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Abstract Fruit color substantially affects consumer preferences, with darker red strawberries being economically more valuable due to their higher anthocyanin content. However, the molecular basis for the dark red coloration remains unclear. Through screening of an ethyl methanesulfonate mutant library, we identified a rg418 mutant, that demonstrated anthocyanin accumulation during early fruit development stages. Furthermore, the ripening fruits of this mutant had higher anthocyanin content than wild-type (WT) fruits. An analysis of flavonoid content in WT and rg418 mutant fruits revealed substantial changes in metabolic fluxes, with the mutant exhibiting increased levels of anthocyanins and flavonols and decreased levels of proanthocyanidins. Bulked sergeant analysis sequencing indicated that the mutant gene was anthocyanidin reductase (ANR), a key gene in the proanthocyanidin synthesis pathway. Furthermore, transcriptome sequencing revealed the increased expression of MYB105 during the early development stage of mutant fruits, which promoted the expression of UFGT (UDP-glucose flavonoid 3-O-glucosyltransferase), a key gene involved in anthocyanin synthesis, thus substantially enhancing the anthocyanin content in the mutant fruits. Additionally, mutating ANR in a white-fruited strawberry variant (myb10 mutant) resulted in appealing pink-colored fruits, suggesting the diverse roles of ANR in fruit color regulation. Our study provides valuable theoretical insights for improving strawberry fruit color.

Anthocyanidin reductase is one of the key enzyme involved in the synthesis of flavonoids. In order to explore the structure of ANR gene, ANR enzyme expression pattern and flavonoid content under drought stress and their correlation, a HrANR gene identified from RNA-seq data of sea buckthorn was screened and analyzed by bioinformatics soft, the expression patterns of HrANR gene in different tissues and of flavonoid contents in leaves were performed. The results were as follows: (1) The ORF of HrANR gene was 1 017 bp, which encoded 338 amino acids. It was a stable hydrophilic protein, and the homologous protein had significant family and genus characteristics. (2) HrANR gene was expressed in roots, stems and leaves of sea buckthorn under drought stress, but the expression trends were different, with an increase followed by a decrease and then an increase in roots, a continuous decrease in stems, and an increase followed by a continuous decrease in leaves. (3) The flavonoid contents in leaves of sea buckthorn under different levels of stress showed a trend that first increased continuously and then decreased slightly, increased to the highest point after rehydration. The above results indicated that the expression of HrANR gene and the changes of flavonoid content were closely related to the drought resistance of sea buckthorn. The flavonoid content in leaves was positively correlated with drought stress at the beginning of drought stress and negatively correlated with drought stress under severe stress. (4) Leaf expression, stem expression and flavonoid content of HrANR gene were negatively correlated (Pleaf= -0.751, Pstem= -0.934) and root expression was positively correlated with flavonoid content (Proot= 0.444). The above results indicate that the expression of HrANR genes and changes of flavonoid content in sea buckthorn are closely related to drought resistance, and it provides a reference for elucidating the drought resistance mechanism of sea buckthorn.

Proanthocyanidins (PAs), one of the most abundant natural polymers found in plants, are gaining increasing attention because of their beneficial effects for agriculture and human health. The study of PA biosynthesis has been active for decades, and progress has been drastically accelerated since the discovery of key enzymes such as Anthocyanidin Reductase (ANR), Leucoanthocyanidin Reductase (LAR), and key transcription factors such as Transparent Testa 2 (TT2) and Transparent Testa 8 (TT8) in the early 2000s. Scientists raised some compelling questions regarding PA biosynthesis about two decades ago in the hope that addressing these questions would lead to an enhanced understanding of PA biosynthesis in plants. These questions focus on the nature of starter and extension units for PA biosynthesis, the stereochemistry of PA monomers and intermediates, and how and where the polymerization or condensation steps work subcellularly. Here, I revisit these long-standing questions and provide an update on progress made toward answering them. Because of advanced technologies in genomics, bioinformatics and metabolomics, we now have a much-improved understanding of functionalities of key enzymes and identities of key intermediates in the PA biosynthesis and polymerization pathway. Still, several questions, particularly the ones related to intracellular PA transportation and deposition, as well as enzyme subcellular localization, largely remain to be explored. Our increasing understanding of PA biosynthesis in various plant species has led to a new set of compelling open questions, suggesting future research directions to gain a more comprehensive understanding of PA biosynthesis.

Seed coat color is an important consumer-related trait of the cowpea (Vigna unguiculata [L.] Walp.) and has been a subject of study for over a century. Utilizing newly available resources, including mapping populations, a high-density genotyping platform, and several genome assemblies, the red seed coat color has been mapped to two loci, Red-1 (R-1) and Red-2 (R-2), on Vu03 and Vu07, respectively. A gene model (Vigun03g118700) encoding a dihydroflavonol 4-reductase, a homolog of anthocyanidin reductase 1, which catalyzes the biosynthesis of epicatechin from cyanidin, has been identified as a candidate gene for R-1. Possible causative variants have also been identified for Vigun03g118700. A gene model on Vu07 (Vigun07g118500), with predicted nucleolar function and high relative expression in the developing seed, has been identified as a candidate for R-2. The observed red color is believed to be the result of a buildup of cyanidins in the seed coat.

