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Flavan-3-ols are a group of flavonoids that exert beneficial effects. This study aimed to enhance key metabolic processes related to flavan-3-ols biosynthesis. The engineered Saccharomyces cerevisiae strain E32 that produces naringenin from glucose was further engineered for de novo production of two basic flavan-3-ols, afzelechin (AFZ) and catechin (CAT). Through introduction of flavonoid 3-hydroxylase, flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR), de novo production of AFZ and CAT can be achieved. The combination of FaDFR from Fragaria × ananassa and VvLAR from Vitis vinifera was optimal. (GGGGS)2 and (EAAAK)2 linkers between DFR and LAR proved optimal for the production of AFZ and CAT, respectively. Optimization of promoters and the enhanced supply of NADPH further increased the production. By combining the best engineering strategies, the optimum strains produced 500.5 mg/L AFZ and 321.3 mg/L CAT, respectively, after fermentation for 90 h in a 5 L bioreactor. The strategies presented could be applied for a more efficient production of flavan-3-ols by various microorganisms.

The mechanism regulating procyanidin (PA) accumulation in banana (Musa acuminata) fruit is not understood. During this study, the effects of PA treatment on the activities of banana PA biosynthetic enzymes and transcriptomic profiles were investigated. The results showed that PA treatment delayed the decreases in leucoanthocyanidin reductase and anthocyanidin reductase activities, which affected the accumulation of PA. Furthermore, the peel samples of the control fruit and the PA-treated fruit on day 1 were selected for transcriptomic analysis. The results revealed that PA treatment induced 1086 differentially expressed genes. Twenty-one key genes, including those encoding biosynthetic enzymes and regulatory factors involved in PA biosynthesis, were validated using a quantitative real-time polymerase chain reaction. The results showed that these genes were upregulated by PA treatment during banana storage. Taken together, our study improves current understanding of the mechanism underlying PA-regulated banana senescence and provide new clues for investigating specific gene functions.

Background Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. Results The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. Conclusion The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

Arbuscular mycorrhizal (AM) fungi may help protect plants against herbivores; however, their use for the pest control of woody plants requires further study. Here, we investigated the effect of Glomus mosseae colonization on the interactions between gypsy moth larvae and Populus alba × P. berolinensis seedlings and deciphered the regulatory mechanisms underlying the mycorrhizal-induced resistance in the leaves of mycorrhizal poplar using RNA-seq and nontargeted metabolomics. The resistance assay showed that AM fungus inoculation protected poplar seedlings against gypsy moth larvae, as evidenced by the decreased larval growth and reduced larval survival. A transcriptome analysis revealed that differentially expressed genes (DEGs) were involved in jasmonic acid biosynthesis (lipoxygenase, hydroperoxide dehydratase, and allene oxide cyclase) and signal transduction (jasmonate-ZIM domain and transcription factor MYC2) and identified the genes that were upregulated in mycorrhizal seedlings. Except for chalcone synthase and anthocyanidin synthase, which were downregulated in mycorrhizal seedlings, all DEGs related to flavonoid biosynthesis were upregulated, including 4-coumarate-CoA ligase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, and leucoanthocyanidin reductase. The metabolome analysis showed that several metabolites with insecticidal properties, including coumarin, stachydrine, artocarpin, norizalpinin, abietic acid, 6-formylumbelliferone, and vanillic acid, were significantly accumulated in the mycorrhizal seedlings. These findings suggest the potential of mycorrhiza-induced resistance for use in pest management of woody plants and demonstrate that the priming of JA-dependent responses in poplar seedlings contributes to mycorrhiza-induced resistance to insect pests.

Tea (Camellia sinensis) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid–phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-O-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-O-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (DAHPS; arogenate dehydratase/prephenatedehydratase) and the synthesis of AP (phenylacetaldehyde reductase; short-chain dehydrogenase/reductase), Dp (F3′H; F3′5′H), and DpG (BZ1), but inhibited the formation of flavones (flavonol synthase) and catechins (leucoanthocyanidin reductase). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.

