Genome-wide characterization and analysis of bHLH transcription factors related to anthocyanin biosynthesis in spine grapes (Vitis davidii).
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ABSTRACT: As one of the largest transcription factor family, basic helix-loop-helix (bHLH) transcription factor family plays an important role in plant metabolism, physiology and growth. Berry color is one of the important factors that determine grape quality. However, the bHLH transcription factor family's function in anthocyanin synthesis of grape berry has not been studied systematically. We identified 115 bHLH transcription factors in grape genome and phylogenetic analysis indicated that bHLH family could be classified into 25 subfamilies. First, we screened six candidate genes by bioinformatics analysis and expression analysis. We found one of the candidate genes VdbHLH037 belonged to III (f) subfamily and interacted with genes related to anthocyanin synthesis through phylogenetic analysis and interaction network prediction. Therefore, we speculated that VdbHLH037 participated in the anthocyanin synthesis process. To confirm this, we transiently expressed VdbHLH037 in grape and Arabidopsis transformation. Compared with the control, transgenic materials can accumulate more anthocyanins. These results provide a good base to study the function of the VdbHLH family in anthocyanin synthesis of grape berry.
Project description:Background: Spine grape has gained attention in the field of wine science due to its good growth characteristics. Spine grape wine has been made by local residents for a long time. However, the scientific evaluation of spine wine has not been systemically documented compared to Vitis vinifera grape wines Methods: We compared 11 spine wines from south China (W1-W11) with 7 high-quality international wines (W12-W18). The total phenolic content, the total anothcyanin content and the antioxidant activity of these wines were analyzed and compared. Meanwhile, anthocyanin profiles of these wines were also documented. Results: Compared with other wines most of the spine wines had a strong red intensity with a blue hue. Malvidin-3,5-O-diglucoside and malvidin-3-O-(6-O-coumaroyl)-glucoside-5-glucoside appeared to be the major anthocyanins in these wines. The scavenging capacity analyses of these wines using ABTS, DPPH, and CUPRAC assays indicated that spine wines possessed high antioxidant properties, especially spine wine W3, W4, W6 and W8. Their high antioxidant properties were mainly related to the high levels of the total phenolic content and anthocyanins. Conclusion: These results suggested that spine wine might be considered a good wine source for the Chinese wine industry and provided useful information on the knowledge of spine grape.
Project description:The color of berry skin is an important economic trait for grape and is essentially determined by the components and content of anthocyanins. The fruit color of Chinese wild grapes is generally black, and the profile of anthocyanins in Chinese wild grapes is significantly different from that of Vitis vinifera. However, V. davidii is the only species that possesses white berry varieties among Chinese wild grape species. Thus, we performed a transcriptomic analysis to compare the difference of transcriptional level in black and white V. davidii, in order to find some key genes that are related to anthocyanins accumulation in V. davidii.The results of anthocyanins detection revealed that 3,5-O-diglucoside anthocyanins is the predominant anthocyanins in V. davidii. It showed obvious differences from V. vinifera in the profile of the composition of anthocyanins. The transcriptome sequencing by Illumina mRNA-Seq technology generated an average of 57 million 100-base pair clean reads from each sample. Differential gene expression analysis revealed thousands of differential expression genes (DEGs) in the pairwise comparison of different fruit developmental stages between and within black and white V. davidii. After the analysis of functional category enrichment and differential expression patterns of DEGs, 46 genes were selected as the candidate genes. Some genes have been reported as being related to anthocyanins accumulation, and some genes were newly found in our study as probably being related to anthocyanins accumulation. We inferred that 3AT (VIT_03s0017g00870) played an important role in anthocyanin acylation, GST4 (VIT_04s0079g00690) and AM2 (VIT_16s0050g00910) played important roles in anthocyanins transport in V. davidii. The expression of some selected DEGs was further confirmed by quantitative real-time PCR (qRT-PCR).The present study investigated the transcriptomic profiles of berry skin from black and white spine grapes at three fruit developmental stages by Illumina mRNA-Seq technology. It revealed the variety specificity of anthocyanins accumulation in V. davidi at the transcriptional level. The data reported here will provide a valuable resource for understanding anthocyanins accumulation in grapes, especially in V. davidii.
Project description:Cinnamomum camphora is one of the most commonly used tree species in landscaping. Improving its ornamental traits, particularly bark and leaf colors, is one of the key breeding goals. The basic helix-loop-helix (bHLH) transcription factors (TFs) are crucial in controlling anthocyanin biosynthesis in many plants. However, their role in C. camphora remains largely unknown. In this study, we identified 150 bHLH TFs (CcbHLHs) using natural mutant C. camphora 'Gantong 1', which has unusual bark and leaf colors. Phylogenetic analysis revealed that 150 CcbHLHs were divided into 26 subfamilies which shared similar gene structures and conserved motifs. According to the protein homology analysis, we identified four candidate CcbHLHs that were highly conserved compared to the TT8 protein in A. thaliana. These TFs are potentially involved in anthocyanin biosynthesis in C. camphora. RNA-seq analysis revealed specific expression patterns of CcbHLHs in different tissue types. Furthermore, we verified expression patterns of seven CcbHLHs (CcbHLH001, CcbHLH015, CcbHLH017, CcbHLH022, CcbHLH101, CcbHLH118, and CcbHLH134) in various tissue types at different growth stages using qRT-PCR. This study opens a new avenue for subsequent research on anthocyanin biosynthesis regulated by CcbHLH TFs in C. camphora.
Project description:Basic helix-loop-helix (bHLH) proteins are transcription factors (TFs) that have been shown to regulate anthocyanin biosynthesis in many plant species. However, the bHLH gene family in walnut (Juglans regia L.) has not yet been reported. In this study, 102 bHLH genes were identified in the walnut genome and were classified into 15 subfamilies according to sequence similarity and phylogenetic relationships. The gene structure, conserved domains, and chromosome location of the genes were analyzed by bioinformatic methods. Gene duplication analyses revealed that 42 JrbHLHs were involved in the expansion of the walnut bHLH gene family. We also characterized cis-regulatory elements of these genes and performed Gene Ontology enrichment analysis of gene functions, and examined protein-protein interactions. Four candidate genes (JrEGL1a, JrEGL1b, JrbHLHA1, and JrbHLHA2) were found to have high homology to genes encoding bHLH TFs involved in anthocyanin biosynthesis in other plants. RNA sequencing revealed tissue- and developmental stage-specific expression profiles and distinct expression patterns of JrbHLHs according to phenotype (red vs. green leaves) and developmental stage in red walnut hybrid progeny, which were confirmed by quantitative real-time PCR analysis. All four of the candidate JrbHLH proteins localized to the nucleus, consistent with a TF function. These results provide a basis for the functional characterization of bHLH genes and investigations on the molecular mechanisms of anthocyanin biosynthesis in red walnut.
Project description:The basic helix-loop-helix (bHLH) proteins compose one of the largest transcription factor (TF) families in plants, which play a vital role in regulating plant biological processes including growth and development, stress response, and secondary metabolite biosynthesis. Ipomoea aquatica is one of the most important nutrient-rich vegetables. Compared to the common green-stemmed I. aquatica, purple-stemmed I. aquatica has extremely high contents of anthocyanins. However, the information on bHLH genes in I. aquatica and their role in regulating anthocyanin accumulation is still unclear. In this study, we confirmed a total of 157 bHLH genes in the I. aquatica genome, which were classified into 23 subgroups according to their phylogenetic relationship with the bHLH of Arabidopsis thaliana (AtbHLH). Of these, 129 IabHLH genes were unevenly distributed across 15 chromosomes, while 28 IabHLH genes were spread on the scaffolds. Subcellular localization prediction revealed that most IabHLH proteins were localized in the nucleus, while some were in the chloroplast, extracellular space, and endomembrane system. Sequence analysis revealed conserved motif distribution and similar patterns of gene structure within IabHLH genes of the same subfamily. Analysis of gene duplication events indicated that DSD and WGD played a vital role in the IabHLH gene family expansion. Transcriptome analysis showed that the expression levels of 13 IabHLH genes were significantly different between the two varieties. Of these, the IabHLH027 had the highest expression fold change, and its expression level was dramatically higher in purple-stemmed I. aquatica than that in green-stemmed I. aquatica. All upregulated DEGs in purple-stemmed I. aquatica exhibited the same expression trends in both qRT-PCR and RNA-seq. Three downregulated genes including IabHLH142, IabHLH057, and IabHLH043 determined by RNA-seq had opposite expression trends of those detected by qRT-PCR. Analysis of the cis-acting elements in the promoter region of 13 differentially expressed genes indicated that light-responsive elements were the most, followed by phytohormone-responsive elements and stress-responsive elements, while plant growth and development-responsive elements were the least. Taken together, this work provides valuable clues for further exploring IabHLH function and facilitating the breeding of anthocyanin-rich functional varieties of I. aquatica.
Project description:Hybrid (Vitis vinifera ×Vitis labrusca) table grape cultivars grown in the subtropics often fail to accumulate sufficient anthocyanins to achieve good uniform berry color. Growers of V. vinifera table grapes in temperate regions generally use ethephon and, more recently, (S)-cis-abscisic acid (S-ABA) to overcome this problem. The objective of this study was to determine if S-ABA applications at different timings and concentrations have an effect on anthocyanin regulatory and biosynthetic genes, pigment accumulation, and berry color of the Selection 21 cultivar, a new V. vinifera ×V. labrusca hybrid seedless grape that presents lack of red color when grown in subtropical areas. Applications of S-ABA 400 mg/L resulted in a higher accumulation of total anthocyanins and of the individual anthocyaninsanthocyanins: delphinidin-3-glucoside, cyanidin-3-glucoside, peonidin-3-glucoside, and malvidin-3-glucoside in the berry skin and improved the color attributes of the berries. Treatment with two applications at 7 days after véraison (DAV) and 21 DAV of S-ABA 400 mg/L resulted in a higher accumulation of total anthocyanins in the skin of berries and increased the gene expression of CHI, F3H, DFR, and UFGT and of the VvMYBA1 and VvMYBA2 transcription factors in the seedless grape cultivar.
Project description:Salvia miltiorrhiza Bunge (Labiatae) is an emerging model plant for traditional medicine, and tanshinones are among the pharmacologically active constituents of this plant. Although extensive chemical and pharmaceutical studies of these compounds have been performed, studies on the basic helix-loop-helix (bHLH) transcription factors that regulate tanshinone biosynthesis are limited. In our study, 127 bHLH transcription factor genes were identified in the genome of S. miltiorrhiza, and phylogenetic analysis indicated that these SmbHLHs could be classified into 25 subfamilies. A total of 19 sequencing libraries were constructed for expression pattern analyses using RNA-Seq. Based on gene-specific expression patterns and up-regulated expression patterns in response to MeJA treatment, 7 bHLH genes were revealed as potentially involved in the regulation of tanshinone biosynthesis. Among them, the gene expression of SmbHLH37, SmbHLH74 and SmbHLH92 perfectly matches the accumulation pattern of tanshinone biosynthesis in S. miltiorrhiza. Our results provide a foundation for understanding the molecular basis and regulatory mechanisms of bHLH transcription factors in S. miltiorrhiza.
Project description:Hydroxycinnamylated anthocyanins (or simply 'acylated anthocyanins') increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families, Vitis rupestris B38 × 'Horizon' and 'Horizon' × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from 'Horizon' on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85-17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.
Project description:Native to China, spine grapes (Vitis davidii Foex) are an important wild grape species. Here, the quality characteristics of one white and three red spine grape clones were evaluated via targeted metabolomic and transcription level analysis. Xiangzhenzhu (XZZ) had the highest soluble sugar and organic acid content. Malvidin-3-acetyl-glucoside and cyanidin-3-glucoside were the characteristic anthocyanins in spine grapes, and significant differences in anthocyanin composition between different clones were detected. Anthocyanins were not detected in Baiyu (BY) grapes. The transcript levels of VdGST, VdF3'H, VdOMT, VdLDOX, and VdUFGT were significantly related to the anthocyanin biosynthesis and proportions. A total of 27 kinds of glycosidically bound volatiles (including alcohols, monoterpenes, esters, aldehydes, ketones, and phenolic acid) were identified in spine grapes, with Gaoshan #4 (G4) and BY grapes having the highest concentrations. The VdGT expression levels were closely related to glycosidically bound volatile concentrations. These results increase our understanding of the quality of wild spine grapes and further promote the development and use of wild grape resources.
Project description:Chrysanthemums (Chrysanthemum morifolium Ramat.) exhibit a variety of flower colors due to their differing abilities to accumulate anthocyanins. One MYB member, CmMYB6, has been verified as a transcription regulator of chrysanthemum genes involved in anthocyanin biosynthesis; however, the co-regulators for CmMYB6 remain unclear in chrysanthemum. Here, the expression pattern of CmbHLH2, which is clustered in the IIIf bHLH subgroup, was shown to be positively correlated with the anthocyanin content of cultivars with red, pink and yellow flower colors, respectively. CmbHLH2 significantly upregulated the CmDFR promoter and triggered anthocyanin accumulation when co-expressed with CmMYB6. Yeast one-hybrid analyses indicated that CmbHLH2 was able to bind directly to the CmDFR promoter. Moreover, yeast two-hybrid assays indicated protein-protein interaction between CmbHLH2 and CmMYB6. These results suggest that CmbHLH2 is the essential partner for CmMYB6 in regulating anthocyanin biosynthesis in chrysanthemum.