Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Abstract

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants

“…Mesophyll protoplasts were isolated from in vitro-grown tobacco plants, as in Negrutiu et al (1987). Freshly isolated protoplasts (4 · 10 4 ppt ll )1 ) were transfected with 10 lg of plasmid DNA by polyethylene glycol-mediated DNA uptake (Walden et al, 1994). Five hours before the analysis, Hoechst 33258 (Sigma-Aldrich) was added (5 lg ml )1 ) to the protoplast culture in order to stain the nuclear DNA.…”

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

“…Such gene activation may produce novel phenotypes that are either redundant members of a gene family or are essential for survival. Activation tagging strategy, developed by Walden et al (1994) has been used to generate collections of morphologically diverse dominant mutants resulting in cloning of corresponding genes (Table 4). Although the paper published by Walden et al (1994) was later retracted as the major findings of the work could not be substantiated.…”

“…Activation tagging strategy, developed by Walden et al (1994) has been used to generate collections of morphologically diverse dominant mutants resulting in cloning of corresponding genes (Table 4). Although the paper published by Walden et al (1994) was later retracted as the major findings of the work could not be substantiated. However, the basic procedure, namely activation tagging described by these workers is a valid technique and is has been used by other workers (Balter, 1999).…”

Analysis of the SHP2 enhancer for the use of tissue specific activation tagging in Arabidopsis thaliana

“…It is an alternative approach for gene function analysis, because loss-of-function mutations has its limitation in cases of functional gene redundancy (Arabidopsis Genome Initiative, 2000). Activation tagging was proposed as a novel gene isolation method in which a gene is either ectopically or constitutively overexpressed compared to normal expression levels (Walden et al, 1994). Walden et al (1994) designed a T-DNA based activation tagging approach to identify and isolate novel genes from tobacco, and since then it has been largely applied using either T-DNA insertion strategies Ito and Meyerowitz, 2000;Lee et al, 2000;van der Graaff et al, 2000;Weigel et al, 2000;Huang et al, 2001) or transposon based approaches (Wilson et al, 1996;Marsch-Martinez et al, 2002).…”

“…Activation tagging was proposed as a novel gene isolation method in which a gene is either ectopically or constitutively overexpressed compared to normal expression levels (Walden et al, 1994). Walden et al (1994) designed a T-DNA based activation tagging approach to identify and isolate novel genes from tobacco, and since then it has been largely applied using either T-DNA insertion strategies Ito and Meyerowitz, 2000;Lee et al, 2000;van der Graaff et al, 2000;Weigel et al, 2000;Huang et al, 2001) or transposon based approaches (Wilson et al, 1996;Marsch-Martinez et al, 2002). This technology has been applied successfully to many plant species like Arabidopsis, rice, tomato, petunia and tobacco (Weigel et al, 2000;Jeong et al, 2002;Zubko et al, 2002;Ahad et al, 2003;Mathews et al, 2003).…”