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Measuring PLD Activity In Vivo

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Book cover Plant Lipid Signaling Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1009))

Abstract

Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927–935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349–2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo.

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References

  1. Li M, Hong Y, Wang X (2009) Phospholipase D- and phosphatidic acid-mediated signaling in plants. Biochim Biophys Acta 1791:927–935

    Article  CAS  PubMed  Google Scholar 

  2. Testerink C, Munnik T (2011) Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J Exp Bot 62:2349–2361

    Article  CAS  PubMed  Google Scholar 

  3. Pappan KL, Wang X (2012) Assaying different types of plant phospholipase D activities in vitro. Meth Mol Biol. 1009:205–217

    Article  Google Scholar 

  4. Arisz SA, Testerink C, Munnik T (2009) Plant PA signaling via diacylglycerol kinase. Biochim Biophys Acta 1791:869–875

    Article  CAS  PubMed  Google Scholar 

  5. Heller M (1978) Phospholipase D. Adv Lipid Res 16:267–326

    Article  CAS  PubMed  Google Scholar 

  6. Quarles RH, Dawson RM (1969) The distribution of phospholipase D in developing and mature plants. Biochem J 112:787–794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Walker SJ, Brown HA (2004) Measurement of G protein-coupled receptor-stimulated phospholipase D activity in intact cells. Methods Mol Biol 237:89–97

    CAS  PubMed  Google Scholar 

  8. Munnik T, Arisz SA, De Vrije T, Musgrave A (1995) G protein activation stimulates phospholipase D signaling in plants. Plant Cell 7:2197–2210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Frank W, Munnik T, Kerkmann K, Salamini F, Bartels D (2000) Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagineum. Plant Cell 12:111–124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. den Hartog M, Musgrave A, Munnik T (2001) Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation. Plant J 25:55–65

    Google Scholar 

  11. Bargmann BO, Laxalt AM, ter Riet B, Testerink C, Merquiol E, Mosblech A, Leon-Reyes A, Pieterse CM, Haring MA, Heilmann I, Bartels D, Munnik T (2009) Reassessing the role of phospholipase D in the Arabidopsis wounding response. Plant Cell Environ 32:837–850

    Article  CAS  PubMed  Google Scholar 

  12. Bargmann BO, Laxalt AM, ter Riet B, van Schooten B, Merquiol E, Testerink C, Haring MA, Bartels D, Munnik T (2009) Multiple PLDs required for high salinity and water deficit tolerance in plants. Plant Cell Physiol 50:78–89

    Article  CAS  PubMed  Google Scholar 

  13. Darwish E, Testerink C, Khaleil M, El-Shihy O, Munnik T (2009) Phospholipid-signaling responses in salt stressed rice leaves. Plant Cell Physiol 50:986–987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Van der Luit AH, Piatti T, van Doorn A, Musgrave A, Felix G, Boller T, Munnik T (2000) Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate. Plant Physiol 123:1507–1516

    Article  PubMed  PubMed Central  Google Scholar 

  15. Munnik T, de Vrije T, Irvine RF, Musgrave A (1996) Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants. J Biol Chem 271:15708–15715

    Article  CAS  PubMed  Google Scholar 

  16. Ramos-Diaz A, Brito-Argaez L, Munnik T, Hernandez-Sotomayor SM (2007) Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway. Planta 225:393–401

    Article  CAS  PubMed  Google Scholar 

  17. Munnik T, Meijer HJ, Ter Riet B, Hirt H, Frank W, Bartels D, Musgrave A (2000) Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. Plant J 22:147–154

    Article  CAS  PubMed  Google Scholar 

  18. den Hartog M, Verhoef N, Munnik T (2003) Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells. Plant Physiol 132:311–317

    Article  Google Scholar 

  19. Meijer HJ, ter Riet B, van Himbergen JA, Musgrave A, Munnik T (2002) KCl activates phospholipase D at two different concentration ranges: distinguishing between hyperosmotic stress and membrane depolarization. Plant J 31:51–59

    Article  CAS  PubMed  Google Scholar 

  20. Zonia L, Munnik T (2004) Osmotically induced cell swelling versus cell shrinking elicits specific changes in phospholipid signals in tobacco pollen tubes. Plant Physiol 134:813–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lanteri ML, Lamattina L, Laxalt AM (2011) Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells. Planta 234:845–855

    Article  CAS  PubMed  Google Scholar 

  22. De Vrije T, Munnik T (1997) Activation of phospholipase D by calmodulin antagonists and mastoparan in carnation petal tissue. J Exp Bot 48:1631–1637

    Article  Google Scholar 

  23. Distefano AM, Garcia-Mata C, Lamattina L, Laxalt AM (2008) Nitric oxide-induced phosphatidic acid accumulation: a role for phospholipases C and D in stomatal closure. Plant Cell Environ 31:187–194

    Article  CAS  PubMed  Google Scholar 

  24. Munnik T, Zarza X (2013) Rapid analysis of plant signaling phospholipids through 32Pi-labeling and TLC. Meth Mol Biol. 1009:3–15

    Article  CAS  Google Scholar 

  25. Munnik T (2001) Phosphatidic acid: an emerging plant lipid second messenger. Trends Plant Sci 6:227–233

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by grants from the Netherlands Organization for Scientific Research (NWO; VIDI 864.05.001), the EU (COST FA0605), the Universidad Nacional de Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT). We thank Jiorgos Kourelis for performing the experiment described in Fig. 1.

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Authors and Affiliations

  1. Swammerdam Institute for Life Sciences, Section Plant Physiology, University of Amsterdam, Amsterdam, The Netherlands

    Teun Munnik

  2. IIB-CONICET-UNMdP, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina

    Ana M. Laxalt

Authors
  1. Teun Munnik
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  2. Ana M. Laxalt
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Editors and Affiliations

  1. Swammerdam Inst. Life Science, Sect. Plant Pathology, University of Amsterdam, Kruislaan 318, Amsterdam, 1098 SM, The Netherlands

    Teun Munnik

  2. Institute for Biochemistry and Biotechno, Department of Cellular Biochemistry, Martin-Luther-Univ Halle-Wittenberg, Halle, 37077, Germany

    Ingo Heilmann

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Munnik, T., Laxalt, A.M. (2013). Measuring PLD Activity In Vivo. In: Munnik, T., Heilmann, I. (eds) Plant Lipid Signaling Protocols. Methods in Molecular Biology, vol 1009. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-62703-401-2_20

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  • DOI: https://doi.org/10.1007/978-1-62703-401-2_20

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  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-62703-400-5

  • Online ISBN: 978-1-62703-401-2

  • eBook Packages: Springer Protocols

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