Secondary metabolic pathways in grape berries are tightly regulated by an array of molecular mechanisms, including microRNA-mediated post-transcriptional regulation. As recently discovered, before being processed into mature microRNAs (miRNAs), the primary transcripts of miRNAs (pri-miRNAs) can encode for small miRNA-encoded peptides (micropeptides miPEPs) that ultimately lead to an accentuated downregulation of the respective miRNA-targeted genes. Although few studies about miPEPs are available, the discovery of miPEPs reveals a new layer of gene regulation at the post-transcriptional level that opens the possibility to regulate plant metabolism without resorting to gene manipulation. Here, we identified a miPEP encoded in non-mature miR164c putatively targeting grapevine transcription factor VvMYBPA1 (miPEP164c/miPEP-MYBPA1), a positive regulator of key genes in the proanthocyanidin (PA)-biosynthetic pathway, a pathway that competes directly for substrate with the anthocyanin-biosynthetic pathway. Thus, the objective of this work was to test the hypothesis that the exogenous application of miPEP164c (miPEP-MYBPA1) can modulate the secondary metabolism of grape berry cells by inhibiting PA biosynthetic pathway while simultaneously stimulating anthocyanin synthesis. The exogenous application of miPEP164c to suspension-cultured cells from grape berry (cv. Gamay) enhanced the transcription of its corresponding non-mature miR164c, with a maximum effect at 1 M and after a period of 10 days, thus leading to a more pronounced post-transcriptional silencing of its target VvMYBPA1. This led to a significant inhibition of the PA pathway, mostly via inhibition of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) enzymatic activities and VvLAR1 downregulation. In parallel, the anthocyanin-biosynthetic route was stimulated. Anthocyanin content was 31% higher in miPEP164c-treated cells, in agreement with the observed upregulation of VvUFGT1 transcripts and UFGT enzyme activity levels., This work was supported by Fundação para a Ciência e Tecnologia (FCT), under the strategic programs UID/AGR/04033/2020 and UID/BIA/04050/2020. This work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project “MitiVineDrought—Combining “omics” with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145- FEDER-030341, respectively; through the research project “BerryPlastid—Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid” with ref. POCI 01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project “GrapeInfectomics” (PTDC/ASPHOR/28485/2017). AC was supported with a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER 030341). FEDER/COMPETE also supported this work through the project “AgriFoodXXI” (ref. NORTE-01-0145-FEDER 000041). MV was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144637/2019). HB was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144638/2019). This work also benefited from the networking activities within the European COST Action CA17111 “INTEGRAPE—Data Integration to maximize the power of omics for grapevine improvement,” the CoLAB VINES & WINES, and the CoLAB 4FOOD – Collaborative Laboratory for Innovation in the Food Industry., info:eu-repo/semantics/publishedVersion

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.

Background The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. Results The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. Conclusion The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Proanthocyanidins (PAs) from plants are a nutritionally valuable component of the human diet and play important roles in defense against pests and diseases. PAs are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. The enzymes leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are involved in PA biosynthesis. The PA biosynthetic pathway has been characterized in several plant species, but the relationship between its expression and PA accumulation in Malus crabapple remains unclear. Here, we cloned the LAR genes MrLAR1, 2, and the ANR genes MrANR1, 2, from the red leaved Malus crabapple cultivar 'Royalty'. The contents of PAs and the expression levels of the LAR and ANR genes were investigated in different organs of the two crabapple cultivars. The transcript levels of two LAR genes and two ANR genes correlated with the contents of the catechin and epicatechin, which are proanthocyanidin precursors. Over-expression of the MrLAR1, 2 and MrANR1, 2 in tobacco (Nicotiana tabacum) promoted the accumulation of PAs, while transient silencing of their expression in crabapple resulted in reduced PA levels. In addition, a negative correlation between quercetin, anthocyanin, and PA biosynthesis was also found during crabapple leaf and fruit peel development. We also found that MrLAR1 and 2 may contribute to epicatechin biosynthesis. In summary, the LAR and ANR genes are critical factors in PA biosynthesis, and there is competition between the quercetin, anthocyanin, and PA biosynthetic pathways during leaf and fruit peel development in Malus crabapple.

An Agrobacterium-mediated transient transformation system was established for gene functional analysis in persimmon (Diospyros kaki Thunb.) utilizing sonication followed by vacuum infiltration of seedlings in vitro. The optimal vacuum conditions for agroinfiltration of persimmon were evaluated by monitoring expression of GUS reporter gene. The technique was further applied to evaluate five different genotypes. Among the genotypes examined in vitro, ‘Xiaoguo-tianshi’ (Chinese PCNA type) produced the highest expression levels of both GUS and GFP, and was the most ideal genotype. Furthermore, the developed system was successfully utilized for DkLAR (leucoanthocyanidin reductase) gene transient silencing. DMACA (4- dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay revealed that the proanthocyanidins (PAs) levels was markedly decrease in ‘Xiaoguo-tianshi’ and ‘Mopanshi’ (Chinese PCA type) leaves. These results confirmed that DkLAR plays an important role in the accumulation of PAs in persimmon. In conclusion, based on the Agrobacterium-mediated vacuum infiltration, we developed a reliable and highly efficient transient transformation system that can be used for gene functional analysis through the seedlings in vitro.

