A serine carboxypeptidase-like acyltransferase catalyzes consecutive four-step reactions of hydrolyzable tannin biosynthesis in Camellia oleifera.

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Deep learning and targeted metabolomics-based monitoring of chewing insects in tea plants and screening defense compounds.

Tea is an important cash crop that is often consumed by chewing pests, resulting in reduced yields and economic losses. It is important to establish a method to quickly identify the degree of damage to tea plants caused by leaf-eating insects and screen green control compounds. This study was performed through the combination of deep learning and targeted metabolomics, in vitro feeding experiment, enzymic analysis and transient genetic transformation. A small target damage detection model based on YOLOv5 with Transformer Prediction Head (TPH-YOLOv5) algorithm for the tea canopy level was established. Orthogonal partial least squares (OPLS) was used to analyze the correlation between the degree of damage and the phenolic metabolites. A potential defensive compound, (-)-epicatechin-3-O-caffeoate (EC-CA), was screened. In vitro feeding experiments showed that compared with EC and epicatechin gallate, Ectropis grisescens exhibited more significant antifeeding against EC-CA. In vitro enzymatic experiments showed that the hydroxycinnamoyl transferase (CsHCTs) recombinant protein has substrate promiscuity and can catalyze the synthesis of EC-CA. Transient overexpression of CsHCTs in tea leaves effectively reduced the degree of damage to tea leaves. This study provides important reference values and application prospects for the effective monitoring of pests in tea gardens and screening of green chemical control substances.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

Impact of sex on treatment-related adverse effects and prognosis in nasopharyngeal carcinoma.

BACKGROUND: In nasopharyngeal cancer (NPC), women have a lower incidence and mortality rate than men. Whether sex influences the prognosis of NPC patients remains debatable. We retrospectively examined the influence of sex on treatment-related side effects and prognosis in NPC. METHODS: Clinical data of 1,462 patients with NPC treated at the Southern Hospital of Southern Medical University from January 2004 to December 2015 were retrospectively examined. Statistical analysis was performed to assess differences in overall survival (OS), distant metastasis-free survival (DMFS), local recurrence-free survival(LRFS), and progression-free survival(PFS), as well as treatment-related adverse effects, including myelosuppression, gastrointestinal responses, and radiation pharyngitis and dermatitis, between men and women. RESULTS: Women had better 5-year OS (81.5% vs. 87.1%, P = 0.032) and DMFS (76.2% vs. 83.9%, P = 0.004) than men. Analysis by age showed that the prognoses of premenopausal and menopausal women were better than those of men, whereas prognoses of postmenopausal women and men were not significantly different. Additionally, women had a better prognosis when stratified by treatment regimen. Furthermore, chemotherapy-related adverse effects were more severe in women than in men; however, the incidences of radiation laryngitis and dermatitis were not significantly different between the sexes. Logistic regression analysis revealed that the female sex was an independent risk factor for severe myelosuppression and gastrointestinal reactions. CONCLUSIONS: Chemotherapy-related side effects are more severe but the overall prognosis is better in women with NPC than in men with NPC. Patients may benefit from a personalized treatment approach for NPC. TRIAL REGISTRATION: This study was approved by the Medical Ethics Committee of Nanfang Hospital of the Southern Medical University (NFEC-201,710-K3).

Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis).

KEY MESSAGE: Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.

The Functional Characterization of Carboxylesterases Involved in the Degradation of Volatile Esters Produced in Strawberry Fruits.

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters.

Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins.

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.

Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality.

Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins.

Plant tannins, including condensed tannins (CTs) and hydrolyzable tannins (HTs), are widely distributed in the plant kingdom. To date, tannase (TA) - is a type of tannin acyl-hydrolase hydrolyzing HTs, CT monomer gallates and depsides - has been reported in microbes only. Whether plants express TA remains unknown. Herein, we report plant TA genes. A native Camellia sinensis TA (CsTA) is identified from leaves. Six TAs are cloned from tea, strawberry (Fragaria × ananassa, Fa) and four other crops. Biochemical analysis shows that the native CsTA and six recombinant TAs hydrolyze tannin compounds, depsides and phenolic glycosides. Transcriptional and metabolic analyses reveal that the expression of CsTA is oppositely associated with the accumulation of galloylated catechins. Moreover, the transient overexpression and RNA interference of FaTA are positively associated with the accumulation of ellagitannins in strawberry fruit. Phylogenetic analysis across different kingdoms shows that 29 plant TA homologs are clustered as a plant-specific TA clade in class I carboxylesterases. Further analysis across the angiosperms reveals that these TA genes are dispersed in tannin-rich plants, which share a single phylogenetic origin c. 120 million yr ago. Plant TA is discovered for the first time in the plant kingdom and is shown to be valuable to improve tannin compositions in plants.

