Proanthocyanidins Biosynthetic Genes Research Articles

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.

Transcriptome Analysis Identifies Two Ethylene Response Factors That Regulate Proanthocyanidin Biosynthesis During Malus Crabapple Fruit Development.

Proanthocyanidins (PAs) are a class of flavonoid compounds in plants that play many important roles in pest and disease resistance and are beneficial components of the human diet. The crabapple (Malus) provides an excellent model to study PA biosynthesis and metabolism; therefore, to gain insights into the PA regulatory network in Malus plants, we performed RNA-seq profiling of fruits of the ‘Flame’ cultivar at five sequential developmental stages. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that differentially expressed genes (DEGs) related to the functional category ‘plant hormone signal transduction’ were significantly enriched during fruit development. Further analysis showed that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ETHYLENE INSENSITIVE 3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis. Additionally, 12 DEGs, including 10 ERFs, 1 MYB, and 1 bHLH transcription factor, associated with PA biosynthesis were identified using WGCNA. The expression patterns of these genes correlated with PA accumulation trends and transcriptome data from qRT-PCR analysis. The expression of RAP2-4 (RELATED TO APETALA 2-4) and RAV1 (related to ABI3/VP1), which belong to the ERF transcription factor family, showed the greatest correlations with PAs accumulation among the 12 identified TFs. Agrobacterium mediated-transient overexpression of the RAP2-4 led to an increase in PA abundance in crabapple leaves and apple fruits, and the opposite results were observed in RAV1-overexpressed crabapple leaves and apple fruits. Moreover, a yeast one-hybrid assay showed that RAP2-4 and RAV1 specifically bound the promoters of the PA biosynthetic genes McLAR1 and McANR2, respectively. These results indicate that RAP2-4 act as an inducer and RAV1 act as a repressor of PA biosynthesis by regulating the expression of the PA biosynthetic genes McLAR1 and McANR2. Taken together, we identified two potential regulators of PA biosynthesis and provide new insights into the ethylene-PA regulatory network.

Application of a JA-Ile Biosynthesis Inhibitor to Methyl Jasmonate-Treated Strawberry Fruit Induces Upregulation of Specific MBW Complex-Related Genes and Accumulation of Proanthocyanidins.

Fleshy fruits are an important source of anthocyanins and proanthocyanidins (PAs), which protect plants against stress, and their consumption provides beneficial effects for human health. In strawberry fruit, the application of exogenous methyl jasmonate (MeJA) upregulates anthocyanin accumulation, although the relationship between the jasmonate pathway and anthocyanin and PA biosynthesis in fruits remains to be understood. Anthocyanin and PA accumulation is mainly regulated at the transcriptional level through R2R3-MYB and bHLH transcription factors in different plant species and organs. Here, the effect of jarin-1, a specific inhibitor of bioactive JA (jasmonoyl-isoleucine, JA-Ile) biosynthesis, on anthocyanin and PA accumulation was evaluated during strawberry (Fragaria × ananassa) fruit development using an in vitro ripening system for 48 h. Also, we observed the effects of MeJA and the application of jarin-1 to MeJA-treated fruits (MeJA + jarin-1 treatment). We assessed changes of expression levels for the JA-Ile and MeJA biosynthetic (FaJAR1.2 and FaJMT), JA signaling-related (FaMYC2 and FaJAZ1), MYB-bHLH-WD40 (MBW) complex-related (FabHLH3/33, FaMYB9/10/11, and repressor FaMYB1), and anthocyanin and PA biosynthetic (FaANS, FaUFGT, FaANR, and FaLAR) genes. In addition, the promoter region of MBW complex-related MYB genes was isolated and sequenced. We found a higher redness of strawberry fruit skin and anthocyanin content in MeJA-treated fruits with respect to jarin-1-treated ones concomitant with an upregulation of FaANS and FaUFGT genes. Inversely, the PA content was higher in jarin-1- and MeJA + jarin-1-treated than in MeJA-treated fruits. MeJA + jarin-1 treatment resulted in an upregulation of FaANR and associated transcription factors such as FabHLH33 and FaMYB9/11 along with FaJMT and FaJAR1.2. Finally, we found JA-responsive elements in the promoter regions of FaMYB1/9/10/11 genes. It is proposed that PA biosynthesis-related genes can be upregulated by the application of jarin-1 to MeJA-treated fruit, thus increasing PA accumulation in strawberry.

Overexpression of the LoMYB29 gene of Larix olgensis contributes to the regulation of proanthocyanidin biosynthesis in Arabidopsis thaliana

Proanthocyanidins (PAs) are the most broadly distributed secondary metabolites that play important roles in various aspects of plant development and response to biotic and abiotic stresses. In this study, we cloned a R2R3 MYB gene LoMYB29, which has a full-length coding sequence of 921 bp identified in Larix olgensis. Quantitative real-time reverse transcription polymerase chain reaction analysis indicates that LoMYB29 is expressed under mechanical wounding, high light intensity, and NaCl, PEG6000, Methyl Jasmonate, and abscisic acid treatments. Subcellular localization analysis and yeast two-hybrid assay localized LoMYB29 to the nucleus, acting as a transcriptional activator. Staining with 4-dimethylaminocinnamaldehyde showed a darker blue-purple color in LoMYB29-overexpressing Arabidopsis seeds compared to that of wild seeds. LoMYB29-overexpression resulted in a significant increase in leaf PA content. The expression of early flavonoid biosynthesis-related gene CHI and late flavonoid biosynthesis-related genes, including DFR, LDOX, and ANR (PA branch gene), were also activated in transgenic plants overexpressing LoMYB29. The results indicate that LoMYB29 plays a positive role in the regulation of PA biosynthesis by activating the expression of PA biosynthetic genes.

Characterization of a gene regulatory network underlying astringency loss in persimmon fruit.

Transcriptome analysis of a persimmon population segregating for an astringency trait in fruit suggested central roles for a limited number of transcriptional regulators in the loss of proanthocyanidin accumulation. Persimmon (Diospyros kaki; 2n=6x=90) accumulates a large amount of proanthocyanidins (PAs) in its fruit, resulting in an astringent taste. Persimmon cultivars are classified into four types based on the nature of astringency loss and the amount of PAs at maturity. Pollination constant and non-astringent (PCNA)-type cultivars stop accumulating PAs in the early stages of fruit development and their fruit can be consumed when still firm without the need for artificial deastringency treatments. While the PCNA trait has been shown to be conferred by a recessive allele at a single locus (ASTRINGENCY; AST), the exact genetic determinant remains unidentified. Here, we conducted transcriptome analyses to elucidate the regulatory mechanism underlying this trait using developing fruits of an F1 population segregating for the PCNA trait. Comparisons of the transcriptomes of PCNA and non-PCNA individuals and hierarchical clustering revealed that genes related to the flavonoid pathway and to abiotic stress responses involving light stimulation were expressed coordinately with PA accumulation. Furthermore, coexpression network analyses suggested that three putative transcription factors were central to the PA regulatory network and that at least DkMYB4 and/or DkMYC1, which have been reported to form a protein complex with each other for PA regulation, may have a central role in the differential expression of PA biosynthetic pathway genes between PCNA and non-PCNA.

Transcriptomic Analysis of Seed Coats in Yellow-Seeded Brassica napus Reveals Novel Genes That Influence Proanthocyanidin Biosynthesis.

Yellow seeds are a favorable trait for Brassica crops breeding due to better quality than their black-seeded counterparts. Here, we compared the Brassica napus seed coat transcriptomes between yellow- and brown-seeded near-isogenic lines (Y-NIL and B-NIL) that were developed from the resynthesized yellow-seeded line No. 2127-17. A total of 4,974 differentially expressed genes (DEG) were identified during seed development, involving 3,128 up-regulated and 1,835 down-regulated genes in yellow seed coats. Phenylpropanoid and flavonoid biosynthesis pathways were enriched in down-regulated genes, whereas the top two pathways for up-regulated genes were plant–pathogen interaction and plant hormone signal transduction. Twelve biosynthetic genes and three regulatory genes involved in the flavonoid pathway exhibited similar expression patterns in seed coats during seed development, of which the down-regulation mainly contributed to the reduction of proanthocyanidins (PAs) in yellow seed coats, indicating that these genes associated with PA biosynthesis may be regulated by an unreported common regulator, possibly corresponding to the candidate for the dominant black-seeded gene D in the NILs. Three transcription factor (TF) genes, including one bHLH gene and two MYB-related genes that are located within the previous seed coat color quantitative trait locus (QTL) region on chromosome A09, also showed similar developmental expression patterns to the key PA biosynthetic genes and they might thus potentially involved participate in flavonoid biosynthesis regulation. Our study identified novel potential TFs involved in PAs accumulation and will provide pivotal information for identifying the candidate genes for seed coat color in B. napus.

The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar.

