Light- and Temperature-Induced Expression of an R2R3-MYB Gene Regulates Anthocyanin Biosynthesis in Red-Fleshed Kiwifruit

The R2R3 MYB genes associated with the flavonoid/anthocyanidin pathway feature two repeats, and represent the most abundant classes of MYB genes in plants; however, the physiological role and regulatory function of most R2R3 MYBs remain poorly understood in kiwifruit (Actinidia). Here, genome-wide analysis identified 155 R2R3-MYBs in the ‘Red 5′ version of the Actinidia chinensis genome. Out of 36 anthocyanin-related AccR2R3-MYBs, AcMYB10 was the most highly expressed in inner pericarp of red-fleshed kiwifruit. The expression of AcMYB10 was highly correlated with anthocyanin accumulation in natural pigmentation during fruit ripening and light-/temperature-induced pigmentation in the callus. AcMYB10 is localized in the nuclei and has transcriptional activation activity. Overexpression of AcMYB10 elevates anthocyanin accumulation in transgenic A. chinensis. In comparison, A. chinensis fruit infiltrated with virus-induced gene silencing showed delayed red coloration, lower anthocyanin content, and lower expression of AcMYB10. The transient expression experiment in Nicotiana tabacum leaves and Actinidia arguta fruit indicated the interaction of AcMYB10 with AcbHLH42 might strongly activate anthocyanin biosynthesis by activating the transcription of AcLDOX and AcF3GT. In conclusion, this study provides novel molecular information about R2R3-MYBs in kiwifruit, advances our understanding of light- and temperature-induced anthocyanin accumulation, and demonstrates the important function of AcMYB10 in the biosynthesis of anthocyanin in kiwifruit.

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Ectopic Expression of Multiple Chrysanthemum (Chrysanthemum × morifolium) R2R3-MYB Transcription Factor Genes Regulates Anthocyanin Accumulation in Tobacco

Genes ◽

10.3390/genes10100777 ◽

2019 ◽

Vol 10 (10) ◽

pp. 777 ◽

Cited By ~ 3

Author(s):

Yan Hong ◽

Mengling Li ◽

Silan Dai

Keyword(s):

Transcription Factor ◽

Anthocyanin Biosynthesis ◽

Functional Divergence ◽

Ectopic Expression ◽

Chrysanthemum Morifolium ◽

Amino Acid Sequences ◽

Regulatory Function ◽

Myb Transcription Factor ◽

Anthocyanin Accumulation ◽

R2r3 Myb

The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p < 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p < 0.05); on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.

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The involvement of PybZIPa in light-induced anthocyanin accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter

Horticulture Research ◽

10.1038/s41438-019-0217-4 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 3

Author(s):

Hainan Liu ◽

Jun Su ◽

Yangfan Zhu ◽

Gaifang Yao ◽

Andrew C. Allan ◽

...

