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SUMMARY 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. 

ABSTRACT The plant shikimate pathway directs a significant portion of photosynthetically assimilated carbon into the downstream biosynthetic pathways of aromatic amino acids (AAA) and aromatic natural products. 3‐Deoxy‐d‐arabino‐heptulosonate 7‐phosphate (DAHP) synthase (hereafter DHS) catalyzes the first step of the shikimate pathway, playing a critical role in controlling the carbon flux from central carbon metabolism into the AAA biosynthesis. Previous biochemical studies suggested the presence of manganese‐ and cobalt‐dependent DHS enzymes (DHS‐Mn and DHS‐Co, respectively) in various plant species. Unlike well‐studied DHS‐Mn, however, the identity of DHS‐Co is still unknown. Here, we show that all three DHS isoforms ofArabidopsis thalianaexhibit both DHS‐Mn and DHS‐Co activities in vitro. A phylogenetic analysis of various DHS orthologs and related sequences showed that Arabidopsis 3‐deoxy‐D‐manno‐octulosonate‐8‐phosphate synthase (KDOPS) proteins were closely related to microbial Type I DHSs. Despite their sequence similarity, these Arabidopsis KDOPS proteins showed no DHS activity. Meanwhile, optimization of the DHS assay conditions led to the successful detection of DHS‐Co activity from Arabidopsis DHS recombinant proteins. Compared with DHS‐Mn, DHS‐Co activity displayed the same redox dependency but distinct optimal pH and cofactor sensitivity. Our work provides biochemical evidence that the DHS isoforms of Arabidopsis possess DHS‐Co activity.