VvMYB15 and VvWRKY40 Positively Co-regulated Anthocyanin Biosynthesis in Grape Berries in Response to Root Restriction

In most grapevine planting regions, especially in south of China, plenty of rainfall and high water level underground are the characteristic of the area, a series of problem during fruit ripening easily caused poor color quality. Thereby affecting fruit quality, yield and economic benefits. The accumulation of anthocyanin is regulated by transcriptional regulatory factor and a series of cultivation measures, root restriction can make plants in the environment of stress and stress relief, root restriction induced the higher expression of VvMYB15 and VvWRKY40, and consistent with anthocyanin accumulation. Whether and how root restriction-inducible VvMYB15 and VvWRKY40 transcription factor regulate anthocyanin synthesis in grape berry is still unclear. In this study, we identified that the transient overexpression of VvMYB15 and VvWRKY40 alone or both in strawberry fruits and grape berries can promote anthocyanin accumulation and increase the expression level of anthocyanin biosynthetic genes, indicating VvMYB15 and VvWRKY40 play a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both VvMYB15 and VvWRKY40 specifically bind to the promoter region of VvF3′5′H and VvUFGT, and the expression of VvF3′5′H and VvUFGT is further activated through the heterodimer formation between VvMYB15 and VvWRKY40. Finally, we confirmed that VvMYB15 promoted anthocyanin accumulation by interacting with VvWRKY40 in grape berries, our findings provide insights into a mechanism involving the synergistic regulation of root restriction-dependent coloration and biosynthesis via a VvMYB15 and VvWRKY40 alone or both in grape berries.

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FvbHLH9 Functions as a Positive Regulator of Anthocyanin Biosynthesis by Forming a HY5–bHLH9 Transcription Complex in Strawberry Fruits

Plant and Cell Physiology ◽

10.1093/pcp/pcaa010 ◽

2020 ◽

Vol 61 (4) ◽

pp. 826-837 ◽

Cited By ~ 4

Author(s):

Yang Li ◽

Pengbo Xu ◽

Guanqun Chen ◽

Jun Wu ◽

Zhongchi Liu ◽

...

Keyword(s):

Transcription Factor ◽

Promoter Region ◽

Anthocyanin Biosynthesis ◽

Anthocyanin Synthesis ◽

Bhlh Transcription Factor ◽

Anthocyanin Accumulation ◽

Positive Regulator ◽

Key Enzyme ◽

Synergistic Regulation ◽

Transient Overexpression

Abstract Anthocyanin accumulation is transcriptionally regulated by the MYB–bHLH–WD40 complex. Light is indispensable for anthocyanin accumulation, and light-inducible MYB and HY5 were considered to promote anthocyanin accumulation in many fruits. Whether and how light-inducible bHLH transcription factor and HY5 regulate anthocyanin synthesis in strawberry is unknown. In this study, we identified a bHLH transcription factor, FvbHLH9, which was induced by light as well as FvHY5, and found that, similar to FvHY5, the transient overexpression and interference FvbHLH9 in strawberry fruits can promote and decrease anthocyanin accumulation, respectively, indicating FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis. Furthermore, we confirmed that both FvHY5 and FvbHLH9 specifically bind to the promoter region of some key enzyme genes, including FvDFR, and the expression of FvDFR was activated through the heterodimer formation between FvHY5 and FvbHLH9. Finally, we confirmed that FvbHLH9-promoted anthocyanin accumulation is dependent on HY5–bHLH heterodimerisation in Arabidopsis. Our findings provide insights into a mechanism involving the synergistic regulation of light-dependent coloration and anthocyanin biosynthesis via a HY5–bHLH heterodimer formed by the interaction of FvHY5 and FvbHLH9 in strawberry fruits.

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Characterization and Action Mechanism Analysis of VvmiR156b/c/d-VvSPL9 Module Responding to Multiple-Hormone Signals in the Modulation of Grape Berry Color Formation

Foods ◽

10.3390/foods10040896 ◽

2021 ◽

Vol 10 (4) ◽

pp. 896

Author(s):

Ziwen Su ◽

Xicheng Wang ◽

Xuxian Xuan ◽

Zilu Sheng ◽

Haoran Jia ◽

...

Keyword(s):

Color Change ◽

Grape Berry ◽

Anthocyanin Synthesis ◽

Structural Genes ◽

Expression Levels ◽

Color Formation ◽

Grape Berries ◽

Ripening Stages ◽

Color Conversion ◽

Hormone Signals

In recent years, more and more reports have shown that the miR156-SPL module can participate in the regulation of anthocyanin synthesis in plants. However, little is known about how this module responds to hormonal signals manipulating this process in grapes. In this study, exogenous GA, ABA, MeJA, and NAA were used to treat the ‘Wink’ grape berries before color conversion, anthocyanin and other related quality physiological indexes (such as sugar, aroma) were determined, and spatio-temporal expression patterns of related genes were analyzed. The results showed that the expression levels of VvmiR156b/c/d showed a gradually rising trend with the ripening and color formation of grape berries, and the highest expression levels were detected at day 28 after treatment, while the expression level of VvSPL9 exhibited an opposite trend as a whole, which further verifies that VvmiR156b/c/d can negatively regulate VvSPL9. Besides, VvmiR156b/c/d was positively correlated with anthocyanin content and related genes levels, while the expression pattern of VvSPL9 showed a negative correlation. Analysis of promoter cis-elements and GUS staining showed that VvmiR156b/c/d contained a large number of hormone response cis-elements (ABA, GA, SA, MeJA, and NAA) and were involved in hormone regulation. Exogenous ABA and MeJA treatments significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion and made grape berries quickly colored. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated in the middle color-change and ripening stages, therefore GA mainly modulated grape berry coloring in the middle- and late-ripening stages. Furthermore, NAA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes and delayed the peak expression of genes. Meanwhile, to further recognize the potential functions of VvmiR156b/c/d, the mature tomato transient trangenetic system was utilized in this work. Results showed that transient overexpression of VvmiR156b/c/d in tomato promoted fruit coloring and overexpression of VvSPL9 inhibited fruit coloration. Finally, a regulatory network of the VvmiR156b/c/d-VvSPL9 module responsive to hormones modulating anthocyanin synthesis was developed. In conclusion, VvmiR156b/c/d-mediated VvSPL9 participated in the formation of grape color in response to multi-hormone signals.

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The Isolation and Identification of Anthocyanin-Related GSTs in Chrysanthemum

Horticulturae ◽

10.3390/horticulturae7080231 ◽

2021 ◽

Vol 7 (8) ◽

pp. 231

Author(s):

Yajing Li ◽

Xiaofen Liu ◽

Fang Li ◽

Lili Xiang ◽

Kunsong Chen

Keyword(s):

Anthocyanin Biosynthesis ◽

Vital Role ◽

Luciferase Assay ◽

Amino Acid Residues ◽

Anthocyanin Accumulation ◽

Isolation And Identification ◽

Specific Amino Acid ◽

Protein Sequence Alignment ◽

Glutathione S Transferases ◽

Transient Overexpression

Anthocyanin is the crucial pigment for the coloration of red chrysanthemum flowers, which synthesizes in the cytosol and is transported to the vacuole for stable storage. In general, glutathione S-transferases (GSTs) play a vital role in this transport. To date, there is no functional GST reported in chrysanthemums. Here, a total of 94 CmGSTs were isolated from the chrysanthemum genome, with phylogenetic analysis suggesting that 16 members of them were clustered into the Phi subgroup which was related to anthocyanin transport. Among them, the expression of CmGST1 was positively correlated with anthocyanin accumulation. Protein sequence alignment revealed that CmGST1 included anthocyanin-related GST-specific amino acid residues. Further transient overexpression experiments in tobacco leaves showed that CmGST1 could promote anthocyanin accumulation. In addition, a dual-luciferase assay demonstrated that CmGST1 could be regulated by CmMYB6, CmbHLH2 and CmMYB#7, which was reported to be related to anthocyanin biosynthesis. Taken together, we suggested that CmGST1 played a key role in anthocyanin transport and accumulation in chrysanthemums.

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Improving the Anthocyanin Accumulation of Hypocotyls in Radish Sprouts by Hemin-induced NO

10.21203/rs.3.rs-117868/v1 ◽

2020 ◽

Author(s):

Nana Su ◽

Ze Liu ◽

Hui Chen ◽

Mengyang Niu ◽

Jin Cui

Keyword(s):

Nitric Oxide ◽

Transcription Factors ◽

Anthocyanin Biosynthesis ◽

Reductase Activity ◽

Specific Inhibitor ◽

Anthocyanin Synthesis ◽

Zinc Protoporphyrin ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Radish Sprouts

Abstract Background: The biosynthesis of anthocyanin in the hypocotyls of radish (Raphanus sativus L.) sprouts was enhanced by hemin in our preliminary experiments, but the underlying mechanism is unclear. Here, we found that NO (nitric oxide) exerted an essential role in Hemin-regulated anthocyanin biosynthesis, which was supported by the following results.Results: Hemin boosted anthocyanin as well as NO content. NO-scavenger cPTIO (carboxy-PTIO) significantly attenuated hemin-induced increase of anthocyanin content, transcripts of anthocyanin synthesis related genes and positive transcription factors, implying that NO played a prominent role during hemin-induced anthocyanin biosynthesis. Hemin specific inhibitor ZnPP (Zinc Protoporphyrin) strongly reduced anthocyanin content, while, NO donor SNP (Sodium Nitroprusside) addition considerably reversed this inhibition and by contrast, resulted in a significant increase in anthocyanin accumulation, closely paralleling the transcripts of structural genes and transcription factors. Moreover, NO content, NR (nitrate reductase) activity and expression level of NOA (nitric oxide associated factor) were up-regulated by Hemin. Conclusions:Those consequences indicated that NO might work downstream in Hemin-heightened anthocyanin accumulation in radish sprouts.

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Transcription Factor VviMYB86 Oppositely Regulates Proanthocyanidin and Anthocyanin Biosynthesis in Grape Berries

Frontiers in Plant Science ◽

10.3389/fpls.2020.613677 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jing Cheng ◽

Keji Yu ◽

Ying Shi ◽

Jun Wang ◽

Changqing Duan

Keyword(s):

Anthocyanin Biosynthesis ◽

Gene Expression Pattern ◽

Anthocyanin Synthesis ◽

Grape Berries ◽

Flavonoid Synthesis ◽

Leucoanthocyanidin Reductase ◽

Arabidopsis Protoplast ◽

R2r3 Myb

Proanthocyanidins (PAs) and anthocyanins are two vital groups of flavonoid compounds for grape berries and red wines. Several transcription factors (TFs) have been identified to be involved in regulating PA and anthocyanin biosynthesis in grape berries. However, research on TFs with different regulatory mechanisms for these two biosynthesis branches in grapes remains limited. In this study, we identified an R2R3-MYB TF, VviMYB86, whose spatiotemporal gene expression pattern in grape berries coincided well with PA accumulation but contrasted with anthocyanin synthesis. Both in vivo and in vitro experiments verified that VviMYB86 positively regulated PA biosynthesis, primarily by upregulating the expression of the two leucoanthocyanidin reductase (LAR) genes in the Arabidopsis protoplast system, as well as in VviMYB86-overexpressing grape callus cultured under 24 h of darkness. Moreover, VviMYB86 was observed to repress the anthocyanin biosynthesis branch in grapes by downregulating the transcript levels of VviANS and VviUFGT. Overall, VviMYB86 is indicated to have a broad effect on flavonoid synthesis in grape berries. The results of this study will help elucidate the regulatory mechanism governing the expression of the two LAR genes in grape berries and provide new insights into the regulation of PA and anthocyanin biosynthesis in grape berries.

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Calcium Positively Mediates Blue Light-Induced Anthocyanin Accumulation in Hypocotyl of Soybean Sprouts

Frontiers in Plant Science ◽

10.3389/fpls.2021.662091 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gang Hu ◽

Xiaomeng Yue ◽

Jinxue Song ◽

Guipei Xing ◽

Jun Chen ◽

...

Keyword(s):

Blue Light ◽

Anthocyanin Biosynthesis ◽

Calcium Supplementation ◽

Distribution Patterns ◽

Anthocyanin Synthesis ◽

Regulation Mechanism ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Soybean Sprouts ◽

Expression Of Genes

Soybean sprouts are a flavorful microgreen that can be eaten all year round and are widely favored in Southeast Asia. In this study, the regulatory mechanism of calcium on anthocyanin biosynthesis in soybean sprouts under blue light was investigated. The results showed that blue light, with a short wavelength, effectively induced anthocyanin accumulation in the hypocotyl of soybean sprout cultivar “Dongnong 690.” Calcium supplementation further enhanced anthocyanin content, which was obviously inhibited by LaCl3 and neomycin treatment. Moreover, exogenous calcium changed the metabolism of anthocyanins, and seven anthocyanin compounds were detected. The trend of calcium fluorescence intensity in hypocotyl cells, as well as that of the inositol 1,4,5-trisphosphate and calmodulin content, was consistent with that of anthocyanins content. Specific spatial distribution patterns of calcium antimonate precipitation were observed in the ultrastructure of hypocotyl cells under different conditions. Furthermore, calcium application upregulated the expression of genes related to anthocyanin biosynthesis, and calcium inhibitors suppressed these genes. Finally, transcriptomics was performed to gain global insights into the molecular regulation mechanism of calcium-associated anthocyanin production. Genes from the flavonoid biosynthesis pathway were distinctly enriched among the differentially expressed genes, and weighted gene co-expression network analysis showed that two MYBs were related to the accumulation of anthocyanins. These results indicated that calcium released from apoplast and intracellular stores in specific spatial-temporal features promote blue light-induced anthocyanin accumulation by upregulation of the expression of genes related to anthocyanin synthesis of “Dongnong 690” hypocotyl. The findings deepen the understanding of the calcium regulation mechanism of blue light-induced anthocyanin accumulation in soybean sprouts, which will help growers produce high-quality foods beneficial for human health.

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Transcriptome Analysis Reveals Candidate Genes Related to Anthocyanin Biosynthesis in Different Carrot Genotypes and Tissues

Plants ◽

10.3390/plants9030344 ◽

2020 ◽

Vol 9 (3) ◽

pp. 344 ◽

Cited By ~ 1

Author(s):

Geng Meng ◽

Sabine K. Clausen ◽

Søren K. Rasmussen

Keyword(s):

Candidate Genes ◽

High Performance ◽

Anthocyanin Biosynthesis ◽

High Performance Liquid Chromatographic ◽

Anthocyanin Synthesis ◽

Natural Food ◽

Sequencing Analysis ◽

Anthocyanin Accumulation ◽

Glutathione S Transferase ◽

The Individual

Black carrots are characterized by a significant amount of anthocyanins, which are not only a good source of natural food colorant, but can also provide many health benefits to humans. In the present work, taproots of different carrot genotypes were used to identify the candidate genes related to anthocyanin synthesis, with particular a focus on R2R3MYB, bHLH transcription factors, and glutathione S-transferase gene (GST). The RNA-sequencing analysis (RNA-Seq) showed that DcMYB6 and DcMYB7 had a genotypic dependent expression and they are likely involved in the regulation of anthocyanin biosynthesis. They were specifically upregulated in solid black taproots, including both black phloem and xylem. DcbHLH3 (LOC108204485) was upregulated in all black samples compared with the orange ones. We also found that GST1 (LOC108205254) might be an important anthocyanin transporter, and its upregulated expression resulted in the increasing of vacuolar anthocyanin accumulation in black samples. Moreover, high performance liquid chromatographic (HPLC) analysis and liquid chromatography coupled to mass spectrometry (LC-MS) were used to identify the individual anthocyanin in the purple tissues of two carrot cultivars. The results showed that five main anthocyanin compounds and the most abundant anthocyanin were the same in different tissues, while the second-highest anthocyanin between three tissues was different, even in the same cultivar. In conclusion, this study combined anthocyanin profiles and comparative transcriptomic analysis to identify candidate genes involved in anthocyanin biosynthesis in carrots, thus providing a better foundation for improving anthocyanin accumulation in carrots as a source of colorants.

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Dynamic Changes in Anthocyanin Accumulation and Cellular Antioxidant Activities in Two Varieties of Grape Berries during Fruit Maturation under Different Climates

Molecules ◽

10.3390/molecules27020384 ◽

2022 ◽

Vol 27 (2) ◽

pp. 384

Author(s):

Liuwei Qin ◽

Hui Xie ◽

Nan Xiang ◽

Min Wang ◽

Shouan Han ◽

...

Keyword(s):

Antioxidant Activity ◽

Developmental Stages ◽

Antioxidant Activities ◽

Anthocyanin Biosynthesis ◽

Climatic Conditions ◽

Anthocyanin Accumulation ◽

Grape Berries ◽

Different Climates ◽

Quality Formation

As popularly consumed fruit berries, grapes are widely planted and processed into products, such as raisins and wine. In order to identify the influences of different climatic conditions on grape coloring and quality formation, we selected two common varieties of grape berries, ‘Red Globe’ and ‘Xin Yu’, for investigation. Grapes were separately grown in different climates, such as a temperate continental arid climate and a temperate continental desert climate, in Urumqi and Turpan, China, for five developmental stages. As measured, the average daily temperature and light intensity were lower in Urumqi. Urumqi grape berries had a lower brightness value (L*) and a higher red-green value (a*) when compared to Turpan’s. A RT-qPCR analysis revealed higher transcriptions of key genes related to anthocyanin biosynthesis in Urumqi grape berries, which was consistent with the more abundant phenolic substances, especially anthocyanins. The maximum antioxidant activity in vitro and cellular antioxidant activity of grape berries were also observed in Urumqi grape berries. These findings enclosed the influence of climate on anthocyanin accumulation and the antioxidant capacity of grapes, which might enlarge our knowledge on the quality formation of grape berries and might also be helpful for cultivating grapes with higher nutritional value.

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The Light-Induced WD40-Repeat Transcription Factor DcTTG1 Regulates Anthocyanin Biosynthesis in Dendrobium candidum

Frontiers in Plant Science ◽

10.3389/fpls.2021.633333 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ning Jia ◽

Jingjing Wang ◽

Yajuan Wang ◽

Wei Ye ◽

Jiameng Liu ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Anthocyanin Biosynthesis ◽

Raw Material ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Wd40 Repeat ◽

Strong Light ◽

Dendrobium Candidum

Dendrobium candidum is used as a traditional Chinese medicine and as a raw material in functional foods. D. candidum stems are green or red, and red stems are richer in anthocyanins. Light is an important environmental factor that induces anthocyanin accumulation in D. candidum. However, the underlying molecular mechanisms have not been fully unraveled. In this study, we exposed D. candidum seedlings to two different light intensities and found that strong light increased the anthocyanin content and the expression of genes involved in anthocyanin biosynthesis. Through transcriptome profiling and expression analysis, we identified a WD40-repeat transcription factor, DcTTG1, whose expression is induced by light. Yeast one-hybrid assays showed that DcTTG1 binds to the promoters of DcCHS2, DcCHI, DcF3H, and DcF3′H, and a transient GUS activity assay indicated that DcTTG1 can induce their expression. In addition, DcTTG1 complemented the anthocyanin deficiency phenotype of the Arabidopsis thaliana ttg1-13 mutant. Collectively, our results suggest that light promotes anthocyanin accumulation in D. candidum seedlings via the upregulation of DcTTG1, which induces anthocyanin synthesis-related gene expression.

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MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple

Tree Physiology ◽

10.1093/treephys/tpaa156 ◽

2020 ◽

Author(s):

Wen-Fang Li ◽

Gai-Xing Ning ◽

Cun-Wu Zuo ◽

Ming-Yu Chu ◽

Shi-Jin Yang ◽

...

Keyword(s):

Dna Methylation ◽

Transcription Factors ◽

Promoter Region ◽

Anthocyanin Biosynthesis ◽

Apple Fruit ◽

Anthocyanin Synthesis ◽

Mrna Levels ◽

Anthocyanin Accumulation ◽

Dna Hypomethylation ◽

Fruit Skin

Abstract Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. ‘Red Delicious’ (G0) and its four continuous-generation bud sport mutants including ‘Starking Red’ (G1), ‘Starkrimson’ (G2), ‘Campbell Redchief’ (G3) and ‘Vallee spur’ (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin colour traits remain elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in ‘Golden Delicious’ fruits, ‘Gala’ calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in ‘Red Fuji’ apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

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