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Anthocyanin Biosynthesis ◽

Integrative Approach ◽

Pyrus Pyrifolia ◽

Anthocyanin Content ◽

Strongly Correlated ◽

Anthocyanin Accumulation ◽

Correlation Networks ◽

Transcriptional Changes

AbstractTo gain insight into how anthocyanin biosynthesis is controlled by light in fruit, transcriptome and metabolome analyses were performed in the Chinese sand pear cultivar “Mantianhong” (Pyrus pyrifolia) after bagging and bag removal. We investigated transcriptional and metabolic changes and gene-metabolite correlation networks. Correlation tests of anthocyanin content and transcriptional changes revealed that 1,530 transcripts were strongly correlated with 15 anthocyanin derivatives (R2 > 0.9, P-value < 0.05), with the top 130 transcripts categorized as being associated with flavonoid metabolism, transcriptional regulation, and light signaling. The connection network revealed a new photosensitive transcription factor, PybZIPa, that might play an important role during light-induced anthocyanin accumulation. The overexpression of PybZIPa promoted anthocyanin accumulation in pear and strawberry fruit as well as tobacco leaves. Dual luciferase and Y1H assays further verified that PybZIPa directly activated the expression of PyUFGT by binding to tandem G-box motifs in the promoter, which was key to differential anthocyanin accumulation in debagged pear skin, and the number of G-box motifs affected the transcriptional activation of PyUFGT by PybZIPa. The results indicate that the light-induced anthocyanin biosynthesis regulatory mechanism in pear differs from that described in previous reports suggesting that a bZIP family member co-regulates anthocyanin biosynthesis with other transcription factors in apple and Arabidopsis. It was found that, in response to light, PybZIPa promoted anthocyanin biosynthesis by regulating important transcription factors (PyMYB114, PyMYB10, and PyBBX22) as well as structural genes (PyUFGT) via binding to G-boxes within promoters. This activation was amplified by the self-binding of PybZIPa to activate its own promoter. Overall, we demonstrate the utility of a multiomics integrative approach for discovering new functional genes and pathways underlying light-induced anthocyanin biosynthesis.

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The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in ”Red Zaosu” (PyrusBretschneideri Rehd.) Pear Fruit

International Journal of Molecular Sciences ◽

10.3390/ijms22063026 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3026

Author(s):

Xieyu Li ◽

Fangxin Xiang ◽

Wei Han ◽

Bingqing Qie ◽

Rui Zhai ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Anthocyanin Biosynthesis ◽

Bimolecular Fluorescence Complementation ◽

Anthocyanin Content ◽

Pear Fruit ◽

Fruit Peel ◽

Interaction Domain ◽

Dihydroflavonol Reductase ◽

Anthocyanin Biosynthetic Pathway ◽

R2r3 Myb

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

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Genomic Analysis of the Glutathione S-Transferase Family in Pear (Pyrus communis) and Functional Identification of PcGST57 in Anthocyanin Accumulation

International Journal of Molecular Sciences ◽

10.3390/ijms23020746 ◽

2022 ◽

Vol 23 (2) ◽

pp. 746

Author(s):

Bo Li ◽

Xiangzhan Zhang ◽

Ruiwei Duan ◽

Chunhong Han ◽

Jian Yang ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Developmental Stages ◽

Genomic Analysis ◽

Pyrus Communis ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Rna Seq ◽

Virus Induced Gene Silencing ◽

Functional Identification ◽

Glutathione S Transferase

Anthocyanin accumulation in vacuoles results in red coloration in pear peels. Glutathione S-transferase (GST) proteins have emerged as important regulators of anthocyanin accumulation. Here, a total of 57 PcGST genes were identified in the European pear ‘Bartlett’ (Pyrus communis) through comprehensive genomic analysis. Phylogenetic analysis showed that PcGST genes were divided into 10 subfamilies. The gene structure, chromosomal localization, collinearity relationship, cis-elements in the promoter region, and conserved motifs of PcGST genes were analyzed. Further research indicated that glutamic acid (Glu) can significantly improve anthocyanin accumulation in pear peels. RNA sequencing (RNA-seq) analysis showed that Glu induced the expression of most PcGST genes, among which PcGST57 was most significantly induced. Further phylogenetic analysis indicated that PcGST57 was closely related to GST genes identified in other species, which were involved in anthocyanin accumulation. Transcript analysis indicated that PcGST57 was expressed in various tissues, other than flesh, and associated with peel coloration at different developmental stages. Silencing of PcGST57 by virus-induced gene silencing (VIGS) inhibited the expression of PcGST57 and reduced the anthocyanin content in pear fruit. In contrast, overexpression of PcGST57 improved anthocyanin accumulation. Collectively, our results demonstrated that PcGST57 was involved in anthocyanin accumulation in pear and provided candidate genes for red pear breeding.

