Proanthocyanidin Biosynthesis Pathway Research Articles

An unconventional proanthocyanidin pathway in maize

Proanthocyanidins (PAs), flavonoid polymers involved in plant defense, are also beneficial to human health and ruminant nutrition. To date, there is little evidence for accumulation of PAs in maize (Zea mays), although maize makes anthocyanins and possesses the key enzyme of the PA pathway, anthocyanidin reductase (ANR). Here, we explore whether there is a functional PA biosynthesis pathway in maize using a combination of analytical chemistry and genetic approaches. The endogenous PA biosynthetic machinery in maize preferentially produces the unusual PA precursor (+)-epicatechin, as well as 4β-(S-cysteinyl)-catechin, as potential PA starter and extension units. Uncommon procyanidin dimers with (+)-epicatechin as starter unit are also found. Expression of soybean (Glycine max) anthocyanidin reductase 1 (ANR1) in maize seeds increases the levels of 4β-(S-cysteinyl)-epicatechin and procyanidin dimers mainly using (-)-epicatechin as starter units. Introducing a Sorghum bicolor transcription factor (SbTT2) specifically regulating PA biosynthesis into a maize inbred deficient in anthocyanin biosynthesis activates both anthocyanin and PA biosynthesis pathways, suggesting conservation of the PA regulatory machinery across species. Our data support the divergence of PA biosynthesis across plant species and offer perspectives for future agricultrural applications in maize.

Transcriptional regulation of proanthocyanidin biosynthesis pathway genes and transcription factors in Indigofera stachyodes Lindl. roots

BackgroundProanthocyanidins (PAs) have always been considered as important medicinal value component. In order to gain insights into the PA biosynthesis regulatory network in I. stachyodes roots, we analyzed the transcriptome of the I. stachyodes in Leaf, Stem, RootI (one-year-old root), and RootII (two-year-old root).ResultsIn this study, a total of 110,779 non-redundant unigenes were obtained, of which 63,863 could be functionally annotated. Simultaneously, 75 structural genes that regulate PA biosynthesis were identified, of these 6 structural genes (IsF3′H1, IsANR2, IsLAR2, IsUGT72L1-3, IsMATE2, IsMATE3) may play an important role in the synthesis of PAs in I. stachyodes roots. Furthermore, co-expression network analysis revealed that 34 IsMYBs, 18 IsbHLHs, 15 IsWRKYs, 9 IsMADSs, and 3 IsWIPs hub TFs are potential regulators for PA accumulation. Among them, IsMYB24 and IsMYB79 may be closely involved in the PA biosynthesis in I. stachyodes roots.ConclusionsThe biosynthesis of PAs in I. stachyodes roots is mainly produced by the subsequent pathway of cyanidin. Our work provides new insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout different tissues.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed, and anthocyanin and proanthocyanidins (PAs) are major seed-coat pigments. Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed. In this study, we identified a transcription factor gene BnbHLH92a (BnaA06T0441000ZS) in rapeseed. Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs. Correspondingly, the expression profiles of anthocyanin and PA biosynthesis genes (TT3, BAN, TT8, TT18, and TTG1) were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds, indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis. BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed. BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression. These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B. napus.

Transcriptome Co-expression Network and Metabolome Analysis Identifies Key Genes and Regulators of Proanthocyanidins Biosynthesis in Brown Cotton.

Brown cotton fiber (BCF) is a unique raw material of naturally colored cotton (NCC). But characteristics of the regulatory gene network and metabolic components related to the proanthocyanidins biosynthesis pathway at various stages of its fiber development remain unclear. Here, the dynamic changes in proanthocyanidins biosynthesis components and transcripts in the BCF variety “Zong 1-61” and its white near-isogenic lines (NILs) “RT” were characterized at five fiber developmental stages (0, 5, 10, 15, and 20 days post-anthesis; DPA). Enrichment analysis of differentially expressed genes (DEGs), comparison of metabolome differences, and pathway enrichment analysis of a weighted gene correlation network analysis together revealed the dominant gene expression of flavonoid biosynthesis (FB), phenylpropanoid metabolisms, and some carbohydrate metabolisms at 15 or 20 DPA than white cotton. Eventually, 63 genes were identified from five modules putatively related to FB. Three R2R3-MYB and two bHLH transcription factors were predicted as the core genes. Further, GhANS, GhANR1, and GhUFGT2 were preliminarily regulated by GhMYB46, GhMYB6, and GhMYB3, respectively, according to yeast one-hybrid assays in vitro. Our findings provide an important transcriptional regulatory network of proanthocyanidins biosynthesis pathway and dynamic flavonoid metabolism profiles.

