Accumulation Of Proanthocyanidins Research Articles

Overexpression of LAR1 Suppresses Anthocyanin Biosynthesis by Enhancing Catechin Competition Leading to Promotion of Proanthocyanidin Pathway in Spine Grape (Vitis davidii) Cells

Proanthocyanidins (PAs) are a class of polyphenolic compounds recognized for their potent antioxidant, anti-cancer, anti-inflammatory, and cardioprotective properties. However, the production of PAs from natural sources is often limited by high costs, resource wastage, and environmental damage. In this study, we investigated the overexpression of VdLAR1, along with phenotypic observation, metabolite determination, light quality treatment, and RT-qPCR analysis, in spine grape cells. The results demonstrated a significant increase in the contents of proanthocyanidins and flavonoids in pVdLAR1-overexpressing transgenic cell lines, while anthocyanin levels showed a decreasing trend. Furthermore, the treatment with white and blue light on the T5 cell line resulted in enhanced accumulation of proanthocyanidins, catechins, and flavonoids, whereas anthocyanins and epicatechins exhibited a declining pattern. Thus, short-wavelength light promoted the accumulation of metabolites, with the proanthocyanidin content in the T5 transformed cell line reaching 2512.0 μg/g (FW) during blue light incubation. RT-qPCR analysis revealed that the key genes involved in the biosynthesis of proanthocyanidin and anthocyanin were upregulated in the transgenic spine grape cell lines, with VdLAR1 expression increasing by several hundredfold, far surpassing the expression levels of LDOX and ANR. The VdLAR1 overexpression markedly improved substrate competitiveness within the metabolic pathway, promoting catechin biosynthesis while inhibiting the production of epicatechins and anthocyanins. This finding provides compelling evidence that LAR1 is a crucial gene for catechin biosynthesis. This research establishes both theoretical and practical foundations for the regulation and development of natural proanthocyanidins, addressing issues related to high costs, safety concerns, resource wastage, and environmental damage associated with their production.

Strigolactones Regulate Secondary Metabolism and Nitrogen/Phosphate Signaling in Tea Plants via Transcriptional Reprogramming and Hormonal Interactions.

Strigolactones (SLs) are known to regulate plant architecture formation, nitrogen (N) and phosphorus (P) responses, and secondary metabolism, but their effects in tea plants remain unclear. We demonstrated that the application of a bioactive SL analogue GR24 either to tea roots or leaves initially stimulated but later inhibited catechins, theanine, and caffeine biosynthesis. GR24 treatment also promoted the accumulation of flavonols and insoluble proanthocyanidins in a time- and dose-dependent manner. GR24 influenced flavonoid and theanine biosynthesis genes, such as up-regulating CsTT2c, CsMYB12, and CsbZIP1, modulating N-responsive and assimilation genes (CsNRT1,1, CsGSI/TS1, CsHRS1, CsPHR1, CsNLA1, and CsLBD37/38/39), and repressing N/P transport and signaling genes (CsPHO2, CsPHT1s, CsNRT2,2, CsHHO1, and CsWRKY38). GR24-induced changes in secondary metabolites were also observed in the leaves of tea plants. GR24-regulated CsLBD37a interacted with CsTT8a and CsTT2c, repressing catechins biosynthesis by interrupting MBW complex formation. GR24 regulated caffeine biosynthesis and regulator genes CsS40 and CsNAC7 and may thereby suppress caffeine production. GR24 altered the transcriptomic profiles of multiple hormone biosynthesis and signaling genes that potentially regulate tea characteristic metabolism and N/P signaling. This study provides new insights into SL-induced transcriptional reprogramming that leads to changes in N/P nutrition, secondary metabolism, and hormone signaling in tea plants.

Effects of phosphorylation on CsTT12 transport function: Acomparative phosphoproteomic analysis of flavonoid biosynthesis in tea plants (Camellia sinensis).

Monomeric flavan-3-ols and their oligomeric forms, proanthocyanidins (PAs), are closely related to the bitterness of tea beverages. Monomeric flavan-3-ols are characteristic flavor compounds in tea. Increasing the content of PAs and anthocyanins enhances the resistance of tea plants to pathogen invasion but decreases the quality of tea beverages. MATE family transporters play a critical role in transferring monomeric flavan-3-ols and anthocyanins into vacuoles for storage or subsequent condensation into PAs. Their activities modulate the ratio of monomeric flavan-3-ols to PAs and increase anthocyanin content in tea plants. In this study, it was observed that the gene expression and protein phosphorylation level of the MATE transporter CsTT12, a vacuole-localized flavonoid transporter, were notably upregulated following exogenous sucrose treatment, promoting PA synthesis in tea plants. Further analysis revealed that overexpression of CsTT12 and CsTT12S17D significantly increased the content of anthocyanins and PAs in plants, whereas CsTT12S17A did not. In CsTT12 knockdown plants, PA's accumulation decreased significantly, while monomeric catechin content increased. Moreover, phosphorylation modification enhanced the vacuolar membrane localization of CsTT12, whereas dephosphorylation weakened its vacuolar membrane localization. This study uncovers the crucial role of phosphorylation in flavonoid biosynthesis and provides insights into balancing quality improvements and resistance enhancement.