Food as medicine: A possible preventive measure against coronavirus disease (COVID-19).

The recent and ongoing outbreak of coronavirus disease (COVID-19) is a huge global challenge. The outbreak, which first occurred in Wuhan City, Hubei Province, China and then rapidly spread to other provinces and to more than 200 countries abroad, has been declared a global pandemic by the World Health Organization. Those with compromised immune systems and/or existing respiratory, metabolic or cardiac problems are more susceptible to the infection and are at higher risk of serious illness or even death. The present review was designed to report important functional food plants with immunomodulatory and anti-viral properties. Data on medicinal food plants were retrieved and downloaded from English-language journals using online search engines. The functional food plants herein documented might not only enhance the immune system and cure respiratory tract infections but can also greatly impact the overall health of the general public. As many people in the world are now confined to their homes, inclusion of these easily accessible plants in the daily diet may help to strengthen the immune system and guard against infection by SARS-CoV-2. This might reduce the risk of COVID-19 and initiate a rapid recovery in cases of SARS-CoV-2 infection.

Three Camellia sinensis glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanidins.

MAIN CONCLUSION: Biochemical, transgenic, and genetic complementation data demonstrate that three glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanins in plant cells. Flavonoids are compounds in tea (Camellia sinensis) that confer the characteristic astringent taste of tea beverages; these compounds have numerous benefits for human health. In plant cells, flavonoids are synthesized in different locations within the cytoplasm and are then transported and finally stored in vacuoles. To date, the mechanism involved in the intracellular transport of flavonoids in tea has not been well elucidated. In this study, we report the functional characterization of three cDNAs encoding glutathione S-transferases (CsGSTs) of C. sinensis, namely, CsGSTa, CsGSTb, and CsGSTc. The expression profiles of CsGSTa and CsGSTb were positively correlated with the accumulation of flavonols, anthocyanins and proanthocyanins in tea tissues and cultivars. These three recombinant CsGSTs showed a high affinity for flavonols (kaempferol-3-O-glucoside and quercetin-3-O-glucoside) and anthocyanin (cyanidin-3-O-glucoside) in vitro but had no or weak affinity for epicatechin. In vivo, CsGSTa, CsGSTb and CsGSTc fully or partially restored the storage of anthocyanins and proanthocyanidins in transgenic tt19 mutants. Metabolic profiling revealed that the contents of anthocyanins, flavonols, and proanthocyanidins were increased in the transgenic petals of Nicotiana tabacum. Taken together, all data showed that CsGSTa, CsGSTb, and CsGSTc are associated with the storage of anthocyanins, flavonols, and proanthocyanins in C. sinensis cells.

AtHB2, a class II HD-ZIP protein, negatively regulates the expression of CsANS, which encodes a key enzyme in Camellia sinensis catechin biosynthesis.

Tea (Camellia sinensis) is an important cash crop that is beneficial to human health because of its remarkable content of catechins. The biosynthesis of catechins follows the flavonoid pathway, which is highly branched. Among the enzymes involved in catechin biosynthesis, ANTHOCYANIDIN SYNTHASE (CsANS) functions at a branch point and play a critical role. Our previous work has showed that the gene encoding CsANS is regulated by light signals; however, the molecular mechanism behind remains unclear. Here, we cloned a full-length CsANS promoter and found that it contained a cis-element recognized by Arabidopsis thaliana HOMEOBOX2 (AtHB2). AtHB2 constitutes one of the class II HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) proteins, which accumulate in the dark and mediate the shade avoidance response in most angiosperms. To analyze the transcription of CsANS in vivo, ß-glucuronidase and luciferase reporter genes driven by the obtained promoter were introduced into A. thaliana and Nicotiana attenuata, respectively. In both expression systems there were indications that the A. thaliana PRODUCTION OF ANTHOCYANIN PIGMENT1 (AtPAP1), a MYB transcription factor of flavonoid biosynthesis, increased the activity of the CsANS promoter, while AtHB2 could significantly undermine the effect of AtPAP1. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that AtHB2 interacted with the A. thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1). A yeast three-hybrid assay further suggested that AtHB2 represses the expression of CsANS and regulates its response to light signals through competitive interactions with AtTTG1. These results show that HD-ZIP II proteins participate in light regulation of flavonoid biosynthesis.

