PMID: 12753593May 20, 2003Paper

Nanolitre-scale assays to determine the activities of enzymes in individual plant cells

The Plant Journal : for Cell and Molecular Biology
Stuart J RoyRoger A Leigh

Abstract

There are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single-cell sampling to extract 10-100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre-scale fluorescence-based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole-leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successf...Continue Reading

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Citations

Nov 13, 2003·Current Opinion in Plant Biology·Julia Kehr
Jan 14, 2009·The New Phytologist·Sakiko OkumotoWolf B Frommer
Mar 28, 2012·The Plant Journal : for Cell and Molecular Biology·Sakiko Okumoto
Apr 23, 2010·Annals of Botany·Simon Conn, Matthew Gilliham
Nov 25, 2003·Phytochemical Analysis : PCA
Mar 16, 2005·Annals of Botany·Achim Walter, Ulrich Schurr

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