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Utilization of GC-MS untargeted metabolomics to assess the delayed response of glufosinate treatment of transgenic herbicide resistant (HR) buffalo grasses (Stenotaphrum secundatum L.).
Metabolomics ( IF 3.6 ) Pub Date : 2020-01-27 , DOI: 10.1007/s11306-020-1644-9
Siriwat Boonchaisri 1 , Trevor Stevenson 1 , Daniel A Dias 2
Affiliation  

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.

中文翻译:

利用GC-MS非靶向代谢组学评估草铵膦处理转基因抗除草剂(HR)的水牛草(Stenotaphrum secundatum L.)的延迟反应。

引言抗除草剂(HR)的野牛草经过基因改造,可以抵抗草铵膦非选择性除草剂,以促进现代化的“除草程序”,该程序在降低成本和劳动力方面非常有效。通过插入pat基因来赋予抗性性状,以允许产生膦丝菌素乙酰转移酶(PAT)来解除草铵膦的抑制作用。迄今为止,只有很少的报道使用代谢组学以及抗草铵膦的农作物的分子表征,而没有关于草皮草的报道。因此,本研究首次研究了抗草铵膦的水牛草的代谢组,这不仅对将来的种植者有用,对科学界也很有用。目的本研究的主要目的是通过综合使用基于气相色谱-质谱(GC-MS)的非靶向代谢组学来表征和评估可能因水牛水草的遗传转化而引起的代谢变化。方法八周大的植物,其中有4种HR水牛草(93-1A,93-2B,93-3C和93-5A)和3种野生型变种(WT 8-4A,WT 9-1B和WT 9-1B)选择进行生理,分子和代谢组学实验。在基于GC-MS的非目标代谢组学分析之前3天,向植物喷洒1%,5%,10%和15%v / v的草铵膦以评估视觉损伤,或浸没在5%v / v的草铵膦中。相反,对照组用蒸馏水处理。叶片用1:1甲醇:水提取,然后进行分析,使用内部GC-MS非目标工作流程。结果结果鉴定出199种代谢产物,其中只有6种(顺式乌头酸,尿囊素,纤维二糖,甘油酸,麦芽糖和十八碳酸)在HR和野生型水牛草品种之间具有统计学意义(p <0.05)。对照实验。在这些代谢产物中,尿囊素的异常积累是突出的,并且是pat基因插入的意想不到的作用。如预期的那样,草铵膦处理在敏感的野生型中引起了显着的代谢改变,几种氨基酸(例如苯丙氨酸和异亮氨酸)的上调可能是由于草铵膦诱导的衰老。由于草铵膦的作用,氨酰基-tRNA生物合成途径被确定为最重要的富集途径,因为其许多中间体是氨基酸。结论HR水牛草与基于基于非靶向代谢组学的综合GC-MS的野生型比较器非常相似,因此应保证这些HR水牛草的安全使用。当前的代谢组学分析不仅证实了草铵膦在敏感野生型中上调游离氨基酸库的作用,而且还报道了糖,糖磷酸和有机酸代谢的几种改变。应该保证这些HR水牛草的安全使用。当前的代谢组学分析不仅证实了草铵膦在敏感野生型中上调游离氨基酸库的作用,而且还报道了糖,糖磷酸和有机酸代谢的几种改变。应该保证这些HR水牛草的安全使用。当前的代谢组学分析不仅证实了草铵膦在敏感野生型中上调游离氨基酸库的作用,而且还报道了糖,糖磷酸和有机酸代谢的几种改变。
更新日期:2020-01-29
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