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An L1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibre development.

Zhang F, Zuo K, Zhang J, Liu X, Zhang L, Sun X, Tang K - J. Exp. Bot. (2010)

Bottom Line: GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves.Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants.Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.

ABSTRACT
Transcription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

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Effect of point mutations of GbML1 on protein interaction. (A) Effect of point mutations on the interaction between GbML1 and GbMYB25 in the yeast two-hybrid assay. (Left) Diagram of point mutation sites. m1 indicates a G to D mutation at the 291 site; m2 indicates an H to D mutation at the 429 site; m3 indicates an S to C mutation at the 655 amino acid site. (Right) The START–SAD domain and its mutations were fused with the BD domain and introduced into yeast with the AD-GbMYB25 construct. Yeast cells harbouring BD-START–SAD/AD were used as negative control. The growth on selective medium was shown. (B) In vitro pull-down assay to confirm the yeast two-hybrid results. MBP-GbML1-START–SAD, MBP-START–SAD-m1, MBP-START–SAD-m2, and MBP-START–SAD-m3 fusion proteins are used as baits to pull down the HIS-GbMYB25 fusion protein from the induced cell extracts. Immunoblot detection of prey protein is with the His antibody. (C) Deduced three-dimensional structure of START domains indicates the pocket structure is important for the binding. 1, Modelled structure of the native START domain from GbML1. The pocket structure is obvious on the upper side. The No. 54 amino acid (Gly) and the No. 192 amino acid (His) of the START domain are shown. 2, The G291D mutation affects the structure of the third loop. The changed conformation is shown by a white arrow. 3, The H429D mutation changes the open pocket to be closed. The white arrow indicates the changed loop.
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fig5: Effect of point mutations of GbML1 on protein interaction. (A) Effect of point mutations on the interaction between GbML1 and GbMYB25 in the yeast two-hybrid assay. (Left) Diagram of point mutation sites. m1 indicates a G to D mutation at the 291 site; m2 indicates an H to D mutation at the 429 site; m3 indicates an S to C mutation at the 655 amino acid site. (Right) The START–SAD domain and its mutations were fused with the BD domain and introduced into yeast with the AD-GbMYB25 construct. Yeast cells harbouring BD-START–SAD/AD were used as negative control. The growth on selective medium was shown. (B) In vitro pull-down assay to confirm the yeast two-hybrid results. MBP-GbML1-START–SAD, MBP-START–SAD-m1, MBP-START–SAD-m2, and MBP-START–SAD-m3 fusion proteins are used as baits to pull down the HIS-GbMYB25 fusion protein from the induced cell extracts. Immunoblot detection of prey protein is with the His antibody. (C) Deduced three-dimensional structure of START domains indicates the pocket structure is important for the binding. 1, Modelled structure of the native START domain from GbML1. The pocket structure is obvious on the upper side. The No. 54 amino acid (Gly) and the No. 192 amino acid (His) of the START domain are shown. 2, The G291D mutation affects the structure of the third loop. The changed conformation is shown by a white arrow. 3, The H429D mutation changes the open pocket to be closed. The white arrow indicates the changed loop.

Mentions: Since the START–SAD domain of GbML1 was required and was sufficient for the binding to GbMYB25, it was investigated if point mutations in some conserved regulatory amino acids might affect the interaction. Three mutations were made by the alteration of one amino acid in different positions (as indicated in Fig. 5A, left). In the yeast two-hybrid assay, the G291D mutation reduced the interaction, H429D completely abolished the interaction, and S665C slightly reduced the interaction (Fig. 5A, right). An in vitro pull-down assay was performed to assess the effect of point mutations on binding to GbMYB25. While the S665C mutation retained less amount of the GbMYB25 protein compared with the wild-type part, the G291D and H429D mutations greatly reduced the amount of bound GbMYB25 protein (Fig. 5B). This pull-down result was consistent with the results gained from the yeast two-hybrid assay, further demonstrating that the START domain was critical for the binding.


An L1 box binding protein, GbML1, interacts with GbMYB25 to control cotton fibre development.

