Proanthocyanidin Biosynthesis Research Articles

Ubiquitin ligase VvPUB26 in grapevine promotes proanthocyanidin synthesis and resistance to powdery mildew.

Proanthocyanidins (PAs) are an important group of flavonoids that contribute to astringency, color, and flavor in grape (Vitis vinifera) and wines. They also play a crucial role in enhancing plant resistance to various stresses. However, the underlying regulatory mechanism governing PAs biosynthesis, particularly in relation to conferring resistance to powdery mildew, has not been extensively explored. This study focused on identifying a key player in PAs biosynthesis, namely the plant U-box (PUB) E3 ubiquitin ligase VvPUB26. We discovered that overexpression of VvPUB26 in grape leads to a significant increase in PAs content, whereas interfering with VvPUB26 has the opposite effect. Additionally, our findings demonstrated that overexpression of VvPUB26 in transgenic grapevines enhances defense against powdery mildew, while interfering with VvPUB26 results in increased susceptibility to the pathogen. Interestingly, we observed that VvPUB26 interacts with the WRKY transcription factor VvWRKY24, thereby facilitating ubiquitination and degradation processes. Through RNA-Seq analysis, we found that VvWRKY24 primarily participates in secondary metabolites biosynthesis, metabolic pathways, and plant-pathogen interaction. Notably, VvWRKY24 directly interacts with the promoters of dihydroflavonol-4-reductase (DFR) and leucoanthocyanidin reductase (LAR) to inhibit PAs biosynthesis. Meanwhile, VvWRKY24 also influences the expression of MYB transcription factor genes related to PAs synthesis. In conclusion, our results unveil a regulatory module involving VvPUB26-VvWRKY24-VvDFR/VvLAR that plays a fundamental role in governing PAs biosynthesis in grapevines. These findings enhance our understanding of the relationship between PAs biosynthesis and defense mechanisms against powdery mildew.

Exogenous application of sucrose promotes the repartitioning of anthocyanin and proanthocyanidin in ‘Cabernet Sauvignon’ grapevine berries

Sugar and its metabolism play a critical role in fruit quality and determine the accumulation characteristics of secondary metabolites. In this study, the dynamic changes in anthocyanin and proanthocyanidin (PA) accumulation during exogenous sugar application were investigated throughout the grape ripening stage, and the underlying biosynthesis mechanism was monitored. Results showed that exogenous sucrose and N-acetyl-d-glucosamine (hexokinase inhibitor, HI) promoted grape berry ripening. Exogenous application of sucrose and HI significantly increased the levels of soluble sugars and anthocyanins, while significantly decreased the levels of acids and PAs in grape berries. Exogenous sucrose and HI activated the transcription of the VvSPS, VvAI and VvNI genes but inhibited the transcription of VvHXK gene to alter sugar metabolism. Notably, the accumulation of anthocyanin compounds, particularly peonidin derivatives, significantly increased in the sucrose- and HI-treated berries by upregulating the transcription of anthocyanin biosynthesis genes, including VvCHS2, VvF3′H, VvDFR, VvLDOX, and VvUFGT, whereas the accumulation of PA units, such as (‒)-epicatechin, (+)-gallocatechin, and (‒)-epigallocatechin, was repressed by downregulating the transcription of the VvLAR2 and VvANR genes, which boosted anthocyanin and PA reprogramming. These results provide preliminary information on how to modulate the biosynthesis and accumulation of anthocyanins and PAs in grape berries by the exogenous application of sucrose and thus, to modify berry composition.

Comparative Genomics of Lotus japonicus Reveals Insights into Proanthocyanidin Accumulation and Abiotic Stress Response.

