Abstract
Measurement of lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) activity in tissue extracts often poses problems due to the inhibitors released during homogenization. The present investigation provides a first report on the localization of lipase activity in viable plant protoplasts isolated from the cotyledons of germinating seeds of sunflower, cotton and peanut by fluorescence microscopy, using a lipase specific, 'glycerol-derived' synthetic substrate, i.e., 1,2-O-dilauryl-rac-3-glycero-glutaric acid-resorufin ester, commonly used for in vitro assays. Due to its lipophilicity, this chromogenic substrate readily permeates plasma membrane of plant protoplasts. Subsequent lipase action leads to cleavage of this substrate to release resorufin, which can be visualized due to emission of red fluorescence (λmax excitation – 567 nm; λmax emission – 584.6 nm) at its intracellular locations.
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References
Beisson F., Tiss A., Riviere C. and Verger R. 2000a. Methods for lipase detection and assay: A critical review. Eur. J. Lipid Sci. Technol. 2: 133–153.
Beisson F., Arondel V. and Verger R. 2000b. Assaying Arabidopsis lipase activity. Biochem. Soc. Trans. 28: 773–775.
Boehringer Mannheim, 1996. Biochemicals Catalogue. Germany.
Bothner C., Chavez R., Wei J., Strupp C., Phung Q., Schneemann A. and Siuzdak A. 2000. Monitoring enzymes catalysis with mass spectrometry. J. Biol. Chem. 275: 13455–13459.
Breedveld B., Schoonderwoerd K., Verhoeven A., Willemsen R. and Jensen H. 1997. Hepatic lipase is localized at the parenchymal cell microvilli in rate liver. Biochem. J. 321: 425–430.
Chapman G.W. Jr. 1987. A proteinaceous competitive inhibitor of lipase isolated from Helianthus annuus seeds. Phytochemistry 26: 3127–3131.
Dousset N., Negre A., Salvayre R., Rogalle P., Dang Q.Q. and Douste-Blazy L. 1988. Use of a fluorescent radiolabeled triaclyglycerol as substrate for lipoprotein lipase and hepatic triacylglyceride lipase. Lipids 23: 605–608.
Greenspan P., Mayer E.P. and Fowler S.D. 1985. Nile red: A fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100: 965–973.
Haugland R. 1996. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes, Eugene, Oregon, USA.
Kuhn D.N. and Stumpf P.K. 1981. Preparation and use of protoplasts for studies of lipid metabolism. Methods Enzymol. 72: 774–783.
Lehner R., Cui Z. and Vance D. 1999. Subcellular localization, developmental expression and characterization of a liver triacylglycerol hydrolase. Biochem. J. 338: 761–768.
Ncube I., Adlercreutz P., Read J. and Mattiasson B. 1993. Purification of rape (Brassica napus) seedling lipase and its use in organic media. Biotechnol. Appl. Biochem. 17: 327–336.
Ruzin S.E. 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York.
Sadeghipour H.R. and Bhatla S.C. 2002. Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings. Plant Cell Physiol. 43: 1117–1126.
Swanson S.J., Bethke P.C. and Jones R.L. 1998. Barley aleurone cells contain two types of vacuoles: Characterization of lytic organelle by use of fluorescent probes. Plant Cell 10: 685–698.
Teissere M., Borel M., Caillol B., Nari J., Gardies A.M. and Noat G. 1995. Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds. Biochim. Biophys. Acta 1255: 105–112.
Wang S.M. and Huang A.H.C. 1984. Inhibitors of lipase activities in soybean and other oilseeds. Plant Physiol. 76: 929–934.
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Gupta, A., Sadeghipour, H.R. & Bhatla, S.C. Subcellular detection of lipase activity in plant protoplasts using fluorescence microscopy. Plant Growth Regulation 41, 259–264 (2003). https://doi.org/10.1023/B:GROW.0000007525.48895.f5
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DOI: https://doi.org/10.1023/B:GROW.0000007525.48895.f5