Understanding the chemical foundation and genetic mechanism of the black grain trait in quinoa by integrating metabolome and transcriptome analyses

Quinoa (Chenopodium quinoa Willd.) cultivars that produce black grains are thought to be more nutritious and contain powerful antioxidant properties, but little is known about the chemical foundation and the genetic mechanism behind this trait. In this research, widely-targeted metabolome and transcriptome analyses were employed to unveil the chemical and genetic differences between black and white quinoa cultivars. A total of 157 differentially expressed metabolites were found in the black and white quinoa cultivars. The tannins and flavonoids were present at high levels in the black quinoa grains. The tannins comprised 13 proanthocyanins, all of which could barely be detected in white grains. Proanthocyanidins are suggested to cause the black grain trait in Arabidopsis thaliana and Brassica species and are thought to produce the black grain trait, while contributing to antioxidant activity, in black grain quinoa. A total of 4620 differentially expressed unigenes were identified in the black and white quinoa grains. Structural genes associated with proanthocyanin biosynthesis, particularly DFR, LAR, LDOX and ANR, were more highly expressed in black than white grains. Genes involved in the betalain biosynthetic pathway, which competes with anthocyanin biosynthesis, showed relatively lower levels of expression in black grains. Two MYB and one MYC transcription factor related to the phenylpropane metabolic pathway displayed higher expression levels in black grains, while BvMYB1, associated with betalain biosynthesis, was not detected. This study suggested that the accumulation of proanthocyanins produces the black grain trait in quinoa, and the activation of transcription factors triggers the proanthocyanidin biosynthesis pathway.

Proanthocyanidin accumulation and transcriptional responses in the seed coat of cranberry beans (Phaseolus vulgaris L.) with different susceptibility to postharvest darkening

BackgroundEdible 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.ResultsSeed 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.ConclusionProanthocyanidin 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.

Characterization of two TT2-type MYB transcription factors regulating proanthocyanidin biosynthesis in tetraploid cotton, Gossypium hirsutum.

Two TT2-type MYB transcription factors identified from tetraploid cotton are involved in regulating proanthocyanidin biosynthesis, providing new strategies for engineering condensed tannins in crops. Proanthocyanidins (PAs), also known as condensed tannins, are important secondary metabolites involved in stress resistance in plants, and are health supplements that help to reduce cholesterol levels. As one of the most widely grown crops in the world, cotton provides the majority of natural fabrics and is a supplemental food for ruminant animals. The previous studies have suggested that PAs present in cotton are a major contributor to fiber color. However, the biosynthesis of PAs in cotton still remains to be elucidated. AtTT2 (transparent testa 2) is a MYB family transcription factor from Arabidopsis that initiates the biosynthesis of PAs by inducing the expression of multiple genes in the pathway. In this study, we isolated two R2R3-type MYB transcription factors from Gossypium hirsutum that are homologous to AtTT2. Expression analysis showed that both genes were expressed at different levels in various cotton tissues, including leaf, seed coat, and fiber. Protoplast transactivation assays revealed that these two GhMYBs were able to activate promoters of genes encoding enzymes in the PA biosynthesis pathway, namely anthocyanidin reductase and leucoanthocyanidin reductase. Complementation experiments showed that both of the GhMYBs were able to recover the transparent testa seed coat phenotype of the Arabidopsis tt2 mutant by restoring PA biosynthesis. Ectopic expression of either of the two GhMYBs in Medicago truncatula hairy roots increased the contents of anthocyanins and PAs compared to control lines expressing the GUS gene, and expression levels of MtDFR, MtLAR, and MtANR were also elevated in lines expressing GhMYBs. Together, these data provide new insights into engineering condensed tannins in cotton.

Functional Characterization of a Dihydroflavanol 4-Reductase from the Fiber of Upland Cotton (Gossypium hirsutum).

Dihydroflavanol 4-reductase (DFR) is a key later enzyme involved in two polyphenols’ (anthocyanins and proanthocyanidins (PAs)) biosynthesis, however it is not characterized in cotton yet. In present reports, a DFR cDNA homolog (designated as GhDFR1) was cloned from developing fibers of upland cotton. Silencing GhDFR1 in cotton by virus-induced gene silencing led to significant decrease in accumulation of anthocyanins and PAs. More interestingly, based on LC-MS analysis, two PA monomers, (–)-epicatachin and (–)-epigallocatachin, remarkably decreased in content in fibers of GhDFR1-silenced plants, but two new monomers, (–)-catachin and (–)-gallocatachin were present compared to the control plants infected with empty vector. The ectopic expression of GhDFR1 in an Arabidopsis TT3 mutant allowed for reconstruction of PAs biosynthesis pathway and led to accumulation of PAs in seed coat. Taken together, these data demonstrate that GhDFR1 contributes to the biosynthesis of anthocyanins and PAs in cotton.

