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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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EAR motif is essential for BZR1 function in BR responses(a) Glutamine (Q) at position 285 in BZR1 is converted to stop codon in the bzs285 mutant. BIN2-DM: BIN2 docking motif. EAR: EAR motif.(b) Intragenic mutant (bzs285) suppresses the constitutive activation of BR response of bzr1-1D. Seedlings were grown in the dark on the medium containing 2 µM propiconazole (PPZ). Scale bar: 5 mm.(c) Both BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs show severe dwarfism. Plants were grown in the long-day condition (16 hr of light and 8 hr of dark) for 4 weeks. Two independent lines (1 and 2) for the BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs are shown.(d) The hypocotyl elongation of bzr1-1DΔEAR-OX is inhibited by PPZ treatment. Seedlings were grown in the dark for 5 days on the medium containing either the mock or 2 µM PPZ. Error bars indicate the s.d. (n=10 plants). Scale bar: 5 mm.(e) Axillary branches and cauline leaves are not fused in bzr1-1DΔEAR-OX.(f,g) bzr1-1DΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation (f) and the inhibition of primary root growth (g). Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL). Scale bar: 5 mm.(h) BZR1ΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation. Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL).Error bars in (f–h) indicate the s.d. (n=10 plants).
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Figure 1: EAR motif is essential for BZR1 function in BR responses(a) Glutamine (Q) at position 285 in BZR1 is converted to stop codon in the bzs285 mutant. BIN2-DM: BIN2 docking motif. EAR: EAR motif.(b) Intragenic mutant (bzs285) suppresses the constitutive activation of BR response of bzr1-1D. Seedlings were grown in the dark on the medium containing 2 µM propiconazole (PPZ). Scale bar: 5 mm.(c) Both BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs show severe dwarfism. Plants were grown in the long-day condition (16 hr of light and 8 hr of dark) for 4 weeks. Two independent lines (1 and 2) for the BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs are shown.(d) The hypocotyl elongation of bzr1-1DΔEAR-OX is inhibited by PPZ treatment. Seedlings were grown in the dark for 5 days on the medium containing either the mock or 2 µM PPZ. Error bars indicate the s.d. (n=10 plants). Scale bar: 5 mm.(e) Axillary branches and cauline leaves are not fused in bzr1-1DΔEAR-OX.(f,g) bzr1-1DΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation (f) and the inhibition of primary root growth (g). Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL). Scale bar: 5 mm.(h) BZR1ΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation. Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL).Error bars in (f–h) indicate the s.d. (n=10 plants).

Mentions: The gain-of-function mutant BZR1 protein, bzr1-1D, is constitutively dephosphorylated and active even in the BR deficiency condition due to a strong interaction with PP2A (ref. 12). Thus, the hypocotyl elongation of bzr1-1D seedling is resistant to BR biosynthesis inhibitors, brassinazole (BRZ) and propiconazole (PPZ) (ref. 12). We identified suppressors of the bzr1-1D mutant through an EMS mutagenesis screen. One of the mutants, bzs285, carried a mutation that creates a stop codon after amino acid 284, resulting in deletion of 52 amino acids at the C-terminus of bzr1-1D protein (Fig. 1a–b). The truncated region includes a BIN2 docking motif and an LxLxL type of EAR motif. Since the BIN2 docking motif mediates inactivation of BZR1 through BIN2-mediated phosphorylation8, the suppression of the bzr1-1D phenotype by the bzs285 mutation was most likely due to loss of the EAR motif. The EAR motif is highly conserved in the BZR1 family of transcription factors across land plants from bryophytes to angiosperms (Supplementary Fig. 1a). In addition, all other BZR1-like proteins in Arabidopsis have the EAR motif at their C-terminus (Supplementary Fig. 1b), implicating an indispensable role of the EAR motif in BZR1 functions.


