β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background

Abstract

Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the V m values and a greater affinity ( K m) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.