Lotus japonicus, is an important perennial model legume, has been widely used for studying biological processes such as symbiotic nitrogen fixation, proanthocyanidin (PA) biosynthesis, and abiotic stress response. High-quality L. japonicus genomes have been reported recently; however, the genetic basis of genes associated with specific characters including proanthocyanidin distribution in most tissues and tolerance to stress has not been systematically explored yet. Here, based on our previous high-quality L. japonicus genome assembly and annotation, we compared the L. japonicus MG-20 genome with those of other legume species. We revealed the expansive and specific gene families enriched in secondary metabolite biosynthesis and the detection of external stimuli. We suggested that increased copy numbers and transcription of PA-related genes contribute to PA accumulation in the stem, petiole, flower, pod, and seed coat of L. japonicus. Meanwhile, According to shared and unique transcription factors responding to five abiotic stresses, we revealed that MYB and AP2/ERF play more crucial roles in abiotic stresses. Our study provides new insights into the key agricultural traits of L. japonicus including PA biosynthesis and response to abiotic stress. This may provide valuable gene resources for legume forage abiotic stress resistance and nutrient improvement.

The crane fly glycosylated triketide δ-lactone cornicinine elicits akinete differentiation of the cyanobiont in aquatic Azolla fern symbioses.

The restriction of plant-symbiont dinitrogen fixation by an insect semiochemical had not been previously described. Here we report on a glycosylated triketide δ-lactone from Nephrotoma cornicina crane flies, cornicinine, that causes chlorosis in the floating-fern symbioses from the genus Azolla. Only the glycosylated trans-A form of chemically synthesized cornicinine was active: 500 nM cornicinine in the growth medium turned all cyanobacterial filaments from Nostoc azollae inside the host leaf-cavities into akinetes typically secretingCTB-bacteriocins. Cornicinine further inhibited akinete germination in Azolla sporelings, precluding re-establishment of the symbiosis during sexual reproduction. It did not impact development of the plant Arabidopsis thaliana or several free-living cyanobacteria from the genera Anabaena or Nostoc but affected the fern host without cyanobiont. Fern-host mRNA sequencing from isolated leaf cavities confirmed high NH4-assimilation and proanthocyanidin biosynthesis in this trichome-rich tissue. After cornicinine treatment, it revealed activation of Cullin-RING ubiquitin-ligase-pathways, known to mediate metabolite signaling and plant elicitation consistent with the chlorosis phenotype, and increased JA-oxidase, sulfate transport and exosome formation. The work begins to uncover molecular mechanisms of cyanobiont differentiation in a seed-free plant symbiosis important for wetland ecology or circular crop-production today, that once caused massive CO2 draw-down during the Eocene geological past.

Revisiting decade-old questions in proanthocyanidin biosynthesis: current understanding and new challenges.

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.

Metabolite and transcriptome analyses reveal the effects of salinity stress on the biosynthesis of proanthocyanidins and anthocyanins in grape suspension cells.

Proanthocyanidins (PAs) and anthocyanins are flavonoids that contribute to the quality and health benefits of grapes and wine. Salinity affects their biosynthesis, but the underlying mechanism is still unclear. We studied the effects of NaCl stress on PA and anthocyanin biosynthesis in grape suspension cells derived from berry skins of Vitis vinifera L. Cabernet Sauvignon using metabolite profiling and transcriptome analysis. We treated the cells with low (75 mM NaCl) and high (150 mM NaCl) salinity for 4 and 7 days. High salinity inhibited cell growth and enhanced PA and anthocyanin accumulation more than low salinity. The salinity-induced PAs and anthocyanins lacked C5'-hydroxylation modification, suggesting the biological significance of delphinidin- and epigallocatechin-derivatives in coping with stress. The genes up-regulated by salinity stress indicated that the anthocyanin pathway was more sensitive to salt concentration than the PA pathway, and WGCNA analysis revealed the coordination between flavonoid biosynthesis and cell wall metabolism under salinity stress. We identified transcription factors potentially involved in regulating NaCl dose- and time-dependent PA and anthocyanin accumulation, showing the dynamic remodeling of flavonoid regulation network under different salinity levels and durations. Our study provides new insights into regulator candidates for tailoring flavonoid composition and molecular indicators of salt stress in grape cells.

Integrated Transcriptomic and Proteomic Analysis Identifies Novel Regulatory Genes Associated with Plant Growth Regulator-Induced Astringency in Grape Berries.

