Proc Natl Acad Sci U S A 103(36): 13549-13554

Protein phosphatase 1 positively regulates stomatal opening in response to blue light in <em>Vicia faba</em>

*Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan; and

^{Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan}

^{To whom correspondence should be addressed. E-mail:}pj.ca.u-uhsuyk.cn.xobm@bcrnek

Edited by Peter H. Quail, University of California, Berkeley, CA, and approved July 20, 2006

^{Present address: RIKEN Genomic Sciences Center, Tsurumi-ku, Yokohama 230-0045, Japan.}

Author contributions: A.T. and K.-i.S. designed research; A.T. and M.A. performed research; T.K. contributed new reagents/analytic tools; A.T. analyzed data; and A.T. and K.-i.S. wrote the paper.

Edited by Peter H. Quail, University of California, Berkeley, CA, and approved July 20, 2006

Received 2006 Mar 28

Abstract

Phototropins, plant blue light receptors, mediate stomatal opening through the activation of the plasma membrane H^{-ATPase by unknown mechanisms. Here we report that type 1 protein phosphatase (PP1) positively regulates the blue light signaling between phototropins and the H}^{-ATPase in guard cells of}Vicia faba. We cloned the four catalytic subunits of PP1 (PP1c) from guard cells and determined the expression of the isoforms in various tissues. Transformation of Vicia guard cells with PP1c isoforms that had lost enzymatic activity by one amino acid mutation, or with human inhibitor-2, a specific inhibitor protein of PP1c, suppressed blue light-induced stomatal opening. Addition of fusicoccin, an activator of the plasma membrane H^{-ATPase, to these transformed guard cells induced normal stomatal opening, suggesting that the transformations did not affect the basic mechanisms for stomatal opening. Tautomycin, an inhibitor of PP1, inhibited blue light-induced H}^{pumping, phosphorylation of the plasma membrane H}^{-ATPase in guard cell protoplasts, and stomatal opening. However, tautomycin did not inhibit the blue light-dependent phosphorylation of phototropins. We conclude that PP1 functions downstream of phototropins and upstream of the H}^{-ATPase in the blue light signaling pathway of guard cells.}

Keywords: phototropin, H^{-ATPase, light signaling, phosphorylation/dephosphorylation}

Abstract

Pairs of guard cells forming stomatal pores regulate gas exchange between plants and the atmosphere, allowing the uptake of CO₂ for photosynthetic carbon fixation and the loss of water vapor by transpiration (1 –3). Stomata open in response to blue light perceived by phototropins (phot1 and phot2), plant-specific Ser/Thr protein kinases with two LOV (light, oxygen, or voltage) domains (4 –6). The light-stimulated phototropins undergo autophosphorylation with a subsequent binding of 14-3-3 proteins (7). Signals from phototropins ultimately activate the plasma membrane H^{-ATPase via phosphorylation of the C terminus with a subsequent binding of 14-3-3 protein (}8). The activated H^{-ATPase creates an inside-negative electrical potential across the plasma membrane, which drives the uptake of K}^{through inward-rectifying voltage-gated K}^{channels (}9 –11); this leads to an increase in turgor pressure that results in stomatal opening. However, the mechanisms of signal transduction from phototropins to the plasma membrane H^{-ATPase are largely unknown. Several components, including cytosolic Ca}^{, RPT2 (ROOT PHOTOTROPISM2), and protein kinase in the plasma membrane, have been suggested to play a role, but the evidence to support these claims is not conclusive (}12 –14).

A previous pharmacological study showed that type 1 or type 2A protein phosphatases (PP1/PP2A) are involved in blue light-dependent stomatal opening in Vicia faba (15). Furthermore, PP1 appears more likely than PP2A to be responsible for this reaction because blue light-dependent H^{pumping was more strongly inhibited by calyculin A than okadaic acid. However, no conclusive evidence for the functional involvement of PP1 in the stomatal response is available so far.}

PP1 is the major class of the PPP family of Ser/Thr protein phosphatases and is ubiquitously distributed throughout higher eukaryotes (16). In animals, PP1 has been shown to regulate a broad range of cellular processes such as glycogen metabolism, protein synthesis, cell cycle progression, muscle contraction, transcription, and neuronal signaling (17). Regulation of these multiple processes is accomplished by a few PP1 catalytic subunits (PP1c) associating with multiple regulatory subunits that specify the catalytic activity, the substrate specificity, and the subcellular localization of the holoenzyme (18, 19). Considering the functional importance of PP1 in animals, it is likely that PP1 also regulates fundamental processes in plant cells; however, very little is known about PP1 function in plants (16).

