A simplified liquid chromatography-mass spectrometry methodology to probe the shikimate and aromatic amino acid biosynthetic pathways in plants.

IF 6.2 1区 生物学 Q1 PLANT SCIENCES The Plant Journal Pub Date : 2024-10-28 DOI:10.1111/tpj.17105
Jorge El-Azaz, Hiroshi A Maeda
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Abstract

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.

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探索植物莽草酸和芳香族氨基酸生物合成途径的简化液相色谱-质谱方法。
植物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,以生产木质素和其他苯丙类化合物。然而,我们对植物如何调节 AAA 代谢的了解十分有限,部分原因是缺乏可靠的分析方法。在此,我们建立了一个简化的工作流程,可同时定量植物组织中的 AAA 及其途径中间产物,该流程基于两种不同 pH 值下的提取,并通过亲水作用液相色谱法结合质谱法进行分析。然后利用这一工作流程分析植物莽草酸途径碳流量增加或减少时的代谢反应。在表达第一个莽草酸途径酶的反馈不敏感异构体时,增加的碳流提高了所有 AAA 和途径中间产物的含量,尤其是氮酸酯,它是酪氨酸和苯丙氨酸生物合成的后氯氨酸途径中最后一种常见的前体。额外过量表达一种原酸脱氢酶会增加酪氨酸的含量,并耗尽苯丙氨酸和原酸库;然而,上游莽草酸途径中间产物的积累水平仍然很高。草甘膦通过抑制莽草酸途径的倒数第二步来限制碳流,从而导致莽草酸及其目标酶上游其他前体的积累,但同时也造成了下游代谢物(包括莽草酸)的意外积累。这些发现突出表明,莽草酸途径和下游氯氨酸后 AAA 途径在植物中作为独立的调控模块发挥作用。本文开发的方法为深入了解植物中的莽草酸和 AAA 生物合成途径铺平了道路。
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来源期刊
The Plant Journal
The Plant Journal 生物-植物科学
CiteScore
13.10
自引率
4.20%
发文量
415
审稿时长
2.3 months
期刊介绍: Publishing the best original research papers in all key areas of modern plant biology from the world"s leading laboratories, The Plant Journal provides a dynamic forum for this ever growing international research community. Plant science research is now at the forefront of research in the biological sciences, with breakthroughs in our understanding of fundamental processes in plants matching those in other organisms. The impact of molecular genetics and the availability of model and crop species can be seen in all aspects of plant biology. For publication in The Plant Journal the research must provide a highly significant new contribution to our understanding of plants and be of general interest to the plant science community.
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