Abstract
The gene families that encode the vesicle trafficking machinery in plants are highly expanded compared to those from protists and animals. As such, classical genetic screens for mutants with lesions in these genes are fraught with issues of redundancy and lethality. A chemical genomics approach can, in theory, circumvent these issues because inhibitory or stimulatory molecules may be applied at any point in development at sublethal concentrations. This chapter describes the protocols for a chemical genomics screen designed to identify components of the plant cell vesicle trafficking machinery. A two-tiered screen was designed where the primary screen assayed for chemicals that modified the gravitropic response, a process that in plant cells is intimately tied to vesicle trafficking; the secondary screen employed fluorescent marker lines that were treated with gravitropic inhibitors or inducers to assay for changes in endomembrane system morphology. We thus identified four compounds by which we can further explore the relationship between gravitropic signal transduction and vesicle trafficking.
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References
Sanderfoot AA, Assaad FF, Raikhel NV (2000) The Arabidopsis genome. An abundance of soluble N-ethylmaleimide-sensitive factor adaptor protein receptors. Plant Physiol 124:1558–1569
Rojo E, Gillmor CS, Kovaleva V, Somerville CR, Raikhel NV (2001) VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis. Dev Cell 1:303–310
Blackwell HE, Zhao Y (2003) Chemical genetic approaches to plant biology. Plant Physiol 133:448–455
Kato T, Morita MT, Tasaka M (2002) Role of endodermal cell vacuoles in shoot gravitropism. J Plant Growth Regul 21:113–119
Kato T, Morita MT, Fukaki H, Yamauchi Y, Uehara M, Niihama M, Tasaka M (2002) SGR2, a phospholipase-like protein, and ZIG/SGR4, a SNARE, are involved in the shoot gravitropism of Arabidopsis. Plant Cell 14:33–46
Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J (1999) The Arabidopsis det3 mutant reveals a central role for the vacuolar H(+)-ATPase in plant growth and development. Genes Dev 13:3259–3270
Surpin M, Rojas-Pierce M, Carter C, Hicks GR, Vasquez J, Raikhel NV (2005) The power of chemical genomics to study the link between endomembrane system components and the gravitropic response. Proc Natl Acad Sci U S A 102:4902–4907
Zouhar J, Hicks GR, Raikhel NV (2004) Sorting inhibitors (Sortins): chemical compounds to study vacuolar sorting in Arabidopsis. Proc Natl Acad Sci U S A 101:9497–9501
Rojas-Pierce M, Titapiwatanakun B, Sohn EJ, Fang F, Larive CK, Blakeslee J, Cheng Y, Cutler SR, Cuttler S, Peer WA, Murphy AS, Raikhel NV (2007) Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin. Chem Biol 14:1366–1376
Surpin M, Zou Y, Xiong C, Raikhel NV, Pirrung MC (2010) Iodine scanning of a phenazine inhibitor of vacuolar sorting. Bioorg Med Chem Lett 20:1496–1499
Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42
Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci U S A 97:3718–3723
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 U S A 105:8464–8469
Girke T, Cheng LC, Raikhel N (2005) ChemMine. A compound mining database for chemical genomics. Plant Physiol 138:573–577
Pirrung MC, Liu Y, Deng L, Halstead DK, Li Z, May JF, Wedel M, Austin DA, Webster NJ (2005) Methyl scanning: total synthesis of demethylasterriquinone B1 and derivatives for identification of sites of interaction with and isolation of its receptor(s). J Am Chem Soc 127:4609–4624
Robert S, Raikhel NV, Hicks GR (2009) Powerful partners: Arabidopsis and chemical genomics. Arabidopsis Book 7:e0109
Bolton EE, Wang Y, Thiessen PA, Bryant SH (2008) PubChem: integrated platform of small molecules and biological activities. In: Annual reports in computational chemistry, vol 4, American Chemical Society, Washington, DC
Acknowledgments
The work described in this chapter was funded by grants from the National Aeronautics and Space Administration (NNA04CC73G) to Glenn R. Hicks and the National Science Foundation (MCB-0515963) to Natasha V. Raikhel. Special thanks are due to Dr. Paul Silverman and Dr. Clay Carter for helpful comments.
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Surpin, M. (2014). Plant Chemical Genomics: Gravity Sensing and Response. In: Hicks, G., Robert, S. (eds) Plant Chemical Genomics. Methods in Molecular Biology, vol 1056. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-592-7_12
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DOI: https://doi.org/10.1007/978-1-62703-592-7_12
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