Identification and expression analysis of the KNOX genes during organogenesis and stress responseness in Camellia sinensis (L.) O. Kuntze.

Tea plant (Camellia sinensis L.), whose leaves are the major reproductive organs, has been cultivated and consumed widely for its economic and health benefits. The Knotted1-like Homeobox (KNOX) proteins play significant roles in leaf morphology formation and development. However, the functions of KNOX proteins in tea plants are still unknown. Here, 11 CsKNOX genes from the tea plants were cloned and divided into Class I, II, and KNATM clades based on their protein sequences. These 11 CsKNOX genes were mapped on 8 out of 15 tea plant chromosomes, all localized in the nucleus. Specific spatiotemporal expression patterns of CsKNOX genes were found in various tissues and different development periods of buds, flowers, and roots of tea plants. Meanwhile, transcript levels of CsKNOX in tea leaves were strongly correlated with the accumulation of flavan-3-ols and proanthocyanidins. It was found that most of the CsKNOX genes could respond to drought, salt, cold, and exogenous MeJA and GA3 by analysis of transcriptomics data and promoter elements. The protein interaction analysis showed that CsKNOX could cooperate with CsAS1 and other critical functional proteins. In conclusion, this research provided the basic information for the functions of the CsKNOX family during organogenesis and stress response in tea plants, which was necessary for further functional characterization verification.

Characteristics of Leaf Spot Disease Caused by Didymella Species and the Influence of Infection on Tea Quality.

Leaf spots are the most damaging and common foliar diseases of tea and are caused by several species of fungi. During 2018 to 2020, leaf spot diseases showing different symptoms (large and small spots) were observed in commercial tea plantations in Guizhou and Sichuan provinces of China. The pathogen causing the two different sized leaf spots was identified as the same species (Didymella segeticola) based on morphological characteristics, pathogenicity, and multilocus phylogenetic analysis using the combined ITS, TUB, LSU, and RPB2 gene regions. Microbial diversity analysis of lesion tissues from small spots on naturally infected tea leaves further confirmed Didymella to be present as the main pathogen. Results of sensory evaluation and quality-related metabolite analysis of tea shoots infected with the small leaf spot symptom indicated that D. segeticola negatively affected the quality and flavor of tea by changing the composition and content of caffeine, catechins, and amino acids. In addition, the significantly reduced amino acid derivatives in tea are confirmed to be positively associated with the enhanced bitter taste. The results improve our understanding of the pathogenicity of Didymella species and the influence of Didymella on the host plant, Camellia sinensis.

An overview of bitter compounds in foodstuffs: Classifications, evaluation methods for sensory contribution, separation and identification techniques, and mechanism of bitter taste transduction.

The bitter taste is generally considered an undesirable sensory attribute. However, bitter-tasting compounds can significantly affect the overall flavor of many foods and beverages and endow them with various beneficial effects on human health. To better understand the relationship between chemical structure and bitterness, this paper has summarized the bitter compounds in foodstuffs and classified them based on the basic skeletons. Only those bitter compounds that are confirmed by human sensory evaluation have been included in this paper. To develop food products that satisfy consumer preferences, correctly ranking the key bitter compounds in foodstuffs according to their contributions to the overall bitterness intensity is the precondition. Generally, three methods were applied to screen out the key bitter compounds in foods and beverages and evaluate their sensory contributions, including dose-over-threshold factors, taste dilution analysis, and spectrum descriptive analysis method. This paper has discussed in detail the mechanisms and applications of these three methods. Typical procedures for separating and identifying the main bitter compounds in foodstuffs have also been summarized. Additionally, the activation of human bitter taste receptors (TAS2Rs) and the mechanisms of bitter taste transduction are outlined. Ultimately, a conclusion has been drawn to highlight the current problems and propose potential directions for further research.

A New Colletotrichum Species Associated with Brown Blight Disease on Camellia sinensis.

Brown blight, as the most damaging and common foliar disease of the tea plant (Camellia sinensis) in China, has been recently reported to be caused by different species of the genus Colletotrichum. During the years 2016 to 2017, tea plants in commercial tea cultivation areas of Chongqing City that reported significant incidences of brown blight disease were investigated and then analyzed using both morphological characteristics and multilocus phylogenetic analysis. The results showed that at least five species of Colletotrichum were identified, including four well-known species (Colletotrichum gloeosporioides, C. camelliae, C. fioriniae, and C. karstii) and one novel species (C. chongqingense), indicating that there is remarkable species diversity in Colletotrichum spp. present as pathogens. Results of pathogenicity analyses confirmed that C. chongqingense was the causal agent of brown blight and different isolates differed in virulence. C. chongqingense, as a novel pathogen, has never been reported as being associated with brown blight disease in tea plants or anthracnose in other host plants anywhere in the world. Knowledge of the Colletotrichum populations will facilitate further studies addressing the relationships between Colletotrichum spp. and their host plant Camellia sinensis.

