Functional diversity of subgroup 5 R2R3-MYBs promoting proanthocyanidin biosynthesis and their key residues and motifs in tea plant

The tea plant(Camellia sinensis)is rich in polyphenolic compounds.Particularly,flavan-3-ols and proanthocyanidins(PAs)are essential for the flavor and disease-resistance property of tea leaves.The fifth subgroup of R2R3-MYB transcription factors comprises the primary activators of PA biosynthesis.This study showed that subgroup 5 R2R3-MYBs in tea plants contained at least nine genes belonging to the TT2,MYB5,and MYBPA types.Tannin-rich plants showed an expansion in the number of subgroup 5 R2R3-MYB genes compared with other dicotyledonous and monocot plants.The MYBPA-type genes of tea plant were slightly expanded.qRT–PCR analysis and GUS staining analysis of promoter activity under a series of treatments revealed the differential responses of CsMYB5s to biotic and abiotic stresses.In particular,CsMYB5a,CsMYB5b,and CsMYB5e responded to high-intensity light,high temperature,MeJA,and mechanical wounding,whereas CsMYB5f and CsMYB5g were only induced by wounding.Three genetic transformation systems(C.sinensis,Nicotiana tabacum,and Arabidopsis thaliana)were used to verify the biological function of CsMYB5s.The results show that CsMYB5a,CsMYB5b,and CsMYB5e could promote the gene expression of CsLAR and CsANR.However,CsMYB5f and CsMYB5g could only upregulate the gene expression of CsLAR but not CsANR.A series of site-directed mutation and domain-swapping experiments were used to verify functional domains and key amino acids of CsMYB5s responsible for the regulation of PA biosynthesis.This study aimed to provide insight into the induced expression and functional diversity model of PA biosynthesis regulation in tea plants.

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.

The MtRGF6 Peptide Differentially Regulates Root Development and Symbiotic Nodulation of Medicago truncatula and Lotus japonicus

Rhizobia induces nitrogen-fixing nodules in legumes used in agricultural production,providing a direct source of combined nitrogen to leguminous crops.Small peptides,such as CLAVATA3/EMBRYO SURROUNDING REGION peptides(CLE),are known to regulate the formation and development of nitrogen-fixing nodules in legumes.Root meristem growth factor(RGF)peptides from Medicago truncatula not only regulate root develop-ment but also modulate nodulation symbiosis with Sinorhizobium meliloti.However,the impact of RGF peptides from one leguminous species on the others remains unclear.In this study,we investigate the effects of the RGF family peptide MtRGF6p from M.truncatula on nodulation symbiosis and root development in Lotus japonicus.The MtRGF6 gene is predominantly expressed in the root nodules of M.truncatula and shows low identity with RGF homologous genes from L.japonicus.The gene promoter is active in the primordia of root nodules and lat-eral roots,as well as in young nodules and roots,and the meristem,infection,and nitrogen-fixing regions of the mature nodule.Chemically synthesized MtRGF6p promoted primary root growth in M.truncatula but sup-pressed the growth of L.japonicus primary roots.The peptide negatively affected the initiation of nodule primor-dia,the formation of infection threads,and nodulation in both legumes,with a low dosage showing effects on L.japonicus compared to M.truncatula.These results suggest that the MtRGF6 peptide from M.truncatula may serve as an inter-species signal affecting the root organ development of L.japonicus.

Molecular mechanism of lycopene cyclases regulating carotenoids ratio in different branches during tea leaf and flower development

Carotenoids are essential components in tea quality, contributing to leaf color and aroma. However, little information about carotenoids in different tea cultivars and their biosynthesis regulation mechanism during leaf development is known. Here we analyzed carotenoids by HPLC in the buds and leaves of 113 tea cultivars harvested on the same day. By profile clustering, carotenoids were divided into five groups. Same group cultivars displayed divergence in the total content of carotenoids but a similar molar ratio. To figure out the molecular mechanisms of this phenomenon, we further characterized all functional lycopene cyclases, which are the branch point of the carotenoid biosynthesis pathway. Two β-lycopene cyclases(CsLCYB1 and CsLCYB2) and one ε-lycopene cyclase(CsLCYE1) were cloned. Subcellular localization analysis showed that all cloned CsLCYs were localized in plastids. Enzyme activity assays in E. coli indicated both CsLCYBs catalyzed lycopene into β-carotene, and CsLCYE1 produced δ-carotene and ε-carotene. We found CsLCYB1 and CsLCYE1 predominantly expressed in leaf, while CsLCYB2was mainly expressed during flowering stages. Suppression by antisense oligonucleotides reduced CsLCYB1 and CsLCYE1 transcripts and led to reduction of both β,β-branch and β,ε-branch carotenoids in leaf. The expression levels of CsLCYB1 showed a significant positive correlation withβ,β-branch carotenoids in leaf. Our study provides carotenoid profiles of different tea cultivars, which can assist tea producers in selecting cultivars of interest. Meanwhile, we proposed the molecular mechanism of carotenoids reflecting the tenderness of tea plant leaf from a metabolic flux perspective, and suggested lycopene cyclase that could be applied to the breeding of tea varieties with different branch carotenoids.