Chinese narcissus (Narcissus tazetta L. var. chinensis Roem.) is a popular flower in Asia. However, flower colors are limited with all cultivars having a white perianth and yellow corona. Previous studies have shown no anthocyanin accumulation in this species. The reason for an absence of anthocyanins remains unknown, with the flavonoid biosynthetic pathway in this flower recently being investigated. In this study, a transcriptomic approach combined with gene expression and biochemical analysis was used to predict and annotate genes in the flavonoid biosynthetic pathway. Results showed that the major flavonoid metabolites are flavonols and proanthocyanidins. Proanthocyanidin biosynthesis is exclusively catechin-based, with the gene encoding leucoanthocyanidin reductase (LAR) being well expressed. We identified 16 unigenes encoding key enzymes involved in flavonoid biosynthesis. Flavonol synthase (FLS) and dihydroflavonol reductase (DFR) appear to play important roles in regulating proanthocyanidin and flavonol levels, while anthocyanidin synthase (ANS) expression is not detected. Our results suggest that the absence of anthocyanins in Chinese narcissus is due to high expression of LAR and FLS, and a lack of ANS expression.

Cistus creticus L. subsp. creticus (rockrose) is a shrub widespread in Greece and the Mediterranean basin and has been used in traditional medicine as herb tea for colds, for healing and digestive hitches, for the treatment of maladies, as perfumes, and for other purposes. Compounds from its flavonoid fraction have recently drawn attention due to antiviral action against influenza virus and HIV. Although several bioactive metabolites belonging to this group have been chemically characterized in the leaves, the genes involved in their biosynthesis in Cistus remain largely unknown. Flavonoid metabolism during C. creticus fruit development was studied by adopting comparative metabolomic and transcriptomic approaches. The present study highlights the fruit of C. creticus subsp. creticus as a rich source of flavonols, flavan-3-ols, and proanthocyanidins, all of which displayed a decreasing trend during fruit development. The majority of proanthocyanidins recorded in Cistus fruit are B-type procyanidins and prodelphinidins, while gallocatechin and catechin are the dominant flavan-3-ols. The expression patterns of biosynthetic genes and transcription factors were analyzed in flowers and throughout three fruit development stages. Flavonoid biosynthetic genes were developmentally regulated, showing a decrease in transcript levels during fruit maturation. A high degree of positive correlations between the content of targeted metabolites and the expression of biosynthetic genes indicated the transcriptional regulation of flavonoid biosynthesis during C. creticus fruit development. This is further supported by the high degree of significant positive correlations between the expression of biosynthetic genes and transcription factors. The results suggest that leucoanthocyanidin reductase predominates the biosynthetic pathway in the control of flavan-3-ol formation, which results in catechin and gallocatechin as two of the major building blocks for Cistus proanthocyanidins. Additionally, there is a decline in ethylene production rates during non-climacteric Cistus fruit maturation, which coincides with the downregulation of the majority of flavonoid- and ethylene-related biosynthetic genes and corresponding transcription factors as well as with the decline in flavonoid content. Finally, functional characterization of a Cistus flavonoid hydroxylase (F3′5′H) was performed for the first time.