Analysis of taste characteristics and identification of key chemical components of fifteen Chinese yellow tea samples.

In order to explore the taste characteristics and molecular sensory basis of Chinese yellow tea, in this study, quantitative descriptive analysis (QDA) and partial least squares regression (PLSR) were used to analyze the sensory characteristics and chemical components of 15 yellow tea samples from different regions of China. The results showed that: 11 sensory descriptors and their definitions were obtained by QDA, namely, sweet, umami, bitter, sour, astringent, sweet after taste, mellow, neutral, after-taste, thick and tainted taste. The results of variance indicated that there were significant variation in taste sub-attributes of different samples (p <0.05). Principal component analysis indicated that there was a positive correlation between bitter and astringent, between sweet, umami and sour, and between mellow, thick, after-taste and neutral. All yellow tea samples were divided into four categories according to cluster analysis. The results of PLSR showed that there were 22 chemical components that had an important contribution to the taste characteristics of yellow tea, and the chemical components that had an important influence on each taste component were obtained. The identification of key contribution components of taste characteristics in yellow teas will provide a theoretical basis for further research on the directional adjustment and control of tea taste quality.

Improved geographical origin discrimination for tea using ICP-MS and ICP-OES techniques in combination with chemometric approach.

BACKGROUND: There is an urgent need to strengthen the testing and certification of geographically iconic foods, as well as to use discriminatory science and technology for their regulation and verification. Multi-element and stable isotope analyses were combined to provide a new chemometric approach for improving the discrimination tea samples from different geographical origins. Different stoichiometric methods [principal component analysis (PCA), hierarchical cluster analysis (HCA), partial least squares-discriminant analysis (PLS-DA), back propagation based artificial neural network (BP-ANN) and linear discriminant analysis (LDA)] were used to demonstrate this discrimination approach using Yongchuanxiuya tea samples in an experimental test. RESULTS: Multi-element and stable isotope analyses of tea samples using inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry easily distinguished the geographical origins. However, the clustering ability of the two unsupervised learning methods (PCA and HCA) were worse compared to that of the three supervised learning methods (PLS-DA, BP-ANN and LDA). BP-ANN and LDA, with 100% recognition and prediction abilities, were found to be better than PLS-DA. 86 Sr and 112 Cd were the markers enabling the successful classification of tea samples according to their geographical origins. Under the validation by 'blind' dataset, the prediction accuracies of the BP-ANN and LDA methods were all greater than 90%. The LDA method showed the best performance, with an accuracy of 100%. CONCLUSION: In summary, determination of mineral elements and stable isotopes using inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry techniques coupled with chemometric methods, especially the LDA method, is a good approach for improving the authentication of a diverse range of tea. The present study contributes toward generalizing the use of fingerprinting mineral elements and stable isotopes as a promising tool for testing the geographic roots of tea and food worldwide. © 2020 Society of Chemical Industry.

Palladium-Catalyzed Amide-Directed Enantioselective Hydrocarbofunctionalization of Unactivated Alkenes Using a Chiral Monodentate Oxazoline Ligand.

A Pd-catalyzed amide-directed enantioselective hydrocarbofunctionalization of unactivated alkenes with C-H nucleophiles has been developed using a chiral monodentate oxazoline (MOXin) ligand. Various indoles react at C3 position with aminoquinoline-coupled 3-alkenamides to give γ addition products in good to excellent yield and enantioselectivity. This study represents an important advance of the development of chiral monodentate oxazoline ligands, which have been underexplored for asymmetric catalysis.

The Effect of Different Treatments of (-)-Epigallocatechin-3-Gallate on Colorectal Carcinoma Cell Lines.

