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A Novel bHLH Transcription Factor Involved in Regulating Anthocyanin Biosynthesis in Chrysanthemums (Chrysanthemum morifolium Ramat.).

Xiang LL, Liu XF, Li X, Yin XR, Grierson D, Li F, Chen KS - PLoS ONE (2015)

Bottom Line: 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.These results suggest that CmbHLH2 is the essential partner for CmMYB6 in regulating anthocyanin biosynthesis in chrysanthemum.

View Article: PubMed Central - PubMed

Affiliation: College of Agriculture & Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou, 310058, PR China.

ABSTRACT
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.

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Characterization of the interaction of three TFs with the CmDFR promoter by yeast one-hybrid assays.P53 and its promoter supplied with the kit were used as a positive control to verify the stability of these assays. The auto-activity of CmDFR promoter bait stain was tested on SD media lacking Ura in presence of AbA. CmbHLH1, CmbHLH2 and CmMYB6 were fused into pGADT7 (AD) and transformed separately into the bait stain. The binding was screened on SD media lacking Leu in presence of AbA175.
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pone.0143892.g005: Characterization of the interaction of three TFs with the CmDFR promoter by yeast one-hybrid assays.P53 and its promoter supplied with the kit were used as a positive control to verify the stability of these assays. The auto-activity of CmDFR promoter bait stain was tested on SD media lacking Ura in presence of AbA. CmbHLH1, CmbHLH2 and CmMYB6 were fused into pGADT7 (AD) and transformed separately into the bait stain. The binding was screened on SD media lacking Leu in presence of AbA175.

Mentions: The relationship between CmbHLH2 and the CmDFR promoter was further analyzed by yeast one-hybrid assay. Successful generation of the CmDFR-pAbAi bait strain was confirmed by screening colony growth on SD/-Ura media and auto-activation was inhibited with 175 ng/ml AbA antibiotic (Fig 5). When the bait strains were transformed with the fusion protein AD-CmMYB6, AD-CmbHLH1 or AD-CmbHLH2, which has the activation domain, different colony growing abilities were detected on the testing media. Strains transformed with AD-CmMYB6 or AD-CmbHLH2 could grow on SD/-Ura+AbA175, while those with either empty vector or AD-CmbHLH1 could not (Fig 5).


A Novel bHLH Transcription Factor Involved in Regulating Anthocyanin Biosynthesis in Chrysanthemums (Chrysanthemum morifolium Ramat.).

Xiang LL, Liu XF, Li X, Yin XR, Grierson D, Li F, Chen KS - PLoS ONE (2015)

Characterization of the interaction of three TFs with the CmDFR promoter by yeast one-hybrid assays.P53 and its promoter supplied with the kit were used as a positive control to verify the stability of these assays. The auto-activity of CmDFR promoter bait stain was tested on SD media lacking Ura in presence of AbA. CmbHLH1, CmbHLH2 and CmMYB6 were fused into pGADT7 (AD) and transformed separately into the bait stain. The binding was screened on SD media lacking Leu in presence of AbA175.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664390&req=5

pone.0143892.g005: Characterization of the interaction of three TFs with the CmDFR promoter by yeast one-hybrid assays.P53 and its promoter supplied with the kit were used as a positive control to verify the stability of these assays. The auto-activity of CmDFR promoter bait stain was tested on SD media lacking Ura in presence of AbA. CmbHLH1, CmbHLH2 and CmMYB6 were fused into pGADT7 (AD) and transformed separately into the bait stain. The binding was screened on SD media lacking Leu in presence of AbA175.
Mentions: The relationship between CmbHLH2 and the CmDFR promoter was further analyzed by yeast one-hybrid assay. Successful generation of the CmDFR-pAbAi bait strain was confirmed by screening colony growth on SD/-Ura media and auto-activation was inhibited with 175 ng/ml AbA antibiotic (Fig 5). When the bait strains were transformed with the fusion protein AD-CmMYB6, AD-CmbHLH1 or AD-CmbHLH2, which has the activation domain, different colony growing abilities were detected on the testing media. Strains transformed with AD-CmMYB6 or AD-CmbHLH2 could grow on SD/-Ura+AbA175, while those with either empty vector or AD-CmbHLH1 could not (Fig 5).

Bottom Line: 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.These results suggest that CmbHLH2 is the essential partner for CmMYB6 in regulating anthocyanin biosynthesis in chrysanthemum.

View Article: PubMed Central - PubMed

Affiliation: College of Agriculture & Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou, 310058, PR China.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus