In vitro reconstitution of an abscisic acid signalling pathway.

Nature
Hiroaki FujiiJian-Kang Zhu

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 ...Continue Reading

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Citations

Jul 26, 2012·Cellular and Molecular Life Sciences : CMLS·Hiroaki Fujii, Jian-Kang Zhu
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Methods Mentioned

BETA
transfection
two-hybrid
co-immunoprecipitation assay
co-immunoprecipitation
affinity purification
electrophoresis
PCR
immunoprecipitation
transgenic

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