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Small-Molecule Dissection of Brassinosteroid Signaling

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 876)

Abstract

The growth-promoting hormones, the brassinosteroids (BRs), are perceived at the plant cell surface by receptor kinases that transduce the signal to the nucleus by an intracellular cascade of phosphorylation-mediated protein–protein interactions. BR signaling is also regulated by the plant endocytic machinery because the increased endosomal localization of the BR receptor enhances the BR responses. Chemical genetics is a powerful approach to identify new components in redundant signaling networks and to characterize highly dynamic processes, such as endocytosis. Here, we describe a screen in Arabidopsis thaliana seedlings for small molecules that affect hypocotyl elongation under continuous light conditions, indicative for an effect on BR responses. The compounds identified in this screen were used to dissect endomembrane trafficking of the BR receptor, BR INSENSITIVE1, a process that is essential for BR signal transduction.

Key words

Arabidopsis Brassinosteroids Endocytosis Chemical genetics BRI1 
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Notes

Acknowledgments

We thank Karin Schumacher, Jiří Friml, Natasha Raikhel, Niko Geldner, and Daniël Van Damme for providing seeds of marker lines and Martine De Cock for help in preparing the manuscript. This work was supported by a grant from the Research Foundation—Flanders (G.0065.08). DA is a postdoctoral fellow of the Research Foundation—Flanders.

References

  1. 1.
    Clouse SD, Sasse JM (1998) Brassinosteroids: essential regulators of plant growth and development. Annu Rev Plant Physiol Plant Mol Biol 49:427–451PubMedCrossRefGoogle Scholar
  2. 2.
    Kinoshita T, Caño-Delgado A, Seto H, Hiranuma S, Fujioka S, Yoshida S, Chory J (2005) Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433:167–171PubMedCrossRefGoogle Scholar
  3. 3.
    Caño-Delgado A, Yin Y, Yu C, Vafeados D, Mora-García S, Cheng J-C, Nam KH, Li J, Chory J (2004) BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131:5341–5351PubMedCrossRefGoogle Scholar
  4. 4.
    Kim T-W, Wang Z-Y (2010) Brassinosteroid signal transduction from receptor kinases to transcription factors. Annu Rev Plant Biol 61:681–704PubMedCrossRefGoogle Scholar
  5. 5.
    Russinova E, Borst J-W, Kwaaitaal M, Caño-Delgado A, Yin Y, Chory J, de Vries SC (2004) Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16:3216–3229PubMedCrossRefGoogle Scholar
  6. 6.
    Geldner N, Hyman DL, Wang X, Schumacher K, Chory J (2007) Endosomal signaling of plant steroid receptor kinase BRI1. Genes Dev 21:1598–1602PubMedCrossRefGoogle Scholar
  7. 7.
    Robert S, Chary SN, Drakakaki G, Li S, Yang Z, Raikhel NV, Hicks GR (2008) Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1. Proc Natl Acad Sci USA 105:8464–8469PubMedCrossRefGoogle Scholar
  8. 8.
    Tang W, Deng Z, Wang Z-Y (2010) Proteomics shed light on the brassinosteroid signaling mechanisms. Curr Opin Plant Biol 13:27–33PubMedCrossRefGoogle Scholar
  9. 9.
    Hicks GR, Raikhel NV (2009) Opportunities and challenges in plant chemical biology. Nat Chem Biol 5:268–272PubMedCrossRefGoogle Scholar
  10. 10.
    Tóth R, van der Hoorn RAL (2009) Emerging principles in plant chemical genetics. Trends Plant Sci 15:81–88PubMedCrossRefGoogle Scholar
  11. 11.
    De Rybel B, Audenaert D, Beeckman T, Kepinski S (2009) The past, present and future of chemical biology in auxin research. ACS Chem Biol 4:987–998PubMedCrossRefGoogle Scholar
  12. 12.
    Szekeres M, Németh K, Koncz-Kálmán Z, Mathur J, Kauschmann A, Altmann T, Rédei GP, Nagy F, Schell J, Koncz C (1996) Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85:171–182PubMedCrossRefGoogle Scholar
  13. 13.
    Kauschmann A, Jessop A, Koncz C, Szekeres M, Willmitzer L, Altmann T (1996) Genetic evidence for an essential role of brassinosteroids in plant development. Plant J 9:701–713CrossRefGoogle Scholar
  14. 14.
    Gendron JM, Haque A, Gendron N, Chang T, Asami T, Wang Z-Y (2008) Chemical genetic dissection of brassinosteroid–ethylene interaction. Mol Plant 1:368–379PubMedCrossRefGoogle Scholar
  15. 15.
    De Rybel B, Audenaert D, Vert G, Rozhon W, Mayerhofer J, Peelman F, Coutuer S, Denayer T, Jansen L, Nguyen L, Vanhoutte I, Beemster GTS, Vleminckx K, Jonak C, Chory J, Inzé D, Russinova E, Beeckman T (2009) Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling. Chem Biol 16:594–604PubMedCrossRefGoogle Scholar
  16. 16.
    Nemhauser JL, Hong F, Chory J (2006) Different plant hormones regulate similar processes through largely nonoverlapping transcriptional responses. Cell 126:467–475PubMedCrossRefGoogle Scholar
  17. 17.
    Mathur J, Molnár G, Fujioka S, Takasutso S, Sakurai A, Yokota T, Adam G, Voigt B, Nagy F, Maas C, Schell J, Koncz C, Szekeres M (1998) Transcription of the Arabidopsis CPD gene, encoding a steroidogenic cytochrome P450, is negatively controlled by brassinosteroids. Plant J 14:593–602PubMedCrossRefGoogle Scholar
  18. 18.
    Vert G, Nemhauser JL, Geldner N, Hong F, Chory J (2005) Molecular mechanisms of steroid hormone signaling in plants. Annu Rev Cell Dev Biol 21:177–201PubMedCrossRefGoogle Scholar
  19. 19.
    Li J, Nam KH (2002) Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science 295:1299–1301PubMedGoogle Scholar
  20. 20.
    Friedrichsen DM, Joazeiro CAP, Li J, Hunter T, Chory J (2000) Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol 123:1247–1255PubMedCrossRefGoogle Scholar
  21. 21.
    Dettmer J, Hong-Hermesdorf A, Stierhof Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18:715–730PubMedCrossRefGoogle Scholar
  22. 22.
    Marc J, Granger CL, Brincat J, Fisher DD, Kao T-h, McCubbin AG, Cyr RJ (1998) A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10:1927–1939PubMedCrossRefGoogle Scholar
  23. 23.
    Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW (2004) A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiol 136:3968–3978PubMedCrossRefGoogle Scholar
  24. 24.
    Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof Y-D, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169–178PubMedCrossRefGoogle Scholar
  25. 25.
    Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Plant Systems BiologyVIBGhentBelgium
  2. 2.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  3. 3.VIB Compound Screening FacilityGhentBelgium

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