Molecular analysis of proanthocyanidins related to pigmentation in brown cotton fibre (Gossypium hirsutum L.).

The structural characteristics and component differences of proanthocyanidins in brown and white cotton fibres were identified by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. Proanthocyanidins in brown and white cotton fibres were found to contain mainly procyanidin (PC) and prodelphidin (PD) units with 2, 3-cis form (epigallocatechin and epicatechin). However, part of the proanthocyanidins in the white cotton fibres were modified by acylation and were constitutively different from the proanthocyanidins in brown cotton fibres. The relative amount of PD was similar to that of PC in white cotton fibres, while proanthocyanidins in brown cotton fibres consisted mainly of PD units with a relative ratio of 9:1. In brown cotton fibres, the proanthocyanidin monomeric composition was consistent with the expression profiles of proanthocyanidin synthase genes, suggesting that anthocyanidin reductase represented the major flow of the proanthocyanidin biosynthesis pathway. In addition, the structural characteristics and component differences of proanthocanidins in brown and white cotton fibres suggested that quinones, the oxidation products of proanthocyanidins, were the direct contributors to colour development in brown cotton fibre. This was demonstrated by vanillin-HCl staining and Borntrager's test. Collectively, these data demonstrated that the biosynthesis of proanthocyanidins is a crucial pigmentation process in brown cotton fibre, and that quinones may represent the main pigments contributing to formation of the the brown colour. This study revealed the molecular basis of pigmentation in brown cotton fibres, and provided important insights for genetic manipulation of pigment production in cotton fibres.

Transcriptome and Biochemical Analyses Revealed a Detailed Proanthocyanidin Biosynthesis Pathway in Brown Cotton Fiber

Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs). To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols) were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form (gallocatechin and catechin), and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates (dihydroflavonol 4-reductase and anthocyanidin synthase, respectively). Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber.

Expression Analysis of the ANR and LAR Gene in Fragaria × ananassa cv. Toyonaka

Strawberry is an economically valuable crop all over the word. Proanthocyanidin is one polyphenol compound rich in strawberry fruits owned significant antioxidant capacity and claimed beneficial health effects. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are both key enzymes of the branch of proanthocyanidins biosynthesis pathway, which are responsible for the production of (-)-epicatechin and (+)-catechin, respectively. In this study, the expression levels of the Fa-ANR and Fa-LAR gene and the total PAs concentration at seven developmental stages of the strawberry fruit were investigated. The results showed that the PAs contents gradually reduced along with the fruit maturation; while the expression patterns of Fa-ANR and Fa-LAR were consistent with the PAs. These results indicated that LAR and ANR were both key enzymes in proanthocyanidin biosynthesis.

Chemical and transcriptional responses of Norway spruce genotypes with different susceptibility to Heterobasidion spp. infection

BackgroundNorway spruce [Picea abies (L.) Karst.] is one of the most important conifer species in Europe. The wood is economically important and infections by wood-rotting fungi cause substantial losses to the industry.The first line of defence in a Norway spruce tree is the bark. It is a very efficient barrier against infection based on its mechanical and chemical properties. Once an injury or an infection is recognized by the tree, induced defences are activated. In this study we examined transcriptional response, using 454-sequencing, and chemical profiles in bark of Norway spruce trees with different susceptibility to Heterobasidion annosum s.l. infection. The aim was to find associations between the transcriptome and chemical profiles to the level of susceptibility to Heterobasidion spp. in Norway spruce genotypes.ResultsBoth terpene and phenol compositions were analysed and at 28 days post inoculation (dpi) high levels of 3-carene was produced in response to H. annosum. However, significant patterns relating to inoculation or to genotypes with higher or lower susceptibility could only be found in the phenol fraction. The levels of the flavonoid catechin, which is polymerized into proanthocyanidins (PA), showed a temporal variation; it accumulated between 5 and 15 dpi in response to H. annosum infection in the less susceptible genotypes. The transcriptome data suggested that the accumulation of free catechin was preceded by an induction of genes in the flavonoid and PA biosynthesis pathway such as leucoanthocyanidin reductase. Quantitative PCR analyses verified the induction of genes in the phenylpropanoid and flavonoid pathway. The qPCR data also highlighted genotype-dependent differences in the transcriptional regulation of these pathways.ConclusionsThe varying dynamics in transcriptional and chemical patterns displayed by the less susceptible genotypes suggest that there is a genotypic variation in successful spruce defence strategies against Heterobasidion. However, both high levels of piceasides and flavonoids in the less susceptible genotypes suggested the importance of the phenolic compounds in the defence. Clearly an extended comparison of the transcriptional responses in the interaction with Heterobasidion between several independent genotypes exhibiting reduced susceptibility is needed to catalogue mechanisms of successful host defence strategies.

