Siriwat Boonchaisri1,2, Simone Rochfort3,4, Trevor Stevenson2, Daniel A Dias5. 1. Division of Biology, School of Sciences, University of Phayao, Phayao, 56000, Thailand. 2. School of Science, RMIT University, Bundoora, VIC, 3083, Australia. 3. Agriculture Research Victoria, AgriBio, Bundoora, VIC, 3083, Australia. 4. School of Applied Systems Biology, La Trobe University, Bundoora, VIC, 3083, Australia. 5. 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: 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.
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.
Authors: Eugenia Barros; Sabine Lezar; Mikko J Anttonen; Jeroen P van Dijk; Richard M Röhlig; Esther J Kok; Karl-Heinz Engel Journal: Plant Biotechnol J Date: 2010-02-03 Impact factor: 9.803
Authors: Beth L Fatland; Jinshan Ke; Marc D Anderson; Wieslawa I Mentzen; Li Wei Cui; C Christy Allred; Jerry L Johnston; Basil J Nikolau; Eve Syrkin Wurtele Journal: Plant Physiol Date: 2002-10 Impact factor: 8.340