HPLC and colourimetric quantification of shikimic acid levels in crops after glyphosate treatment

Abstract

The shikimic acid pathway is one of the major biosynthetic pathways in higher plants responsible for the biosynthesis of aromatic amino acids (tryptophan, tyrosine and phenylalanine) and multiple secondary metabolites, such as lignin, phytoalexins and indoleacetic acid (IAA). The herbicide glyphosate [N-(phosohonomethyl)glycine] is a non-selective, broad spectrum, post emergence, foliar applied, systemic herbicide that is used globally to control over 180 weed species. Glyphosate is a potent inhibitor of a key enzyme in the shikimic acid pathway namely; 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which is the only cellular target for this herbicide. Upon inhibition of EPSPS by glyphosate, shikimate, the dephosphorylated substrate of EPSPS which is upstream of this enzyme in the pathway, cannot be converted to 5-enolpyruvylshikimate-3-phosphate (EPSP). Blockage of the shikimic acid pathway consequently results in the accumulation of high levels of shikimate. Since shikimate accumulation is a direct result of herbicide inhibition of EPSPS, shikimate can be used as a convenient biomarker to measure glyphosate exposure, glyphosate damage as well as the degree of glyphosate resistance. Glyphosate resistance is conferred in glyphosate resistant (GR) crops also known as Roundup Ready® (RR) crops by incorporating a glyphosate tolerant CP4-EPSPS gene from the CP4 strain of Agrobacterium which encodes for a bacterial version of the EPSPS that is highly insensitive to glyphosate. When this enzyme (CP4-EPSPS) is expressed and present in RR crops it enables the plant to bypass the glyphosate inhibited native EPSPS in the shikimic acid pathway, thereby allowing the plant to complete the shikimic acid pathway (aromatic amino acid biosynthesis) by making use of the alternative enzyme, thus preventing aromatic amino acid and protein starvation and deregulation of this metabolic route, both of which follow glyphosate treatment in susceptible plants. Thus, RR crops are unaffected by herbicide treatment. Since glyphosate inhibits the EPSPS in susceptible (non-RR) crops, but not in a RR crop line, differences in the shikimate levels occur between these crop lines after glyphosate exposure. The main aims of this study were to quantify shikimic acid levels in Roundup Ready® and non-RR crops after being treated with glyphosate (Roundup Turbo®) by making use of high performance liquid chromatographic (HPLC) analysis as well as a colourimetric assay, and to use these two assays to differentiate between glyphosate resistant and susceptible plants after being exposed to glyphosate. These assays were also used to indicate whether glyphosate was responsible for herbicide damage in maize plants due to drift. Plant tissues sensitive to glyphosate accumulate shikimic acid to high levels after glyphosate treatment. The detection of shikimic acid has been shown to be a useful marker as a measure of glyphosate injury or to score for glyphosate sensitive and resistant weed biotypes. Up to now, the most common methods for shikimic acid assay include: spectrophotometry, capillary zone electrophoresis, HPLC with UV detection, and the periodate oxidation, or Cromartie and Polge, method. Here we introduce a new method for shikimic acid detection which has a broad application, is colourimetric, sensitive, simple and very quick to use. The method can be used for quantification in plant extracts using a microtiter plate, and can be further adapted for detection of shikimic acid in intact leaf discs or other plant tissues.