Astringency influences the sensory characteristics and flavor quality of table grapes. We tested the astringency sensory attributes of berries and investigated the concentration of flavan-3-ols/proanthocyanidins (PAs) in skins after the application of the plant growth regulators CPPU and GA3 to the flowers and young berries of the "Summer Black" grape. Our results showed that CPPU and GA3 applications increase sensory astringency perception scores and flavan-3-ol/proanthocyanidin concentrations. Using integrated transcriptomic and proteomic analysis, differentially expressed transcripts and proteins associated with growth regulator treatment were identified, including those for flavonoid biosynthesis that contribute to the changes in sensory astringency levels. Transient overexpression of candidate astringency-related regulatory genes in grape leaves revealed that VvWRKY71, in combination with VvMYBPA1 and VvMYC1, could promote the biosynthesis of proanthocyanidins, while overexpression of VvNAC83 reduced the accumulation of proanthocyanidins. However, in transient promoter studies in Nicotiana benthamiana, VvWRKY71 repressed the promoter of VvMYBPA2, while VvNAC83 had no significant effect on the promoter activity of four PA-related genes, and VvMYBPA1 was shown to activate its own promoter. This study provides new insights into the molecular mechanisms of sensory astringency formation induced by plant growth regulators in grape berries.

Molecular evolution analysis of MYB5 in Brassicaceae with specific focus on seed coat color of Brassica napus

BackgroundMYB transcription factors are splay a vital role in plant biology, with previous research highlighting the significant impact of the R2R3-MYB-like transcription factor MYB5 on seed mucilage biosynthesis, trichome branching, and seed coat development. However, there is a dearth of studies investigating its role in the regulation of proanthocyanidin (PA) biosynthesis.ResultsIn this study, a total of 51 MYB5 homologous genes were identified across 31 species belonging to the Brassicaceae family, with particular emphasis on Brassica napus for subsequent investigation. Through phylogenetic analysis, these genes were categorized into four distinct subclasses. Protein sequence similarity and identity analysis demonstrated a high degree of conservation of MYB5 among species within the Brassicaceae family. Additionally, the examination of selection pressure revealed that MYB5 predominantly underwent purifying selection during its evolutionary history, as indicated by the Ka/Ks values of all MYB5 homologous gene pairs being less than one. Notably, we observed a higher rate of non-synonymous mutations in orthologous genes compared to paralogous genes, and the Ka/Ks value displayed a stronger correlation with Ka. In B. napus, an examination of expression patterns in five tissues revealed that MYB5 exhibited particularly high expression in the black seed coat. The findings from the WGCNA demonstrated a robust correlation between MYB5 and BAN(ANR) associated with PA biosynthesis in the black seed coat, providing further evidence of their close association and co-expression. Furthermore, the results obtained from of the analysis of protein interaction networks offer supplementary support for the proposition that MYB5 possesses the capability to interact with transcriptional regulatory proteins, specifically TT8 and TT2, alongside catalytic enzymes implicated in the synthesis of PAs, thereby making a contribution to the biosynthesis of PAs. These findings imply a plausible and significant correlation between the nuique expression pattern of MYB5 and the pigmentation of rapeseed coats. Nevertheless, additional research endeavors are imperative to authenticate and substantiate these findings.ConclusionsThis study offers valuable insights into the genetic evolution of Brassicaceae plants, thereby serving as a significant reference for the genetic enhancement of Brassicaceae seed coat color.

Anthocyanin gene enrichment in the distal region of cotton chromosome A07: mechanisms of reproductive organ coloration.

The biosynthesis of secondary metabolites like anthocyanins is often governed by metabolic gene clusters (MGCs) in the plant ancestral genome. However, the existence of gene clusters specifically regulating anthocyanin accumulation in certain organs is not well understood. In this study, we identify MGCs linked to the coloration of cotton reproductive organs, such as petals, spots, and fibers. Through genetic analysis and map-based cloning, we pinpointed key genes on chromosome A07, such as PCC/GhTT19, which is involved in anthocyanin transport, and GbBM and GhTT2-3A, which are associated with the regulation of anthocyanin and proanthocyanidin biosynthesis. Our results demonstrate the coordinated control of anthocyanin and proanthocyanidin pathways, highlighting the evolutionary significance of MGCs in plant adaptation. The conservation of these clusters in cotton chromosome A07 across species underscores their importance in reproductive development and color variation. Our study sheds light on the complex biosynthesis and transport mechanisms for plant pigments, emphasizing the role of transcription factors and transport proteins in pigment accumulation. This research offers insights into the genetic basis of color variation in cotton reproductive organs and the potential of MGCs to enhance our comprehension of plant secondary metabolism.

