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In vitro reconstitution of an abscisic acid signalling pathway.

Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, Rodriguez PL, Zhu JK - Nature (2009)

Bottom Line: Introduction of these four components into plant protoplasts results in ABA-responsive gene expression.We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases.Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California at Riverside, Riverside, California 92521, USA.

ABSTRACT
The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

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ABI1 and ABI2 inhibit SnRK2.6 by dephosphorylationa, SnRK2.6 is deactivated by ABI1. MBP or MBP-SnRK2.6 treated without (−) or with GST-ABI1 or GST was incubated with GST-ABF2 fragment (amino acids Gly73 to Gln119) in the presence of [γ32P]-ATP. In the furthest right lane (post), GST-ABI1 was added after phosphorylation of GST-ABF2 fragment by MBP-SnRK2.6. Bands of GST-ABF2 fragment and MBP-SnRK2.6 are indicated by an arrow and an arrowhead, respectively. Radioactivities of GST-ABF2 fragment bands were measured with a phospho-imager and were normalized, taking the radioactivity of the band by MBP-SnRK2.6 without ABI1 treatment as 1 (mean ± s.e.m., n = 5). b, Coomassie staining of purified MBP, SnRK2.6, ABF2, GST and GST-ABI1. c, FLAG-SnRK2.6 extracted from transgenic plants before and after ABA treatment was used instead of MBP-SnRK2.6 in (a). Coomassie staining, autoradiography and relative radioactivities (mean ± s.e.m. n = 5) of GST-ABF2 fragment are shown. Western blotting with anti-FLAG antibody shows FLAG-SnRK2.6 protein amount. d, Autoradiography of autophosphorylated SnRK2.6 showing dephosphorylation of SnRK2.6 by MBP-ABI1 and MBP-ABI2 and the effect of PYL8 and PYL5, respectively, in the presence of 1 μM ABA. e, Phosphate release from synthetic peptide HSQPKpSTVGTP, corresponding to amino acids 170-180 of SnRK2.6.
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Figure 2: ABI1 and ABI2 inhibit SnRK2.6 by dephosphorylationa, SnRK2.6 is deactivated by ABI1. MBP or MBP-SnRK2.6 treated without (−) or with GST-ABI1 or GST was incubated with GST-ABF2 fragment (amino acids Gly73 to Gln119) in the presence of [γ32P]-ATP. In the furthest right lane (post), GST-ABI1 was added after phosphorylation of GST-ABF2 fragment by MBP-SnRK2.6. Bands of GST-ABF2 fragment and MBP-SnRK2.6 are indicated by an arrow and an arrowhead, respectively. Radioactivities of GST-ABF2 fragment bands were measured with a phospho-imager and were normalized, taking the radioactivity of the band by MBP-SnRK2.6 without ABI1 treatment as 1 (mean ± s.e.m., n = 5). b, Coomassie staining of purified MBP, SnRK2.6, ABF2, GST and GST-ABI1. c, FLAG-SnRK2.6 extracted from transgenic plants before and after ABA treatment was used instead of MBP-SnRK2.6 in (a). Coomassie staining, autoradiography and relative radioactivities (mean ± s.e.m. n = 5) of GST-ABF2 fragment are shown. Western blotting with anti-FLAG antibody shows FLAG-SnRK2.6 protein amount. d, Autoradiography of autophosphorylated SnRK2.6 showing dephosphorylation of SnRK2.6 by MBP-ABI1 and MBP-ABI2 and the effect of PYL8 and PYL5, respectively, in the presence of 1 μM ABA. e, Phosphate release from synthetic peptide HSQPKpSTVGTP, corresponding to amino acids 170-180 of SnRK2.6.

Mentions: Author Contributions: HF contributed Figures 2a-c & e, Figure 3, Figures 4a-c and Supplementary Figure 4. VC contributed Figures 1a-d, and Supplementary Figures 1a & b. AR, SR, RA and PLR contributed Figure 2d, and Supplementary figures 2 & 3. SYP and SRC assisted with the generation of recombinant proteins, and SRC helped edit the manuscript. JS assisted with protoplast assays. JKZ designed the experiments, and wrote the paper together with VC and HF.


