Siriwat Boonchaisri1, Trevor Stevenson1, Daniel A Dias2. 1. School of Science, RMIT University, Bundoora, VIC, 3083, Australia. 2. School of Health and Biomedical Sciences, Discipline of Laboratory Medicine, RMIT University, PO Box 71, Bundoora, VIC, 3083, Australia. daniel.dias@rmit.edu.au.
Abstract
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 accumulation of allantoin was prominent and was an unanticipated effect of the pat gene insertion. As expected, glufosinate treatment caused significant metabolic alterations in the sensitive wild type, with the up-regulation of several amino acids (e.g. phenylalanine and isoleucine) which was likely due to glufosinate-induced senescence. The aminoacyl-tRNA biosynthetic pathway was identified as the most significant enriched pathway as a result of glufosinate effects because a number of its intermediates were amino acids. CONCLUSION: HR buffalo grasses were very similar to its wild type comparator based on a comprehensive GC-MS based untargeted metabolomics and therefore, should guarantee the safe use of these HR buffalo grasses. The current metabolomics analyses not only confirmed the effects of glufosinate to up-regulate free amino acid pools in the sensitive wild type but also several alterations in sugar, sugar phosphate and organic acid metabolism have been reported.
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 accumulation of allantoin was prominent and was an unanticipated effect of the pat gene insertion. As expected, glufosinate treatment caused significant metabolic alterations in the sensitive wild type, with the up-regulation of several amino acids (e.g. phenylalanine and isoleucine) which was likely due to glufosinate-induced senescence. The aminoacyl-tRNA biosynthetic pathway was identified as the most significant enriched pathway as a result of glufosinate effects because a number of its intermediates were amino acids. CONCLUSION: HR buffalo grasses were very similar to its wild type comparator based on a comprehensive GC-MS based untargeted metabolomics and therefore, should guarantee the safe use of these HR buffalo grasses. The current metabolomics analyses not only confirmed the effects of glufosinate to up-regulate free amino acid pools in the sensitive wild type but also several alterations in sugar, sugar phosphate and organic acid metabolism have been reported.
Authors: George G Harrigan; Tyamagondlu V Venkatesh; Mark Leibman; Jonathan Blankenship; Timothy Perez; Steven Halls; Alexander W Chassy; Oliver Fiehn; Yun Xu; Royston Goodacre Journal: Metabolomics Date: 2016-03-15 Impact factor: 4.290