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

Citation

Roy, S and Cuin, TA and Leigh, RA, Nanolitre-scale assays to determine the activities of enzymes in individual plant cells, The Plant Journal, 34, (4) pp. 555-564. ISSN 0960-7412 (2003) [Refereed Article]

DOI: doi:10.1046/j.1365-313X.2003.01744.x

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 successfully to Arabidopsis and, as in barley, revealed cell-specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single-cell level.

Item Details

Item Type:Refereed Article
Research Division:Agricultural and Veterinary Sciences
Research Group:Crop and Pasture Production
Research Field:Crop and Pasture Biochemistry and Physiology
Objective Division:Plant Production and Plant Primary Products
Objective Group:Winter Grains and Oilseeds
Objective Field:Barley
Author:Cuin, TA (Dr Tracey Cuin)
ID Code:39814
Year Published:2003
Web of Science® Times Cited:9
Deposited By:Agricultural Science
Deposited On:2006-07-04
Last Modified:2006-07-04
Downloads:0

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