Ligand fishing of anti-neurodegenerative components from Lonicera japonica using magnetic nanoparticles immobilised with monoamine oxidase B.

In this work, monoamine oxidase B was immobilised onto magnetic nanoparticles to prepare a new type of affinity solid-phase extraction adsorbent, which was used to extract the possible anti-neurodegenerative components from the Lonicera japonica flower extracts. Coupled with high-performance liquid chromatography with mass spectrometry, two monoamine oxidase B ligands were fished-out and identified as isochlorogenic acid A and isochlorogenic acid C, which were found to be inhibitors of the enzyme for the first time, with similar half maximal inhibitory concentration values of 29.05 ± 0.49 and 29.77 ± 1.03 µM, respectively. Furthermore, equilibrium-dialysis dissociation assay of enzyme-inhibitor complex showed that both compounds have reversible binding patterns to monoamine oxidase B, and kinetic analysis demonstrated that they were mixed-type inhibitors for monoamine oxidase B, with Ki and Kis values of 9.55 and 37.24 µM for isochlorogenic acid A, 9.53 and 35.50 µM for isochlorogenic acid C, respectively. The results indicated that isochlorogenic acid A and isochlorogenic acid C were the major active components responsible for the anti-degenerative activity of the flowers of L. japonica, while magnetic nanoparticles immobilised monoamine oxidase B could serve as an efficient solid-phase extraction adsorbent to specifically extract monoamine oxidase B inhibitors from complex herbal extracts.

Effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis.

BACKGROUND: Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea's quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants. RESULTS: Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment. CONCLUSIONS: Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant.

Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis.

MAIN CONCLUSION: LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of NtLAR and NtANR s in CsMYB5b transgenic tobacco. Tea is rich in polyphenolic compounds, and catechins are the major polyphenols in tea. The biosynthesis of polyphenols is closely related to the expression of the leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes. In this paper, an evolutionary analysis and functional characterization of three CsLARs were performed. The phylogenetic tree showed that plant LARs could be grouped into three, including gymnosperms, monocotyledons and dicotyledons (clusters I and II). The eighth amino acid residue in a conserved LAR-specific motif is changeable due to a transversion (G â†’ T) and transition (G â†’ C) that occur in the corresponding codon. Therefore, plant LARs can be classified as G-type, A-type and S-type LARs due to this variable amino acid residue. Although (2R, 3S)-trans-flavan-3-ols were the products of recombinant CsLARs proteins expressed in Escherichia coli, both (2R, 3S)-trans and (2R, 3R)-cis-flavan-3-ols were detected in tobacco overexpressing CsLARs. However, a butanol/HCl hydrolysis assay indicated that overexpression of the CsLARs caused a decrease in polymerized catechins. A hybridization experiment with CsLARc + AtPAP1 also showed that no polymers other than epicatechin, catechin and glycoside were detected, although the accumulation of anthocyanins was markedly decreased. CsMYB5b promoted the biosynthesis of both flavan-3-ols and proanthocyanidins (PAs). Therefore, LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of the NtLAR and NtANRs in CsMYB5b transgenic tobacco.

Bombyx mori Dihydroorotate Dehydrogenase: Knockdown Inhibits Cell Growth and Proliferation via Inducing Cell Cycle Arrest.