Targeted mutagenesis of BnTTG1 homologues generated yellow-seeded rapeseed with increased oil content and seed germination under abiotic stress

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses.

Tartary Buckwheat (Fagopyrum tataricum) FtTT8 Inhibits Anthocyanin Biosynthesis and Promotes Proanthocyanidin Biosynthesis.

Tartary buckwheat (Fagopyrum tataricum) is an important plant, utilized for both medicine and food. It has become a current research hotspot due to its rich content of flavonoids, which are beneficial for human health. Anthocyanins (ATs) and proanthocyanidins (PAs) are the two main kinds of flavonoid compounds in Tartary buckwheat, which participate in the pigmentation of some tissue as well as rendering resistance to many biotic and abiotic stresses. Additionally, Tartary buckwheat anthocyanins and PAs have many health benefits for humans and the plant itself. However, little is known about the regulation mechanism of the biosynthesis of anthocyanin and PA in Tartary buckwheat. In the present study, a bHLH transcription factor (TF) FtTT8 was characterized to be homologous with AtTT8 and phylogenetically close to bHLH proteins from other plant species. Subcellular location and yeast two-hybrid assays suggested that FtTT8 locates in the nucleus and plays a role as a transcription factor. Complementation analysis in Arabidopsis tt8 mutant showed that FtTT8 could not recover anthocyanin deficiency but could promote PAs accumulation. Overexpression of FtTT8 in red-flowering tobacco showed that FtTT8 inhibits anthocyanin biosynthesis and accelerates proanthocyanidin biosynthesis. QRT-PCR and yeast one-hybrid assay revealed that FtTT8 might bind to the promoter of NtUFGT and suppress its expression, while binding to the promoter of NtLAR and upregulating its expression in K326 tobacco. This displayed the bidirectional regulating function of FtTT8 that negatively regulates anthocyanin biosynthesis and positively regulates proanthocyanidin biosynthesis. The results provide new insights on TT8 in Tartary buckwheat, which is inconsistent with TT8 from other plant species, and FtTT8 might be a high-quality gene resource for Tartary buckwheat breeding.

The APETALA2–MYBL2 module represses proanthocyanidin biosynthesis by affecting formation of the MBW complex in seeds of Arabidopsis thaliana

Proanthocyanidins (PAs) are the second most abundant plant phenolic natural products. PA biosynthesis is regulated by the well-documented MYB/bHLH/WD40 (MBW) complex, but how this complex itself is regulated remains ill defined. Here, in situ hybridization and β-glucuronidase staining show that APETALA2 (AP2), a well-defined regulator of flower and seed development, is strongly expressed in the seed coat endothelium, where PAs accumulate. AP2 negatively regulates PA content and expression levels of key PA pathway genes. AP2 activates MYBL2 transcription and interacts with MYBL2, a key suppressor of the PA pathway. AP2 exerts its function by directly binding to the AT-rich motifs near the promoter region of MYBL2. Molecular and biochemical analyses revealed that AP2 forms AP2–MYBL2–TT8/EGL3 complexes, disrupting the MBW complex and thereby repressing expression of ANR, TT12, TT19, and AHA10. Genetic analyses revealed that AP2 functions upstream of MYBL2, TT2, and TT8 in PA regulation. Our work reveals a new role of AP2 as a key regulator of PA biosynthesis in Arabidopsis. Overall, this study sheds new light on the comprehensive regulation network of PA biosynthesis as well as the dual regulatory roles of AP2 in seed development and accumulation of major secondary metabolites in Arabidopsis.

