Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

In vitro reconstitution of an abscisic acid signalling pathway

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

This article has been updated

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Change history

  • 03 December 2009

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

References

  1. Zhu, J. K. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247–273 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hetherington, A. M. Guard cell signaling. Cell 107, 711–714 (2001)

    Article  CAS  PubMed  Google Scholar 

  3. Schroeder, J. I., Kwak, J. M. & Allen, G. J. Guard cell abscisic acid signalling and engineering of drought hardiness in plants. Nature 410, 327–330 (2001)

    Article  CAS  ADS  PubMed  Google Scholar 

  4. Finkelstein, R. R., Gampala, S. S. & Rock, C. D. Abscisic acid signaling in seeds and seedlings. Plant Cell 14 (Suppl). S15–S45 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Koornneef, M., Bentsink, L. & Hilhorst, H. Seed dormancy and germination. Curr. Opin. Plant Biol. 5, 33–36 (2002)

    Article  CAS  PubMed  Google Scholar 

  6. Roelfsema, M. R. G. & Hedrich, R. In the light of stomatal opening—new insights into ‘the Watergate’. New Phytol. 167, 665–691 (2005)

    Article  CAS  PubMed  Google Scholar 

  7. Nemhauser, J. L., Hong, F. & Chory, J. Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses. Cell 126, 467–475 (2006)

    Article  CAS  PubMed  Google Scholar 

  8. Shen, Y. Y. et al. The Mg-chelatase H subunit is an abscisic acid receptor. Nature 443, 823–826 (2006)

    Article  CAS  ADS  PubMed  Google Scholar 

  9. Liu, X. et al. G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid. Science 315, 1712–1716 (2007)

    Article  CAS  ADS  PubMed  Google Scholar 

  10. Pandey, S., Nelson, D. C. & Assmann, S. M. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis . Cell 136, 136–148 (2009)

    Article  CAS  PubMed  Google Scholar 

  11. Ma, Y. et al. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324, 1064–1068 (2009)

    CAS  ADS  PubMed  Google Scholar 

  12. Park, S. Y. et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324, 1068–1071 (2009)

    CAS  ADS  PubMed  PubMed Central  Google Scholar 

  13. Santiago, J. et al. Modulation of drought resistance by the abscisic acid-receptor PYL5 through inhibition of clade A PP2Cs.Plant J. 10.1111/j.1365–313X.2009.03981.x (2009)

  14. Li, S., Assmann, S. M. & Albert, R. Predicting essential components of signal transduction networks: a dynamic model of guard cell abscisic acid signaling. PLoS Biol. 4, e312 (2006)

    Article  PubMed  PubMed Central  Google Scholar 

  15. McCourt, P. & Creelman, R. The ABA receptors–we report you decide. Curr. Opin. Plant Biol. 11, 474–478 (2008)

    Article  CAS  PubMed  Google Scholar 

  16. Pennisi, E. Stressed out over a stress hormone. Science 324, 1012–1013 (2009)

    Article  CAS  PubMed  Google Scholar 

  17. Mustilli, A. C., Merlot, S., Vavasseur, A., Fenzi, F. & Giraudat, J. Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089–3099 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yoshida, R. et al. ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis . Plant Cell Physiol. 43, 1473–1483 (2002)

    Article  CAS  PubMed  Google Scholar 

  19. Yoshida, R. et al. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis . J. Biol. Chem. 281, 5310–5318 (2006)

    Article  CAS  PubMed  Google Scholar 

  20. Fujii, H., Verslues, P. E. & Zhu, J. K. Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis . Plant Cell 19, 485–494 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fujii, H. & Zhu, J. K. Arabidopsis mutant deficient in three abscisic acid-activated protein kinases reveals critical roles in growth, reproduction and stress. Proc. Natl Acad. Sci. USA 106, 8380–8385 (2009)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  22. Johnson, R. R., Wagner, R. L., Verhey, S. D. & Walker-Simmons, M. K. The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences. Plant Physiol. 130, 837–846 (2002)

    Article  PubMed  PubMed Central  Google Scholar 

  23. Furihata, T. et al. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc. Natl Acad. Sci. USA 103, 1988–1993 (2006)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  24. Belin, C. et al. Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant Physiol. 141, 1316–1327 (2006)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

  25. Rubio, S. et al. Triple loss of function of protein phosphatases type 2C leads to partial constitutive response to endogenous abscisic acid. Plant Physiol. 150, 1345–1355 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kuhn, J. M. & Schroeder, J. I. Impacts of altered RNA metabolism on abscisic acid signaling. Curr. Opin. Plant Biol. 6, 463–469 (2003)

    Article  CAS  PubMed  Google Scholar 

  27. Hirayama, T. & Shinozaki, K. Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone ABA. Trends Plant Sci. 12, 343–351 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565–1572 (2007)

    Article  CAS  PubMed  Google Scholar 

  29. Van Veldhoven, P. P. & Mannaerts, G. P. Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161, 45–48 (1987)

    Article  CAS  PubMed  Google Scholar 

  30. Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004)

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Hiroaki Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Viswanathan Chinnusamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Americo Rodrigues
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Silvia Rubio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Regina Antoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sang-Youl Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sean R. Cutler
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jen Sheen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Pedro L. Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jian-Kang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jian-Kang Zhu.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-4 with Legends. (PDF 571 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08599

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing