Abstract
Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC activity, to phosphatidic acid (PA) that participates in ABA signaling pathways.
Here we describe a step-by-step protocol, which can be used to determine in situ or in vitro NPC activity. Determination is based on quantification of fluorescently labeled DAG as a product of cleavage of the fluorescently labeled substrate lipid, phosphatidylcholine. High-performance thin-layer chromatography is used for separation of fluorescent DAG. The spot is visualized with a laser scanner and the relative amounts of fluorescent DAG are quantified using imaging software.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scherer GFE, Paul RU, Holk A et al (2002) Down-regulation by elicitors of phosphatidylcholine-hydrolyzing phospholipase C and up-regulation of phospholipase A in plant cells. Biochem Biophys Res Commun 293:766–770
Nakamura Y, Awai K, Masuda T et al (2005) A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis. J Biol Chem 280:7469–7476
Gaude N, Nakamura Y, Scheible WR et al (2008) Phospholipase C5 (NPC5) is involved in galactolipid accumulation during phosphate limitation in leaves of Arabidopsis. Plant J 56:28–39
Wimalasekera R, Pejchar P, Holk A et al (2010) Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signalling in Arabidopsis thaliana. Mol Plant 3:610–625
Pejchar P, Potocký M, Novotná Z et al (2010) Aluminium ions inhibit the formation of diacylglycerol generated by phosphatidylcholine-hydrolysing phospholipase C in tobacco cells. New Phytol 188:150–160
Peters C, Li MY, Narasimhan R et al (2010) Nonspecific phospholipase C NPC4 promotes responses to abscisic acid and tolerance to hyperosmotic stress in Arabidopsis. Plant Cell 22:2642–2659
Kocourková D, Krčková Z, Pejchar P et al (2011) The phosphatidylcholine-hydrolyzing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress. J Exp Bot 62:3753–3763
Wang X (2001) Plant phospholipases. Annu Rev Plant Physiol Plant Mol Biol 52:211–231
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Bligh EG (1978) Citation classic—rapid method of total lipid extraction and purification, Commentary on ref. (10). http://www.garfield.library.upenn.edu/classics1978/A1978FZ82000002_OnlinePDF.pdf
Novotná Z, Martinec J, Profotová B et al (2003) In vitro distribution and characterization of membrane-associated PLD and PI-PLC in Brassica napus. J Exp Bot 54:691–698
Munnik T, Irvine RF, Musgrave A (1998) Phospholipid signalling in plants. Biochim Biophys Acta Lipids Lipid Metab 1389:222–272
Pokotylo I, Pejchar P, Potocký M et al (2013)The plant non-specific phospholipase C gene family. Novel competitors in lipid signalling. Prog Lipid Res 52:62–79
Acknowledgments
This work was supported by the Czech Science Foundation (grants no. P501/12/1942 and no. P501/12/P950).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pejchar, P., Scherer, G.F.E., Martinec, J. (2013). Assaying Nonspecific Phospholipase C Activity. 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_18
Download citation
DOI: https://doi.org/10.1007/978-1-62703-401-2_18
Published:
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-1-62703-400-5
Online ISBN: 978-1-62703-401-2
eBook Packages: Springer Protocols