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This study investigates the volatile compounds of green tea produced with different leaves from spring, summer, and autumn in high−latitude region. A total of 95 volatile compounds were identified by gas chromatography–mass spectrometry (GC–MS). Spring, summer and autumn green tea contained 68, 72 and 82 volatile compounds, respectively. Principal component analysis (PCA), partial least squares−discrimination analysis (PLS−DA), and hierarchical cluster analysis (HCA) classified the samples and showed the difference. And 32 key characteristic components were screened out based on variable importance in the projection (VIP) values higher than 1.0. The characteristic volatile compounds of spring green tea including 18 components, such as geranylacetone, phenethyl alcohol, geraniol, β−ionone, jasmone, 1−octen−3−ol and longifolene. 13 components such as 2−methylfuran, indole, 1−octanol, D−limonene and ethanethiol were the key compounds in summer green tea. And 2,4,6−trimethylstyrene was the major differential volatile compounds in autumn green tea. The results increase our knowledge of green tea in different seasons and provide a theoretical basis for production control of green tea. [ABSTRACT FROM AUTHOR]

Shandong matcha has the quality characteristics of bright green color, seaweed-like aroma and strong, fresh and brisk taste. In order to identify the characteristic aroma components and clarify the contribution of the grinding process to the aroma of Shandong matcha. Three grades of Shandong matcha and corresponding tencha material were firstly tested with sensory evaluation, and the volatile components were extracted with headspace solid-phase microextraction (HS-SPME) and solvent-assisted flavor evaporation (SAFE) and analyzed using GC–MS. The sensory evaluation results showed that high-grade matcha (M-GS) had prominent seaweed-like, fresh and roasted notes, whereas medium and low-grade matcha (M-G1, M-G2) were gradually coupled with grassy, fatty and high-fired aromas. GC–MS results showed that in the HS-SPME method, heterocyclic compounds (45.84–65.35%) were the highest in Shandong matcha, followed by terpenoids (7.44–16.92%) and esters (6.91–15.27%), while in the safe method, esters were the highest (12.96–24.99%), followed by terpenoids (10.76–25.09%) and heterocyclic compounds (12.12–17.07%). As a whole, the composition of volatile components between M-G1 and M-G2 is relatively close, and there are more differences in volatile components between them and M-GS. The volatile components unique to M-GS were screened using the odor activity value (OAV) evaluation method, with components such as 3-methyl-2-butene-1-thiol, 3-ethyl-Phenol, 2-thiophenemethanethiol, 2,4-undecadienal, (E,E)-2,6-nonadienal, (E,Z)- being evaluated. There were other differentially volatile components, that is, volatile components that coexist in the three grades of matcha, but with different concentrations and proportions. M-G1 and M-G2 contained more volatile substances with high-fired aroma, such as 2-ethyl-3-methyl-pyrazine, coumarin and 5,6,7,8-tetrahydroquinoxaline. The grinding process not only changes the appearance of tencha, but also increases the content of volatile components of matcha as a whole, enhancing the aroma and flavor characteristics of matcha. In this study, the contents of 24 volatile components in matcha were mainly increased, such as benzene, (2,2-dimethoxyethyl)-, cis-7-decen-1-al, safranal and fenchyl acetate. The dual factors of material tencha and matcha grinding technology are indispensable in forming the differences in aroma and flavor of Shandong matcha at different levels. [ABSTRACT FROM AUTHOR]

