El-Azaz Jorge, Maeda Hiroshi A
Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Plant J. 2024 Dec;120(5):2286-2304. doi: 10.1111/tpj.17105. Epub 2024 Oct 28.
Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback-insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post-chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post-chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants.
植物会将大量碳用于芳香族氨基酸（AAA）的生物合成，尤其是苯丙氨酸，以产生木质素和其他苯丙烷类化合物。然而，我们对植物如何调节AAA代谢的了解有限，部分原因是缺乏可靠的分析方法。在此，我们基于在两种不同pH值下的提取以及两性离子亲水相互作用液相色谱-质谱联用分析，建立了一种用于同时定量植物组织中AAA及其途径中间体的简化工作流程。然后，该工作流程被用于分析植物中莽草酸途径碳流量增加或减少时的代谢反应。在表达莽草酸途径首个酶的反馈不敏感同工型后，流量增加使所有AAA和途径中间体升高，尤其是预苯酸，它是酪氨酸和苯丙氨酸生物合成的分支酸途径之后的最后一个共同前体。额外过表达预苯酸脱氢酶会增加酪氨酸水平，并耗尽苯丙氨酸和预苯酸库；然而，上游莽草酸途径中间体仍大量积累。草甘膦处理通过抑制莽草酸途径的倒数第二步来限制碳流量，导致其靶酶上游的莽草酸和其他前体可预测地积累，但也导致下游代谢物意外积累，包括预苯酸。这些发现突出表明，莽草酸途径和下游分支酸后的AAA途径在植物中作为独立调节的模块发挥作用。这里开发的方法为更深入了解植物中的莽草酸和AAA生物合成途径铺平了道路。