Multiple bHLH/MYB-based protein complexes regulate proanthocyanidin biosynthesis in the herbage of Lotus spp.

Main conclusionThe complexes involving MYBPA2, TT2b, and TT8 proteins are the critical regulators of ANR and LAR genes to promote the biosynthesis of proanthocyanidins in the leaves of Lotus spp.The environmental impact and health of ruminants fed with forage legumes depend on the herbage's concentration and structure of proanthocyanidins (PAs). Unfortunately, the primary forage legumes (alfalfa and clover) do not contain substantial levels of PAs. No significant progress has been made to induce PAs to agronomically valuable levels in their edible organs by biotechnological approaches thus far. Building this trait requires a profound knowledge of PA regulators and their interplay in species naturally committed to accumulating these metabolites in the target organs. Against this background, we compared the shoot transcriptomes of two inter-fertile Lotus species, namely Lotus tenuis and Lotus corniculatus, polymorphic for this trait, to search for differentially expressed MYB and bHLH genes. We then tested the expression of the above-reported regulators in L. tenuis x L. corniculatus interspecific hybrids, several Lotus spp., and different L. corniculatus organs with contrasting PA levels. We identified a novel MYB activator and MYB-bHLH-based complexes that, when expressed in Nicotiana benthamiana, trans-activated the promoters of L. corniculatus anthocyanidin reductase and leucoanthocyanidin reductase 1 genes. The last are the two critical structural genes for the biosynthesis of PAs in Lotus spp. Competition between MYB activators for the transactivation of these promoters also emerged. Overall, by employing Lotus as a model genus, we refined the transcriptional network underlying PA biosynthesis in the herbage of legumes. These findings are crucial to engineering this trait in pasture legumes.

Transcriptome and weighted gene co-expression network analysis reveal key genes involved in the proanthocyanidin biosynthesis in Cerasus humilis

Proanthocyanidins (PAs), contributing greatly to the antioxidant activity, are among the most abundant flavonoid components in Cerasus humilis fruits. In this study, contents of six different PA components and total PAs in fruits of two C. humilis varieties (‘Jinou1’ and ‘DS-1′) were determined at four different ripening stages. Results showed that, five PA components and the total PAs contents of ‘Jinou1’ were notedly higher than that of ‘DS-1′ except proanthocyanidins A1. All PA components showed a general decreasing trend during ripening, and dropped to their lowest levels at the fully ripe stage. In ripe C. humilis fruits, the major PA was catechin, the content of which undergoes considerable changes during fruit ripening. The key genes involved in PA biosynthesis were explored based on the transcriptome profiles, and their co-expression networks were constructed. Transcriptomic analysis identified 19 PA biosynthesis-related structural genes as differentially expressed genes (DEGs). The highly expressed MSTRG.938 (LAR, leucoanthocyanidin reductase), MSTRG.4502 (ANR, anthocyanidin reductase), and MSTRG.4956 (PAL, phenylalanine ammonia lyase) genes may play key roles in regulating PA accumulation. Moreover, through weighted gene co-expression network analysis (WGCNA), we constructed a gene regulatory network of two modules involving 15 PA biosynthesis-related DEGs, and the expression changes in 10 of them were validated using quantitative real-time PCR. These results indicated that they are key contributors to the PA biosynthesis in C. humilis fruits. Our study will aid in elucidating the molecular mechanisms underlying PAs accumulation in C. humilis fruits.

Transcription factor CsTT8 promotes fruit coloration by positively regulating the methylerythritol 4-phosphate pathway and carotenoid biosynthesis pathway in citrus (Citrus spp.).

