Abstract
BackgroundThe β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has thus far been underestimated.ResultsWe report that inhibitors of GUS activity are ubiquitous in organ tissues of Arabidopsis, tobacco and rice, and significantly bias quantitative assessment of GUS activity in plant transformation experiments. Combined with previous literature reports on non-model species, our findings suggest that inhibitors may be common components of plant cells, with variable affinity towards the E. coli enzyme. The reduced inhibitory capacity towards the plant endogenous GUS discredits the hypothesis of a regulatory role of these compounds in plant cells, and their effect on the bacterial enzyme is better interpreted as a side effect due to their interaction with GUS during the assay. This is likely to have a bearing also on histochemical analyses, leading to inaccurate evaluations of GUS expression.ConclusionsIn order to achieve reliable results, inhibitor activity should be routinely tested during quantitative GUS assays. Two separate methods to correct the measured activity of the transgenic and endogenous GUS are presented.
Highlights
The β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology
Analysis of GUS assays is often complex in plant cells, as GUS gene expression is affected by various biochemical, molecular and biological factors that render its use as a reporter gene susceptible to limitations
GUS inhibitors represent a source of potential artifacts in the use of the uidA reporter gene, and the supposed ubiquity of such components in plant tissues urges for careful examination of their interference in GUS expression analyses
Summary
The β-glucuronidase (GUS) gene reporter system is one of the most effective and employed techniques in the study of gene regulation in plant molecular biology. Improving protocols for GUS assays have rendered the original method described by Jefferson amenable to various requirements and conditions, but the serious limitation caused by inhibitors of the enzyme activity in plant tissues has far been underestimated. The Escherichia coli uidA gene encoding β-glucuronidase (GUS) is one of the most effective reporter gene systems used for evaluating transient and stable transformation in plants. Since its description by Jefferson [1], the GUS gene fusion system has found extensive application in plant gene expression studies because of the enzyme stability and the high sensitivity and suitability of the assay to detection by fluorometric, spectrophotometric, or histochemical techniques.
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