Glufosinate ammonium is the active ingredient in a number of proprietary broad-spectrum herbicides that have been registered for use in Australia, including Basta®, Finale® and Liberty®. These herbicides function by inhibiting the plant enzyme glutamine synthetase, which is a key enzyme involved in plant nitrogen metabolism. In the absence of glutamine synthetase activity, ammonia accumulates in plant tissues causing inhibition of amino acid biosynthesis, inhibition of photosynthesis and rapid death of the plant (Evstigneeva et al. 2003).
The herbicidal component of glufosinate ammonium is the L-isoform of phosphinothricin (PPT). PPT is a component of the antibiotic bialaphos, which is produced naturally by the soil bacteria Streptomyces hygroscopicus and Streptomyces viridochromogenes. To avoid the toxicity associated with biaphalos production, S. hygroscopicus and S. viridochromogenes express the biaphalos resistance genes bar and pat, respectively (Murakami et al. 1986; Thompson et al. 1987; Wohlleben et al. 1988; Strauch et al. 1988). Both the bar and pat genes encode phosphinothricin acetyl transferase (PAT), an enzyme that acetylates the free amino groups of PPT with high affinity and specificity to render it inactive (Wohlleben et al. 1988; Droge-Laser et al. 1994; OECD 1999b).
The bar and pat genes and their encoded proteins
Each of the GM canola lines authorised under licence DIR 021/2002 was modified for tolerance to glufosinate ammonium by the introduction of either the bargene from S.hygroscopicus or the pat gene from S. viridochromogenes. The bar and pat genes are very similar with an overall identity of 87% at the nucleotide sequence level. Both genes encode PAT proteins of 183 amino acids with 85% amino acid sequence identity, comparable molecular weights (~22 kDa) and similar substrate affinity and biochemical activity (Wehrmann et al. 1996). In fact, the PAT proteins encoded by bar and pat are so similar as to be functionally equivalent for the purpose of conferring tolerance to glufosinate ammonium (Wehrmann et al. 1996; OECD 1999b).
The DNA sequences of both the pat and bar genes introduced into the GM canola lines approved under DIR 021/2002 were modified for plant-preferred codon usage to ensure optimal expression in canola (European Scientific Committee on Plants 1998a; European Scientific Committee on Plants 1998b; EFSA 2008).
The PAT protein produced from the pat gene in GM canola lines T45 and Topas 19/2 has exactly the same amino acid sequence as the native protein from S. viridochromogenes (European Scientific Committee on Plants 1998a; OECD 1999b).
The bar gene introduced into the MS and RF GM canola lines was modified by the substitution of the N terminal two codons of the bacterial gene, GTG and AGC, with the codons ATG and GAC, respectively (OECD 1999b; Japanese Biosafety Clearing House 2007). The modification from GTG to ATG does not result in an amino acid change, but serine changes to aspartic acid in the modification from AGC to GAC. However, the function of the PAT gene with this single amino acid substitution remains unchanged (Japanese Biosafety Clearing House 2007).
Glyphosate tolerance
Glyphosate is the active ingredient in a number of broad-spectrum systemic herbicides that have been approved for use in Australia and was first marketed as the proprietary herbicide Roundup®. The herbicidal activity of glyphosate is derived from its ability to inhibit the function of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme involved in the shikimate biosynthetic pathway present in all plants, bacteria and fungi.
The shikimate pathway enables biosynthesis of aromatic compounds from carbohydrate precursors in a series of seven biosynthetic steps. The penultimate step in the pathway is the condensation of shikimate 3-phosphate and phosphoenol pyruvate to form 5-enolpyruvylshikimate 3-phosphate, a reaction catalysed by EPSPS (reviewed by Herrmann & Weaver 1999). Glyphosate competes with phosphoenol pyruvate for binding to the complex formed between EPSPS and shikimate 3-phosphate. Upon glyphosate binding, the EPSPS:shikimate 3-phosphate complex is very stable and has a slow reversal rate, effectively terminating the shikimate pathway prematurely and preventing biosynthesis of essential aromatic compounds required for plant growth and development, including the amino acids phenylalanine, tyrosine and tryptophan (Dill 2005).
Two main approaches have been utilised to generate GM plants that are tolerant to glyphosate-based herbicides: introduction of genes that encode proteins capable of detoxifying the glyphosate molecule; and introduction of genes that encode EPSPS enzymes with reduced affinity for glyphosate (Dill 2005).
Roundup Ready® canola line GT73, approved under DIR 020/2002, was modified to contain both a glyphosate detoxifying enzyme, encoded by the goxv247 gene, and an EPSPS protein with naturally reduced affinity for glyphosate, encoded by the cp4 epsps gene.
The goxv247 gene and its encoded protein
The goxv247 gene introduced into Roundup Ready® canola GT73 was isolated from the common soil bacterium Ochrobactrum anthropi strain LBAA (formerly Achromobacter sp.). It encodes a glyphosate oxidoreductase (GOX) enzyme that inactivates glyphosate by converting it into aminomethylphosphonic acid (AMPA) and glyoxylate (Pipke & Amrhein 1988; Duke 2010). Glyoxylate is a common plant metabolite and AMPA is degraded by several microorganisms (ANZFA 2000).
The goxv247 gene encodes a single polypeptide of 431 amino acids with a molecular mass of 46.1 kD. This gene is a variant of the O. anthropigox gene and has improved affinity for glyphosate and therefore degrades the herbicide more efficiently. The DNA sequence of goxv247 was modified for plant-preferred codon usage. The goxv247 gene varies from the gox gene by only 5 nucleotides, and the variant GOXv247 protein is 99% identical to the native GOX enzyme, differing by 3 amino acids.