Carotenoids directly influence citrus fruit color and nutritional value, which is critical to consumer acceptance. Elucidating the potential molecular mechanism underlying carotenoid metabolism is of great importance for improving fruit quality. Despite the well-established carotenoid biosynthetic pathways, the molecular regulatory mechanism underlying carotenoid metabolism remains poorly understood. Our previous studies have reported that the Myc-type basic helix-loop-helix (bHLH) transcription factor (TF) regulates citrus proanthocyanidin biosynthesis. Transgenic analyses further showed that overexpression of CsTT8 could significantly promote carotenoid accumulation in transgenic citrus calli, but its regulatory mechanism is still unclear. In the present study, we found that overexpression of CsTT8 enhances carotenoid content in citrus fruit and calli by increasing the expression of CsDXR, CsHDS, CsHDR, CsPDS, CsLCYE, CsZEP, and CsNCED2, which was accompanied by changes in the contents of abscisic acid and gibberellin. The in vitro and in vivo assays indicated that CsTT8 directly bound to the promoters of CsDXR, CsHDS, and CsHDR, the key metabolic enzymes of the methylerythritol 4-phosphate (MEP) pathway, thus providing precursors for carotenoid biosynthesis and transcriptionally activating the expression of these three genes. In addition, CsTT8 activated the promoters of four key carotenoid biosynthesis pathway genes, CsPDS, CsLCYE, CsZEP, and CsNCED2, directly promoting carotenoid biosynthesis. This study reveals a novel network of carotenoid metabolism regulated by CsTT8. Our findings will contribute to manipulating carotenoid metabolic engineering to improve the quality of citrus fruit and other crops.

Anthocyanin Metabolite and Transcriptome Sequencing Analysis Reveals White Flowers in Rhododendron dauricum var. albiflorum

Rhododendron dauricum is a cold-hardy shrub integrating ornamental, medicinal, and aromatic functions. Flower color is an important feature related to ornamental value for breeders and consumers. Nevertheless, the coloration mechanism of flower color in R. dauricum is still unclear. R. dauricum var. albiflorum is a white flower variety of R. dauricum. In this study, an integrative analysis of the metabolome and transcriptome was conducted between R. dauricum var. albiflorum and R. dauricum. A total of nine anthocyanins and two proanthocyanidins were differentially accumulated between R. dauricum var. albiflorum and R. dauricum. A decrease in malvidin-, delphinidin-, cyanidin-, peonidin-, and petunidin-based anthocyanins and an increase in procyanidin A2 and procyanidin B2 were responsible for the white flowers of R. dauricum var. albiflorum. Furthermore, a total of 4376 differentially expressed genes (DEGs) were identified using transcriptome sequencing. Integrated analysis of the metabolome and transcriptome showed that 21 DEGs encoding 9 enzymes (PAL, C4H, CHS, CHI, F3H, F3′5′H, DFR, ANR, and UFGT) were identified as structural genes involved in anthocyanin and proanthocyanidin biosynthesis, and 15 MYBs and 10 bHLHs were the transcriptional regulators of the anthocyanin biosynthesis pathways in R. dauricum var. albiflorum. Our results deepen the understanding of variations in azalea flower color, which is helpful for identifying important genes in the genetic engineering of azalea shrubs.

Genome-Wide Identification of R2R3-MYB Transcription Factor Family in Tartary Buckwheat (Fagopyrum tataricum) Identifies a Member Involved in Anthocyanin Biosynthesis

Tartary buckwheat (Fagopyrum tataricum Gaertn.) belongs to the family of Polygonaceae and is used as a multi-functional plant. R2R3-MYB transcription factors play a crucial part in plant growth and are involved in many biological processes where they regulate their internal environment. To date, there is no documented systematic research on the R2R3-MYB gene family in Tartary buckwheat. Here, domain features, chromosomal location, motif prediction, gene structure, cis-acting elements, as well as the expression pattern of R2R3-MYB transcription factors were analyzed comprehensively in Tartary buckwheat using a bioinformatic approach. Additionally, one R2R3-MYB gene was verified by transgenic Arabidopsis. Results indicate that a total of 152 R2R3-MYB genes were identified with special R2R3 domains and were distributed on 8 chromosomes of Tartary buckwheat. They were further classified into 25 sub-categories via phylogenetic analysis in terms of the R2R3-MYB gene family classification principles of Arabidopsis thaliana. This classification was further supported by analysis of exon–intron structure, motif, and cis-elements. Tandem and segmental duplication existed among the R2R3-MYB gene family of Tartary buckwheat, and there were 5, 8, 27, and 36 FtR2R3-MYB homologous genes, respectively, when comparing with Zea mays, Oryza sativa, Arabidopsis thaliana, and Solanum melongena by synteny analysis. The expression pattern of FtR2R3-MYB genes in different tissue and under salt stress and different light condition showed that members had tissue-specific expression levels and that these members may play diverse functions in plant growth and adaptation to varying environments. In addition, one of the FtR2R3-MYB gene families, FtMYB43, a homologue of AtTT2, clustered with R2R3-MYB from other plant species, which were reported to be involved in the regulation of anthocyanin or proanthocyanidin biosynthesis. This gene was located in the nucleus, and had transcriptional activation activity, indicating that FtMYB43 may be a positive transcript factor of anthocyanin or proanthocyadin biosynthesis. Moreover, the function of FtMYB43 was further verified to improve the production of anthocyanin in transgenic Arabidopsis by overexpression, and qRT-PCR assay implied that FtMYB43 may regulate the expression levels of most structural genes of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. These results provide more insights into the structure and function of the R2R3-MYB gene family and may accelerate the breeding of ornamental buckwheat cultivars.