Backgroud: (-)-Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer by targeting multiple signal transduction pathways. However, EGCG is extremely unstable in general culture conditions and rapidly degraded. So, to what extent EGCG or which degradation products of EGCG play a role in anti-tumor is still unknown. In this study, we evaluated the effect of different treatments of EGCG on HCT116 cells. DESIGN: MTT assay was applied to evaluated the inhibitory effect of different treatments of EGCG on HCT116 cells. Cell cycle and apoptosis were performed by flow cytometry. Finally, western blot analysis was used to elucidate the molecular mechanism associated with cell cycle arrest and apoptosis. RESULTS: Compared with control, both EGCG and O-EGCG (i.e., EGCG being pre-incubated at 37°C for 3 h) significantly inhibited HCT116 cells growth. Surprisingly, we found that the inhibitory effect of O-EGCG was stronger than that of EGCG. The IC50 values of EGCG and O-EGCG were 8.75 and 5.40 µM, respectively. Cell cycle analysis showed that 20 µM of EGCG simultaneously caused cell cycle arrest at G1 and G2 phase in HCT116 cells, differing from O-EGCG which exclusively caused cell cycle arrest at G2. This result suggested that parent EGCG at the early treatment might cause cell cycle arrest at G1. As time went on, EGCG disappeared and degraded products of EGCG were formed which might cause cell cycle arrest at G2. Further studies revealed that EGCG induced cell cycle arrest at G1 by downregulation of cyclin E and cyclin D1 and upregulation of p21. On the other hand, O-EGCG induced HCT116 cells apoptosis mainly by increasing the expression of p53 and cleaved caspase-3, which might be the underlying reason why O-EGCG had stronger inhibitory effect on HCT116 cells line than EGCG. CONCLUSIONS: The pretreatment of EGCG may be an effective way to enhance its antitumor effect.

Pestalotiopsis-Like Species Causing Gray Blight Disease on Camellia sinensis in China.

Gray blight of tea, caused by several Pestalotiopsis-like species, is one of the most destructive foliar diseases in tea cultivation yet the characteristics of these pathogens have not been confirmed until now. With morphological and multigene phylogenetic analyses, we have identified the gray blight fungi as Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α gene regions successfully resolved most of the Pestalotiopsis-like species used in this study with high bootstrap supports and revealed three major clusters representing these three species. Differences in colony appearance and conidia morphology (shape, size, septation, color and length of median cells, and length and number of apical and basal appendages) were consistent with the phylogenetic grouping. Pathogenicity tests validated that all three species isolated from tea leaves were causal agents of gray blight disease on tea plant (Camellia sinensis). This is the first description of the characteristics of the three species Pseudopestalotiopsis camelliae-sinensis, N. clavispora, and Pestalotiopsis camelliae as causal agents of tea gray blight disease in China.

Identification of key aromatic compounds in Congou black tea by partial least-square regression with variable importance of projection scores and gas chromatography-mass spectrometry/gas chromatography-olfactometry.

BACKGROUND: Gas chromatography-olfactometry (GC-O) is the most frequently used method to estimate the sensory contribution of single odorant, but disregards the interactions between volatiles. In order to select the key volatiles responsible for the aroma attributes of Congou black tea (Camellia sinensis), instrumental, sensory and multivariate statistical approaches were applied. RESULTS: Using sensory analysis, nine panellists developed eight descriptors: floral, sweet, fruity, green, roasted, oil, spicy, and off-odour. Linalool, (E)-furan linalool oxide, (Z)-pyran linalool oxide, methyl salicylate, ß-myrcene, and phenylethyl alcohol, which were identified from the most representative samples by the GC-O procedure, were the essential aroma-active compounds in the formation of basic Congou black tea aroma. In addition, 136 volatiles were identified by gas chromatography-mass spectrometry (GC-MS), among which 55 compounds were determined as the key factors for six sensory attributes by partial least-square regression (PLSR) with variable importance of projection scores. CONCLUSION: Our results demonstrated that headspace solid-phase microextraction/GC-MS/GC-O was a fast approach for isolation and quantification aroma-active compounds. The PLSR method was also considered to be a useful tool in selecting important variables for sensory attributes. These two strategies, which allowed us to comprehensively evaluate the sensorial contribution of a single volatile from different perspectives, can be applied to related products for comprehensive quality control. © 2018 Society of Chemical Industry.

Characterization, Pathogenicity, and Phylogenetic Analyses of Colletotrichum Species Associated with Brown Blight Disease on Camellia sinensis in China.

Brown blight disease caused by Colletotrichum species is a common and serious foliar disease of tea (Camellia sinensis). Fungal isolates from several tea plantations causing typical brown blight symptoms were identified as belonging to the Colletotrichum acutatum species complex and the Colletotrichum gloeosporioides species complex based on morphological characteristics as well as DNA analysis of the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Colletotrichum acutatum, a new causal agent associated with C. sinensis, showed high phenotypic and genotypic diversity compared with the more commonly reported C. gloeosporioides. Phylogenetic analysis derived from individual and combined ITS and GAPDH sequences clearly clustered C. acutatum and C. gloeosporioides into separate species. Pathogenicity tests validated that both species were causal agents of tea brown blight disease and were highly pathogenic to tea leaves. However, the two groups of C. gloeosporioides with low levels of variability within their ITS and GAPDH regions differed in their virulence. This study reports for the first time the characterization of C. acutatum and C. gloeosporioides causing brown blight disease on tea (Camellia sinensis (L.) O. Kuntze) in China.