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.

Al-induced CsUGT84J2 enhances flavonol and auxin accumulation to promote root growth in tea plants

Although Al is not necessary or even toxic to most plants,it is beneficial for the growth of tea plants.However,the mechanism through which Al promotes root growth in tea plants remains unclear.In the present study,we found that flavonol glycoside levels in tea roots increased following Al treatment,and the Al-induced UDP glycosyltransferase CsUGT84J2 was involved in this mechanism.Enzyme activity assays revealed that rCsUGT84J2 exhibited catalytic activity on multiple types of substrates,including phenolic acids,flavonols,and auxins in vitro.Furthermore,metabolic analysis with UPLC-QqQ-MS/MS revealed significantly increased flavonol and auxin glycoside accumulation in CsUGT84J2-overexpressing Arabidopsis thaliana.In addition,the expression of genes involved in the flavonol pathway as well as in the auxin metabolism,transport,and signaling pathways was remarkably enhanced.Additionally,lateral root growth and exogenous Al stress tolerance were significantly improved in transgenic A.thaliana.Moreover,gene expression and metabolic accumulation related to phenolic acids,flavonols,and auxin were upregulated in CsUGT84J2-overexpressing tea plants but downregulated in CsUGT84J2-silenced tea plants.In conclusion,Al treatment induced CsUGT84J2 expression,mediated flavonol and auxin glycosylation,and regulated endogenous auxin homeostasis in tea roots,thereby promoting the growth of tea plants.Our findings lay the foundation for studying the precise mechanisms through which Al promotes the growth of tea plants.

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.

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.

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.

Core collection construction of tea plant germplasm in Anhui Province based on genetic diversity analysis using simple sequence repeat markers

The tea plant[Camellia sinensis(L.)O.Kuntze]is an industrial crop in China.The Anhui Province has a long history of tea cultivation and has a large resource of tea germplasm with abundant genetic diversity.To reduce the cost of conservation and utilization of germplasm resources,a core collection needs to be constructed.To this end,573 representative tea accessions were collected from six major tea-producing areas in Anhui Province.Based on 60 pairs of simple sequence repeat(SSR)markers,phylogenetic relationships,population structure and principal coordinate analysis(PCoA)were conducted.Phylogenetic analysis indicated that the 573 tea individuals clustered into five groups were related to geographical location and were consistent with the results of the PCoA.Finally,we constructed a core collection consisting of 115 tea individuals,accounting for 20%of the whole collection.The 115 core collections were considered to have a 90.9%retention rate for the observed number of alleles(Na),and Shannon’s information index(I)of the core and whole collections were highly consistent.Of these,39 individuals were preserved in the Huangshan area,accounting for 33.9%of the core collection,while only 10 individuals were reserved in the Jinzhai County,accounting for 8.9%of the core set.PCoA of the accessions in the tea plant core collection exhibited a pattern nearly identical to that of the accessions in the entire collection,further supporting the broad representation of the core germplasm in Anhui Province.The results demonstrated that the core collection could represent the genetic diversity of the original collection.Our present work is valuable for the high-efficiency conservation and utilization of tea plant germplasms in Anhui Province.

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.

Mycorrhiza improves plant growth and photosynthetic characteristics of tea plants in response to drought stress

Tea plants are sensitive to soil moisture deficit,with the level of soil water being a critical factor affecting their growth and quality.Arbuscular mycorrhizal fungi(AMF)can improve water and nutrient absorption,but it is not clear whether AMF can improve the photosynthetic characteristics of tea plants.A potted study was conducted to determine the effects of Claroideoglomus etunicatum on plant growth,leaf water status,pigment content,gas exchange,and chlorophyll fluorescence parameters in Camellia sinensis cv.Fuding Dabaicha under well-watered(WW)and drought stress(DS)conditions.Root mycorrhizal colonization and soil hyphal length were significantly reduced by the eightweek DS treatment.AMF inoculation displayed a significant increase in shoot and root biomass production.The relative water content,leaf water potential,nitrogen balance index,pigment content,maximum photometric effect(Fv/Fm,QY_max),and steady-state photometric effect Y(II)(QY_Lss)decreased dramatically,while the leaf water saturation deficit and steady-state non-photochemical fluorescence quenching(NPQ_Lss)generally increased under DS conditions.Mycorrhizal treatment induced significantly higher relative water content,leaf water potential,nitrogen balance index,pigment(chlorophyll,flavonoid,and anthocyanin)content,net photosynthesis rate,transpiration rate,stomatal conductance,intercellular CO_(2)concentration,QY_max,and QY_Lss;however,it resulted in a lower leaf water saturation deficit and NPQ_Lss under both WW and DS conditions,as compared with nonmycorrhizal plants.These results imply that AMF promoted tea plant growth and alleviated negative effects of DS by promoting gas exchange,regulating the water status of leaves,and regulating photosynthetic parameters.