Targeted regulation of 5-aminolevulinic acid enhances flavonoids, anthocyanins and proanthocyanidins accumulation in Vitis davidii callus

BackgroundSpine grape (Vitis davidii) is a promising source of high-quality anthocyanins, with vast potential for application in food, pharmaceutical, and cosmetic industries. However, their availability is limited by resource constraints. Plant cell culture has emerged as a valuable approach for anthocyanin production and serves as an ideal model to investigate the regulation of anthocyanin biosynthesis. Elicitors are employed to achieve targeted enhancement of anthocyanin biosynthesis. The present study investigated the impact of 5-aminolevulinic acid (ALA) as an elicitor on the accumulation of anthocyanins and flavonoids during spine grape callus growth. Specifically, we examined the effects of ALA on anthocyanin and its component accumulation in callus, and biosynthetic anthocyanin gene expression.ResultsALA at 25 µg/L increased the biomass of spine grape callus. ALA induction enhanced the levels of flavonoids, anthocyanins and proanthocyanidins in callus, with maximum values reaching 911.11 mg/100 g DW, 604.60 mg/100 g DW, and 5357.00 mg/100 g DW, respectively, after callus culture for 45 days. Notably, those levels were 1.47-, 1.93- and 1.83-fold higher than controls. ALA induction modulated the flavonoid profile, and among 97 differential flavonoid metabolites differing from controls, 77 were upregulated and 20 were downregulated. Six kinds of anthocyanins, namely cyanidin (8), delphinidin (6), peonidin (5), malvidin (4), petunidin (3) and pelargonidin (3), were detected in callus, with peonidin most abundant. Compared with controls, anthocyanin components were increased in ALA-treated callus. The key genes PAL1, PAL2, PAL4, CHI, CHS3, F3’H, F3H, FLS, DFR, UFGT, MYBA1, LDOX, OMT3, GT1 and ACT involved in anthocyanin biosynthesis were upregulated following ALA treatment, resulting in anthocyanin accumulation.ConclusionThis study revealed a novel mode of ALA-mediated promotion of plant anthocyanin biosynthesis and accumulation at the cellular level, and a strategy for enhancing anthocyanin content in spine grape callus. The findings advance commercial-scale production of anthocyanins via spine grape callus culture. we also explored the accumulation patterns of flavonoids and anthocyanins under ALA treatment. Augmentation of anthocyanins coincided with elevated expression levels of most genes involved in anthocyanin biosynthesis within spine grape callus following ALA treatment.

Integrated Metabolome and Transcriptome Analyses Reveal the Mechanisms Regulating Flavonoid Biosynthesis in Blueberry Leaves under Salt Stress

The flavonoids play important roles in plant salt tolerance. Blueberries (Vaccinium spp.) are extremely sensitive to soil salt increases. Therefore, improving the salt resistance of blueberries by increasing the flavonoid content is crucial for the development of the blueberry industry. To explore the underlying molecular mechanism, we performed an integrated analysis of the metabolome and transcriptome of blueberry leaves under salt stress. We identified 525 differentially accumulated metabolites (DAMs) under salt stress vs. control treatment, primarily including members of the flavonoid class. We also identified 20,920 differentially expressed genes (DEGs) based on transcriptome data; of these, 568 differentially expressed transcription factors (TFs) were annotated, and bHLH123, OsHSP20, and HSP20 TFs might be responsible for blueberry leaf salt tolerance. DEGs involved in the flavonoid biosynthesis pathway were significantly enriched at almost all stages of salt stress. Salt treatment upregulated the expression of most flavonoid biosynthetic pathway genes and promoted the accumulation of flavonols, flavonol glycosides, flavans, proanthocyanidins, and anthocyanins. Correlation analysis suggested that 4-coumarate CoA ligases (4CL5 and 4CL1) play important roles in the accumulation of flavonols (quercetin and pinoquercetin) and flavan-3-ol (epicatechin and prodelphinidin C2) under salt stress, respectively. The flavonoid 3′5′-hydroxylases (F3′5′H) regulate anthocyanin (cyanidin 3-O-beta-D-sambubioside and delphinidin-3-O-glucoside chloride) biosynthesis, and leucoanthocyanidin reductases (LAR) are crucial for the biosynthesis of epicatechin and prodelphinidin C2 during salt stress. Taken together, it is one of the future breeding goals to cultivate salt-resistant blueberry varieties by increasing the expression of flavonoid biosynthetic genes, especially 4CL, F3′5′H, and LAR genes, to promote flavonoid content in blueberry leaves.

