Phosphinothricin (l-PPT), also known as glufosinate-ammonium, is the active ingredient in several non-selective herbicide products, such as Basta, Liberty, Finale, Buster and Herbiace. Because l-PPT is structurally similar to l-glutamate, it is able to bind to the active site of glutamine synthetase (GS) and act as a competitive inhibitor of the enzyme (Block et al., 1987; Wohlleben et al., 1988). GS is an essential enzyme in plant nitrogen metabolism, because it assimilates ammonium through the conversion of l-glutamate to l-glutamine. Ammonium is generated in a variety of metabolic processes and serves as the nitrogen donor for the biosynthesis of all nitrogenous organic compounds needed for plant growth and development (Crawford and Arst, 1993; Daniel-Vedele et al., 1998). The mechanism of l-PPT toxicity is believed to involve ammonia accumulation to phytotoxic levels, followed by the impairment of photosynthesis causing plant death (Wendler et al., 1990; Wehrmann et al., 1996; Dan Hess, 2000).
The bialaphos resistance (bar) and phosphinothricin acetyltransferase (pat) genes from Streptomyces hygroscopicus and S. viridochromogenes, respectively, encode the enzyme phosphinothricin acetyltransferase (PAT) which inactivates l-PPT by transferring the acetyl group from acetyl-coenzyme A (acetyl-CoA) to the free amino group of l-PPT, yielding N-acetyl-l-PPT (Thompson et al., 1987; Wohlleben et al., 1988; D’Halluin et al., 1992). The enzymes perform comparably in plants and are highly specific for l-PPT (Wehrmann et al., 1996). Crops resistant to l-PPT have been created with the bar and pat genes using Agrobacterium- and particle bombardment-mediated transformation, and are now widely grown. In addition to Arabidopsis, these include transgenic maize, rice, barley, oat, wheat and cotton (Fromm et al., 1990; Christou et al., 1991; Somers et al., 1992; Weeks et al., 1993; Akama et al., 1995; Cheng et al., 1997; Keller et al., 1997; Tingay et al., 1997). Furthermore, both the bar and pat genes are preferred plant-selectable marker genes in several plant species (Wehrmann et al., 1996). Among the herbicide-resistance genes, they are the most extensively used as selectable marker genes in the scientific literature (Miki and McHugh, 2004).
An understanding of the unintended effects of genes (Cellini et al., 2004), such as pat or bar, is essential for a true comparison of transgenic plants with their non-transgenic progenitors. The functional analyses of unknown genes in transgenic plants in which pat or bar have been co-transformed and co-expressed as selectable marker genes are dependent on this knowledge. Studies with other selectable marker genes and reporter genes have shown that the insertion and expression of neomycin phosphotransferase type II (nptII) for kanamycin resistance and UidA for β-glucuronidase (GUS) reporter activity do not produce unintended effects on the transcriptome of Arabidopsis plants under normal growth conditions or under abiotic stresses, such as heat, cold, salt and drought (El Ouakfaoui and Miki, 2005). These data show that transgenic plants created with these marker genes are transcriptionally identical to non-transgenic plants. Comparable data are needed for each marker gene used in the study of transgenic plants.
The effects of the pat and bar genes on the transcriptome have not yet been investigated in the presence or absence of l-PPT. Therefore, a large number of questions related to the detailed mechanism of action, the range of pleiotropic effects and the progression of events following exposure to l-PPT remain largely unanswered, despite the widespread use of the genes and herbicides containing l-PPT. In this work, we used the Affymetrix ATH1 GeneChip to profile global gene expression patterns associated with the insertion and expression of the bar gene in the model plant Arabidopsis thaliana. We found that the expression and insertion of the bar gene produced changes in global gene expression in both the absence and presence of l-PPT. We report the identification of genes that are differentially expressed between wild-type (WT) and bar-expressing plants in response to 6-h and 48-h treatments with glufosinate-ammonium, providing new knowledge on the mechanisms behind l-PPT toxicity and plant responses to its presence.