[Progress in catechins oxidation products and their formation mechanism].

Catechins are the key components of tea and have a great impact on its quality. Catechins can be oxidized to form a new black tea polyphenols, some of which have better pharmacological effect. However, the formation mechanism of these new polyphenols is still unclear. In this paper, oxidation products coming from catechins and the formation mechanism of the new compounds are reviewed.It is the base of further study on theaflavins, thearubigins and theabrownines.

An Enantioselective Bidentate Auxiliary Directed Palladium-Catalyzed Benzylic C-H Arylation of Amines Using a BINOL Phosphate Ligand.

A new enantioselective palladium(II)-catalyzed benzylic C-H arylation reaction of amines is enabled by the bidentate picolinamide (PA) directing group. This reaction provides the first example of enantioselective benzylic γ-C-H arylations of alkyl amines, and proceeds with up to 97 % ee. The 2,2'-dihydroxy-1,1'-binaphthyl (BINOL) phosphoric acid ligand, Cs2 CO3 , and solvent-free conditions are essential for high enantioselectivity. Mechanistic studies suggest that multiple BINOL ligands are involved in the stereodetermining C-H palladation step.

Amino acids and flavonoids analysis reveals quality constituents difference among different albino tea resources.

Albino tea has attracted increased attention due to its unique flavor. To reveal the difference in key metabolites constituting the important quality of different tea resources, amino acids and flavonoids profiles in three albino resources with different degrees of albinism and one normal green variety were comprehensively investigated. K-means analysis revealed 35 amino acids were significantly enriched in 'Jibai', while 3 and 2 were specifically accumulated in 'Huangjinya' and 'Anjibaicha', respectively. Based on OPLS-DA models, 40, 31 and 45 significantly differential flavonoids were determined in 'Huangjinya', 'Anjibaicha' and 'Jibai' compared to 'Fudingdabaicha', and most were down-regulated. Among them, 10, 5 and 13 differential flavonoids were exclusively found in 'Huangjinya', 'Anjibaicha' and 'Jibai', respectively, which may contribute to unique quality for different resources. The differential flavonoids and amino acids involved in their metabolic pathways were obviously different among four resources, resulting in the difference in tea quality and flavor.

Effect of blister blight disease caused by Exobasidium on tea quality.

Blister blight, as one of the most threatening and damaging disease worldwide, mainly infects young organs and tissues seriously affecting tea growth and quality. In this study, the spread of pathogen on tea leaves were examined by toluidine blue staining, scanning electron microscope and transmission electron microscope analysis. The composition and abundance of fungal community on leaf tissues were firstly analyzed. Sensory evaluation and metabolites analysis indicated that diseased tea leaves had strong sweet taste and soluble sugars contributed significantly to the taste, while metabolites showing bitter and astringent taste (caffeine, catechins) were significantly decreased. According to the biological functions of differential metabolites, sugars including 7 monosaccharides (d-xylose, d-arabinose, d-mannose, d-glucuronic acid, glucose, d-galactose and d-fructose), 2 disaccharide (sucrose and maltose) and 1 trisaccharide (raffinose) were the main differential sugars increased in content (>2 fold change), which was of great significance to sweet taste of diseased tea.

The unique aroma of ripened Pu-erh tea, Liupao tea and Tietban tea: Associated post-fermentation condition and dominant microorganism with key aroma-active compound.

Dark tea with a unique aroma is a particular type of tea. Post-fermentation is the key manufacturing process that forms a unique aroma, distinguishing dark tea from other types of tea. Different post-fermentation conditions may be the reason for forming unique aromas in various dark teas. Microbial community and composition during post-fermentation are the main contributors to the formation of characteristic aroma in various dark teas. In this paper, we illustrated the differences in fermentation length, pile temperature and water content of ripened Pu-erh tea, Liupao tea and Tibetan tea during post-fermentation, and only six dominant microorganisms and six key aroma-active compounds were found to be consistent among the three dark teas. Furthermore, we elaborated on the correlations between key aroma-active compounds and core functional microorganisms during post-fermentation. Exploring these interrelationships during post-fermentation is crucial and worthwhile to maintain the stability of the aroma quality of the three dark teas.

Evaluation of Chongqing Tuo Tea at Different Grades: An Integrated Approach by Artificial and Intelligent Sensory, Non-Volatile, and Volatile Compounds Analysis.

This study aims to investigate the relationship between the grades of Tuo tea and the quality of compounds. A combination of artificial sensory evaluation, intelligent sensory technologies (electronic nose and electronic tongue), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), chemical-physical analysis, and multivariate statistical analysis were employed to examine the differences among three grades of Tuo tea (SG, 1G, and 2G). The results of artificial sensory evaluation, electronic tongue, and electronic nose revealed that the aroma and taste of different grades of Tuo tea varied greatly. A total of 112 volatile compounds and 44 non-volatile compounds were identified. In order to elucidate the key components that cause differences in the quality of Tuo tea, 2 partial least squares discriminant analysis (PLS-DA) models with excellent parameters (volatile, R2Y = 0.999 and Q2 = 0.996; non-volatile, R2Y = 0.992 and Q2 = 0.972) were established. A total of 80 key differential volatile compounds were identified with the double selection criterion of variable importance in projection (VIP) greater than 1 and p < 0.05. Among these, 43 compounds with OAV > 1 were further identified as the odor-active compounds in all three grades of Tuo. Moreover, 22 key non-volatile compounds that contribute to the quality differences have been screened out. This investigation implied that the volatile and non-volatile compounds of Tuo tea could serve as indicators of its quality. The results provided a new approach to distinguish the grades of Tuo tea.

Palladium-Catalyzed Enantioselective Directed C(sp3)-H Functionalization Using C5-Substituted 8-Aminoquinoline Auxiliaries.

8-Aminoquinoline (AQ) has proven to be a highly effective bidentate directing group for palladium-catalyzed C-H functionalization reactions. However, enantiocontrol of AQ-directed C(sp3)-H functionalization reactions has been challenging. Herein, a new protocol is presented for the Pd-catalyzed enantioselective arylation of unactivated ß C(sp3)-H bonds of alkyl carboxamides with aryl iodides using a C5-iodinated 8-aminoquinolines (IQ) auxiliary in conjugation with a BINOL ligand. Additionally, a C5-aryl substituted 8-aminoquinoline auxiliary can facilitate enantioselective alkenylation and alkynylation of benzylic C(sp3)-H bonds of 3-arylpropanamides with the corresponding bromide reagents under similar conditions.

Evaluation of the quality grade of Congou black tea by the fusion of GC-E-Nose, E-tongue, and E-eye.

In the present study, the comprehensive quality of Congou black tea (CBT) including aroma, taste, and liquid color was investigated by a combination of gas chromatography electronic nose (GC-E-Nose), electronic tongue (E-tongue), and electronic eye (E-eye). An excellent discrimination of different quality grades of CBT was accomplished through the fusion of GC-E-Nose, E-tongue, and E-eye combined with orthogonal partial least squares discriminant analysis, with parameters of R2Y = 0.803 and Q2 = 0.740. Moreover, the quantitative evaluation of CBT quality was successfully achieved by partial least squares regression analysis, with the absolute error within 1.39 point, and the relative error within 1.62%. Additionally, 12 key variables were screened out by stepwise multiple linear regression analysis, which significantly contributed to the comprehensive quality score of CBT. Our results suggest that the fusion of multiple intelligent sensory technologies offers great potential and practicability in the quality evaluation of black tea.

Non-volatile metabolites profiling analysis reveals the tea flavor of "Zijuan" in different tea plantations.

Characteristic metabolites including tea polyphenols, amino acids, catechins, caffeine, sugars and anthocyanins were fully analyzed by high performance liquid chromatography (HPLC), gas chromatography tandem mass spectrometry (GC-MS) and ultra-high performance liquid chromatography (UHPLC)-ESI-tandem mass spectrometry (MS/MS), and showed significant differences among Zijuan tea from different plantations in Yunnan province (YN-ZJ), Qijiang (QJ-ZJ) and Ersheng (ES-ZJ) district, China, indicating that Zijuan is significantly influenced by growth conditions. Monosaccharides were the most abundant soluble sugars in YN-ZJ and ES-ZJ, while disaccharides was abundant in QJ-ZJ. d-galactose, d-mannose, d-sorbitol, inositol, d-glucose, d-galacturonic acid and raffinose involved in galactose metabolism were significantly changed (P < 0.05). Delphinidin, cyanidin, pelargonidin and their glycoside derivatives were the major anthocyanins, and showed significant differences among Zijuan samples. Flavonoids and procyanidins abundant in Zijuan provided more substrates for anthocyanins accumulation. This study presented comprehensive chemical profiling and characterized metabolites of Zijuan in different tea plantations.