Rubus chingii, is widely distributed in many Asian countries and well known for its medicinal and dietary properties. Diversity of fruit color in raspberry has been attributed to the presence of either anthocyanins or carotenoids. In this study, we investigated anthocyanins and carotenoids, and their biosynthesis by LC-MS/MS. Six anthocyanins mainly consisted of flavanol-anthocyanins while five carotenoids mainly consisted of β-citraurin esters. Flavanol-anthocyanins were produced from an offshoot of the anthocyanin biosynthesis, which started with biosynthesis of flavanols and anthocyanidin by leucoanthocyanidin reductase (LAR)/anthocyanidin reductase (ANR) and anthocyanidin synthase (ANS/LDOX) respectively. β-citraurin esters were produced from cleavage of zeaxanthin and esterification by organic acid, which was an offshoot of the carotenoid biosynthesis. The offshoot started with biosynthesis of zeaxanthin and β-citraurin by carotene β-hydroxylase (CHYB/LUT5) and carotenoid cleavage dioxygenase (CCD) respectively. During fruit ripening, biosynthesis of flavanols and anthocyanins was down-regulated by genes/proteins involved in phenylpropanoid and flavonoid biosynthesis, while biosynthesis of β-citraurin esters was up-regulated by imbalanced expression of genes/proteins involved in β,β-ring and β, e-ring hydroxylation. Thus, β-citraurin esters, instead of anthocyanins imparted reddish color to the ripe fruit. These pigments and their biosynthesis in R. chingii are totally different from what occurs in other raspberry species.

Flesh browning after cutting or bruising reduces the commercial value of apple fruit. Here, we investigated the genetic factors that regulate flesh browning (at 15 min and 24 h after grating) by a genome-wide association study (GWAS) of 24 sib-families (468 genotypes). Three quantitative trait loci (QTLs) were detected on chr5, chr16, and chr17. Their effects were estimated in a germplasm collection of 86 cultivars and selection lines: flesh browning differed with the genotypes of SNPs linked to these QTLs, respectively explaining 31.3%, 3.3%, and 23.7% of phenotypic variance at 15 min, and 36.7%, 11.0%, and 14.8% of variance at 24 h. Homozygous recessive alleles at these QTLs were associated with low browning. ‘Aori 27’, a rare cultivar that never browns even 5 days after grating, was homozygous recessive at all three QTLs. Browning was clearly lower in cultivars that were homozygous recessive at two QTLs, rather than one or none. We analyzed browning-related traits—polyphenol oxidase (PPO) activity, total polyphenol content, and juice browning—by GWAS using the germplasm collection. A QTL for PPO activity was detected on chr5, where PPO genes are located; and QTLs for total polyphenol content and juice browning were detected on chr16, where leucoanthocyanidin reductase gene is located. No QTLs for the browning-related traits were detected on chr17. These results indicate that the juice browning is mainly regulated by polyphenol content (chr16 QTL), whereas the flesh browning is also regulated by additional factors, including PPO activity (chr5 QTLs). These findings will facilitate development of non-browning apple cultivars.

Summary Proanthocyanidins (PAs), also known as condensed tannins, are plant natural products that are beneficial for human and livestock health. As one of the largest grown crops in the world, soybean (Glycine max) is widely used as human food and animal feed. Many cultivated soybeans with yellow seed coats lack PAs or anthocyanins, although some soybean cultivars have coloured seed coats that contain these compounds. Here, we analyse the transcriptional control of PA and anthocyanin biosynthesis in soybean. Ectopic expression of the transcription factors (TFs) GmTT2A, GmTT2B, GmMYB5A or R in soybean hairy roots induced the accumulation of PAs (primarily in phloem tissues) or anthocyanins and led to up‐regulation of 1775, 856, 1411 and 1766 genes, respectively, several of which encode enzymes involved in PA biosynthesis. The genes regulated by GmTT2A and GmTT2B partially overlapped, suggesting conserved but potentially divergent roles for these two TFs in regulating PA accumulation in soybean. The two key enzymes anthocyanidin reductase and leucoanthocyanidin reductase were differentially upregulated, by GmTT2A/GmTT2B and GmMYB5A, respectively. Transgenic soybean plants overexpressing GmTT2B or MtLAP1 (a proven up‐regulator of the upstream reactions for production of precursors for PA biosynthesis in legumes) showed increased accumulation of PAs and anthocyanins, respectively, associated with transcriptional reprogramming paralleling the RNA‐seq data collected in soybean hairy roots. Collectively, our results show that engineered PA biosynthesis in soybean exhibits qualitative and spatial differences from the better‐studied model systems Arabidopsis thaliana and Medicago truncatula, and suggest targets for engineering PAs in soybean plants.

