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TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1.

Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY - Nat Commun (2014)

Bottom Line: Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1.A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype.Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA [2].

ABSTRACT
Brassinosteroid (BR) regulates plant development by activating the transcription factor brassinazole resistant 1 (BZR1), which activates and represses different target genes to switch cellular programmes. The mechanisms that determine BZR1's transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

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TOPLESS is required for BR responses(a) The BZR1 transcriptional repression activity is compromised in tpl;tpr1;tpr4. Total RNA were extracted from etiolated seedlings grown on the medium containing 2 µM PPZ. The relative gene expression levels were determined by qRT-PCR. Numbers indicate the ratios of expression levels (bzr1-1D/Col-0 or bzr1-1D;tpl;tpr1;tpr4/tpl;tpr1;tpr4). Error bars indicate the s.d. (n=3).(b) The tpl;tpr1;tpr4 triple mutant is less sensitive to BR. Seedlings were grown for 5 days on the medium containing either mock or 1000 nM BL. Numbers indicate ratio of hypocotyl lengths (BL-treated / mock-treated). Error bars indicate the s.d. (n=10 plants) and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(c) The tpl;tpr1;tpr4 triple mutation suppresses the long hypocotyl phenotype of bzr1-1D. Seedlings were grown on the medium containing 2 µM PPZ for 6 days in the dark. Error bars indicate the s.d. (n=10 plants). n.s.=not significant and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(d) Scatter plot of fresh weight measurements of 3-week-old plants grown on the medium containing 2 µM PPZ. This experiment was repeated with similar results. n.s.=not significant and ** : P<0.01 by Student’s t-test (n=10 plants).(e) TPL fusion at the C-terminus restores the transcriptional repression activity of bzr1-1DmEAR. Relative expression fold changes of CPD and DWF4 were determined by qRT-PCR in Arabidopsis plants transformed with the indicated constructs, compared to wild type (Col-0). Error bars indicate the s.e. (n=4).(f) TPL fusion restores the activity of bzr1-1DmEAR in promoting hypocotyl elongation. Seedlings were grown in the dark on the medium containing 1 µM BRZ for 5 days. Error bars indicate the s.e. (n=10)
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Figure 5: TOPLESS is required for BR responses(a) The BZR1 transcriptional repression activity is compromised in tpl;tpr1;tpr4. Total RNA were extracted from etiolated seedlings grown on the medium containing 2 µM PPZ. The relative gene expression levels were determined by qRT-PCR. Numbers indicate the ratios of expression levels (bzr1-1D/Col-0 or bzr1-1D;tpl;tpr1;tpr4/tpl;tpr1;tpr4). Error bars indicate the s.d. (n=3).(b) The tpl;tpr1;tpr4 triple mutant is less sensitive to BR. Seedlings were grown for 5 days on the medium containing either mock or 1000 nM BL. Numbers indicate ratio of hypocotyl lengths (BL-treated / mock-treated). Error bars indicate the s.d. (n=10 plants) and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(c) The tpl;tpr1;tpr4 triple mutation suppresses the long hypocotyl phenotype of bzr1-1D. Seedlings were grown on the medium containing 2 µM PPZ for 6 days in the dark. Error bars indicate the s.d. (n=10 plants). n.s.=not significant and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(d) Scatter plot of fresh weight measurements of 3-week-old plants grown on the medium containing 2 µM PPZ. This experiment was repeated with similar results. n.s.=not significant and ** : P<0.01 by Student’s t-test (n=10 plants).(e) TPL fusion at the C-terminus restores the transcriptional repression activity of bzr1-1DmEAR. Relative expression fold changes of CPD and DWF4 were determined by qRT-PCR in Arabidopsis plants transformed with the indicated constructs, compared to wild type (Col-0). Error bars indicate the s.e. (n=4).(f) TPL fusion restores the activity of bzr1-1DmEAR in promoting hypocotyl elongation. Seedlings were grown in the dark on the medium containing 1 µM BRZ for 5 days. Error bars indicate the s.e. (n=10)

Mentions: To further verify the requirement of TPL/TPRs activities in BR responses, we tested the BZR1-repressed genes in the tpl;tpr1;tpr4 triple mutant lacking TPL and two TOPLESS RELATED (TPR) genes28. As expected, bzr1-1D caused a much milder repression of CPD and DWF4 in tpl;tpr1;tpr4 than in wild type background (Fig. 5a), supporting that the full TPL/TPRs activities are required for BZR1 repression of these target genes. In contrast, bzr1-1D activation of IAA19 and EXP8 was not significantly affected by the tpl;tpr1;tpr4 mutation, and SAUR15 activation by bzr1-1D was only slightly decreased in tpl;tpr1;tpr4 (Supplementary Fig. 3). These results indicate that TPL/TPRs play a major role in BZR1 repression of target genes but not in BZR1 activation of gene expression. Consistent with the defect in the BZR1 repression of gene expression, the tpl;tpr1;tpr4 seedlings were significantly less sensitive to BR treatment (Fig. 5b) in hypocotyl elongation compared to wild type. In addition, the tpl;tpr1;tpr4 triple mutation partially suppressed the PPZ-insensitive phenotypes of the bzr1-1D mutation both in the dark and under light (Fig. 5c,d). These results demonstrate that the normal level of TPL/TPR activity is required for BZR1-mediated gene repression and hypocotyl cell elongation.


TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1.

Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY - Nat Commun (2014)

TOPLESS is required for BR responses(a) The BZR1 transcriptional repression activity is compromised in tpl;tpr1;tpr4. Total RNA were extracted from etiolated seedlings grown on the medium containing 2 µM PPZ. The relative gene expression levels were determined by qRT-PCR. Numbers indicate the ratios of expression levels (bzr1-1D/Col-0 or bzr1-1D;tpl;tpr1;tpr4/tpl;tpr1;tpr4). Error bars indicate the s.d. (n=3).(b) The tpl;tpr1;tpr4 triple mutant is less sensitive to BR. Seedlings were grown for 5 days on the medium containing either mock or 1000 nM BL. Numbers indicate ratio of hypocotyl lengths (BL-treated / mock-treated). Error bars indicate the s.d. (n=10 plants) and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(c) The tpl;tpr1;tpr4 triple mutation suppresses the long hypocotyl phenotype of bzr1-1D. Seedlings were grown on the medium containing 2 µM PPZ for 6 days in the dark. Error bars indicate the s.d. (n=10 plants). n.s.=not significant and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(d) Scatter plot of fresh weight measurements of 3-week-old plants grown on the medium containing 2 µM PPZ. This experiment was repeated with similar results. n.s.=not significant and ** : P<0.01 by Student’s t-test (n=10 plants).(e) TPL fusion at the C-terminus restores the transcriptional repression activity of bzr1-1DmEAR. Relative expression fold changes of CPD and DWF4 were determined by qRT-PCR in Arabidopsis plants transformed with the indicated constructs, compared to wild type (Col-0). Error bars indicate the s.e. (n=4).(f) TPL fusion restores the activity of bzr1-1DmEAR in promoting hypocotyl elongation. Seedlings were grown in the dark on the medium containing 1 µM BRZ for 5 days. Error bars indicate the s.e. (n=10)
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Figure 5: TOPLESS is required for BR responses(a) The BZR1 transcriptional repression activity is compromised in tpl;tpr1;tpr4. Total RNA were extracted from etiolated seedlings grown on the medium containing 2 µM PPZ. The relative gene expression levels were determined by qRT-PCR. Numbers indicate the ratios of expression levels (bzr1-1D/Col-0 or bzr1-1D;tpl;tpr1;tpr4/tpl;tpr1;tpr4). Error bars indicate the s.d. (n=3).(b) The tpl;tpr1;tpr4 triple mutant is less sensitive to BR. Seedlings were grown for 5 days on the medium containing either mock or 1000 nM BL. Numbers indicate ratio of hypocotyl lengths (BL-treated / mock-treated). Error bars indicate the s.d. (n=10 plants) and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(c) The tpl;tpr1;tpr4 triple mutation suppresses the long hypocotyl phenotype of bzr1-1D. Seedlings were grown on the medium containing 2 µM PPZ for 6 days in the dark. Error bars indicate the s.d. (n=10 plants). n.s.=not significant and **: P<0.01 by Student’s t-test. Scale bar: 5 mm.(d) Scatter plot of fresh weight measurements of 3-week-old plants grown on the medium containing 2 µM PPZ. This experiment was repeated with similar results. n.s.=not significant and ** : P<0.01 by Student’s t-test (n=10 plants).(e) TPL fusion at the C-terminus restores the transcriptional repression activity of bzr1-1DmEAR. Relative expression fold changes of CPD and DWF4 were determined by qRT-PCR in Arabidopsis plants transformed with the indicated constructs, compared to wild type (Col-0). Error bars indicate the s.e. (n=4).(f) TPL fusion restores the activity of bzr1-1DmEAR in promoting hypocotyl elongation. Seedlings were grown in the dark on the medium containing 1 µM BRZ for 5 days. Error bars indicate the s.e. (n=10)
Mentions: To further verify the requirement of TPL/TPRs activities in BR responses, we tested the BZR1-repressed genes in the tpl;tpr1;tpr4 triple mutant lacking TPL and two TOPLESS RELATED (TPR) genes28. As expected, bzr1-1D caused a much milder repression of CPD and DWF4 in tpl;tpr1;tpr4 than in wild type background (Fig. 5a), supporting that the full TPL/TPRs activities are required for BZR1 repression of these target genes. In contrast, bzr1-1D activation of IAA19 and EXP8 was not significantly affected by the tpl;tpr1;tpr4 mutation, and SAUR15 activation by bzr1-1D was only slightly decreased in tpl;tpr1;tpr4 (Supplementary Fig. 3). These results indicate that TPL/TPRs play a major role in BZR1 repression of target genes but not in BZR1 activation of gene expression. Consistent with the defect in the BZR1 repression of gene expression, the tpl;tpr1;tpr4 seedlings were significantly less sensitive to BR treatment (Fig. 5b) in hypocotyl elongation compared to wild type. In addition, the tpl;tpr1;tpr4 triple mutation partially suppressed the PPZ-insensitive phenotypes of the bzr1-1D mutation both in the dark and under light (Fig. 5c,d). These results demonstrate that the normal level of TPL/TPR activity is required for BZR1-mediated gene repression and hypocotyl cell elongation.

Bottom Line: Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1.A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype.Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA [2].

ABSTRACT
Brassinosteroid (BR) regulates plant development by activating the transcription factor brassinazole resistant 1 (BZR1), which activates and represses different target genes to switch cellular programmes. The mechanisms that determine BZR1's transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

Show MeSH
Related in: MedlinePlus