In vitro Reconstitution of an ABA Signaling Pathway

Abstract

The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants and plays critical roles in stress resistance, and 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 signaling 3, 4, 14. However, the identities of ABA receptors remain controversial and the mechanism of signaling from perception to downstream gene expression is unclear 15, 16. Here we show that by combining the recently identified ABA receptor PYR1, with the protein phosphatase 2C 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. The 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 receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2Cs-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signaling mechanisms and define a minimal set of core components of a complete major ABA signaling pathway.