Focusing on the recent progress of tea polyphenol chemistry and perspectives

Myriad evidence attests to the health-promoting benefits of tea drinking.While there are multiple factors of tea influencing the effective biological properties,tea polyphenols are the most significant and valuable components.The chemical characterization and physical characteristics of tea polyphenols have been comprehensively studied over the previous years.Still the emergence of new chemistry in tea,particularly the property of scavenging reactive carbonyl species(RCS)and the newly discovered flavoalkaloid compounds,has drawn increasing attention.In this review,we summarize recent findings of a new class of compounds in tea-flavonoid alkaloids(flavoalkaloids),which exist in fresh tea leaves and can be generated during the process of post-harvesting,and also postulate the formation mechanism of flavoalkaloids between catechins and theanine-derived Strecker aldehyde.Additionally,we detail the up-to-date research results of tea polyphenols regarding their ability to trap RCS and their in vivo aminated metabolites to suppress advanced glycation ends products(AGEs).We further raise questions to be addressed in the near future,including the synthetic pathways for the generation of flavoalkaloids and AGEs in fresh tea leaves before processing and the concentrations of tea polyphenols that affect their RCS scavenging capability due to their pro-oxidant nature.More intensive research is warranted to elucidate the mechanisms of action underlying the biological activity of flavoalkaloids and the pharmacological application of tea polyphenols in scavenging RCS and impeding detrimental AGEs.

UGT74AF3 enzymes specifically catalyze the glucosylation of 4-hydroxy-2,5-dimethylfuran-3(2H)-one,an important volatile compound in Camellia sinensis

4-Hydroxy-2,5-dimethylfuran-3(2H)-one(HDMF)is an important odorant in some fruits,and is proposed to play a crucial role in the caramel-like notes of some teas.However,its biosynthesis and metabolism in tea plants are still unknown.Here,HDMF glucoside was unambiguously identified as a native metabolite in tea plants.A novel glucosyltransferase UGT74AF3a and its allelic protein UGT74AF3b specifically catalyzed the glucosylation of HDMF and the commercially important structural homologues 2(or 5)-ethyl-4-hydroxy-5(or 2)-methylfuran-3(2H)-one(EHMF)and 4-hydroxy-5-methylfuran-3(2H)-one(HMF)to their correspondingβ-D-glucosides.Site-directed mutagenesis of UGT74AF3b to introduce a single A456V mutation resulted in improved HDMF and EHMF glucosylation activity and affected the sugar donor preference compared with that of the wild-type control enzyme.The accumulation of HDMF glucoside was consistent with the transcript levels of UGT74AF3 in different tea cultivars.In addition,transient UGT74AF3a overexpression in tobacco significantly increased the HDMF glucoside contents,and downregulation of UGT74AF3 transcripts in tea leaves significantly reduced the concentration of HDMF glucoside compared with the levels in the controls.The identification of HDMF glucoside in the tea plant and the discovery of a novel-specific UDP-glucose:HDMF glucosyltransferase in tea plants provide the foundation for improvement of tea flavor and the biotechnological production of HDMF glucoside.

Maintenance of mesophyll potassium and regulation of plasma membrane H^+-ATPase are associated with physiological responses of tea plants to drought and subsequent rehydration

Drought stress is one of the main factors limiting yield in tea plants. The plant cell's ability to preserve K^+homeostasis is an important strategy for coping with drought stress. Plasma membrane H^+-ATPase in the mesophyll cell is important for maintaining membrane potential to regulate K^+transmembrane transport. However, no research to date has investigated the possible relationship between plasma membrane H^+-ATPase and mesophyll K^+retention in tea plants under drought and subsequent rehydration conditions. In our experiment, drought stress inhibited plasma membrane H^+-ATPase activities and induced net H^+influx, leading to membrane potential depolarization and inducing a massive K^+efflux in tea plant mesophyll cells. Subsequent rehydration increased plasma membrane H^+-ATPase activity and induced net H^+efflux, leading to membrane potential hyperpolarization and thus lowering K^+loss. A first downregulated and then upregulated plasma membrane H^+-ATPase protein expression level was also observed under drought and subsequent rehydration treatment, a finding in agreement with the change of measured plasma membrane H^+-ATPase activities. Taken together, our results suggest that maintenance of mesophyll K^+in tea plants under drought and rehydration is associated with regulation of plasma membrane H^+-ATPase activity.

Comprehensive comparison on the chemical metabolites and taste evaluation of tea after roasting using untargeted and pseudotargeted metabolomics

Roasting is a common manufacture technology for processing various teas.It is not only used in decreasing the water content of finished tea,but also improving the flavor of teas.In the present study,the roasted and non-roasted teas were compared by liquid-chromatography mass spectrometry and sensory evaluation.The roasted tea tasted less bitter and astringent.The content of main galloylated and simple catechins,caffeine and theobromine in roasted were significantly lower than non-roasted teas.Targeted taste-compounds metabolomics revealed that(-)-epigallocatechin gallate,kaempferol-glucose-rhamnose-glucose and(-)-epicatechin gallate were main contributors tightly correlated to astringent intensity.Flavonol glycosides including kaempferol-glucose,quercetin-glucose,kaempferol-glucose-rhamnose-glucose,and quercetin-glucose-rhamnose-glucose in roasted teas were also significantly less than non-roasted teas.To study the chemical changes during roasting,tea with a strong astringency was roasted under 80,100,120,140,and 160°C.With the increase of roasting temperature,the bitter and astringent intensity of tea was gradually decreased,but the main astringent compounds including(-)-epigallocatechin,(-)-epigallocatechin gallate and kaempferol/quercetin glycosides were irregularly varied with temperature.The Pearson correlation coefficient analysis suggested procyanidin B2,coumaroylquinic acids and gallotannins were tightly correlated to the astringent and bitter perceptions,while N-ethyl-2-pyrrolidonesubstituted flavan-3-ols were negatively correlated.

Promoter and coding sequence diversity of CsCCD1 may contribute to the differential accumulation of floralβ-ionone in fresh tea leaves

The carotenoid-derived volatileβ-ionone makes an important contribution to tea fragrance.Here,we qualitatively and quantitatively analysed 15 carotenoids in tea leaves of 13 cultivars by UHPLC-APCI-MS/MS.The 13 cultivars were divided into two groups by PCA(Principal Component Analysis)clustering analysis of their carotenoid content,and OPLS-DA(Orthogonal projections to latent structures)indicated that the levels ofβ-carotene(VIP=2.89)and lutein(VIP=2.30)were responsible for much of the variation between the two groups.Interestingly,theβ-carotene toβ-ionone conversion rates in Group 1 were higher than in Group 2,while theβ-carotene content was significantly lower in Group 1 than in Group 2.Theβ-ionone content was significantly higher in Group 1.Pearson Correlation Coefficient calculation between the transcription level of candidate genes(CsCCD1 and CsCCD4)and the accumulation ofβ-ionone indicated that CsCCD1 may involve in the formation ofβ-ionone in 13 cultivars.Prokaryotic expression and in vitro enzyme activity assays showed that‘Chuanhuang 1’had an amino acid mutation in carotenoid cleavage dioxygenases 1(CsCCD1)compared with‘Shuchazao’,resulting in a significantly higherβ-ionone content in‘Chuanhuang 1’.Sequence analysis showed that‘Chuanhuang 1’and‘Huangdan’had different CsCCD1 promoter sequences,leading to significantly higher CsCCD1 expression andβ-ionone accumulation in‘Chuanhuang 1’.These results indicated that the promoter and coding sequence diversity of CsCCD1 might contribute to the differential accumulation ofβ-ionone in different tea cultivars.

GOLDEN 2-LIKE transcription factors regulate chlorophyll biosynthesis and flavonoid accumulation in response to UV-B in tea plants

Flavonoids are critical secondary metabolites that determine the health benefits and flavor of tea,while chlorophylls are important contributors to the appearance of tea.However,transcription factors(TFs)that can integrate both chlorophyll biosynthesis and flavonoid accumulation in response to specific light signals are rarely identified.In this study,we report that the GOLDEN 2-LIKE TF pair,CsGLK1 and CsGLK2,orchestrate UV-B-induced responses in the chlorophyll biosynthesis and flavonoid accumulation of tea leaves.The absence of solar UV-B reduced the transcriptional expression of CsGLKs in the tea leaves and was highly correlated with a decrease in flavonoid levels(especially flavonol glycosides)and the expression of genes and TFs involved in chlorophyll biosynthesis and flavonoid accumulation.In vivo and in vitro molecular analyses showed that CsGLKs could be regulated by the UV-B signal mediator CsHY5,and could directly bind to the promoters of gene and TF involved in light-harvesting(CsLhcb),chlorophyll biosynthesis(CsCHLH,CsHEMA1,and CsPORA),and flavonoid accumulation(CsMYB12,CsFLSa,CsDFRa,and CsLARa),eventually leading to UV-B-induced responses in the chlorophylls and flavonoids of tea leaves.Furthermore,UV-B exposure increased the levels of total flavonoids,CsGLK1 protein,and expression of CsGLKs and target genes in the tea leaves.These results indicate that CsGLKs may modulate tea leaf characteristics by regulating chlorophyll biosynthesis and flavonoid accumulation in response to solar UV-B.As the first report on UV-B-induced changes in flavonoid and chlorophyll regulation mediated by CsGLKs,this study improves our understanding of the environmental regulations regarding tea quality and sheds new light on UV-B-induced flavonoid responses in higher plants.

Screening ofα-glucosidase inhibitors in large-leaf yellow tea by offline bioassay coupled with liquid chromatography tandem mass spectrometry

Larger-leaf yellow tea(LYT)is a characteristic type of Chinese tea produced in Huoshan County,Anhui Province,which is made by mature leaves with stems.According to recent report,LYT showed competitive effects in anti-hyperglycemia in comparison to other teas such as green or black tea.However,the bioactive compounds of LYT are still undiscovered so far.For this purpose,5 fractions of LYT were prepared by sequential extraction.The in vitro bioassay results indicated that the ethyl acetate fraction of LYT had the strongest inhibitory effects onα-glucosidase andα-amylase.Fluorescence-quenching analysis and proteinbinding test revealed that the compounds of ethyl acetate fraction could inhibitα-glucosidase andα-amylase activities through binding to enzymes or other mechanisms.All chromatographic peaks of high-performance liquid chromatography(HPLC)of ethyl acetate fraction were separated and collected.The purified compounds were identified by liquid chromatography-mass spectrometry(LC-MS),and subsequently screened by calculating their inhibition ratio onα-glucosidase at the real concentration in LYT infusion.The results showed that(-)-epigallocatechin gallate,(-)-gallocatechin gallate,caffeine,N-ethyl-2-pyrrolidone-substituted flavan-3-ols were effective inhibitors forα-glucosidase.

A geraniol synthase regulates plant defense via alternative splicing in tea plants

Geraniol is an important contributor to the pleasant floral scent of tea products and one of the most abundant aroma compounds in tea plants;however,its biosynthesis and physiological function in response to stress in tea plants remain unclear.The proteins encoded by the full-length terpene synthase(CsTPS1)and its alternative splicing isoform(CsTPS1-AS)could catalyze the formation of geraniol when GPP was used as a substrate in vitro,whereas the expression of CsTPS1-AS was only significantly induced by Colletotrichum gloeosporioides and Neopestalotiopsis sp.infection.Silencing of CsTPS1 and CsTPS1-AS resulted in a significant decrease of geraniol content in tea plants.The geraniol content and disease resistance of tea plants were compared when CsTPS1 and CsTPS1-AS were silenced.Down-regulation of the expression of CsTPS1-AS reduced the accumulation of geraniol,and the silenced tea plants exhibited greater susceptibility to pathogen infection than control plants.However,there was no significant difference observed in the geraniol content and pathogen resistance between CsTPS1-silenced plants and control plants in the tea plants infected with two pathogens.Further analysis showed that silencing of CsTPS1-AS led to a decrease in the expression of the defense-related genes PR1 and PR2 and SA pathway-related genes in tea plants,which increased the susceptibility of tea plants to pathogens infections.Both in vitro and in vivo results indicated that CsTPS1 is involved in the regulation of geraniol formation and plant defense via alternative splicing in tea plants.The results of this study provide new insights into geraniol biosynthesis and highlight the role of monoterpene synthases in modulating plant disease resistance via alternative splicing.

Haem Oxygenase 1 is a potential target for creating etiolated/albino tea plants (Camellia sinensis) with high theanine accumulation

Theanine content is highly correlated with sensory quality and health benefits of tea infusion.The tender shoots of etiolated and albino tea plants contain higher theanine than the normal green tea plants and are valuable materials for high quality green tea processing.However,why these etiolated or albino tea plants can highly accumulate theanine is largely unknown.In this study,we observed an Arabidopsis etiolated mutant hy1–100(mutation in Haem Oxygenase 1,HO1)that accumulated higher levels of glutamine(an analog of theanine).We therefore identified CsHO1 in tea plants and found CsHO1 is conserved in amino acid sequences and subcellular localization with its homologs in other plants.Importantly,CsHO1 expression in the new shoots was much lower in an etiolated tea plants‘Huangkui’and an albino tea plant‘Huangshan Baicha’than that in normal green tea plants.The expression levels of CsHO1 were negatively correlated with theanine contents in these green,etiolated and albino shoots.Moreover,CsHO1 expression levels in various organs and different time points were also negatively correlated with theanine accumulation.The hy1–100 was hypersensitive to high levels of theanine and accumulated more theanine under theanine feeding,and these phenotypes were rescued by the expression of CsHO1 in this mutant.Transient knockdown CsHO1 expression in the new shoots of tea plant using antisense oligonucleotides(asODN)increased theanine accumulation.Collectively,these results demonstrated CsHO1 negatively regulates theanine accumulation in tea plants,and that low expression CsHO1 likely contributes to the theanine accumulation in etiolated/albino tea plants.

CsGDH2.1 negatively regulates theanine accumulation in late-spring tea plants (Camellia sinensis var. sinensis)

Theanine,a unique and the most abundant non-proteinogenic amino acid in tea plants,endows tea infusion with the umami taste and anti-stress effects.Its content in tea correlates highly with green tea quality.Theanine content in new shoots of tea plants is high in mid-spring and greatly decreases in late spring.However,how the decrease is regulated is largely unknown.In a genetic screening,we observed that a yeast mutant,glutamate dehydrolase 2(gdh2),was hypersensitive to 40 mM theanine and accumulated more theanine.This result implied a role of CsGDH2s in theanine accumulation in tea plants.Therefore,we identified the two homologs of GDH2,CsGDH2.1 and CsGDH2.2,in tea plants.Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyperaccumulation of this mutant.Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues.Importantly,CsGDH2.1 expression was low in early spring,and increased significantly in late spring,in the new shoots of tea plants.These results all support the idea that CsGDH2.1 regulates theanine accumulation in the new shoots.Moreover,the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity,and knockdown of CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants.These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring,and provides a functional gene for improving late-spring green tea quality.

Theanine, a tea-plant-specific non-proteinogenic amino acid, is involved in the regulation of lateral root development in response to nitrogen status

Glutamine synthetase type I(GSI)-like proteins are proposed to mediate nitrogen signaling and developmental fate by synthesizing yet unidentified metabolites.Theanine,the most abundant non-proteinogenic amino acid in tea plants,is the first identified metabolite synthesized by a GSI-like protein(CsTSI)in a living system.However,the roles of theanine in nitrogen signaling and development are little understood.In this study we found that nitrogen deficiency significantly reduced theanine accumulation and increased lateral root development in tea plant seedlings.Exogenous theanine feeding significantly repressed lateral root development of seedlings of tea plants and the model plant Arabidopsis.The transcriptomic analysis revealed that the differentially expressed genes in the roots under theanine feeding were enriched in the apoplastic pathway and H2O_(2) metabolism.Consistently,theanine feeding reduced H2O_(2) levels in the roots.Importantly,when co-treated with H2O_(2),theanine abolished the promoting effect of H2O_(2) on lateral root development in both tea plant and Arabidopsis seedlings.The results of histochemical assays confirmed that theanine inhibited reactive oxygen species accumulation in the roots.Further transcriptomic analyses suggested the expression of genes encoding enzymes involved in H2O_(2) generation and scavenging was down-and upregulated by theanine,respectively.Moreover,the expression of genes involved in auxin metabolism and signaling,cell division,and cell expansion was also regulated by theanine.Collectively,these results suggested that CsTSI-synthesized theanine is likely involved in the regulation of lateral root development,via modulating H2O_(2) accumulation,in response to nitrogen levels in tea plants.This study also implied that the module consisting of GSI-like protein and theanine-like metabolite is probably conserved in regulating development in response to nitrogen status in plant species.

Strong biosynthesis and weak catabolism of theanine in new shoots contribute to the high theanine accumulation in Albino/etiolated tea plant(Camellia sinensis)

Tea processed from albino/etiolated tea shoots is favored by consumers because of its high theanine accumulation.To explore why theanine accumulates highly in new shoots of albino/etiolated tea cultivars,we compared theanine content in shoots and roots of albino/etiolated and common green tea cultivars.Results suggested that high theanine accumulation in albino/etiolated tea shoots was likely not caused by higher theanine biosynthesis in roots.Further analyses suggested that CsAlaDC-catalyzed ethylamine biosynthesis and CsGOGAT1-catalyzed glutamate biosynthesis were more active,and CsGGT2-catalyzed theanine catabolism was weaker in new shoots of these albino/etiolated tea plant cultivars.Therefore,the high theanine accumulation in albino/etiolated shoots is probably contributed by the strong theanine biosynthesis and weak catabolism in new shoots.These findings provided more comprehensive insights into the high accumulation of theanine in new shoots of albino/etiolated tea cultivars,and the knowledge can be used in plant breeding for new cultivars with higher theanine accumulation.

The effects of circadian rhythm on catechin accumulation in tea leaves

As the major bitter-and astringent-tasting components in tea leaves,catechins play an essential role in determining flavor characteristics and health benefits of tea products.This study analyzed the circadian rhythm effects on the accumulation pattern of catechins in tender leaves of three tea cultivars,'Huangkui'(HK),'Longjing43'(LJ43)and'Fuding Dabaicha'(FD).There were two major factors involved in the catechin regulation in tea plants:genetic background of the cultivar-specific regulation,and temporal effects of the circadian rhythm.Etiolated leaves of'HK'contained lower levels of catechins than'LJ43'and'FD'.The composition of EGCG in total catechins was significantly higher in'LJ43'leaves(65%)when compared with that in'HK'and'FD'(45%).The accumulation of individual catechins increased during the daytime but decreased overnight,especially over the time period of 22:00−02:00.The correlation analysis between catechins and environmental factors indicated catechin accumulation in tea leaves potentially resulted from a combined regulation of light and temperature.These findings provide new insights into our understanding of circadian rhythm regulation on flavor-important secondary metabolites in tea leaves.

茶树茎段不定芽高效发生体系的建立

茶树(Camellia sinensis)是重要的经济作物,杂合度高且变异度大,其高效离体再生体系鲜见报道。以舒茶早茎段为起始外植体,进行不定芽高效发生影响因子研究。结果表明,MS+2 mg·L^(-1)6-BA为定芽诱导的最适配方,定芽诱导率为84.44%,吸收底盘膨大率为80%,利于后续不定芽诱导;MS+2 mg·L^(-1)6-BA+0.2 mg·L^(-1)NAA+0.1 mg·L^(-1)KT+1 mg·L^(-1)脯氨酸为不定芽增殖诱导的适宜配方,不定芽诱导率为88.89%,平均芽数为7.8。不定根诱导的适宜配方为1/2MS+3 mg·L^(-1)IBA,生根率为85.56%。采用RAPD和ISSR技术对再生植株进行分子检测,在连续2代离体再生植株中未发现明显变异。

