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Summary: The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (−)‐epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol−HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (−)‐epicatechin‐4‐O‐methyl (EOM) ether, which resulted from (−)‐epicatechin carbocation and the methyl group of methanol. Acid‐based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)‐catechin‐4‐O‐methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (−)‐epicatechin or (+)‐catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan‐3‐ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan‐3‐ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants. Significance Statement: Although the dogma of plant flavonoids proposes that labile plant flavonoids carbocations (PFCs) exist to associate with the biosynthesis of proanthocyanidins (PAs), supportive evidence is lacking. Data from biochemical analysis, isotope labeling, genetic analysis, metabolic engineering and metabolic profiling demonstrate that PFCs are involved in the biosynthesis of PAs. [ABSTRACT FROM AUTHOR]

Background: The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically important woody crops. Plastic greenhouse covering cultivation has been widely used in tea areas of northern China. Chlorophyll is not only the crucial pigment for green tea, but also plays an important role in the growth and development of tea plants. Currently, little is known about the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves. Results: To investigate the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves, color difference values, chlorophyll contents, gene expression, enzyme activities and photosynthetic parameters were analyzed in our study. Sensory evaluation showed the color of appearance, liquor and infused leaves of greenhouse tea was greener than field tea. Color difference analysis for tea liquor revealed that the value of ∆L, ∆b and b/a of greenhouse tea was significantly higher than field tea. Significant increase in chlorophyll content, intracellular CO2, stomatal conductance, transpiration rate, and net photosynthetic rate was observed in greenhouse tea leaves. The gene expression and activities of chlorophyll-metabolism-related enzymes in tea leaves were also activated by greenhouse covering. Conclusion: The higher contents of chlorophyll a, chlorophyll b and total chlorophyll in greenhouse tea samples were primarily due to higher gene expression and activities of chlorophyll-metabolism-related enzymes especially, chlorophyll a synthetase (chlG), pheophorbide a oxygenase (PAO) and chlorophyllide a oxygenase (CAO) in tea leaves covered by greenhouse. In general, our results revealed the molecular basis of chlorophyll metabolism in tea leaves caused by plastic greenhouse covering cultivation, which had great significance in production of greenhouse tea. [ABSTRACT FROM AUTHOR]

Plants would encounter various biotic and abiotic stresses during the growth and development. WRKY transcription factors (TFs) as plant-specific TFs, play an important role in responding to various adverse circumstances. Despite some advances were achieved in functional studies of WRKY TFs in tea plants, systematic analysis of the involvement of CsWRKY TFs when facing cold, salt, drought stresses and pathogen and insect attack was lacked. In present study, a total of 78 CsWRKY TFs were identified following the genomic and transcript databases. The expression patterns of CsWRKYs in various organs of tea plants and the expression profiles in response to biotic and abiotic stresses were investigated by examining representative RNA-seq data. Moreover, the effects of hormone treatments (SA and MeJA) on the transcription levels of WRKY TFs were also investigated. The phylogenetic tree of CsWRKY TFs from different species indicated the functional diversity of WRKY TFs was not closely related to their protein classification. Concurrently, CsWRKY70-2 TF was identified as a positive regulator in response to drought stress. This study provided solid and valuable information, helping us better understand the functional diversity of CsWRKY TFs, and laid the foundation for further research on the function of key WRKY genes in tea plants. In this study, 78 WRKY transcription factors had been identified in tea plants as accurately as possible based on the transcriptome and genomic data. At the transcriptomic level, the expression patterns of CsWRKYs in various organs of tea plants and the expression profiles in response to biotic (Ectropis oblique, tea leafhopper and gray-blight attack), abiotic stresses (Cold, drought, salt) and exogenous hormones (SA and MeJA) were investigated. Expression pattern analysis showed that most Cs WRKYs were involved in the process of tea plants resisting biotic and abiotic stresses. Phylogenetic analysis indicated that the functional diversity of CsWRKY TFs was not closely related to their protein classification. In addition, CsWRKY70-2 TF had been proved to play a positive regulatory role in drought stress tolerance. This study provided solid and valuable information, helping better understand the functional diversity of CsWRKY TFs, and laid the foundation for further research on the function of key WRKY genes in tea plants. [ABSTRACT FROM AUTHOR]

In this study, gas chromatography–mass spectrometry (GC–MS) and gas chromatography–olfactometry (GC–O) analysis was performed to figure out the characteristic aroma compounds of high-latitude black teas and select the suitable cultivar for producing high-aroma black tea. Sensory evaluation revealed that Jinxuan presented an obviously floral aroma and got the highest score, followed by Longjing Changye, Zhongcha 302, Yingshuang, Baihaozao and Huangshan Quntizhong. Furthermore, a total of 135 volatiles were identified in all black teas and 12 components can be used to distinguish different latitude black teas by principal component analysis (PCA). Cluster analysis (CA) showed six samples were divided into two clusters and Jinxuan was classified separately of cluster B. Based on GC–O analysis, 41 volatile organic compounds (VOCs) were found to promote the aroma of high-latitude black teas. Among them, phenylethyl alcohol, geraniol, linalool, α-ionone, cis-3-hexenyl hexanoate and methyl salicylate were the major contributors to the floral fragrance of black tea and accounted for a relative high proportion in Jinxuan, Longjing Changye and Baihaozao. In summary, Jinxuan and Longjing Changye were suitable for producing high-quality black tea in high-latitude area. [ABSTRACT FROM AUTHOR]

Tea is one of the most widely consumed nonalcoholic beverages worldwide. Polyphenols are nutritional compounds present in the leaves of tea plants. Although numerous genes are functionally characterized to encode enzymes that catalyze the formation of diverse polyphenolic metabolites, transcriptional regulation of those different pathways such as late steps of the proanthcoyanidin (PA) pathway remains unclear. In this study, using different tea transcriptome databases, we screened at least 140 R2R3-MYB transcription factors (TFs) and grouped them according to the basic function domains of the R2R3 MYB TF superfamily. Among 140 R2R3 TFs, CsMYB5a and CsMYB5e were chosen for analysis because they may be involved in PA biosynthesis regulation. CsMYB5a- overexpressing tobacco plants exhibited downregulated anthocyanin accumulation but a high polymeric PA content in the flowers. Overexpression of CsMYB5e in tobacco plants did not change the anthocyanin content but increased the dimethylaminocinnamaldehyde-stained PA content. RNA-seq and qRT-PCR analyses revealed that genes related to PA and anthocyanin biosynthesis pathways were markedly upregulated in both CsMYB5a - and CsMYB5e -overexpressing flowers. Three UGTs and four GSTs were identified as involved in PA and anthocyanin glycosylation and transportation in transgenic plants. These results provide new insights into the regulation of PA and anthocyanin biosynthesis in Camellia sinensis . [ABSTRACT FROM AUTHOR]