Quantitative GUS Activity Assay in Intact Plant Tissue

Abstract

INTRODUCTIONThe activity of β-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl β-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be normalized per unit tissue weight, per unit protein (if it is determined in the tissue after the incubation with 4-MUG), or per sample. The latter is particularly useful if GUS is expressed only in a subset of cells within the tissue assayed. The advantage of this method is that since many samples can be assayed with relatively little effort, it is particularly appropriate for large-scale screens. The following protocol has been adapted for the use of 96-well microtiter plates (200-μl wells).