The present study aimed to evaluate the effect of an arbuscular mycorrhizal fungus (AMF), Clariodeoglomus etunicatum, on leaf food quality and relevant gene expression levels of tea (Camellia sinensis cv. ‘Fuding Dabaicha’) seedlings exposed to 0.5 μM P (P0.5) and 50 μM P (P50) levels. Twenty-four weeks later, the seedlings recorded higher root mycorrhizal fungal colonization in P50 than in P0.5. AMF-inoculated tea plants represented significantly higher leaf fructose and glucose contents and lower sucrose content than non-inoculated plants, irrespective of substate P levels. AMF treatment also increased total amino acids content in P0.5 and P50, accompanied with higher expression of glutamate dehydrogenase (CsGDH) and lower expression of glutamine synthetase (CsGS) and glutamine oxoglutarate aminotransferase (CsGOGAT). The total flavonoid content was higher in mycorrhizal versus non-mycorrhizal plants under P0.5 and P50, together with induced expression of phenylalanine ammonia-lyase (CsPAL) and cinnamic acid 4-hydroxylase (CsC4H). Mycorrhizal fungal inoculation improved catechins content, which is due to the up-regulated expression of flavanone 3-hydroxylase (CsF3H), flavonoid 3'-hydroxylase (CsF3'H), dihydroflavonol 4-reductase (CsDFR), leucoanthocyanidin reductase (CsLAR), anthocyanidin reductase (CsANR), and chalcone isomerase (CsCHI) under P0.5. However, under P50, the gene involved in catechins synthesis was not affected or down-regulated by mycorrhization, implying a complex mechanism (e.g. nutrient improvement). AMF also inhibited the tea caffeine synthase 1 (CsTCS1) expression regardless of P levels. Therefore, the results of this study concluded that inoculation with C. etunicatum improves leaf food quality of tea exposed to P stress, but the improved mechanisms were different between P0.5 and P50.

To investigate the effect of light intensity on flavonoid biosynthesis, grapevine calluses were subjected to high light (HL, 250 &mu, mol m&minus, 2 s&minus, 1) and dark (0 &mu, 1) in comparison to 125 &mu, 1 under controlled conditions (NL). The alteration of flavonoid profiles was determined and was integrated with RNA sequencing (RNA-seq)-based transcriptional changes of the flavonoid pathway genes. Results revealed that dark conditions inhibited flavonoid biosynthesis. Increasing light intensity affected flavonoids differently&mdash, the concentrations of flavonols and anthocyanins as well as the expressions of corresponding genes were less affected, whereas flavan-3-ol concentrations were predominantly increased, which caused enhanced trans-flavan-3-ol concentrations. Moreover, genes encoding leucoanthocyanidin reductase (LAR) exhibited different response patterns to light intensity changes&mdash, VviLAR1 expression increased with an increased light intensity, whereas VviLAR2 expression was insensitive. We further confirmed that the known transcription factors (TFs) involved in regulating flavan-3-ol biosynthesis utilized VviLAR1 as a target gene in grapevine calluses. In addition, VviLAR1 promoter activity was more sensitive to light intensity changes than that of VviLAR2 as determined using a transgenic Arabidopsis leaf system. These results suggested that light intensity had the most prominent effect on trans-flavan-3-ols in grapevine calluses and demonstrated that the two LAR genes had different response patterns to light intensity changes.

Vitis vinifera, one of the most cultivated fruit crops, is susceptible to several diseases particularly caused by fungus and oomycete pathogens. In contrast, other Vitis species (American, Asian) display different degrees of tolerance/resistance to these pathogens, being widely used in breeding programs to introgress resistance traits in elite V. vinifera cultivars. Secondary metabolites are important players in plant defence responses. Therefore, the characterization of the metabolic profiles associated with disease resistance and susceptibility traits in grapevine is a promising approach to identify trait-related biomarkers. In this work, the leaf metabolic composition of eleven Vitis genotypes was analysed using an untargeted metabolomics approach. A total of 190 putative metabolites were found to discriminate resistant/partial resistant from susceptible genotypes. The biological relevance of discriminative compounds was assessed by pathway analysis. Several compounds were selected as promising biomarkers and the expression of genes coding for enzymes associated with their metabolic pathways was analysed. Reference genes for these grapevine genotypes were established for normalisation of candidate gene expression. The leucoanthocyanidin reductase 2 gene (LAR2) presented a significant increase of expression in susceptible genotypes, in accordance with catechin accumulation in this analysis group. Up to our knowledge this is the first time that metabolic constitutive biomarkers are proposed, opening new insights into plant selection on breeding programs.