Dihydroorotate dehydrogenase (DHODH), in the de novo pyrimidine biosynthetic pathway, is the fourth enzyme of pyrimidine synthesis and is used to oxidize dihydroorotate and hence to orotat. We cloned and characterized here the dhod of silkworms, Bombyx mori. The full-length cDNA sequence of dhod is 1339 bp, including an open reading frame (ORF) of 1173 bp that encoded a 390 amino acid protein. And two domains were involved in the Dihydroorotate dehydrogenase amino acid sequence of silkworms, Bombyx mori (BmDHODH), namely a DHO_dh domain and a transmembrane domain in N-termina. The silkworm dhod is expressed throughout development and in nine tissues. Moreover, knockdown of the silkworm dhod gene reduced cell growth and proliferation through G2/M phase cell cycle arrest. Similarly, DHODH inhibitor (leflunomide) also reduced cell growth and proliferation, with a significant decrease of cyclin B and cdk2. DHODH is the fourth enzyme of pyrimidine synthesis, so we also found that leflunomide can inhibit, at least in part, the endomitotic DNA replication in silk glands cells. These findings demonstrate that downregulation of BmDHODH inhibits cell growth and proliferation in silkworm cells, and the endomitotic DNA replication in silk gland cells.

A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB⁻bHLH⁻WD40 Ternary Complexes.

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.

Metabolic Characterization of the Anthocyanidin Reductase Pathway Involved in the Biosynthesis of Flavan-3-ols in Elite Shuchazao Tea (Camellia sinensis) Cultivar in the Field.

Anthocyanidin reductase (ANR) is a key enzyme in the ANR biosynthetic pathway of flavan-3-ols and proanthocyanidins (PAs) in plants. Herein, we report characterization of the ANR pathway of flavan-3-ols in Shuchazao tea (Camellia sinesis), which is an elite and widely grown cultivar in China and is rich in flavan-3-ols providing with high nutritional value to human health. In our study, metabolic profiling was preformed to identify two conjugates and four aglycones of flavan-3-ols: (-)-epigallocatechin-gallate [(-)-EGCG], (-)-epicatechin-gallate [(-)-ECG], (-)-epigallocatechin [(-)-EGC], (-)-epicatechin [(-)-EC], (+)-catechin [(+)-Ca], and (+)-gallocatechin [(+)-GC], of which (-)-EGCG, (-)-ECG, (-)-EGC, and (-)-EC accounted for 70-85% of total flavan-3-ols in different tissues. Crude ANR enzyme was extracted from young leaves. Enzymatic assays showed that crude ANR extracts catalyzed cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively, in which (-)-EC and (-)-EGC were major products. Moreover, two ANR cDNAs were cloned from leaves, namely CssANRa and CssANRb. His-Tag fused recombinant CssANRa and CssANRb converted cyanidin and delphinidin to (-)-EC and (-)-Ca and (-)-EGC and (-)-GC, respectively. In addition, (+)-EC was observed from the catalysis of recombinant CssANRa and CssANRb. Further overexpression of the two genes in tobacco led to the formation of PAs in flowers and the reduction of anthocyanins. Taken together, these data indicate that the majority of leaf flavan-3-ols in Shuchazao's leaves were produced from the ANR pathway.

Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis).

Galloylated catechins and flavonol 3-O-glycosides are characteristic astringent taste compounds in tea (Camellia sinensis). The mechanism involved in the formation of these metabolites remains unknown in tea plants. In this paper, 178 UGT genes (CsUGTs) were identified inC. sinensis based on an analysis of tea transcriptome data. Phylogenetic analysis revealed that 132 of these genes were clustered into 15 previously established phylogenetic groups (A to M, O and P) and a newly identified group R. Three of the 11 recombinant UGT proteins tested were found to be involved in the in vitro biosynthesis of ß-glucogallin and glycosylated flavonols. CsUGT84A22 exhibited catalytic activity toward phenolic acids, in particular gallic acid, to produce ß-glucogallin, which is the immediate precursor of galloylated catechin biosynthesis in tea plants. CsUGT78A14 and CsUGT78A15 were found to be responsible for the biosynthesis of flavonol 3-O-glucosides and flavonol 3-O-galactosides, respectively. Site-directed mutagenesis of the Q373H substitution for CsUGT78A14 indicated that the Q (Gln) residue played a catalytically crucial role for flavonoid 3-O-glucosyltransferase activity. The expression profiles of the CsUGT84A22, CsUGT78A14, and CsUGT78A15 genes were correlated with the accumulation patterns of ß-glucogallin and the glycosylated flavonols which indicated that these three CsUGT genes were involved in the biosynthesis of astringent compounds inC. sinensis.