LC-MS untargeted metabolomics assesses the delayed response of glufosinate treatment of transgenic glufosinate resistant (GR) buffalo grasses (Stenotaphrum secundatum L.).

INTRODUCTION: Glufosinate resistant (GR) buffalo grasses were genetically modified to resist the broad-spectrum herbicide, glufosinate by inserting a novel pat gene into its genome. This modification results in a production of additional phosphinothricin acetyltransferase (PAT) to detoxify the deleterious effects of glufosinate. The GR grasses and its associated herbicide form a modern, weeding program, to eradicate obnoxious weeds in turf lawn without damaging the grasses at relatively low costs and labor. As with several principal crops which are genetically modified to improve agricultural traits, biosafety of the GR buffalo grasses is inevitably expected to become a public concern. For the first time, we had previously examined the metabolome of glufosinate-resistant buffalo grasses, using a GC-MS untargeted approach to assess the risk of GR as well as identify any pleotropic effects arising from the genetically modification process. In this paper, an untargeted high-resolution LC-MS (LC-HRMS) untargeted metabolomics approach was carried out to complement our previous findings with respect to GR and wild type (WT) buffalo grasses. OBJECTIVE: One of the major aims of this present work was to compare GR to WT buffalo grasses by including the detection of the secondary metabolome and determine any unprecedented metabolic changes. METHODS: Eight-week old plants of 4 GR buffalo grasses, (93-1A, 93-2B, 93-3 C and 93-5A) and 3 wild type varieties (WT 8-4A, WT 9-1B and WT 9-1B) were submerged in either 5 % v/v of glufosinate or distilled water 3 days prior to a LC-HRMS based untargeted metabolomics analysis (glufosinate-treated or control, samples, respectively). An Ultra-High-Performance Liquid Chromatography (UHPLC) system coupled to a Velos Pro Orbitrap mass spectrometer system was employed to holistically measure the primary and secondary metabolome of both GR and WT buffalo grasses either treated with or without glufosinate and subsequently apply several bioinformatic tools including the automated pathway analysis algorithm, mummichog. RESULTS: LC-HRMS untargeted based metabolomics clearly identified that the global metabolite pools of both GR and WT cultivars were highly similar, providing strong, supporting evidence of substantial equivalence between the GR and WT varieties. These findings indicate that if any associated risks to these GR grasses were somehow present, the risk would be within those acceptable ranges present in the WT. Additionally, mummichog-based pathway analysis indicated that phenylalanine metabolism and the TCA cycle were significantly impacted by glufosinate treatment in the WT cultivar. It was possible that alterations in the relative concentrations of several intermediates in these pathways were likely due to glufosinate-induced production of secondary metabolites to enhance plant defense mechanisms against herbicidal stress at the expense of primary metabolism. CONCLUSIONS: GR buffalo grasses were found to be near identical to its WT comparator based on this complementary LC-HRMS based untargeted metabolomics. Therefore, these results further support the safe use of these GR buffalo grasses with substantial evidence. Interestingly, despite protected by PAT, GR buffalo grasses still demonstrated the response to glufosinate treatment by up-regulating some secondary metabolite-related pathways.

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 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.

Recent developments in metabolomics-based research in understanding transgenic grass metabolism.

BACKGROUND: Transgenic herbicide-resistant (HR) turfgrass together with its associated, broad spectrum herbicides promise cheap, selective and efficient weed control by excluding infested weeds resulting in turf lawn with high uniformity and aesthetic value. The concept of this "weeding program" initiated from modern biotechnology has been widely implemented in several principal crops including maize, soybean, canola and cotton as early as the 1990s. Transgenic HR turfgrass classified as a genetically modified organism (GMO) has undoubtedly caused public concern with respect to its biosafety and legalities similar to well-established HR crops. Nevertheless, applying metabolomics-based approaches which focuses on the identification of the global metabolic state of a biological system in response to either internal or external stimuli can also provide a comprehensive characterization of transgenic grass metabolism and its involvement in biosecurity and public perception. AIM OF REVIEW: This review summaries the recent applications of metabolomics applied to HR crops to predict the molecular and physiological phenotypes of HR turfgrass species, glyphosate-resistant Kentucky bluegrass (Poa pratensis L.) and glufosinate-resistant creeping bentgrass (Agrotis stonifera L.). Additionally, this review also presents background knowledge with respect to the application of metabolomics, transformation of HR crops and its biosafety concerns, turfgrass botanical knowledge and its economic and aesthetic value. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is to demonstrate the molecular and physiological phenotypes of HR turfgrass based on several lines of evidence primarily derived from metabolomics data applied to HR crops to identify alterations on HR turfgrass metabolism as a result of genetic modification that confers resistant traits.