title: Measuring PLD activity in vivo.
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.
authors.affiliation: Swammerdam Institute for Life Sciences, Section Plant Physiology, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Teun
authors.initials: T
authors.lastname: Munnik
authors.forename: Ana M
authors.initials: AM
authors.lastname: Laxalt
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D002855:Chromatography, Thin Layer; D057075:Enzyme Assays / Q000379:methods*; D010739:Phospholipase D / Q000378:metabolism*; D059828:Plant Cells / Q000201:enzymology; D054046:Plant Stomata / Q000166:cytology / Q000201:enzymology; D036226:Seedlings / Q000201:enzymology; D013535:Suspensions; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology; D031306:Vicia / Q000201:enzymology
chemical_list: D013535:Suspensions; D010739:Phospholipase D