Proanthocyanidins (PAs) are major defense phenolic compounds in the leaves of poplar (Populus spp.) in response to abiotic and biotic stresses. Transcriptional regulation of PA biosynthetic genes by the MYB-basic helix-loop-helix (bHLH)-WD40 complexes in poplar is not still fully understood. Here, an Arabidopsis TT2-like gene MYB115 was isolated from Populus tomentosa and characterized by various molecular, genetic and biochemical approaches. MYB115 restored PA productions in the seed coat of the Arabidopsis tt2 mutant. Overexpression of MYB115 in poplar activated expression of PA biosynthetic genes, resulting in a significant increase in PA concentrations. By contrast, the CRISPR/Cas9-generated myb115 mutant exhibited reduced PA content and decreased expression of PA biosynthetic genes. MYB115 directly activated the promoters of PA-specific structural genes. MYB115 interacted with poplar TT8. Coexpression of MYB115, TT8 and poplar TTG1 significantly enhanced the expression of ANR1 and LAR3. Additionally, transgenic plants overexpressing MYB115 had increased resistance to the fungal pathogen Dothiorella gregaria, whereas myb115 mutant exhibited greater sensitivity compared with wild-type plants. Our data provide insight into the regulatory mechanisms controlling PA biosynthesis by MYB115 in poplar, which could be effectively employed for metabolic engineering of PAs to improve resistance to fungal pathogens.

McMYB12 Transcription Factors Co-regulate Proanthocyanidin and Anthocyanin Biosynthesis in Malus Crabapple

The flavonoid compounds, proanthocyanidins (PAs), protect plants from biotic stresses, contribute to the taste of many fruits, and are beneficial to human health in the form of dietary antioxidants. In this study, we functionally characterized two Malus crabapple R2R3-MYB transcription factors, McMYB12a and McMYB12b, which co-regulate PAs and anthocyanin biosynthesis. McMYB12a was shown to be mainly responsible for upregulating the expression of anthocyanin biosynthetic genes by binding to their promoters, but to be only partially responsible for regulating PAs biosynthetic genes. In contrast, McMYB12b showed preferential binding to the promoters of PAs biosynthetic genes. Overexpression of McMYB12a and McMYB12b in tobacco (Nicotiana tabacum) altered the expression of flavonoid biosynthetic genes and promoted the accumulation of PAs and anthocyanins in tobacco petals. Conversely, transient silencing their expression in crabapple plants, using a conserved gene region, resulted in reduced PAs and anthocyanin production a green leaf phenotype. Meanwhile, transient overexpression of the two genes and silenced McMYB12s in apple (Malus domestica) fruit had a similar effect as overexpression in tobacco and silenced in crabapple. This study reveals a new mechanism for the coordinated regulation of PAs and anthocyanin accumulation in crabapple leaves, which depends on an auto-regulatory balance involving McMYB12a and McMYB12b expression.

Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

Identification and characterization of MYB‐bHLH‐WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria × ananassa) fruits

Strawberry (Fragaria×ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein). A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators. Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts. Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants

The flavonoid-derived proanthocyanidins (PAs) are one class of the major defence phenolics in poplar leaves. Transcriptional activation of PA biosynthetic genes, resulting in PA accumulation in leaves, was detected following infection by the fungal Marssonina brunnea f.sp. multigermtubi using digital gene expression analysis. In order to study PA biosynthesis and its induction by fungi, a putative leucoanthocyanidin reductase gene, PtrLAR3, was isolated from Populus trichocarpa. Sequence comparison of PtrLAR3 with other known leucoanthocyanidin reductase proteins revealed high amino acid sequence similarity. Semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrLAR3 was expressed in various tissues and the highest level of expression was observed in roots. Overexpression of PtrLAR3 in Chinese white poplar (Populus tomentosa Carr.) led to a significant plant-wide increase in PA levels. In vitro assays showed that crude leaf extracts from 35S:PtrLAR3 transformants were able to inhibit significantly the hyphal growth of M. brunnea f.sp. multigermtubi compared to the extracts from control plants. The transgenic 35S:PtrLAR3 poplar plants displayed a significant (P < 0.05) reduction in their disease symptoms compared with the control. RT-PCR analysis showed that PtrLAR3 expression was up-regulated in all transformants. These results suggested that constitutive expression of endogenous PtrLAR3 could be exploited to improve resistance to fungal pathogens in poplar.

