A simplified liquid chromatography‐mass spectrometry methodology to probe the shikimate and aromatic amino acid biosynthetic pathways in plants
Year: 2024
Type: article
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.
Source: The Plant Journal
DOI: 10.1111/tpj.17105
Authors: Jorge El‐Azaz, Hiroshi A. Maeda
Institution: University of Wisconsin–Madison
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $5,190
Awards: Mechanisms and Impacts of De-regulating Aromatic Amino Acid Biosynthesis in Plants, MCA-PGR: Phylogeny-Guided Biochemical Genomics to Elucidate the Tyrosine-Derived Lignin Metabolic Network of Grasses, Collaborative Research: Metabolic fluxes from the Calvin-Benson cycle through the parallel shikimate and non-shikimate pathways in plants, G8771531156
OpenAlex ID: W4403826272