Data_Sheet_1_Increased Glyphosate-Induced Gene Expression in the Shikimate Pathway Is Abolished in the Presence of Aromatic Amino Acids and Mimicked by Shikimate.docx

The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway, also known as the shikimate pathway. Amaranthus palmeri is a fast-growing weed, and several populations have evolved resistance to glyphosate through increased EPSPS gene copy number. The main objective of this study was to elucidate the regulation of the shikimate pathway and determine whether the regulatory mechanisms of glyphosate-sensitive and glyphosate-resistant plants were different. Leaf disks of sensitive and resistant (due to EPSPS gene amplification) A. palmeri plants were incubated for 24 h with glyphosate, AAA, glyphosate + AAA, or several intermediates of the pathway: shikimate, quinate, chorismate and anthranilate. In the sensitive population, glyphosate induced shikimate accumulation and induced the gene expression of the shikimate pathway. While AAA alone did not elicit any change, AAA applied with glyphosate abolished the effects of the herbicide on gene expression. It was not possible to fully mimic the effect of glyphosate by incubation with any of the intermediates, but shikimate was the intermediate that induced the highest increase (three-fold) in the expression level of the genes of the shikimate pathway of the sensitive population. These results suggest that, in this population, the lack of end products (AAA) of the shikimate pathway and shikimate accumulation would be the signals inducing gene expression in the AAA pathway after glyphosate application. In general, the effects on gene expression detected after the application of the intermediates were more severe in the sensitive population than in the resistant population. These results suggest that when EPSPS is overexpressed, as in the resistant population, the regulatory mechanisms of the AAA pathway are disrupted or buffered. The mechanisms underlying this behavior remain to be elucidated.

除草剂草甘膦通过抑制植物酶5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）在芳香族氨基酸（AAA）生物合成途径中的活性，该途径亦称为莽草酸途径。藜麦属植物（Amaranthus palmeri）为一种生长迅速的杂草，多个种群通过EPSPS基因拷贝数的增加，已进化出对草甘膦的抵抗性。本研究的主要目标是阐明莽草酸途径的调控机制，并确定草甘膦敏感型与草甘膦抵抗型植物的调控机制是否存在差异。将敏感型和因EPSPS基因扩增而抵抗的A. palmeri植物的叶片切片与草甘膦、AAA、草甘膦+AAA或途径中多个中间产物：莽草酸、奎宁酸、香草酸和邻氨基苯甲酸共同孵育24小时。在敏感型种群中，草甘膦诱导莽草酸的积累，并引发莽草酸途径基因表达。尽管单独的AAA并未引起任何变化，但AAA与草甘膦共同应用则消除了除草剂对基因表达的影响。通过与任何中间产物的孵育均无法完全模拟草甘膦的效果，但莽草酸是导致敏感型种群莽草酸途径基因表达水平（增加三倍）最高的中间产物。这些结果提示，在该种群中，莽草酸途径的终端产物（AAA）的缺乏和莽草酸的积累可能是诱导草甘膦应用后AAA途径基因表达信号的信号。总体而言，在敏感型种群中，应用中间产物对基因表达的影响比在抵抗型种群中更为严重。这些结果表明，当EPSPS在抵抗型种群中过度表达时，AAA途径的调控机制可能被破坏或缓冲。这一行为背后的机制尚待阐明。