In vitro reconstitution of an abscisic acid signalling pathway.

Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, Rodriguez PL, Zhu JK - Nature (2009)

ABI1 and ABI2 inhibit SnRK2.6 by dephosphorylationa, SnRK2.6 is deactivated by ABI1. MBP or MBP-SnRK2.6 treated without (−) or with GST-ABI1 or GST was incubated with GST-ABF2 fragment (amino acids Gly73 to Gln119) in the presence of [γ32P]-ATP. In the furthest right lane (post), GST-ABI1 was added after phosphorylation of GST-ABF2 fragment by MBP-SnRK2.6. Bands of GST-ABF2 fragment and MBP-SnRK2.6 are indicated by an arrow and an arrowhead, respectively. Radioactivities of GST-ABF2 fragment bands were measured with a phospho-imager and were normalized, taking the radioactivity of the band by MBP-SnRK2.6 without ABI1 treatment as 1 (mean ± s.e.m., n = 5). b, Coomassie staining of purified MBP, SnRK2.6, ABF2, GST and GST-ABI1. c, FLAG-SnRK2.6 extracted from transgenic plants before and after ABA treatment was used instead of MBP-SnRK2.6 in (a). Coomassie staining, autoradiography and relative radioactivities (mean ± s.e.m. n = 5) of GST-ABF2 fragment are shown. Western blotting with anti-FLAG antibody shows FLAG-SnRK2.6 protein amount. d, Autoradiography of autophosphorylated SnRK2.6 showing dephosphorylation of SnRK2.6 by MBP-ABI1 and MBP-ABI2 and the effect of PYL8 and PYL5, respectively, in the presence of 1 μM ABA. e, Phosphate release from synthetic peptide HSQPKpSTVGTP, corresponding to amino acids 170-180 of SnRK2.6.
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Figure 2: ABI1 and ABI2 inhibit SnRK2.6 by dephosphorylationa, SnRK2.6 is deactivated by ABI1. MBP or MBP-SnRK2.6 treated without (−) or with GST-ABI1 or GST was incubated with GST-ABF2 fragment (amino acids Gly73 to Gln119) in the presence of [γ32P]-ATP. In the furthest right lane (post), GST-ABI1 was added after phosphorylation of GST-ABF2 fragment by MBP-SnRK2.6. Bands of GST-ABF2 fragment and MBP-SnRK2.6 are indicated by an arrow and an arrowhead, respectively. Radioactivities of GST-ABF2 fragment bands were measured with a phospho-imager and were normalized, taking the radioactivity of the band by MBP-SnRK2.6 without ABI1 treatment as 1 (mean ± s.e.m., n = 5). b, Coomassie staining of purified MBP, SnRK2.6, ABF2, GST and GST-ABI1. c, FLAG-SnRK2.6 extracted from transgenic plants before and after ABA treatment was used instead of MBP-SnRK2.6 in (a). Coomassie staining, autoradiography and relative radioactivities (mean ± s.e.m. n = 5) of GST-ABF2 fragment are shown. Western blotting with anti-FLAG antibody shows FLAG-SnRK2.6 protein amount. d, Autoradiography of autophosphorylated SnRK2.6 showing dephosphorylation of SnRK2.6 by MBP-ABI1 and MBP-ABI2 and the effect of PYL8 and PYL5, respectively, in the presence of 1 μM ABA. e, Phosphate release from synthetic peptide HSQPKpSTVGTP, corresponding to amino acids 170-180 of SnRK2.6.
Mentions: Author Contributions: HF contributed Figures 2a-c & e, Figure 3, Figures 4a-c and Supplementary Figure 4. VC contributed Figures 1a-d, and Supplementary Figures 1a & b. AR, SR, RA and PLR contributed Figure 2d, and Supplementary figures 2 & 3. SYP and SRC assisted with the generation of recombinant proteins, and SRC helped edit the manuscript. JS assisted with protoplast assays. JKZ designed the experiments, and wrote the paper together with VC and HF.

Bottom Line: Introduction of these four components into plant protoplasts results in ABA-responsive gene expression.We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases.Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Botany and Plant Sciences, University of California at Riverside, Riverside, California 92521, USA.

ABSTRACT
The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

Show MeSH