Spurious localizations of diX-indigo microcrystals generated by the histochemical GUS assay

For several models expressing theuidA orgus reporter gene with or without a presequence for mitochondrial targeting, we have demonstrated that the compartmentation of -glucuronidase (E.C. 3.2.1.31) activity was not in agreement within situ localization of the diX-indigo microcrystals generated by the cytoenzymological GUS assay. These crystals were generally associated with the various cytomembranes and lipid inclusions. Experiments with purified -glucuronidase or withgus-expressing bacteria incubated with 5-bromo-4-chloro-3-indolyl--d-glucuronide and maize oil-phosphate buffer emulsion indicated that the intermediate products resulting from the GUS assay actively diffused and crystallized preferentially in association with lipids, sometimes far from the site of enzyme activity. This phenomenon could not be suppressed by the addition of potassium ferricyanide in the incubation medium. These findings are discussed with regard to previously reported biochemical and histochemical data on animal tissues, and focus on the necessity for caution in studies of tissue-specific gene expression using the GUS assay, particularly for lipid-rich plant models.