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CsMYB3 and CsRuby1 form an ‘Activator-and-Repressor’ Loop for the Regulation of Anthocyanin Biosynthesis in Citrus

Plant and Cell Physiology ◽

10.1093/pcp/pcz198 ◽

2019 ◽

Vol 61 (2) ◽

pp. 318-330 ◽

Cited By ~ 7

Author(s):

Ding Huang ◽

Zhouzhou Tang ◽

Jialing Fu ◽

Yue Yuan ◽

Xiuxin Deng ◽

...

Keyword(s):

Transcription Factor ◽

Anthocyanin Biosynthesis ◽

Myb Transcription Factor ◽

Nitrogen Stress ◽

Loop Model ◽

Anthocyanin Accumulation ◽

Promoter Activation ◽

R2r3 Myb ◽

Activation Capacity ◽

Anthocyanin Biosynthetic Genes

Abstract Anthocyanins are preferentially accumulated in certain tissues of particular species of citrus. A R2R3-MYB transcription factor (named Ruby1) has been well documented as an activator of citrus anthocyanin biosynthesis. In this study, we characterized CsMYB3, a transcriptional repressor that regulates anthocyanin biosynthesis in citrus. CsMYB3 was expressed in anthocyanin-pigmented tissues, and the expression was closely associated with that of Ruby1, which is a key anthocyanin activator. Overexpression of CsMYB3 in Arabidopsis resulted in a decrease in anthocyanins under nitrogen stress. Overexpression of CsMYB3 in the background of CsRuby1-overexpressing strawberry and Arabidopsis reduced the anthocyanin accumulation level. Transient promoter activation assays revealed that CsMYB3 could repress the activation capacity of the complex formed by CsRuby1/CsbHLH1 for the anthocyanin biosynthetic genes. Moreover, CsMYB3 could be transcriptionally activated by CsRuby1 via promoter binding, thus forming an ‘activator-and-repressor’ loop to regulate anthocyanin biosynthesis in citrus. This study shows that CsMYB3 plays a repressor role in the regulation of anthocyanin biosynthesis and proposes an ‘activator-and-repressor’ loop model constituted by CsRuby1 and CsMYB3 in the regulation of anthocyanin biosynthesis in citrus.

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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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Pericarp Pigmentation Correlates with Hormones and Intensifies with Continuation of Bud Sport Generations from ‘Red Delicious’

10.1101/336263 ◽

2018 ◽

Author(s):

Wen-Fang Li ◽

Juan Mao ◽

Shi-Jin Yang ◽

Zhi-Gang Guo ◽

Zong-Huan Ma ◽

...

Keyword(s):

Molecular Level ◽

Anthocyanin Synthesis ◽

Anthocyanin Content ◽

Biosynthesis Pathway ◽

Anthocyanin Accumulation ◽

Structural Genes ◽

Bud Sport ◽

Phenylpropanoid Biosynthesis ◽

Highly Correlated ◽

Flavonoid Biosynthesis Pathway

ABSTRACTBud sport mutants of apple (Malus domestica Borkh.) trees with a highly blushed colouring pattern are mainly caused by the accumulation of anthocyanins in the pericarp. Hormones are important factors modulating anthocyanin accumulation. However, a good understanding of the interplay between hormones and anthocyanin synthesis in apples, especially in mutants at the molecular level, remains elusive. Here, physiological and comparative transcriptome approaches were used to reveal the molecular basis of pericarp pigmentation in ‘Red Delicious’ and its mutants, including ‘Starking Red’, ‘Starkrimson’, ‘Campbell Redchief’ and ‘Vallee spur’, which were designated G0 to G4, respectively. Pericarp pigmentation gradually proliferated from G0 to G4. The anthocyanin content was higher in the mutants than in ‘Red Delicious’. The activation of early phenylpropanoid biosynthesis genes, including ASP3, PAL, 4CL, PER, CHS, CYP98A and F3’H, was responsible for anthocyanin accumulation in mutants. In addition, IAA and ABA had a positive regulatory effect on the synthesis of anthocyanins, while GA had the reverse effect. The down-regulation of AACT1, HMGS, HMGR, MVK, MVD2, IDI1 and FPPS2 involved in terpenoid biosynthesis influences anthocyanin accumulation by positively regulating transcripts of AUX1 and SAUR that contribute to the synthesis of IAA, GID2 to GA, PP2C and SnRK2 to ABA. Furthermore, MYB and bHLH members, which are highly correlated (r=0.882–0.980) with anthocyanin content, modulated anthocyanin accumulation by regulating the transcription of structural genes, including CHS and F3’H, involved in the flavonoid biosynthesis pathway.

