Limits...
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.

Show MeSH

Related in: MedlinePlus

Protein-protein interactions between CmbHLHs and CmMYB6 studied by yeast two-hybrid assay.The yeast strain was co-transformed with the indicated combinations of CmbHLH1 or CmbHLH2 fused into pGBKT7 (BD) and CmMYB6 fused into pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Protein-protein interactions were detected on DDO (SD media lacking Leu and Trp) and QDO/X/A (SD media lacking Leu, Trp, His and Ade, with AbA and X-α-gal) media.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664390&req=5

pone.0143892.g006: Protein-protein interactions between CmbHLHs and CmMYB6 studied by yeast two-hybrid assay.The yeast strain was co-transformed with the indicated combinations of CmbHLH1 or CmbHLH2 fused into pGBKT7 (BD) and CmMYB6 fused into pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Protein-protein interactions were detected on DDO (SD media lacking Leu and Trp) and QDO/X/A (SD media lacking Leu, Trp, His and Ade, with AbA and X-α-gal) media.

Mentions: To study the mode of interaction between CmbHLH2 and CmMYB6 proteins during the transcriptional regulation of CmDFR promoter, Y2H assay were carried out. Based on the testing results, the auto-activation of BD-CmbHLH1 and BD-CmbHLH2 were inhibited with 100 ng/ml and 400 ng/ml AbA, respectively (S2 Fig). However, auto-activation of BD-CmMYB6 could not be inhibited even with 1000 ng/ml AbA (S2 Fig), thus CmMYB6 was used as the prey protein and CmbHLH1 or CmbHLH2 as bait proteins in subsequent experiments. Protein-protein interaction between CmMYB6 and CmbHLH2 was indicated by growth of colonies containing both AD-CmMYB6 and BD-CmbHLH2 proteins on SD/-Ade/-His/-Leu/-Trp/+X-α-Gal/+AbA screening media (Fig 6).


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)

Protein-protein interactions between CmbHLHs and CmMYB6 studied by yeast two-hybrid assay.The yeast strain was co-transformed with the indicated combinations of CmbHLH1 or CmbHLH2 fused into pGBKT7 (BD) and CmMYB6 fused into pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Protein-protein interactions were detected on DDO (SD media lacking Leu and Trp) and QDO/X/A (SD media lacking Leu, Trp, His and Ade, with AbA and X-α-gal) media.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143892.g006: Protein-protein interactions between CmbHLHs and CmMYB6 studied by yeast two-hybrid assay.The yeast strain was co-transformed with the indicated combinations of CmbHLH1 or CmbHLH2 fused into pGBKT7 (BD) and CmMYB6 fused into pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Protein-protein interactions were detected on DDO (SD media lacking Leu and Trp) and QDO/X/A (SD media lacking Leu, Trp, His and Ade, with AbA and X-α-gal) media.
Mentions: To study the mode of interaction between CmbHLH2 and CmMYB6 proteins during the transcriptional regulation of CmDFR promoter, Y2H assay were carried out. Based on the testing results, the auto-activation of BD-CmbHLH1 and BD-CmbHLH2 were inhibited with 100 ng/ml and 400 ng/ml AbA, respectively (S2 Fig). However, auto-activation of BD-CmMYB6 could not be inhibited even with 1000 ng/ml AbA (S2 Fig), thus CmMYB6 was used as the prey protein and CmbHLH1 or CmbHLH2 as bait proteins in subsequent experiments. Protein-protein interaction between CmMYB6 and CmbHLH2 was indicated by growth of colonies containing both AD-CmMYB6 and BD-CmbHLH2 proteins on SD/-Ade/-His/-Leu/-Trp/+X-α-Gal/+AbA screening media (Fig 6).

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