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SUMMARY: 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. [ABSTRACT FROM AUTHOR]

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. Summary statement: Based on deep learning and targeted metabolomics, a model was established and (‐)‐epicatechin‐3‐O‐caffeoate (EC‐CA) was screened to have the function of defense against chewing pests, and hydroxycinnamoyl transferase (HCT) was identified as a substrate promiscuity that could efficiently catalyze the synthesis of EC‐CA. [ABSTRACT FROM AUTHOR]

SUMMARY: 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‐β‐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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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

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, β‐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. [ABSTRACT FROM AUTHOR]