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC50 = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC50 = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

Persimmon (Diospyros kaki Thunb.) is an important fruit crop in East Asia and has great commercial potential due to the high demand for fresh edible fruits or processed dried persimmon, as well as its being a source of raw materials for the chemical industry. Genetic engineering has shown great potential in persimmon fruit improvement and compensated for the limitations of conventional breeding. In this study, we developed an efficient transformation system and in vivo regeneration protocol by using the leaf disc culture of ‘Gongcheng Shuishi’ persimmon through the optimization of different basic media and hormone combinations. Based on the secondary induction system on 1/2DKW (DKW medium) + ZT (2.0 mg/L) + TDZ (0.5 mg/L) and MS (1/2 N) + ZT (2.0 mg/L) + IAA (0.1 mg/L), the callus induction rate and adventitious bud induction rate were 96.0% and 94.0%, respectively, and RNA interference of DkANR (ANR, anthocyanidin reductase) was achieved after transformation, thus resulting in a reduction in proanthocyanidin accumulation. The GFP (green fluorescent protein) fluorescence signal was visualized in the transgenic seedlings with GFP gene cotransformation. A total of 17.9% of the positive regenerated transgenic seedlings with empty vector were confirmed by both hyg gene and 35S promoter PCR analysis. Southern blotting assays showed that 20 positive transgenic persimmon seedlings were obtained from 135 regenerated transformants derived from DkANRi. Altogether, a reliable and highly efficient transformation system was achieved in persimmon, which may contribute to gene function identification and genetic improvement in the future.

Anthocyanins are important dietary and health-promoting substances present in high quantities in the peel and arils of the pomegranate (Punica granatum L.) fruit. Yet, there is a high variation in the content of anthocyanin among different pomegranate varieties. The ‘Black’ pomegranate variety (P.G.127-28) found in Israel contains exceptionally high levels of anthocyanins in its fruit peel which can reach up to two orders of magnitude higher content as compared to that of other pomegranate varieties’ peel anthocyanins. Biochemical analysis reveals that delphinidin is highly abundant in the peel of ‘Black’ variety. The pattern of anthocyanin accumulation in the fruit peel during fruit development of ‘Black’ variety differs from that of other pomegranates. High anthocyanin levels are maintained during all developmental stages. Moreover, the accumulation of anthocyanin in the fruit peel of ‘Black’ variety is not dependent on light. Genetic analysis of an F2 population segregating for the “black” phenotype reveals that it is determined by a single recessive gene. Genetic mapping of the F2 population using single nucleotide polymorphism (SNP) markers identified few markers tightly linked to the “black” phenotype. Recombination analysis of the F2 population and F3 populations narrowed the “black” trait to an area of 178.5 kb on the draft genome sequence of pomegranate cv. ‘Dabenzi.’ A putative anthocyanidin reductase (ANR) gene is located in this area. Only pomegranate varieties displaying the “black” trait carry a base pair deletion toward the end of the gene, causing a frame shift resulting in a shorter protein. We propose that this mutation in the ANR gene is responsible for the different anthocyanin composition and high anthocyanin levels of the “black” trait in pomegranate.

Abstract Background Edible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown. Results Seed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products. Conclusion Proanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.

Proanthocyanidins (PAs) are distributed widely in Chinese bayberry fruit and have been associated with human health benefits, but molecular and biochemical characterization of PA biosynthesis remains unclear. Here, two genes encoding key PA biosynthetic enzymes, anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) were isolated in bayberry fruit. MrANR was highly expressed at the early stage of fruit development when soluble PAs accumulated at high levels. Meanwhile, the transcript abundance of both MrANR and MrLAR observed at the late stage was paralleled with the high amounts of insoluble PAs. LC-MS/MS showed that PAs in developing Chinese bayberry fruits were comprised predominantly of epigallocatechin-3-O-gallate terminal subunits, while the extension subunits were a mixture of epigallocatechin-3-O-gallate, epigallocatechin and catechin. Recombinant MrANR protein converted cyanidin to a mixture of epicatechin and catechin, and delphinidin to a mixture of epigallocatechin and gallocatechin in vitro. Recombinant MrLAR was active with leucocyanidin as substrate to produce catechin. Ectopic expression of MrANR in tobacco reduced anthocyanin levels but increased PA accumulation. The catechin and epicatechin contents in transgenic flowers overexpressed MrANR were significantly higher than those of wild-type. However, overexpression of MrLAR in tobacco led to an increase in catechin levels but had no impact on PA contents. Quantitative real time PCR revealed that the loss of anthocyanin in transgenic flowers overexpressed MrANR or MrLAR is probably attributed to decreased expression of tobacco chalcone isomerase (CHI) gene. Our results not only reveal in vivo and in vitro functions for ANR and LAR but also provide a resource for understanding the mechanism of PA biosynthesis in Chinese bayberry fruit.

Catechin epimerization is an important factor affecting tea catechin compositions and thereby tea quality. However, a lack of tea germplasms with high non-epicatechins limits relative research. Here, a tea cultivar Y510 with high non-epicatechins was firstly reported and used for catechin and RNA sequencing (RNA-Seq) analysis. Results showed that the (-)-gallocatechin gallate and (+)-catechin (C) contents in Y510 were at least 136 and 6 times higher than those in Fudingdabaicha and 0306I, but the epicatechins (-)-epigallocatechin and (-)-epicatechin (EC) were significantly lower. Eleven unigenes potentially involved in catechin epimerization were identified by RNA-Seq analysis. Based on a combination of catechin and gene expression analysis, it was hypothesized that two anthocyanidin reductase genes (CsANR1, CsANR2) and an anthocyanidin synthase gene (CsANS) are the key genes affecting catechin epimerization in tea. Non-epicatechin formations were hypothesized to be mainly influenced by the expression ratio of CsANR2 to CsANR1 and the expression of CsANS. Overexpression of CsANS in an Arabidopsis mutant tds4-2 led to a significant increase of EC accumulation in seeds, revealing CsANS is important for catechin epimerization. These results shed new light on breeding tea cultivars with special catechin compositions.

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (−)-epicatechin, (+)-gallocatechin and (−)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.

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.

Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavour and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.