Recently, several experimental approaches to separate the functions of the PP1 and PP2A families in vivo have been reported. Mutational studies have shown that the expression of a dominant negative-type mutant of the PP2A catalytic subunit causes growth defects and reduction of PP2A activity without affecting its holoenzyme assembly in yeast (20, 21). In Drosophila cells, overexpression of inhibitor-2, which inhibits PP1c activity as a regulatory subunit, specifically reduces PP1 activity without affecting that of PP2A (22).

In the present study, we investigated the function of PP1 in guard cells, a highly developed model system for characterizing signal transduction mechanisms in plants (23). In this system, we can induce the signaling cascade by applying blue light and directly observe specific effects caused by any genetic or pharmacological perturbation. We transformed Vicia guard cells with a dominant negative-form of PP1c as well as with inhibitor-2 and demonstrated that PP1 functions as a positive regulator between phototropins and the H^{-ATPase in the blue light signaling pathway.}

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Acknowledgments

We thank Xiao Zhang (Department of Biology, Henan University, Henan, China) for help with H^{pumping measurements. This research was partially supported by Japanese Ministry of Education, Science, Sports, and Culture Grants-in-Aid for Scientific Research on Priority Areas 17084005, Scientific Research (A) 16207003 (to K.-i.S.), and Young Scientists (A) 14704003 (to T.K.).}

Acknowledgments

Abbreviations

phot	phototropin
PP1	type 1 protein phosphatase
PP1c	catalytic subunit of PP1
VfPP1c	V. faba PP1c.

Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AB038648","term_id":"7209507"}}AB038648 and {"type":"entrez-nucleotide","attrs":{"text":"AB254850","term_id":"114213453"}}AB254850 {"type":"entrez-nucleotide","attrs":{"text":"AB254851","term_id":"114213455"}}AB254851–{"type":"entrez-nucleotide","attrs":{"text":"AB254852","term_id":"114213457"}}AB254852).