Comparative Analysis of the Triplicate Proathocyanidin Regulators in Lotus japonicus

Proanthocyanidins (PAs), which are flavonoid compounds widely distributed in the plant kingdom, protect against environmental stress. The accumulation of PAs is regulated by a ternary transcriptional complex comprising the R2R3-MYB transcription factor, a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat (WDR) protein. Recently, multigene families of the R2R3-MYB-type PA regulators from Lotus japonicus, LjTT2a, b and c, were isolated and characterized. Although their roles as transcription factors that up-regulate PA biosynthetic genes have been elucidated, the significance of their redundancies and functions in planta is unknown. In this study, we characterized LjTT2a, b and c to elucidate their functions in planta and determine differences in transcriptional activation properties. Transgenic studies demonstrated that LjTT2a could induce ectopic PA accumulation in Arabidopsis. Further analysis of the LjTT2 multigene family using a transient expression system revealed differences in transcriptional activities in cooperation with WDR and bHLH proteins isolated from L. japonicus. In-depth characterization of chimeric constructs of three LjTT2s, as well as site-directed mutagenesis in R2-MYB domains, identified the amino acid residues that affect the level of transcriptional activation of LjTT2.

A WD40 Repeat Protein fromMedicago truncatulaIs Necessary for Tissue-Specific Anthocyanin and Proanthocyanidin Biosynthesis But Not for Trichome Development

WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins (PAs), and mucilage in the seed and the development of trichomes and root hairs. We have cloned and characterized a WD40 repeat protein gene from Medicago truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in the seed, reduces epicatechin levels without corresponding effects on other flavonoids in flowers, reduces isoflavone levels in roots, but does not impair trichome or root hair development. MtWD40-1 is expressed constitutively, with highest expression in the seed coat, where its transcript profile temporally parallels those of PA biosynthetic genes. Transcript profile analysis revealed that many genes of flavonoid biosynthesis were down-regulated in a tissue-specific manner in M. truncatula lines harboring retrotransposon insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa glabrous1 mutant. We discuss the function of MtWD40-1 in natural product formation in M. truncatula and the potential use of the gene for engineering PAs in the forage legume alfalfa (Medicago sativa).

The wound-, pathogen-, and ultraviolet B-responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar.

In poplar (Populus spp.), the major defense phenolics produced in leaves are the flavonoid-derived proanthocyanidins (PAs) and the salicin-based phenolic glycosides. Transcriptional activation of PA biosynthetic genes leading to PA accumulation in leaves occurs following herbivore damage and mechanical wounding as well as infection by the fungal biotroph Melampsora medusae. In this study, we have identified a poplar R2R3 MYB transcription factor gene, MYB134, that exhibits close sequence similarity to the Arabidopsis (Arabidopsis thaliana) PA regulator TRANSPARENT TESTA2 and that is coinduced with PA biosynthetic genes following mechanical wounding, M. medusae infection, and exposure to elevated ultraviolet B light. Overexpression of MYB134 in poplar resulted in transcriptional activation of the full PA biosynthetic pathway and a significant plant-wide increase in PA levels, and electrophoretic mobility shift assays showed that recombinant MYB134 protein is able to bind to promoter regions of PA pathway genes. MYB134-overexpressing plants exhibited a concomitant reduction in phenolic glycoside concentrations and other minor alterations to levels of small phenylpropanoid metabolites. Our data provide insight into the regulatory mechanisms controlling stress-induced PA metabolism in poplar, and the identification of a regulator of stress-responsive PA biosynthesis constitutes a valuable tool for manipulating PA metabolism in poplar and investigating the biological functions of PAs in resistance to biotic and abiotic stresses.

Detection and Quantification of Engineered Proanthocyanidins in Transgenic Plants

Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and/or catechin monomers. In studies aimed at engineering PAs into plant tissues that do not naturally make these compounds, we have expressed PA biosynthetic and regulatory genes in tobacco, alfalfa ( Medicago sativa) and the model legume Medicago truncatula. Because engineered tannins may be produced in small quantities and it is often necessary to screen many independent plant lines, we have developed an improved, highly sensitive method to quantify and determine the composition of oligomeric PAs in plant extracts. The method involves normal-phase HPLC separation of semi-purified PAs followed by post-column reaction with the PA-specific reagent DMACA (dimethylaminocinnamaldehyde). This procedure allows for accurate and sensitive quantification of individual oligomeric PAs and, unlike currently used methods, does not require exhaustive sample preparation and clean-up. Compositional data are shown for genetically engineered PAs in tobacco and alfalfa.