful in managing chronic diseases linked to lifestyle. Many of these phytochemicals are reported to be biologically active when combined. Knowledge of the synergistic interactions of tea with other teas or herbs in terms of biological activities will be of benefit for therapeutic enhancement. There is evidence that various types of teas act synergistically in exhibiting health benefits to humans, improving consumer acceptance and economic value. Similar observations have been made when teas and herbs or medicinal drugs were combined. The aim of this review is to highlight potential beneficial synergies between combinations of different types of teas, tea and herbs, and tea and medicinal drugs. © 2017 Society of Chemical Industry.

Determination of volatile organic compounds, catechins, caffeine and theanine in Jukro tea at three growth stages by chromatographic and spectrometric methods.

Tea contains characteristic volatile organic compounds, polyphenols, caffeine and catechins, and is therefore among the most widely consumed beverages all over the world. In this study, fresh Jukro tea leaves collected from Damyang-gun (Jeollanam-do) at 40, 60 and 90day growth stages, were semi-fermented. The volatile organic compounds (VOCs) were extracted by simultaneous distillation-solvent extraction (SDE) and analyzed by gas chromatography/mass spectrometry (GC/MS). Catechins, caffeine and theanine were analyzed by high performance liquid chromatography (HPLC). A total of 159 VOCs were identified in the analyzed Jukro tea leaves. Comparatively, the increase in the concentrations of VOCs was high in 60day leaves. The content of catechins increased along the three growth stages, whereas caffeine, compared to 90day leaves, was higher for 40 and 60day leaves. Based on the results, the 60day leaves were found to be the most suitable and useful for making semi-fermented Jukro tea.