To investigate the effect of light intensity on flavonoid biosynthesis, grapevine calluses were subjected to high light (HL, 250 μmol m−2 s−1) and dark (0 μmol m−2 s−1) in comparison to 125 μmol m−2 s−1 under controlled conditions (NL). The alteration of flavonoid profiles was determined and was integrated with RNA sequencing (RNA-seq)-based transcriptional changes of the flavonoid pathway genes. Results revealed that dark conditions inhibited flavonoid biosynthesis. Increasing light intensity affected flavonoids differently—the concentrations of flavonols and anthocyanins as well as the expressions of corresponding genes were less affected, whereas flavan-3-ol concentrations were predominantly increased, which caused enhanced trans-flavan-3-ol concentrations. Moreover, genes encoding leucoanthocyanidin reductase (LAR) exhibited different response patterns to light intensity changes—VviLAR1 expression increased with an increased light intensity, whereas VviLAR2 expression was insensitive. We further confirmed that the known transcription factors (TFs) involved in regulating flavan-3-ol biosynthesis utilized VviLAR1 as a target gene in grapevine calluses. In addition, VviLAR1 promoter activity was more sensitive to light intensity changes than that of VviLAR2 as determined using a transgenic Arabidopsis leaf system. These results suggested that light intensity had the most prominent effect on trans-flavan-3-ols in grapevine calluses and demonstrated that the two LAR genes had different response patterns to light intensity changes.

&lsquo, Ziyan&rsquo, is a novel anthocyanin-rich tea cultivar with dark purple young shoots. However, how its anthocyanin accumulation is affected by environmental factors, such as ultraviolet (UV), remains unclear. In this study, we observed that UV light treatments stimulated anthocyanin accumulation in &lsquo, leaves, and we further analyzed the underlying mechanisms at gene expression and enzyme activity levels. In addition, the catechins and chlorophyll contents of young shoots under different light treatments were also changed. The results showed that the contents of total anthocyanins and three major anthocyanin molecules, i.e., delphinidin, cyanidin, and pelargonidin, were significantly higher in leaves under UV-A, UV-B, and UV-AB treatments than those under white light treatment alone. However, the total catechins and chlorophyll contents in these purple tea plant leaves displayed the opposite trends. The anthocyanin content was the highest under UV-A treatment, which was higher by about 66% than control. Compared with the white light treatment alone, the enzyme activities of chalcone synthase (CHS), flavonoid 3&prime, 5&prime, hydroxylase (F3&prime, H), and anthocyanidin synthase (ANS) under UV treatments increased significantly, whereas the leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) activities reduced. There was no significant difference in dihydroflavonol 4-reductase (DFR) activity under all treatments. Comparative transcriptome analyses unveiled that there were 565 differentially expressed genes (DEGs) of 29,648 genes in three pair-wise comparisons (white light versus UV-A, W vs. UV-A, white light versus UV-B, W vs. UV-A, white light versus UV-AB, W vs. UV-AB). The structural genes in anthocyanin pathway such as flavanone 3-hydroxylase (F3H), F3&prime, H, DFR, and ANS, and regulatory gene TT8 were upregulated under UV-A treatment, F3&prime, H, DFR, ANS, and UFGT and regulatory genes EGL1 and TT2 were upregulated under UV-AB treatment. However, most structural genes involved in phenylpropanoid and flavonoid pathways were downregulated under UV-B treatment compared with control. The expression of LAR and ANR were repressed in all UV treatments. Our results indicated that UV-A and UV-B radiations can induce anthocyanin accumulation in tea plant &lsquo, by upregulating the structural and regulatory genes involved in anthocyanin biosynthesis. In addition, UV radiation repressed the expression levels of LAR, ANR, and FLS, resulting in reduced ANR activity and a metabolic flux shift toward anthocyanin biosynthesis.

Proanthocyanidins (PAs) are major defense-related phenolics in mulberry, but the mechanism underlying their biosynthesis remains uncharacterized. In this study, the relationship between the expression of genes encoding anthocyanidin reductase (ANR) or leucoanthocyanidin reductase (LAR) and PA biosynthesis was investigated in white and red mulberry fruits. In ripening fruits, the MnANR and MnLAR transcription levels tended to decrease, whereas the catechin and epicatechin contents initially increased and then decreased. In contrast, the PA content exhibited a clearly different trend. The ectopic expression of MnANR and MnLAR in tobacco increased the resistance to Botrytis cinerea, as evidenced by the less extensive disease symptoms of the transgenic plants compared with the wild-type plants. In vitro experiments revealed that the transgenic tobacco crude leaf extract had an obvious inhibitory effect on B. cinerea. Moreover, the ectopic expression of MnANR and MnLAR in tobacco inhibited the expression of anthocyanin biosynthesis genes, resulting in decreased anthocyanin contents in flowers. The results of this study may be useful for elucidating the mechanism underlying PA biosynthesis. Furthermore, ANR and LAR represent potential targets for improving the resistance of mulberry and related plant species to B. cinerea.

Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.

MicroRNAs (miRNAs) are endogenous non-protein-coding small RNAs that play crucial and versatile regulatory roles in plants. Using a computational identification method, we identified 55 conserved miRNAs in tea (Camellia sinensis) by aligning miRNA sequences of different plant species with the transcriptome library of tea strain 1005. We then used quantitative real-time PCR (qRT-PCR) to analyze the expression of 31 identified miRNAs in tea leaves of different ages, thereby verifying the existence of these miRNAs and confirming the reliability of the computational identification method. We predicted which miRNAs were involved in catechin synthesis using psRNAtarget Software based on conserved miRNAs and catechin synthesis pathway-related genes. Then, we used RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) to obtain seven miRNAs cleaving eight catechin synthesis pathway-related genes including chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and flavanone 3-hydroxylase (F3H). An expression analysis of miRNAs and target genes revealed that miR529d and miR156g-3p were negatively correlated with their targets CHI and F3H, respectively. The expression of other miRNAs was not significantly related to their target genes in the catechin synthesis pathway. The RLM-RACE results suggest that catechin synthesis is regulated by miRNAs that can cleave genes involved in catechin synthesis.

To fully elucidate molecular mechanisms of flavonoid biosynthesis in Chinese bayberry, transcriptomes of two genotypes with different colours, white cultivar (Shuijing, SJ) and red cultivar (Biqi, BQ), were compared. During fruit development, ‘BQ’ exhibited substantial increase of total anthocyanins content together with dramatic decrease of total soluble proanthocyanidins (PAs) content, while ‘SJ’ displayed decrease of total soluble PAs levels but failed to accumulate anthocyanin. Based on the sequencing results, 124,265 unigenes were generated with an average length of 708 bp. All genes involved in anthocyanin biosynthesis and glycosylation were identified and their expression patterns were in accordance with total anthocyanin accumulation in developing fruits of ‘BQ’ and ‘SJ’. Expression levels of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) were down-regulated in ‘SJ’ in agreement with the decrease in PAs, while an opposite trend was observed in ‘BQ’. The higher expression levels of LAR and ANR in ‘BQ’ may be an important reason for the higher levels of total soluble PAs as compared to ‘SJ’. Phylogenetic analysis showed that seven MYBs could be identified as putative homologues of PA-specific regulator and exhibited considerable genotypic and temporal specificity of expression. In addition, three WD40 genes in Chinese bayberry clustered with the WD40s related to flavonoid biosynthesis in other species. Finally, RNA-Seq data was validated by using qRT-PCR analysis with a high correlation, suggesting that the RNA-Seq data here are credible. These results provide new insights into the regulation of the complex branching pathway leading to various flavonoid compounds biosynthesis in bayberries.

Salicylic acid (SA) induces flavanol biosynthesis in grapes; however, the underlying molecular mechanisms remain largely unclear. Here, we showed that exogenous SA increases the content of flavan-3-ol, one of the main phenolics in grape leaves, by improving the activities of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) through induction of the expression of the VvANR, VvLAR1, VvLAR2 and VvMYBPA2 genes in grape leaves. Suppression of VvANR expression in TRV-VvANR-infected grape leaves using virus-induced gene silencing (VIGS) resulted in reduced expression of the VvANR, VvLAR1, VvMYBPA1 and VvMYBPA2 genes and a subsequent reduction in the enzyme activities of ANR and LAR and the contents of (-)-epicatechin (EC) and (-)-epigallocatechin (EGC). Supplementation with exogenous SA increased the contents of flavan-3-ol monomers, i.e., (+)-catechin, (-)-epicatechin and (-)-epigallocatechin, and improved ANR activity by upregulating the expression of VvLAR1, VvLAR2, VvMYBPA1 and VvMYBPA2 in TRV-VvANR-infected grape leaves. These results collectively suggested that the application of SA may represent a useful strategy for improving flavan-3-ol biosynthesis in grapes and that ANR plays a vital role in this process.