CRISPR/Cas9 disruption of MYB134 and MYB115 in transgenic poplar leads to differential reduction of proanthocyanidin synthesis in roots and leaves.

Proanthocyanidins (PAs) are common specialized metabolites and particularly abundant in trees and woody plants. In poplar (Populus spp.), PA biosynthesis is stress-induced and regulated by two previously studied transcription factors MYB115 and MYB134. To determine the relative contribution of these regulators to PA biosynthesis, we created single and double knockout mutants for both genes in transgenic poplars using CRISPR/Cas9. Knocking out either MYB134 or MYB115 showed reduced PA accumulation and down-regulated flavonoid genes in leaves, but MYB134 disruption had the greatest impact and reduced PAs to 30% of controls. In roots, by contrast, only the MYB134/MYB115 double knockouts showed a significant change in PA concentration. The loss of PAs paralleled the lower expression of PA biosynthesis genes, and concentrations of flavan-3-ol PA precursors catechin and epicatechin. Interestingly, salicinoids were also affected in double knock-outs, with distinct patterns in roots and shoots. We conclude that the regulatory pathways for PA biosynthesis differs in poplar leaves and roots. The residual PA content in the double-KO plants indicates that other transcription factors must also be involved in control of the PA pathway.

Transcription Regulation of Anthocyanins and Proanthocyanidins Accumulation by Bagging in ‘Ruby’ Red Mango: An RNA-seq Study

The biosynthesis of anthocyanins and proanthocyanidins (PAs), components of two main flavonoids in plants, is regulated by environmental factors such as light. We previously found that bagging significantly repressed the biosynthesis of anthocyanins in red ‘Ruby’ mango fruit peel, but induced the accumulation of PAs. However, the molecular mechanism remains unclear. In the current study, transcriptome sequencing was used for screening the essential genes responsible for the opposite accumulation pattern of anthocyanins and PAs by bagging treatment. According to weighted gene co-expression network analysis (WGCNA), structural genes and transcription factors highly positively correlated to anthocyanins and PAs were identified. One flavanone 3-hydroxylase (F3H) and seven structural genes, including one chalcone synthase (CHS), one flavonoid 3’-hydroxylase (F3’H), one anthocyanidin synthesis (ANS), three leucoanthocyanidin reductase (LARs), and one UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT), are crucial for anthocyanin and PA biosynthesis, respectively. In addition to MYB and bHLH, ERF, C2H2, HD-ZIP, and NAC are important transcription factors that participate in the regulation of anthocyanin and PA biosynthesis in ‘Ruby’ mango fruit peel by bagging treatment. Our results are helpful for revealing the transcription regulation mechanism of light-regulated mango anthocyanin and PA biosynthesis, developing new technologies for inducing flavonoid biosynthesis in mangos, and breeding mango cultivars containing high concentrations of flavonoids.

FaMYB5 Interacts with FaBBX24 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Strawberry (Fragaria × ananassa).

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3'H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5-FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit.