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In vitro reconstitution of an abscisic acid signalling pathway

Nature volume 462, pages 660–664 (2009)Cite this article

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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 development1,2,3,4,5,6,7. Several proteins have been reported to function as ABA receptors8,9,10,11,12,13, and many more are known to be involved in ABA signalling3,4,14. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear15,16. 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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Figure 1: Reconstitution of the ABA signalling pathway for stress-responsive gene expression in Arabidopsis protoplasts.
Figure 2: ABI1 and ABI2 inhibit SnRK2.6 by dephosphorylation.
Figure 3: The combined effect of ABA, PYR1 and ABI1 on the phosphorylation of the GST–ABF2 fragment by SnRK2.6 in vitro.
Figure 4: Effect of PP2C mutations on ABA response phenotypes and kinase activities of SnRK2s.

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  • 03 December 2009

    Two of the labels on Fig. 1b were corrected on 3 December 2009.

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Acknowledgements

We thank R. Bressan for helpful discussions and for editing of the manuscript. This work was supported by National Institutes of Health grants (J.-K.Z.) and MICIIN and CSIC fellowships (S.R. and R.A.).

Author Contributions H.F. contributed Figs 2a–c, e, 3, 4a–c and Supplementary Fig. 4. V.C. contributed Fig. 1a–d and Supplementary Fig. 1a, b. A.R., S.R., R.A. and P.L.R. contributed Fig. 2d and Supplementary Figs 2 and 3. S.-Y.P. and S.R.C. assisted with the generation of recombinant proteins, and S.R.C. helped edit the manuscript. J.S. assisted with protoplast assays. J.-K.Z. designed the experiments, and wrote the paper together with V.C. and H.F.

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Author notes

  1. Hiroaki Fujii and Viswanathan Chinnusamy: These authors contributed equally to this work.

Authors and Affiliations

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

    Hiroaki Fujii, Viswanathan Chinnusamy, Sang-Youl Park, Sean R. Cutler & Jian-Kang Zhu

  2. Center for Plant Stress Genomics and Technology, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

    Hiroaki Fujii, Viswanathan Chinnusamy & Jian-Kang Zhu

  3. Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-UPV, ES-46022 Valencia, Spain ,

    Americo Rodrigues, Silvia Rubio, Regina Antoni & Pedro L. Rodriguez

  4. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA,

    Jen Sheen

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Correspondence to Jian-Kang Zhu.

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Fujii, H., Chinnusamy, V., Rodrigues, A. et al. In vitro reconstitution of an abscisic acid signalling pathway . Nature 462, 660–664 (2009). https://doi.org/10.1038/nature08599

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