Utilization of GC–MS untargeted metabolomics to assess the delayed response of glufosinate treatment of transgenic herbicide resistant (HR) buffalo grasses (Stenotaphrum secundatum L.)

Introduction: Herbicide resistant (HR) buffalo grasses were genetically engineered to resist the non-selective herbicide, glufosinate in order to facilitate a modern, ‘weeding program’ which is highly effective in terms of minimizing costs and labor. The resistant trait was conferred by an insertion of the pat gene to allow for the production of the enzyme phosphinothricin acetyltransferase (PAT) to detoxify the glufosinate inhibitive effect. To date, there are only a few reports using metabolomics as well as molecular characterizations published for glufosinate resistant crops with no reports on HR turfgrass. Therefore, for the first time, this study examines the metabolome of glufosinate-resistant buffalo grasses which not only will be useful to future growers but also the scientific community. Objective: A major aim of this present work is to characterize and evaluate the metabolic alterations which may arise from a genetic transformation of HR buffalo grasses by comprehensively using gas chromatography–mass spectrometry (GC–MS) based untargeted metabolomics. Methods: Eight-week old plants of 4 HR buffalo grasses, (93-1A, 93-2B, 93-3C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were selected for physiological, molecular and metabolomics experiments. Plants were either sprayed with 1, 5, 10 and 15% v/v of glufosinate to evaluate the visual injuries or submerged in 5% v/v of glufosinate 3 days prior to a GC–MS based untargeted metabolomics analysis. In contrast, the control group was treated with distilled water. Leaves were extracted in 1:1 methanol:water and then analysed, using an in-house GC–MS untargeted workflow. Results: Results identified 199 metabolites with only 6 of them (cis-aconitic acid, allantoin, cellobiose, glyceric acid, maltose and octadecanoic acid) found to be statistically significant (p < 0.05) between the HR and wild type buffalo grass varieties compared to the control experiment. Among these metabolites, unusual accu