An official website of the United States government
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.
more »
« less
Coordinated regulation of the entry and exit steps of aromatic amino acid biosynthesis supports the dual lignin pathway in grasses
Abstract Vascular plants direct large amounts of carbon to produce the aromatic amino acid phenylalanine to support the production of lignin and other phenylpropanoids. Uniquely, grasses, which include many major crops, can synthesize lignin and phenylpropanoids from both phenylalanine and tyrosine. However, how grasses regulate aromatic amino acid biosynthesis to feed this dual lignin pathway is unknown. Here we show, by stable-isotope labeling, that grasses produce tyrosine >10-times faster than Arabidopsis without compromising phenylalanine biosynthesis. Detailed in vitro enzyme characterization and combinatorialin plantaexpression uncovered that coordinated expression of specific enzyme isoforms at the entry and exit steps of the aromatic amino acid pathway enables grasses to maintain high production of both tyrosine and phenylalanine, the precursors of the dual lignin pathway. These findings highlight the complex regulation of plant aromatic amino acid biosynthesis and provide novel genetic tools to engineer the interface of primary and specialized metabolism in plants.
more »
« less
- PAR ID:
- 10475958
- Publisher / Repository:
- Springer Nature
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Abstract The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.more » « less
-
Abstract In plants, embryo size is determined via interactions between metabolic and developmental signals. Maize (Zea mays) big embryo 6 (bige6) enhances embryo size while sharply reducing plant growth. Here, we show that BigE6 encodes a plastidial prephenate aminotransferase (PPA-AT), a key enzyme in the arogenate pathway for L-phenylalanine (Phe) and L-tyrosine (Tyr) biosynthesis. The maize BigE6 paralog, BigE6Like, encodes a cytosol-localized PPA-AT, revealing Phe and Tyr biosynthesis via cytosolic arogenate as a potential alternative to the known cytosolic phenylpyruvate pathway. Moreover, the single PPA-AT gene of Arabidopsis (Arabidopsis thaliana) encodes plastidial and cytosolic enzymes by alternative splicing. Transgenic rescue of a ppa-at mutant in Arabidopsis demonstrates that the plastidial PPA-AT is indispensable for seed formation due, in part, to its essential role in the female gametophyte. Leaves of bige6 maize maintained overall homeostasis for aromatic amino acids and downstream metabolites, revealing a resilience of mechanisms that scale growth to a limiting supply of Phe and Tyr. In bige6 seeds, broad perturbation of amino acid homeostasis is associated with transcriptomic upregulation of growth processes in the embryo and endosperm, implicating amino acid signaling in the regulation of embryo size. Our findings reveal the complexity and developmental dependence of growth responses to limiting amino acid biosynthesis.more » « less
-
SUMMARY Phenylpropanoids are specialized metabolites derived from phenylalanine. Glucosinolates are defense compounds derived mainly from methionine and tryptophan in Arabidopsis. It was previously shown that the phenylpropanoid pathway and glucosinolate production are metabolically linked. The accumulation of indole‐3‐acetaldoxime (IAOx), the precursor of tryptophan‐derived glucosinolates, represses phenylpropanoid biosynthesis through accelerated degradation of phenylalanine ammonia lyase (PAL). As PAL functions at the entry point of the phenylpropanoid pathway, which produces indispensable specialized metabolites such as lignin, aldoxime‐mediated phenylpropanoid repression is detrimental to plant survival. Although methionine‐derived glucosinolates in Arabidopsis are abundant, any impact of aliphatic aldoximes (AAOx) derived from aliphatic amino acids such as methionine on phenylpropanoid production remains unclear. Here, we investigate the impact of AAOx accumulation on phenylpropanoid production using Arabidopsis aldoxime mutants,ref2andref5. REF2 and REF5 metabolize aldoximes to respective nitrile oxides redundantly, but with different substrate specificities.ref2andref5mutants have decreased phenylpropanoid contents due to the accumulation of aldoximes. As REF2 and REF5 have high substrate specificity toward AAOx and IAOx, respectively, it was assumed thatref2accumulates AAOx, not IAOx. Our study indicates thatref2accumulates both AAOx and IAOx. Removing IAOx partially restored phenylpropanoid content inref2, but not to the wild‐type level. However, when AAOx biosynthesis was silenced, phenylpropanoid production and PAL activity inref2were completely restored, suggesting an inhibitory effect of AAOx on phenylpropanoid production. Further feeding studies revealed that the abnormal growth phenotype commonly observed in Arabidopsis mutants lacking AAOx production is a consequence of methionine accumulation.more » « less
-
While mammals require the essential amino acid tryptophan (Trp) in their diet, plants and microorganisms synthesize Trp de novo. The five-step Trp pathway starts with the shikimate pathway product, chorismate. Chorismate is converted to the aromatic compound anthranilate, which is then conjugated to a phosphoribosyl sugar in the second step by anthranilate phosphoribosyltransferase (PAT1). As a single-copy gene in plants, all fixed carbon flux to indole and Trp for protein synthesis, specialized metabolism, and auxin hormone biosynthesis proceeds through PAT1. While bacterial PAT1s have been studied extensively, plant PAT1s have escaped biochemical characterization. Using a structure model, we identified putative active site residues that were variable across plants and kinetically characterized six PAT1s (Arabidopsis thaliana (thale cress), Citrus sinensis (sweet orange), Pistacia vera (pistachio), Juglans regia (English walnut), Selaginella moellendorffii (spike moss), and Physcomitrium patens (spreading earth-moss)). We probed the catalytic efficiency, substrate promiscuity, and regulation of these six enzymes and found that the C. sinensis PAT1 is highly specific for its cognate substrate, anthranilate. Investigations of site-directed mutants of the A. thaliana PAT1 uncovered an active site residue that contributes to promiscuity. While Trp inhibits bacterial PAT1 enzymes, the six plant PAT1s that we tested were not modu- lated by Trp. Instead, the P. patens PAT1 was inhibited by tyrosine, and the S. moellendorffii PAT1 was inhibited by phenylalanine. This structure-informed biochemical examina- tion identified variations in activity, efficiency, specificity, and enzyme-level regulation across PAT1s from evolutionarily diverse plants.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1038/s41467-023-42587-7