Zhang F, Zuo K, Zhang J, Liu X, Zhang L, Sun X, Tang K - J. Exp. Bot. (2010)

Effect of point mutations of GbML1 on protein interaction. (A) Effect of point mutations on the interaction between GbML1 and GbMYB25 in the yeast two-hybrid assay. (Left) Diagram of point mutation sites. m1 indicates a G to D mutation at the 291 site; m2 indicates an H to D mutation at the 429 site; m3 indicates an S to C mutation at the 655 amino acid site. (Right) The START–SAD domain and its mutations were fused with the BD domain and introduced into yeast with the AD-GbMYB25 construct. Yeast cells harbouring BD-START–SAD/AD were used as negative control. The growth on selective medium was shown. (B) In vitro pull-down assay to confirm the yeast two-hybrid results. MBP-GbML1-START–SAD, MBP-START–SAD-m1, MBP-START–SAD-m2, and MBP-START–SAD-m3 fusion proteins are used as baits to pull down the HIS-GbMYB25 fusion protein from the induced cell extracts. Immunoblot detection of prey protein is with the His antibody. (C) Deduced three-dimensional structure of START domains indicates the pocket structure is important for the binding. 1, Modelled structure of the native START domain from GbML1. The pocket structure is obvious on the upper side. The No. 54 amino acid (Gly) and the No. 192 amino acid (His) of the START domain are shown. 2, The G291D mutation affects the structure of the third loop. The changed conformation is shown by a white arrow. 3, The H429D mutation changes the open pocket to be closed. The white arrow indicates the changed loop.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921199&req=5

fig5: Effect of point mutations of GbML1 on protein interaction. (A) Effect of point mutations on the interaction between GbML1 and GbMYB25 in the yeast two-hybrid assay. (Left) Diagram of point mutation sites. m1 indicates a G to D mutation at the 291 site; m2 indicates an H to D mutation at the 429 site; m3 indicates an S to C mutation at the 655 amino acid site. (Right) The START–SAD domain and its mutations were fused with the BD domain and introduced into yeast with the AD-GbMYB25 construct. Yeast cells harbouring BD-START–SAD/AD were used as negative control. The growth on selective medium was shown. (B) In vitro pull-down assay to confirm the yeast two-hybrid results. MBP-GbML1-START–SAD, MBP-START–SAD-m1, MBP-START–SAD-m2, and MBP-START–SAD-m3 fusion proteins are used as baits to pull down the HIS-GbMYB25 fusion protein from the induced cell extracts. Immunoblot detection of prey protein is with the His antibody. (C) Deduced three-dimensional structure of START domains indicates the pocket structure is important for the binding. 1, Modelled structure of the native START domain from GbML1. The pocket structure is obvious on the upper side. The No. 54 amino acid (Gly) and the No. 192 amino acid (His) of the START domain are shown. 2, The G291D mutation affects the structure of the third loop. The changed conformation is shown by a white arrow. 3, The H429D mutation changes the open pocket to be closed. The white arrow indicates the changed loop.
Mentions: Since the START–SAD domain of GbML1 was required and was sufficient for the binding to GbMYB25, it was investigated if point mutations in some conserved regulatory amino acids might affect the interaction. Three mutations were made by the alteration of one amino acid in different positions (as indicated in Fig. 5A, left). In the yeast two-hybrid assay, the G291D mutation reduced the interaction, H429D completely abolished the interaction, and S665C slightly reduced the interaction (Fig. 5A, right). An in vitro pull-down assay was performed to assess the effect of point mutations on binding to GbMYB25. While the S665C mutation retained less amount of the GbMYB25 protein compared with the wild-type part, the G291D and H429D mutations greatly reduced the amount of bound GbMYB25 protein (Fig. 5B). This pull-down result was consistent with the results gained from the yeast two-hybrid assay, further demonstrating that the START domain was critical for the binding.

Bottom Line: GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves.Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants.Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.

ABSTRACT
Transcription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, Gossypium barbadense Meristem Layer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants.

Show MeSH