Involvement of Abscisic Acid in Controlling the Proanthocyanidin Biosynthesis Pathway in Grape Skin: New Elements Regarding the Regulation of Tannin Composition and Leucoanthocyanidin Reductase (LAR) and Anthocyanidin Reductase (ANR) Activities and Expression

Proanthocyanidins, or condensed tannins, are crucial polyphenolic compounds for grape and wine quality. Recently, significant advances were achieved in understanding the biosynthesis of their main subunits, (+)-catechin and (-)-epicatechin, produced by leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively. Expression and enzyme activity studies have been published but no data were previously available on their regulation by hormonal status. As abscisic acid (ABA) is involved in controlling anthocyanin biosynthesis, we examined the effects of the ABA signal on the proanthocyanidin pathway. The aim of this research was to determine whether ABA application on green grapes affected tannin content and composition, LAR and ANR activity, and the expression patterns of the genes encoding them (VvANR, VvLAR1, VvLAR2) and the associated transcription regulators (VvMYB5a, VvMYBPA1) in grape skin during ripening. Our results show that ABA affects tannin content and is involved in the tannin biosynthesis pathway in grape skin by decreasing LAR and ANR activity and repressing the expression of related genes a few days after application. This treatment reduced the tannin content of green grapes without modifying their composition but had a positive impact on tannin biosynthesis during veraison, as previously demonstrated for anthocyanins, suggesting that ANR and LAR were coregulated by ABA.

Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport

The colour of the red wine is essentially due to the release of anthocyanins from the red skin of grape berries during the process of wine making. Anthocyanins are synthesized during ripening of the berries under the control of VvMYBA1 transcription factor that controls the expression of UFGT. In order to identify the whole set of downstream regulated genes, we targeted constitutive ectopic expression of VlmybA1-2 into grapevine hairy roots and plants. The ectopic expression of VlmybA1-2 triggered de novo production and storage of anthocyanins in all transgenic vegetative organs, leading to a very intense red coloration, and did not interfere with proanthocyanidin (PA) biosynthesis. The ectopic red pigmentation was due to the accumulation of anthocyanins in vacuoles and anthocyanin vacuolar inclusion (AVIs) in all organs but only in specific tissues. A transcriptomic analysis using a 14 K oligoarray revealed that the ectopic expression of VlmybA1-2 activated only few genes, most of which are involved in both PA and anthocyanin biosynthesis, while the expression of BAN and LAR (two specific genes of the PA biosynthesis pathway) was unaffected. Among these, 4 genes emerged given the amplitude of their up-regulation, quantitatively similar to VlmybA1-2 itself. In addition to the previously described UFGT, this set comprised an isogen of GST, an O-methyltransferase, both of which are supposed to play a role in the anthocyanin biosynthesis pathway, as well as a candidate gene putatively involved in the vacuolar anthocyanin transport in grapevine (anthoMATE). Together, these results suggest that MybA1 activates the last steps of anthocyanin synthesis and transport through the regulation of a narrow, specific spectrum of genes regulated as a cluster.

Cloning and Expression Analysis of Dihydroflavonol 4-Reductase Gene in Brassica juncea

Dihydroflavonol 4-reductase ( DFR) gene is a key gene of proanthocyanidins biosynthesis pathway in seed coat of Arabidopsis thaliana. The mutation of this gene brings about a transparent testa. To study molecular mechanism of seed coat color in Brassica, DFR gene was cloned from B. juncea using homology-based clone strategy. The cloned gene 1,612 bp in length contains 5 introns. The complementary DNA (cDNA) consists of 1,214 bp and has a 1,158 bp open reading frame encoding a deduced polypeptide of 385 amino acids with a predicted molecular weight of 42,886 Da and an estimated isoelectric point of 5.54. RT-PCR analysis showed that DFR was expressed in leaves, embryos, and seed coats of Purple-Leaf Mustard and 2 black-seeded near-isogenic lines developed from backcross breeding using Sichuan Yellow as a recurrent parent. Gene DFR was expressed only in the leaves and embryos of Sichuan Yellow, but not in seed coats. No expression of DFR blocked the biosynthesis of anthocyanidins and proanthocyanidins in the yellow seed coat, and seeds appeared yellow because of transparent testa. Gene DFR is essential in the formation of seed coat color in B. juncea. This study provided a foundation for understanding the molecular mechanism of seed coat color and developing novel yellow-seeded rapeseed germplasm through antisense expression or RNAi suppression of DFR gene in black-seeded cultivars using a seed- or seed-coat-specific promoter.

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development.

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.