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Histochemical assay to detect class III ADH activity in situ in Arabidopsis seedlings

Abstract:

Whole-mount detection methods are quick, inexpensive and offer the possibility of studying the temporal and spatial patterns of gene expression in a morphological context. These methods have been used widely to detect messenger RNAs and to measure enzymatic activity of reporter genes, such as β-galactosidase or β-glucuronidase. Taking advantage of the fact that NADH generated during the oxidation of formaldehyde by class III alcohol dehydrogenase can reduce the compound nitroblue tetrazolium to form a blue precipitate, we have developed a new method to detect class III alcohol dehydrogenase activity in situ in whole Arabidopsis plants. This reaction has been used earlier for in situ electrophoresis detection and for histochemical analysis in animal tissue sections. With a few modifications, it can be used in whole Arabidopsis plants or excised plant tissues to allow a rapid analysis of class III ADH activity during development or in response to elicitors. The method might be extended to other dehydrogenases by using specific substrates. © Biological Stain Commission.