Candidate Genes for Brown Fiber in Cotton Revealed Among the R2R3-Myb and bHLH-Myc Gene Families

ABSTRACT Naturally colored cotton varieties attract investigators due to their eco-friendly and hypoallergenic properties. Genetic mechanisms, determining control of pigment biosynthesis pathway in cotton fiber, are being intensively studied nowadays. However, the knowledge, explaining control of proanthocyanidins accumulation in cotton fiber by the MBW complex (R2R3-Myb, bHLH-Myc and WD40), is insufficient. In this connection, we focused on the regulatory genes from a number of the most likely ones involved in proanthocyanidins biosynthesis. We used the Sanger sequencing approach to reveal the allelic diversity of the R2R3-Myb and bHLH-Myc genes among contrast genotype accessions including various fiber shades and hypocotyl color. Having used the number of bioinformatics tools, we concluded that the replacement of conserved tryptophan residue abolished the DNA-binding activity of the GhMYB10-A transcription factor and this, probably, can be associated with the light brown fiber phenotype. Deeper studies are required to definitely establish the GhMYB10-A function. However, our achievements can be practically applied in marker-assisted breeding of cotton varieties with naturally colored fiber.

DkDTX1/MATE1 mediates the accumulation of proanthocyanidin and affects astringency in persimmon.

Proanthocyanidins (PAs) is a kind of polyphenols widely distributed in plants, and their astringent properties can protect plants from herbivores and regulate fruit taste. There is a great difference in PA composition between astringent (A)-type and nonastringent (NA)-type persimmon. Here, we studied the potential of DkDTX1/MATE1 in regulating PAs composition through its preferred transport in persimmon fruit. The results of fluorescence microscope showed that the DkDTX1/MATE1 green fluorescence overlapped with the blue light emitted by PA. Overexpression of DkDTX1/MATE1 in persimmon leaves not only significantly increase the concentrations of PA, but also upregulated the expression of PA biosynthesis pathway genes. Further overexpression of DkDTX1/MATE1 in persimmon fruit discs and stable genetic transformation of DkDTX1/MATE1 also led to PA concentrations increased. Molecular docking and transporter assays showed that DkDTX1/MATE1 preferentially transported catechin, epicatechin gallate and epigallocatechin gallate. DkDTX1/MATE1 mainly bound to the PA precursors via serine at position 68. Our findings indicate that DkDTX1/MATE1 play a role in the accumulation of PAs in early stage of fruit development and affects the astringency of persimmon through preferential transport PA precursors, which provided a theoretical basis for the future use of metabolic engineering to regulate the composition of PAs in persimmon.

Functional characterization of TrGSTF15, a glutathione S-transferase gene family member, on the transport and accumulation of anthocyanins and proanthocyanidins in Trifolium repens

Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites in plants, high contents of which are an important goal for quality breeding of white clover (Trifolium repens). However, the involvement of glutathione S-transferase (GST) in the transport of anthocyanins and PAs remains unexplored in white clover. This study identified 153 different TrGSTs in white clover. At the transcriptional level, compared to other TrGSTFs, TrGSTF10 and TrGSTF15 are highly expressed in the ‘Purple’ white clover, and they may work with the anthocyanin biosynthesis structural genes CHS and CHI to contribute to pigment buildup in white clover. Subcellular localization confirmed that TrGSTF10 and TrGSTF15 are located in the cytoplasm. Additionally, molecular docking experiments showed that TrGSTF10 and TrGSTF15 have similar binding affinity with two flavonoid monomers. Overexpression of TrGSTF15 complemented the deficiency of anthocyanin coloring and PA accumulation in the Arabidopsis tt19 mutant. The initial findings of this research indicate that TrGSTF15 encodes an important transporter of anthocyanin and PA in white clover, thus providing a new perspective for the further exploration of related transport and regulatory mechanisms.

The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat.

Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB-bHLH-WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain-leucine zipper (HD-ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production.

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.

Regulatory Mechanism of Proanthocyanidins in Grape Peels Using vvi-miR828a and Its Target Gene VvMYBPA1.

Anthocyanins and proanthocyanidins are considered to be essential secondary metabolites in grapes and are used to regulate metabolic processes, while miRNAs are involved in their synthesis of anthocyanins and proanthocyanidins to regulate metabolic processes. The present research work was carried out to investigate the underlying regulatory mechanism of target genes in the grape cultivars 'Italia' and 'Benitaka'. miRNA and transnscriptomic sequencing technology were employed to characterize both the profiles of miRNAs and the transcripts of grape peels at 10 and 11 weeks post flowering (10 wpf and 11 wpf). The results revealed that the expression level of vvi-miR828a in 'Italia' at 10 and 11 wpf was significantly higher than that in 'Benitaka'. miRNA-seq analysis predicted MYBPA1 to be the target gene of vvi-miR828a. In transcriptome analysis, the expression level of the VvMYBPA1 gene in 'Benitaka' was significantly higher than that in 'Italia'; in addition, the TPM values (expression levels) of VvMYBPA1 and miR828a also showed an evident negative correlation. The determination of the proanthocyanidin (PA) content in 'Italia' and 'Benitaka' peels at 11 wpf demonstrated that the PA content of 'Benitaka' was significantly higher than that of 'Italia'. The outcomes of RT-qRCR analysis exhibited that the expression levels of the VdPAL, VdCHS, VdCHI, VdDFR, VdMYB5b, VdANR, and VdMYBPA1 genes related anthocyanin and proanthocyanidin pathways were reduced, while the expression levels of all of the above genes were increased after the transient expression of the VvMYBPA1 vector into grape leaves. The results of the transient overexpression experiment of vvi-miR828a before the veraison period of strawberry fruits showed that vvi-miR828a can significantly slow down the coloration of strawberries. The vvi-miR828a negatively regulates the accumulation of proanthocyanidins in grape fruits by inhibiting the expression of VvMYBPA1.

A structural variation in the promoter of the leucoanthocyanidin reductase gene AaLAR1 enhances freezing tolerance by modulating proanthocyanidin accumulation in kiwifruit (Actinidia arguta).

Proanthocyanidins (PAs) are importantmetabolites that enhance freezing tolerance of plants. Actinidia arguta, especially freezing-tolerant germplasms, accumulate abundant PAs in dormant shoots and thereby enhance freezing tolerance, but the underlying mechanism is unknown. In this study, we used two A. arguta with contrasting cold-resistant phenotypes,KL and RB, to explore the mechanisms in response to cold tolerance. We determined that a leucoanthocyanidin reductase gene (AaLAR1) was more highly expressed in freezing-tolerant KL than in freezing-sensitive RB. Moreover, overexpressing AaLAR1 in kiwifruit promoted PAs biosynthesis and enhanced cold tolerance. The AaLAR1 promoters of various A. arguta germplasms differ due to the presence of a 60-bp deletion in cold-tolerant genotypes that forms a functional binding site for MYC-type transcription factor. Yeast one-hybrid and two-hybrid, dual-luciferase reporter, bimolecular fluorescence complementationand coimmunoprecipitation assays indicated that the AaMYC2a binds to the MYC-core cis-element in the AaLAR1 promoter with the assistance of AaMYB5a, thereby promoting PAs accumulation in the shoots of cold-tolerant kiwifruit. We conclude that the variation in the AaLAR1 promoter and the AaMYC2a-AaMYB5a-AaLAR1 module shape freezing tolerance in A. arguta. The identification of a key structural variation in the AaLAR1 promoter offers a new target for resistance breeding of kiwifruit.

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.

BrTTG1 regulates seed coat proanthocyanidin formation through a direct interaction with structural gene promoters of flavonoid pathway and glutathione S-transferases in Brassica rapa L.

Seed coat color is a significant agronomic trait in horticultural crops such as Brassica rapa which is characterized by brown or yellow seed coat coloration. Previous Brassica rapa studies have shown that BrTTG1 is responsible for seed coat proanthocyanidin formation, which is dependent on the MYB-bHLH-WD40 complex, whereas some studies have reported that TRANSPARENT TESTA GLABRA 1 (TTG1) directly interacts with the structural gene promoters of the flavonoid pathway. Herein, the brown-seeded inbred B147 and ttg1 yellow-seeded inbred B80 mutants were used as plant materials for gene expression level analysis, gene promoter clone and transient overexpression. The analysis identified eleven structural genes involved in the flavonoid biosynthesis pathway, which are potentially responsible for BrTTG1- dependent seed coat proanthocyanidin formation. The promoters of these genes were cloned and cis-acting elements were identified. Yeast one-hybrid and dual-luciferase assays confirmed that BrTTG1 directly and independently interacted with proCHS-Bra008792, proDFR-Bra027457, proTT12-Bra003361, proTT19-Bra008570, proTT19-Bra023602 and proAHA10-Bra016610. A TTG1-binding motif (RTWWGTRGM) was also identified. Overexpression of TTG1 in the yellow-seed B. rapa inbred induced proanthocyanidin accumulation by increasing the expression levels of related genes. Our study unveiled, for the first time, the direct interaction between TTG1 and the promoters of the flavonoid biosynthesis pathway structural genes and glutathione S-transferases in Brassica rapa. Additionally, we have identified a novel TTG1-binding motif, providing a basis for further exploration into the function of TTG1 and the accumulation of proanthocyanidins in seed coats.

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.

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.

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.