Chemistry and health beneficial effects of oolong tea and theasinensins

Among six major types of tea(white,green,oolong,yellow,black,and dark teas)from Camellia sinensis,oolong tea,a semi-fermented tea,with its own unique aroma and taste,has become a popular consumption as indicated by the increasing production.Representing the characteristic flavonoids of oolong tea,theasinensins are dimeric flavan-3-ols.Many recent studies have indicated that oolong tea and theasinensins possess several health benefit properties.We consider it significant and necessary to have a comprehensive review in the recent advances of oolong tea.Therefore,the aim of the present review is to provide a new perspective on oolong tea and its characteristic phytochemicals,theasinensins associated with health benefits,molecular action pathway,and chemical mechanism of theasinensin formation from scientific evidences available on the literature.Furthermore,the chemical characterization of the oxidation products and the model oxidation system to the chemical changes of theasinensins are also discussed.

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.

Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant(Camellia sinensis)

Kunitz protease inhibitors(KPIs)are ubiquitous in plants and act as crucial compounds in defense responses against insect attack and pathogen infection.However,the influence of gene duplication on the postdivergence of the CsKPI genes involved in biotic stresses in tea plant is not well known.Here,we identified three CsKPI genes from tea plant(Camellia sinensis)and characterized their expression and evolutionary patterns among plant species.We found that CsKPI1,CsKPI2,and CsKPI3 diverged from their common ancestor 72.94 million years ago(MYA),and the tandem duplication of CsKPI2 and CsKPI3 occurred 26.78 MYA.An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of p-nitroanilide(PNA)from an artificial substrate.The three CsKPI-GFP fusion proteins localized to the cytoplasm.We showed that salicylic acid(SA)and transcripts of CsKPI2 and CsKPI3 significantly accumulated after infection with Glomerella cingulata.The application of exogenous SA stimulated the high expression of both CsKPI2 and CsKPI3 by activating cis-elements within their promoters.Under Ectropis oblique attack,CsKPI1 expression and jasmonic acid(JA)levels were more abundant in both insect-damaged leaf tissues and undamaged neighboring leaves.The application of jasmonic acid methyl ester elicited high expression levels of CsKPI1,suggesting that CsKPI1 accumulation requires JA production in tea plant.The overall findings suggest that the transcriptional divergence of KPI genes after duplication led to the specialized role of CsKPI1 in the physiological response to insect stress;the functional conservation between CsKPI2 and CsKPI3 confers resistance to pathogen infection in tea plant.

Soil and fine root-associated microbial communities are niche dependent and influenced by copper fungicide treatment during tea plant cultivation

Dear Editor,Fungicide treatment has a profound effect on controlling plant pathogens in modern agriculture,however,it also carries the risk of undesirable outcomes.For decades,scientists have been concerned about the harmful impacts of heavy metals like copper(Cu)on crop performance and soil microorganisms.Use of various copper fungicides,like Bordeaux mixture,have been a component of conventional agricultural practices to control fungal and bacterial pathogens,especially in vineyards,tea gardens,or fruit tree orchards[9,10].This treatment increases the accumulation of high levels of Cu in surface soils,and despite the critical role of Cu as an essential trace element in wide biological and metabolic processes,it becomes toxic to plants when applied at high levels[4].The regular application of copper fungicides has also been linked to affecting microbial communities at the levels of diversity[8],population structure[2],abundance,and growth[1,3].Understanding the undesired effects of fungicides on microorganisms’beneficial activities is therefore important for evaluating the hazards associated with the fungicide used in agriculture.Yet,the effects of copper fungicide on full microbial communities remains relatively understudied,especially in tea plants.Thus,we herein explored the inf luence of Bordeaux mixture under different management regimes(raking or without raking leaf litter)on microbial communities of root,bulk soil,and rhizosphere compartments of tea plants planted in a ten-year-old tea garden.We provided insights into the ecological consequences of tea management practices that might help to identify specific fungicide treatment regimens,environmental characteristics,and microbial community members to minimize the negative environmental outcomes and optimize the positive anti-pathogen aspects of fungicide treatment.