The Reference Genome of Tea Plant and Resequencing of 81 Diverse Accessions Provide Insights into Its Genome Evolution and Adaptation

Tea plant is an important economic crop,which is used to produce the world's oldest and most widely consumed tea beverages.Here,we present a high-quality reference genome assembly of the tea plant(Camellia sinensis var.sinensis)consisting of 15 pseudo-chromosomes.LTR retrotransposons(LTR-RTs)account for 70.38%of the genome,and we present evidence that LTR-RTS play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns.Genes,particularly those coding for terpene biosynthesis pro-teins,associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome.Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations,support-ing the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction.Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome,particularly those associated with tea quality and stress resis-tance.The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cul-tivars of tea plants.

COVID-19:immunopathogenesis and Immunotherapeutics

The recent novel coronavirus disease(COVID-19)outbreak,caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),is seeing a rapid increase in infected patients worldwide.The host immune response to SARS-CoV-2 appears to play a critical role in disease pathogenesis and clinical manifestations.SARS-CoV-2 not only activates antiviral immune responses,but can also cause uncontrolled inflammatory responses characterized by marked pro-inflammatory cytokine release in patients with severe COVID-19,leading to lymphopenia,lymphocyte dysfunction,and granulocyte and monocyte abnormalities.These SARS-CoV-2-induced immune abnormalities may lead to infections by microorganisms,septic shock,and severe multiple organ dysfunction.Therefore,mechanisms underlying immune abnormalities in patients with COVID-19 must be elucidated to guide clinical management of the disease.Moreover,rational management of the immune responses to SARSCoV-2,which includes enhancing anti-viral immunity while inhibiting systemic inflammation,may be key to successful treatment.In this review,we discuss the immunopathology of COVID-19,its potential mechanisms,and clinical implications to aid the development of new therapeutic strategies against COVID-19.

Induction of priming by cold stress via inducible volatile cues in neighboring tea plants^FA

Plants have evolved sophisticated defense mechanisms to overcome their sessile nature.However,if and how volatiles from cold‐stressed plants can trigger interplant communication is still unknown.Here,we provide the first evidence for interplant communication via inducible volatiles in cold stress.The volatiles,including nerolidol,geraniol,linalool,and methyl salicylate,emitted from cold‐stressed tea plants play key role(s)in priming cold tolerance of their neighbors via a C‐repeatbinding factors‐dependent pathway.The knowledge will help us to understand how plants respond to volatile cues in cold stress and agricultural ecosystems.

Efficient iron plaque formation on tea(Camellia sinensis) roots contributes to acidic stress tolerance

Tea plants grow in acidic soil, but to date, their intrinsic mechanisms of acidic stress tolerance have not been elucidated. Here, we assessed the tea plant response to growth on NHt4 nutrient media having different p H and iron levels. When grown in standard NHt4 nutrient solution(iron insufficient, 0.35 mg Là1 Fe2t), tea roots exhibited significantly lower nitrogen accumulation, plasma membrane Ht-ATPase activity, and protein levels; net Htefflux was lower at pH 4.0 and 5.0 than at pH 6.0. Addition of30 mg Là1 Fe2t(iron sufficient, mimicking normal soil Fe2tconcentrations) to the NHt4 nutrient solution led to more efficient iron plaque formation on roots and increased root plasma membrane Ht-ATPase levels and activities at p H 4.0 eland 5.0, compared to the p H 6.0 condition. Furthermore,plants grown at pH 4.0 and 5.0, with sufficient iron,exhibited significantly higher nitrogen accumulation than those grown at pH 6.0. Together, these results support the hypothesis that efficient iron plaque formation, on tea roots, is important for acidic stress tolerance. Furthermore,our findings establish that efficient iron plaque formation is linked to increased levels and activities of the tea root plasma membrane Ht-ATPase, under low pH conditions.