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.

In vitro reconstitution of an abscisic acid signalling pathway

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Change history

  • 03 December 2009

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

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 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)

    CAS  ADS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  ADS  Article  Google Scholar 

  9. 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)

    CAS  ADS  Article  Google Scholar 

  10. 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)

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  ADS  PubMed  PubMed Central  Google Scholar 

  12. 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. 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. 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  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 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)

    CAS  Article  Google Scholar 

  18. 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)

    CAS  Article  Google Scholar 

  19. 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)

    CAS  Article  Google Scholar 

  20. 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)

    CAS  Article  Google Scholar 

  21. 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)

    CAS  ADS  Article  Google Scholar 

  22. 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  Google Scholar 

  23. 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)

    CAS  ADS  Article  Google Scholar 

  24. 24

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

    CAS  ADS  Article  Google Scholar 

  25. 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)

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 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)

    CAS  Article  Google Scholar 

  28. 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)

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  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

Affiliations

Authors

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

Further reading

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

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