A1 Refereed original research article in a scientific journal
In vitro reconstitution of an abscisic acid signalling pathway
Authors: Fujii, Hiroaki; Chinnusamy, Viswanathan; Rodrigues, Americo; Rubio, Silvia; Antoni, Regina; Park, Sang-Youl; Cutler, Sean R.; Sheen, Jen; Rodriguez, Pedro L.; Zhu, Jian-Kang
Publisher: NATURE PORTFOLIO
Publishing place: BERLIN
Publication year: 2009
Journal: Nature
Journal name in source: NATURE
Journal acronym: NATURE
Volume: 462
Issue: 7273
First page : 660
Last page: U138
Number of pages: 7
ISSN: 0028-0836
eISSN: 1476-4687
DOI: https://doi.org/10.1038/nature08599(external)
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(1-7). Several proteins have been reported to function as ABA receptors(8-13), and many more are known to be involved in ABA signalling(3,4,14). However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear(15,16). 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.
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(1-7). Several proteins have been reported to function as ABA receptors(8-13), and many more are known to be involved in ABA signalling(3,4,14). However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear(15,16). 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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