Footnotes

References

1. Zeiger E. Annu. Rev. Plant Physiol. 1983;34:441–475.[PubMed]
2. Assmann SM., Shimazaki K. Plant Physiol. 1999;119:809–815.[Google Scholar]
3. Schroeder J. I., Allen G. J., Hugouvieux V., Kwak J. M., Waner D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001;52:627–658.[PubMed]
4. Huala E., Oeller P. W., Liscum E., Han I. S., Larsen E., Briggs W. R. Science. 1997;278:2120–2123.[PubMed]
5. Christie J. M., Reymond P., Powell G. K., Bernasconi P., Raibekas A. A., Liscum E., Briggs W. R. Science. 1998;282:1698–1701.[PubMed]
6. Kinoshita T., Doi M., Suetsugu N., Kagawa T., Wada M., Shimazaki K. Nature. 2001;414:656–660.[PubMed]
7. Kinoshita T., Emi T., Tominaga M., Sakamoto K., Shigenaga A., Doi M., Shimazaki K. Plant Physiol. 2003;133:1453–1463.
8. Kinoshita T., Shimazaki K. EMBO J. 1999;18:5548–5558.
9. Assmann S. M., Simoncini L., Schroeder J. I. Nature. 1985;318:285–287.[PubMed]
10. Shimazaki K., Iino M., Zeiger E. Nature. 1986;319:324–326.[PubMed]
11. Schroeder JI., Raschke K., Neher E. Proc. Natl. Acad. Sci. USA. 1987;84:4108–4112.[Google Scholar]
12. Shimazaki K., Goh C.-H., Kinoshita T. Physiol. Plantarum. 1999;105:554–561.[PubMed]
13. Svennelid F., Olsson A., Piotrowski M., Rosenquist M., Ottman C., Larsson C., Oecking C., Sommarin M. Plant Cell. 1999;11:2379–2391.
14. Inada S., Ohgishi M., Mayama T., Okada K., Sakai T. Plant Cell. 2004;16:887–896.
15. Kinoshita T., Shimazaki K. Plant Cell Physiol. 1997;38:1281–1285.[PubMed]
16. Luan S. Annu. Rev. Plant Biol. 2003;54:63–92.[PubMed]
17. Ceulemans H., Bollen M. Physiol. Rev. 2004;84:1–39.[PubMed]
18. Bollen M. Trends Biochem. Sci. 2001;26:426–431.[PubMed]
19. Cohen P. T. W. J. Cell Sci. 2002;115:241–256.[PubMed]
20. Evans D. R. H., Myles T., Hofsteenge J., Hemmings B. A. J. Biol. Chem. 1999;274:24038–24046.[PubMed]
21. Lizotte D. L., McManus D. D., Cohen H. R., DeLong A. Gene. 1999;234:35–44.[PubMed]
22. Bennett D., Szöor B., Gross S., Vereshchagina N., Alphey L. Genetics. 2003;164:235–245.
23. Fan L.-M., Zhao Z., Assmann SM. Curr. Opin. Plant Biol. 2004;7:537–547.[PubMed][Google Scholar]
24. Egloff M.-P., Cohen P. T. W., Reinemer P., Barford D. J. Mol. Biol. 1995;254:942–959.[PubMed]
25. MacKintosh C., Klumpp S. FEBS Lett. 1990;277:137–140.[PubMed]
26. Favre B., Turowski P., Hemmings BA. J. Biol. Chem. 1997;272:13856–13863.[PubMed][Google Scholar]
27. Resjö S., Oknianska A., Zolnierowicz S., Manganiello V., Degerman E. Biochem. J. 1999;341:839–845.
28. Yan Y., Mumby MC. J. Biol. Chem. 1999;274:31917–31924.[PubMed][Google Scholar]
29. Smith R. D., Walker J. C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996;47:101–125.[PubMed]
30. Kim D.-H., Kang J.-G., Yang S.-S., Chung K.-S., Song P.-S., Park C.-M. Plant Cell. 2002;14:3043–3056.
31. Ryu J. S., Kim J.-I., Kunkel T., Kim B. C., Cho D. S., Hong S. H., Kim S.-H., Fernández A. P., Kim Y., Alonso J. M., et al. Cell. 2005;120:395–406.[PubMed]
32. Møller SG., Kim Y.-S., Kunkel T., Chua N.-H. Plant Cell. 2003;15:1111–1119.[Google Scholar]
33. Arabidopsis Genome Initiative. Nature. 2000;408:796–815.[PubMed]
34. Kerk D., Bulgrien J., Smith DW., Barsam B., Veretnik S., Gribskov M. Plant Physiol. 2002;129:908–925.[Google Scholar]
35. Lin Q., Li J., Smith R. D., Walker J. C. Plant Mol. Biol. 1998;37:471–481.[PubMed]
36. Sakai T., Kagawa T., Kasahara M., Swartz T. E., Christie J. M., Briggs W. R., Wada M., Okada K. Proc. Natl. Acad. Sci. USA. 2001;98:6969–6974.
37. Lin C. Plant Cell. 2002;14(Suppl.):S207–S225.
38. Liscum E., Hodgson D. W., Campbell T. J. Plant Physiol. 2003;133:1429–1436.
39. Chen M., Chory J., Fankhauser C. Annu. Rev. Genet. 2004;38:87–117.[PubMed]
40. Motchoulski A., Liscum E. Science. 1999;286:961–964.[PubMed]
41. Sakai T., Wada T., Ishiguro S., Okada K. Plant Cell. 2000;12:225–236.
42. Briggs W. R., Christie J. M. Trends Plant Sci. 2002;7:204–210.[PubMed]
43. Takemiya A., Inoue S., Doi M., Kinoshita T., Shimazaki K. Plant Cell. 2005;17:1120–1127.
44. Jarillo J. A., Gabrys H., Capel J., Alonso J. M., Ecker J. R., Cashmore A. R. Nature. 2001;410:952–954.[PubMed]
45. Kagawa T., Sakai T., Suetsugu N., Oikawa K., Ishiguro S., Kato T., Tabata S., Okada K., Wada M. Science. 2001;291:2138–2141.[PubMed]
46. Sakamoto K., Briggs WR. Plant Cell. 2002;14:1723–1735.[Google Scholar]
47. Inoue S., Kinoshita T., Shimazaki K. Plant Physiol. 2005;138:1994–2004.
48. Shimazaki K., Kinoshita T., Nishimura M. Plant Physiol. 1992;99:1416–1421.
49. Niwa Y., Hirano T., Yoshimoto K., Shimizu M., Kobayashi H. Plant J. 1999;18:455–463.[PubMed]
50. Watanabe T., da Cruz e Silva E. F., Huang H.-B., Starkova N., Kwon Y.-G., Horiuchi A., Greengard P., Nairn A. C. Methods Enzymol. 2003;366:321–338.[PubMed]
51. Zhang X., Wang H., Takemiya A., Song CP., Kinoshita T., Shimazaki K. Plant Physiol. 2004;136:4150–4158.[Google Scholar]