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MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

10.1101/429456 ◽

2018 ◽

Author(s):

Yi-Cheng Wang ◽

Jing-Jing Sun ◽

Yan-Fen Qiu ◽

Xiao-Jun Gong ◽

Li Ma ◽

...

Keyword(s):

Anthocyanin Biosynthesis ◽

Myb Transcription Factor ◽

Anthocyanin Content ◽

Anthocyanin Accumulation ◽

Mobility Shift ◽

In The Wild ◽

Acidic Conditions ◽

Electrophoretic Mobility Shift Assays

AbstractAnthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

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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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Loss of the R2R3 MYB Transcription Factor RsMYB1 Shapes Anthocyanin Biosynthesis and Accumulation in Raphanus sativus

International Journal of Molecular Sciences ◽

10.3390/ijms222010927 ◽

2021 ◽

Vol 22 (20) ◽

pp. 10927

Author(s):

Da-Hye Kim ◽

Jundae Lee ◽

JuHee Rhee ◽

Jong-Yeol Lee ◽

Sun-Hyung Lim

Keyword(s):

Transcription Factor ◽

Raphanus Sativus ◽

Anthocyanin Biosynthesis ◽

Antioxidant Properties ◽

Myb Transcription Factor ◽

Anthocyanin Accumulation ◽

Truncated Protein ◽

Frame Shift ◽

Frame Shift Mutation ◽

R2r3 Myb

The red or purple color of radish (Raphanus sativus L.) taproots is due to anthocyanins, which have nutritional and aesthetic value, as well as antioxidant properties. Moreover, the varied patterns and levels of anthocyanin accumulation in radish roots make them an interesting system for studying the transcriptional regulation of anthocyanin biosynthesis. The R2R3 MYB transcription factor RsMYB1 is a key positive regulator of anthocyanin biosynthesis in radish. Here, we isolated an allele of RsMYB1, named RsMYB1Short, in radish cultivars with white taproots. The RsMYB1Short allele carried a 4 bp insertion in the first exon causing a frame-shift mutation of RsMYB1, generating a truncated protein with only a partial R2 domain at the N-terminus. Unlike RsMYB1Full, RsMYB1Short was localized to the nucleus and the cytoplasm and failed to interact with their cognate partner RsTT8. Transient expression of genomic or cDNA sequences for RsMYB1Short in radish cotyledons failed to induce anthocyanin accumulation, but that for RsMYB1Full activated it. Additionally, RsMYB1Short showed the lost ability to induce pigment accumulation and to enhance the transcript level of anthocyanin biosynthetic genes, while RsMYB1Full promoted both processes when co-expressed with RsTT8 in tobacco leaves. As the result of the transient assay, co-expressing RsTT8 and RsMYB1Full, but not RsMYB1Short, also enhanced the promoter activity of RsCHS and RsDFR. We designed a molecular marker for RsMYB1 genotyping, and revealed that the RsMYB1Short allele is common in white radish cultivars, underscoring the importance of variation at the RsMYB1 locus in anthocyanin biosynthesis in the radish taproot. Together, these results indicate that the nonsense mutation of RsMYB1 generated the truncated protein, RsMYB1Short, that had the loss of ability to regulate anthocyanin biosynthesis. Our findings highlight that the frame shift mutation of RsMYB1 plays a key role in anthocyanin biosynthesis in the radish taproot.

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