Brassinosteroid (BR), a kind of polyhydroxylated steroid hormone, plays an important role in physiological and biochemical processes in plants. Studies were mainly focused on BR signaling and its exogenous spraying to help enhance crop yields. Few research studies are centered on the accumulation pattern of BR and its mechanism. Yet, it is crucial to unlock the mystery of the function of BR and its cross action with other hormones. Tea (Camellia sinensis (L.) O. Kuntze) is one of the important economic crops in some countries, and new shoots are the raw materials for the preparation of various tea products. Different concentrations of exogenous BR were reported to have different effects on growth and development. New shoots of tea plants can thus be considered a valuable research object to study the accumulation pattern of BR. In this study, the quantity of five BR components (brassinolide, 28-norbrassinolide, 28-homobrassinolide, castasterone, and 28-norcastasterone) in different tissues of tea plants, including buds (Bud), different maturity of leaves (L1, L2), and stems (S1, S2) were determined by UPLC-MS/MS. A total of 15 cDNA libraries of the same tissue with three repetitions for each were constructed and sequenced. The BR-accumulation pattern and gene expression pattern were combined together for weighted gene co-expression network analysis (WGCNA). BR-accumulation-relative genes were then screened using two methods, based on the K.in value and BR biosynthetic pathway (ko00905), respectively. The result showed that photosynthesis-related genes and CYP450 family genes were actively involved and might play important roles in BR accumulation and/or its accumulation pattern. First and foremost, feedback inhibition was more likely to dominate the accumulation pattern of BR in the new shoots of tea plants. Moreover, three conserved miRNAs with their target transcriptional factors and target mRNAs had been figured out from negative correlation modules that might be strongly linked to the BR-accumulation pattern. Our study provided an experimental basis for the role of BR in tea plants. The excavation of genes related to the accumulation pattern of BR provided the possibility of cross-action studies on the regulation of BR biosynthesis and the study between BR and other hormones. [ABSTRACT FROM AUTHOR]

It is still a serious challenge to develop a simple synthesized method of SAPO‐34 sieve modified by Cu with high NH3‐SCR performance. The Cu/SAPO‐34 samples with hierarchical structure were synthesized for the first time using cupric citrate as copper source by one‐pot hydrothermal method which showed excellent NH3‐SCR performance for NO removal. Among them, the 0.15Cu/SAPO‐34 catalyst with a Cu/Al ratio of 0.15 exhibited the widest temperature operating window (160–500 °C) and better hydrothermal stability. The physicochemical properties of 0.15Cu/SAPO‐34 was measured to understand the impacts of the Cu species on the NH3‐SCR of NO activity by various characterization methods including XRD, NH3‐TPD, N2 adsorption‐desorption, XPS, EPR, UV‐vis, NH3‐oxidation, NMR and in situ DRIFTS. The results reveal that the good activity is assigned to the isolated Cu species located at the site (I) in SAPO‐34, and the co‐existence of surface Cu2+ and Cu+ species with almost equal amounts. In addition, 0.15Cu/SAPO‐34 also possesses suitable acidic sites which are conducive to catalytic activity. The results of in situ DRIFTS suggest that the NH3‐SCR reaction follows Eley‐Rideal mechanism over 0.15Cu/SAPO‐34. [ABSTRACT FROM AUTHOR]

To save time and economic cost of the synthesis of SAPO-34 zeolites, the effects of hydrothermal time on the structure and methanol-to-olefin (MTO) catalytic activity of SAPO-34 zeolites were investigated in detail. A series of SAPO-34 zeolites (SAPO-34-x h, x = 3, 4, 8, 12, 24 and 48) were obtained using the traditional hydrothermal method by changing the synthesis time at 210 °C. The obtained catalysts were characterized by XRD, N2 adsorption–desorption, SEM, TG-DTA, ICP, 29Si and 27Al MAS NMR and NH3-TPD techniques. Among these samples, SAPO-34-4 h with a higher Si/(Si + Al + P) ratio shows a much longer lifetime (518 min) and higher light olefin selectivity (82.0%) in the MTO reaction, which can be attributed to the unique Si distribution and low acidity. Moreover, the SAPO-34-4 h sample can maintain good MTO performance even under long time storage without complicated processing or after several cycles of regeneration. This facile synthesis procedure may be beneficial for large scale industrial catalyst production because of its short synthesis time. [ABSTRACT FROM AUTHOR]

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]

This paper describes a concise and practical route to (+)-subersic acid from naturally occurring (−)-Sclareol in 7 steps. The key step of this route relied on the cross coupling of the diterpene with the arene fragment using the arylation of allylic acetate followed by β-acetoxy elimination type Heck reaction. [ABSTRACT FROM AUTHOR]

A new method has been developed for the copper-mediated trifluoromethylthiolation of allylic halides by using potassium fluoride, elemental sulfur, and (trifluoromethyl)trimethylsilane in anhydrous N, N-dimethylformamide. This protocol provides facile access to a variety of allylic trifluoromethyl thioethers in moderate to good yields under mild, ligand-free reaction conditions. [ABSTRACT FROM AUTHOR]

The 2-oxoglutarate-dependent dioxygenase (2-OGD) superfamily is one of the largest protein families in plants. The main oxidation reactions they catalyze in plants are hydroxylation, desaturation, demethylation, epimerization, and halogenation. Four members of the 2-OGD superfamily, i.e., flavonone 3β-hydroxylase (F3H), flavones synthase I (FNS I), flavonol synthase (FLS), and anthocyanidin synthase (ANS)/leucoanthocyanidin dioxygenase (LDOX), are present in the flavonoid pathway, catalyzing hydroxylation and desaturation reactions. In this review, we summarize the recent research progress on these proteins, from the discovery of their enzymatic activity, to their functional verification, to the analysis of the response they mediate in plants towards adversity. Substrate diversity analysis indicated that F3H, FNS Ⅰ, ANS/LDOX, and FLS perform their respective dominant functions in the flavonoid pathway, despite the presence of functional redundancy among them. The phylogenetic tree classified two types of FNS Ⅰ, one mainly performing FNS activity, and the other, a new type of FNS present in angiosperms, mainly involved in C-5 hydroxylation of SA. Additionally, a new class of LDOXs is highlighted, which can catalyze the conversion of (+)-catechin to cyanidin, further influencing the starter and extension unit composition of proanthocyanidins (PAs). The systematical description of the functional diversity and evolutionary relationship among these enzymes can facilitate the understanding of their impacts on plant metabolism. On the other hand, it provides molecular genetic evidence of the chemical evolution of flavonoids from lower to higher plants, promoting plant adaptation to harsh environments. [ABSTRACT FROM AUTHOR]

Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea (Camellia sinensis), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 (ttg1) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development. [ABSTRACT FROM AUTHOR]

An abstract of the article "Concise Synthesis of (+)-Subersic Acid from (-)-Sclareol" by P. Wang and colleagues is presented.

A convenient process is described to convert a variety of allylic chlorides into allylic trifluoromethyl thioethers. [ABSTRACT FROM AUTHOR]

Drying is an important factor in the formation of green tea flavor. In this study, metabolomic analysis were used to detect characteristic components of four green teas with clean aroma (CA), chestnut-like aroma (CLA), bean-like aroma (BLA) and high-fired aroma (HFA) produced by adjusting drying temperature. The sensory evaluation results showed that the tea dried at 90°C, 110°C, 140°C and 160°C had a clean aroma, chestnut-like aroma, bean-like aroma and high-fired aroma respectively. A total of 95 volatile compounds were identified. Linalool, naphthalene, 2-methyl-naphthalene, 1-octen-3-ol contributed most to the clean aroma. 1,2-dihydro-1,1,6-trimethyl-naphthalene was the key component of the chestnut-like aroma. 2,3-diethyl-5-methyl-pyrazine, 2-methyl-butanal, 3-ethyl-2,5-dimethyl-pyrazine had the biggest contribution to the bean-like aroma. 2,3-diethyl-5-methyl-pyrazine, 3-ethyl-2,5-dimethyl-pyrazine and 2-ethyl-5-methyl-pyrazine contributed most to the high-fired aroma. A total of 838 non-volatile compounds were detected and amino acids, saccharides decreased while catechins, flavonols increased during the increasing of drying temperature. It's found that they were related to the taste of umami, astringency and bitterness. This result provides a guidance and theoretical basis for the processing of green tea with different aroma types. • Four aroma types of green teas were manufactured by different drying temperatures. • Naphthalene and its derivatives contributed most to clean and chestnut-like aroma. • Pyrazine derivatives contributed most to bean-like and high-fired aroma green teas. • The higher the drying temperature, the more bitter and more astringent of green tea. [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]

In the present work, a serial of Cu 0.02 Fe 0.2 Ce y Ti 1-y O x catalysts are prepared by sol-gel method and applied for NH 3 -SCR of NO, meanwhile Cu 0.02 Fe 0.2 Ce 0.2 Ti 0.8 O x shows good low-temperature NH 3 -SCR performance with/without water and an outstanding water resistance. The bulk structure, redox ability, surface acidity and surface species of Cu 0.02 Fe 0.2 Ce y Ti 1-y O x are measured and discussed by series of characterization in details to illuminate the reasons for the good low-temperature activity and water resistance. The Ce modification can tune the surface acidic distribution, improve the surface oxygen content and surface oxidation reduction cycle (Ce4+ + Fe2+ ↔ Ce3+ + Fe3+), which contribute the good activity. In addition, the effect of water on NH 3 -SCR performance over Cu 0.02 Fe 0.2 TiO x and Cu 0.02 Fe 0·2 Ce 0·2 Ti 0.8 O x are investigated emphatically by in situ DRIFTS. Image 1 • Excellent low-temperature activity for NH 3 -SCR of NO over Cu 0·02 Fe 0·2 Ce 0·2 Ti 0.8 O x. • Ce addition significantly improves the water tolerance for the NH 3 -SCR reaction. • Enhancement of surface acidity for Cu 0·02 Fe 0·2 Ce 0·2 Ti 0.8 O x due to the Ce addition. • Increase in Lewis acid sites contributes the good water tolerance. [ABSTRACT FROM AUTHOR]

• Improving low temperature SCR activity and resistance to H 2 O/SO 2 on Sm a MnNi 2 Ti 7 O x due to Sm addition. • Sm 0.3 MnNi 2 Ti 7 Ox shows over 90% NO conversion and N 2 selectivity in range of 140–329 °C. • The better SCR activity is attributed to the enhanced surface area, redox ability and acid amount for Sm 0.3 MnNi 2 Ti 7 Ox • The excellent activity and resistance to H 2 O/SO 2 of Sm 0.3 MnNi 2 Ti 7 O x is significant for its practical applications in NO removal A series of Sm-modified Sm a MnNi 2 Ti 7 O x (a = 0.1, 0.2, 0.3, 0.4) catalysts were synthesized and showed better NH 3 -SCR activity than MnNi 2 Ti 7 O x , meanwhile, the Sm 0.3 MnNi 2 Ti 7 O x sample exhibited much higher low temperature NH 3 -SCR performance below about 200 °C, and good SO 2 and/or H 2 O durability. The bulk and surface physicochemical properties were measured to investigate the influence of the Sm addition on the activity by various characterization methods including NO/NH 3 oxidation, NH 3 -TPD, H 2 -TPR, XRD, N 2 adsorption-desorption, XPS. The results illustrate that the Sm addition can not only increase the adsorbed NO/NH 3 species, BET surface area, surface acid amount, Mn4+ content, surface adsorbed oxygen and the redox ability but also inhibit the generation of ammonium sulfate / bisulfate, which are beneficial to the NH 3 -SCR activity. Moreover, the in situ diffuse reflectance infrared Fourier transform spectroscopy was applied to analyze the reaction process, which reveals that the NH 3 -SCR reaction follows both E–R and L-H mechanisms over Sm 0.3 MnNi 2 Ti 7 O x. [ABSTRACT FROM AUTHOR]

• Good activity in range of 210–420 °C with 100% NO conversion and N 2 selectivity. • The special activity due to more surface acid amount and suitable redox ability. • Monodentate nitrate and NH 2 species as main active intermediates. • H 2 O mainly restrains L-H reaction pathway leading to the deactivation of catalyst. A serial of novel Ni m Co 0.3-m Ti 0.7 O x (m = 0.05-0.25) mixed-oxide catalysts was synthesized by sol-gel method, and applied for selective catalytic reduction (SCR) of NO x by NH 3. Influences of the Ni/Co ratio on the physi co- chemical properties and SCR performance were characterized by various analytical techniques, including BET surface area, XRD, XPS, NH 3 -TPD, H 2 -TPR and in situ DRIFTS. The Ni 0.1 Co 0.2 Ti 0.7 O x catalyst shows optimum NH 3 -SCR activity and excellent N 2 selectivity with a broader activity temperature window (210–450 °C), which is associated with the much higher BET surface area, more surface acid amounts and the suitable redox ability derived from the strong interaction between Co and Ni (Co3+ + Ni2+ ↔ Ni3+ + Co2+). In situ DRIFTS results reveal that the formed monodentate nitrate and NH 2 species, as main active intermediates, are favor of the NH 3 -SCR reaction. The existence of H 2 O mainly restrains Langmuir-Hinshelwood reaction pathway which may be the main cause for the catalyst deactivation. [ABSTRACT FROM AUTHOR]

Background: Taxaceae, is a class of dioecious and evergreen plant with substantial economic and ecology value. At present many phytochemical analyses have been performed in Taxus plants. And various biological constituents have been isolated from various Taxus species. However, the difference of compounds and antioxidant capacity of different tissues of T. media is not clear., Results: In the present study, we investigated the metabolites and antioxidant activity of four tissues of T. media, including T. media bark (TB), T. media fresh leaves (TFL), T. media seeds (TS), T. media aril (TA). In total, 808 compounds, covering 11 subclasses, were identified by using UPLC-MS/MS. Paclitaxel, the most popular anticancer compound, was found to accumulate most in TS, followed by TB, TFL and TA in order. Further analysis found that 70 key differential metabolites with VIP > 1.0 and p < 0.05, covering 8 subclasses, were screened as the key differential metabolites in four tissues. The characteristic compounds of TFL mainly included flavonoids and tanninsis. Alkaloids and phenolic acids were major characteristic compounds of TS and TB respectively. Amino acids and derivatives, organic acids, saccharides and lipids were the major characteristic compounds of TA. Additionally, based on FRAP and ABTS method, TS and TFL exhibited higher antioxidant activity than TB and TA., Conclusion: There was significant difference in metabolite content among different tissues of T. media. TFL and TS had higher metabolites and antioxidant capacity than other tissues, indicating that TFL and TS were more suitable for the development and utilization of T. media in foods and drinks., (© 2023. The Author(s).)

Matcha has a unique aroma of seaweed-like, which is popular with Chinese consumers. In order to effectively understand and use matcha for drinks and tea products, we roundly analyzed the variation of main quality components of 11 matcha samples from different regions in the Chinese market. Most of matcha samples had lower ratio of tea polyphenols to amino acids (RTA), and the RTA of 9 samples of matcha was less than 10, which is beneficial to the formation of fresh and mellow taste of matcha. The total volatile compounds concentrations by HS-SPME were 1563.59 ~ 2754.09 mg/L, among which terpenoids, esters and alcohols were the top three volatile components. The total volatile compounds concentrations by SAFE was 1009.21 ~ 1661.98 mg/L, among which terpenoids, heterocyclic compounds and esters ranked the top three. The 147 volatile components with high concentration (>1 mg/L) and no difference between samples are the common odorants to the 11 samples of matcha. The 108 distinct odorants had differences among the matcha samples, which were important substances leading to the different aroma characteristics. Hierarchical cluster analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that 11 samples of matcha were well clustered according to different components. Japanese matcha (MT, MY, ML, MR, MJ) could be clustered into two categories. The aroma composition of Guizhou matcha (GM1, GM2) was similar to that of Japanese matcha, 45 volatile components (decanal, pyrazine, 3,5-diethyl-2-methyl-, 1-hexadecanol, etc. were its characteristic aroma components. The aroma characteristics of Shandong matcha and Japanese matcha (ML, MR, MJ) were similar, 15 volatile components (γ-terpinene, myrtenol, cis -3-hexenyl valerate, etc.) were its characteristic aroma components. While Jiangsu matcha and Zhejiang matcha have similar aroma characteristics due to 225 characteristic aroma components (coumarin, furan, 2-pentyl-, etc). In short, the difference of volatile components formed the regional flavor characteristics of matcha. This study clarified the compound basis of the flavor difference of matcha from different regions in the Chinese market, and provided a theoretical basis for the selection and application of matcha in drinks and tea products., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Luo, Zhang, Qu, Qian, Wang, Zhang, Zhang and Hu.)

This study investigates the volatile compounds of green tea produced with different leaves from spring, summer, and autumn in high-latitude region. A total of 95 volatile compounds were identified by gas chromatography-mass spectrometry (GC-MS). Spring, summer and autumn green tea contained 68, 72 and 82 volatile compounds, respectively. Principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA), and hierarchical cluster analysis (HCA) classified the samples and showed the difference. And 32 key characteristic components were screened out based on variable importance in the projection (VIP) values higher than 1.0. The characteristic volatile compounds of spring green tea including 18 components, such as geranylacetone, phenethyl alcohol, geraniol, β-ionone, jasmone, 1-octen-3-ol and longifolene. 13 components such as 2-methylfuran, indole, 1-octanol, D-limonene and ethanethiol were the key compounds in summer green tea. And 2,4,6-trimethylstyrene was the major differential volatile compounds in autumn green tea. The results increase our knowledge of green tea in different seasons and provide a theoretical basis for production control of green tea.

Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo . The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro . Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana , EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa . In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.

Proanthocyanidins (PAs) are mainly composed of epicatechin (EC) or catechin (C) subunits. C-type catechins (C and GC) are generally considered to be catalyzed by leucocyanidin reductase (LAR). In this study, we re-evaluated the function of LAR. LcLAR1 was isolated from Lotus corniculatus , which is rich in C-type catechins. Overexpression of LcLAR1 in tobacco resulted in a significantly increased content of EC and EC-glucoside. Overexpression of LcLAR1 in Arabidopsis thaliana promoted the accumulation of soluble PAs, including EC, PA dimers, and PA trimers. However, in the transgenic ans mutant overexpressing LcLAR1 , the contents of C and C-glucoside were increased. In addition, overexpression of LcLAR1 in L. corniculatus resulted in a significant increase of C levels. Taken together, the products of Lc LAR1 depended on the substrates, which revealed the substrate diversity of Lc LAR1. Our study provides new insights into the flavonoid pathway, especially the role of LAR.

Melatonin and gibberellin are bioactive molecules in plants. In the present study, the role of exogenous melatonin (MT) and gibberellin (GA) in the tea plant was explored by transcriptome and metabolic analysis. Results showed that the growth of tea plant was enhanced by MT treatment. The pathways of terpenoid synthesis and plant-pathogen interaction were significantly strengthened, combined with the upregulation of LRR-RLK and transcription factors which contributed to the growth of tea plant. The internode elongation and leaf enlargement were hastened by GA treatment. Significantly modulated expression occurred in the plant hormonal signal transduction, complemented by the upregulation of phenylpropanoid biosynthesis and expansins to achieve growth acceleration, whereas the flavonoid synthesis was repressed in GA treatment. Therefore, the distinctive effect of MT and GA treatment on tea plant was different. The MT exhibited significant promotion in terpenoid synthesis, especially, TPS14 and TPS1. GA was prominent in coordinated regulation of plant hormonal signal transduction.

Proanthocyanidins (PAs, also called condensed tannins), are an important class of secondary metabolites and exist widely in plants. Tea ( Camellia sinensis) is rich in PAs and their precursors, (-)-epicatechin (EC) and (+)-catechin (C). The biosynthesis of PAs is constantly regulated by many different MBW complexes, consisting of MYB transcription factors (TFs), basic-helix-loop-helix (bHLH) TFs, and WD-repeat (WDR) proteins. These regulatory factors can be environmentally affected, such as by biotic and abiotic stresses. In this study, we revalidated the effect of sucrose treatment on tea branches, and a sucrose-induced MYB (SIMYB) TF was screened and studied. Phylogenetic analysis indicted that this SIMYB TF belonged to MYB subgroup 5, named CsMYB5b. Heterologous expression of CsMYB5b in tobacco strongly induced PA accumulation, through up-regulating the key target genes LAR or ANRs. In addition, CsMYB5b restored PA production in the seed coat of A. thaliana tt2 mutant and rescued its phenotype. Yeast two-hybrid assay demonstrated CsMYB5b can interact directly with CsTT8 (an AtTT8 ortholog) and CsWD40 protein. Linking to the expression profiling of CsMYB5b and the PA accumulation pattern in tea plants suggest that the CsMYB5b acts as an important switch for the synthesis of monomeric catechins and PAs. Therefore, these data provide insight into the regulatory mechanisms controlling the biosynthesis of PAs.

Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea ( Camellia sinensis ), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 ( ttg1 ) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development., Competing Interests: The authors declare no conflict of interest.

Flavonoids are major secondary metabolites in Camellia sinensis . Flavanone-3-hydroxylase (F3H) is a key enzyme in flavonoid biosynthesis in plants. However, its role in the flavonoid metabolism in C. sinensis has not been well studied. In this study, we cloned two F3H s from C. sinensis , named CsF3Ha and CsF3Hb , where CsF3Ha containing 1107 bases encoded 368 amino acids, and CsF3Hb containing 1071 bases encoded 357 amino acids. Enzymatic activity analysis showed both recombinant CsF3H enzymes in Escherichia coli could convert naringenin and eriodictyol into dihydrokaempferol (DHK) and dihydroquercetin (DHQ), respectively. The expression profiles showed that CsF3Ha and CsF3Hb were highly expressed in the tender leaves of tea plants. Under different abiotic stresses, the two CsF3H s were induced remarkably by ultraviolet (UV) radiation, sucrose, and abscisic acid (ABA). In the seeds of CsF3H s transgenic Arabidopsis thaliana , the concentration of most flavonol glycosides and oligomeric proanthocyanidins increased significantly, while the content of monocatechin derivatives decreased. The present study revealed that CsF3H s played critical roles in flavonoid biosynthesis in tea plants., Competing Interests: The authors declare no conflict of interest.

Flavonol glycosides, which are often converted from aglycones in a process catalyzed by UDP-glycosyltransferases (UGTs), play an important role for the health of plants and animals. In the present study, a gene encoding a flavonoid 7-O-glycosyltransferase (CsUGT75L12) was identified in tea plants. Recombinant CsUGT75L12 protein displayed glycosyltransferase activity on the 7-OH position of multiple phenolic compounds. In relative comparison to wild-type seeds, the levels of flavonol-glucosides increased in Arabidopsis seeds overexpressing CsUGT75L12. In order to determine the key amino acid residues responsible for the catalytic activity of the protein, a series of site-directed mutagenesis and enzymatic assays were performed based on the 3D structural modeling and docking analyses. These results suggested that residue Q54 is a double binding site that functions as both a sugar receptor and donor. Residues H56 and T151, corresponding to the basic active residues H20 and D119 of VvGT1, were not irreplaceable for CsUGT75L12. In addition, residues Y182, S223, P238, T239, and F240 were demonstrated to be responsible for a 'reversed' sugar receptor binding model. The results of single and triple substitutions confirmed that the function of residues P238, T239, and F240 may substitute or compensate with each other for the flavonoid 7-O-glycosyltransferase activity.

Tea (Camellia sinensis) is an important commercial crop, in which the high content of flavonoids provides health benefits. A flavonoid glycosyltransferase (CsUGT73A20), belonging to cluster IIIa, was isolated from tea plant. The recombinant CsUGT73A20 in Escherichia coli exhibited a broad substrate tolerance toward multiple flavonoids. Among them, kaempferol was the optimal substrate compared to quercetin, myricetin, naringenin, apigenin, and kaempferide. However, no product was detected when UDP-galactose was used as the sugar donor. The reaction assay indicated that rCsUGT73A20 performed multisite glycosidation toward flavonol compounds, mainly forming 3-O-glucoside and 7-O-glucoside in vitro. The biochemical characterization analysis of CsUGT73A20 showed more K7G product accumulated at pH 8.0, but K3G was the main product at pH 9.0. Kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro. Besides, the content of five flavonol-glucosides was increased in CsUGT73A20-overexpressing tobaccos (Nicotiana tabacum).