The tea plant CsLHT1 and CsLHT6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations

Organic tea is more popular than conventional tea that originates from fertilized plants.Amino acids inorganic soils constitute a substantial pool nitrogen(N)available for plants.However,the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown.In this study,we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation.Glutamate,alanine,valine,and leucine were the most abundant amino acids in the soil of this tea plantation.When 15 N-glutamate was fed to tea plants,it was efficiently absorbed and significantly increased the contents of other amino acids in the roots.We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1,CsLHT2,and CsLHT6 in the roots significantly increased upon glutamate feeding.Moreover,the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant,22Δ10α,enabled growth on media with amino acids constituting the sole N source.Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H^(+)-dependent high-and low-affinity amino-acid transporters,respectively.We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane.Moreover,overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15 N-glutamate and 15 N-glutamine.Taken together,our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil,which is particularly important for tea plants grown inorganic tea plantations.

Potential prebiotic effects of nonabsorptive components of Keemun and Dianhong black tea:an in vitro study

Black tea is a healthy and popular tea beverage.However,its main bioactive compounds(theaflavins and thearubigins)are not easily absorbed.The aim of this study was to investigate the modulation of intestinal microbiota by the nonabsorptive components of Keemun black tea(KBT)and Dianhong black tea(DBT),and fructooligosaccharide(FOS)was selected for use in the control group.KBT,DBT,and FOS significantly increased total short-chain acid production.Specifically,FOS treatment predominantly increased the production of acetic acids and black tea treatments increased the production of acetic,propionic,and butyric acids at similar rates.Moreover,FOS exerted a strong bifidogenic effect after 24 h of fermentation;KBT and DBT increased the abundance of the beneficial genus Bacteroides and Roseburia.In summary,the nonabsorptive components of KBT and DBT could serve as novel prebiotics,the underlying mechanisms of which are quite different from those of FOS.

JA-mediated MYC2/LOX/AOS feedback loop regulates osmotic stress response in tea plant

Osmotic stress caused by low-temperature,drought and salinity was a prevalent abiotic stress in plant that severely inhibited plant development and agricultural yield,particularly in tea plant.Jasmonic acid(JA)is an important phytohormone involving in plant stress.However,underlying molecular mechanisms of JA modulated osmotic stress response remains unclear.In this study,high concentration of mannitol induced JA accumulation and increase of peroxidase activity in tea plant.Integrated transcriptome mined a JA signaling master,MYC2 transcription factor is shown as a hub regulator that induced by mannitol,expression of which positively correlated with JA biosynthetic genes(LOX and AOS)and peroxidase genes(PER).CsMYC2 was determined as a nuclei-localized transcription activator,furthermore,ProteinDNA interaction analysis indicated that CsMYC2 was positive regulator that activated the transcription of CsLOX7,CsAOS2,CsPER1 and CsPER3via bound with their promoters,respectively.Suppression of CsMYC2 expression resulted in a reduced JA content and peroxidase activity and osmotic stress tolerance of tea plant.Overexpression of CsMYC2 in Arabidopsis improved JA content,peroxidase activity and plants tolerance against mannitol stress.Together,we proposed a positive feedback loop mediated by CsMYC2,CsLOX7 and CsAOS2 which constituted to increase the tolerance of osmotic stress through fine-tuning the accumulation of JA levels and increase of POD activity in tea plant.

Characterization of CsWRKY29 and CsWRKY37 transcription factors and their functional roles in cold tolerance of tea plant

The WRKY gene family is most widely known as being the key plant transcription factor family involved in various stress responses and affecting plant growth and development.In this study,a total of 86 members of the CsWRKY genes were identified from the tea plant genome.Most of these genes contain several important Cis-regulatory elements in the promoter regions associated with multiple stress-responses.These genes were further classified into three groups,I,II,and III,each with 21,58,and 7 members,respectively.We showed evidence that tandem duplications,but not the whole genome duplication,are likely to drive the amplification of CsWRKY genes in tea plants.All the 86 CsWRKY genes showed differential expression patterns either in different tissues,or under exposure to diverse abiotic stresses such as drought,cold acclimation,and MeJA treatments.Additionally,the functional roles of two genes,CsWRKY29 and CsWRKY37,were examined under cold stress;and the silencing of these genes resulted in tea plant phenotypes susceptible to cold stress.Moreover,transgenic Arabidopsis lines overexpressing CsWRKY29 and CsWRKY37 genes showed higher survival rates and lower malondialdehyde levels under freezing treatment than the wild type plants.The core findings from this work provide valuable evolutionary pattern of WRKY gene family and underpinning the underlying regulatory roles of CsWRKY29 and CsWRKY37 from tea plants that conferred cold tolerance in transgenic Arabidopsis plants.