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

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

EAR motif is essential for BZR1 function in BR responses(a) Glutamine (Q) at position 285 in BZR1 is converted to stop codon in the bzs285 mutant. BIN2-DM: BIN2 docking motif. EAR: EAR motif.(b) Intragenic mutant (bzs285) suppresses the constitutive activation of BR response of bzr1-1D. Seedlings were grown in the dark on the medium containing 2 µM propiconazole (PPZ). Scale bar: 5 mm.(c) Both BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs show severe dwarfism. Plants were grown in the long-day condition (16 hr of light and 8 hr of dark) for 4 weeks. Two independent lines (1 and 2) for the BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs are shown.(d) The hypocotyl elongation of bzr1-1DΔEAR-OX is inhibited by PPZ treatment. Seedlings were grown in the dark for 5 days on the medium containing either the mock or 2 µM PPZ. Error bars indicate the s.d. (n=10 plants). Scale bar: 5 mm.(e) Axillary branches and cauline leaves are not fused in bzr1-1DΔEAR-OX.(f,g) bzr1-1DΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation (f) and the inhibition of primary root growth (g). Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL). Scale bar: 5 mm.(h) BZR1ΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation. Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL).Error bars in (f–h) indicate the s.d. (n=10 plants).
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Figure 1: EAR motif is essential for BZR1 function in BR responses(a) Glutamine (Q) at position 285 in BZR1 is converted to stop codon in the bzs285 mutant. BIN2-DM: BIN2 docking motif. EAR: EAR motif.(b) Intragenic mutant (bzs285) suppresses the constitutive activation of BR response of bzr1-1D. Seedlings were grown in the dark on the medium containing 2 µM propiconazole (PPZ). Scale bar: 5 mm.(c) Both BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs show severe dwarfism. Plants were grown in the long-day condition (16 hr of light and 8 hr of dark) for 4 weeks. Two independent lines (1 and 2) for the BZR1ΔEAR-OXs and bzr1-1DΔEAR-OXs are shown.(d) The hypocotyl elongation of bzr1-1DΔEAR-OX is inhibited by PPZ treatment. Seedlings were grown in the dark for 5 days on the medium containing either the mock or 2 µM PPZ. Error bars indicate the s.d. (n=10 plants). Scale bar: 5 mm.(e) Axillary branches and cauline leaves are not fused in bzr1-1DΔEAR-OX.(f,g) bzr1-1DΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation (f) and the inhibition of primary root growth (g). Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL). Scale bar: 5 mm.(h) BZR1ΔEAR-OXs are insensitive to BR in the promotion of hypocotyl elongation. Seedlings were grown under the white light for 5 days on the medium containing either mock or 1000 nM brassinolide (BL).Error bars in (f–h) indicate the s.d. (n=10 plants).
Mentions: The gain-of-function mutant BZR1 protein, bzr1-1D, is constitutively dephosphorylated and active even in the BR deficiency condition due to a strong interaction with PP2A (ref. 12). Thus, the hypocotyl elongation of bzr1-1D seedling is resistant to BR biosynthesis inhibitors, brassinazole (BRZ) and propiconazole (PPZ) (ref. 12). We identified suppressors of the bzr1-1D mutant through an EMS mutagenesis screen. One of the mutants, bzs285, carried a mutation that creates a stop codon after amino acid 284, resulting in deletion of 52 amino acids at the C-terminus of bzr1-1D protein (Fig. 1a–b). The truncated region includes a BIN2 docking motif and an LxLxL type of EAR motif. Since the BIN2 docking motif mediates inactivation of BZR1 through BIN2-mediated phosphorylation8, the suppression of the bzr1-1D phenotype by the bzs285 mutation was most likely due to loss of the EAR motif. The EAR motif is highly conserved in the BZR1 family of transcription factors across land plants from bryophytes to angiosperms (Supplementary Fig. 1a). In addition, all other BZR1-like proteins in Arabidopsis have the EAR motif at their C-terminus (Supplementary Fig. 1b), implicating an indispensable role of the EAR motif in BZR1 functions.

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