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Abstract l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrAa), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrAp), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterizedTnt1‐transposon mutants ofMtPDH1(pdh1) inMedicago truncatulato investigate PDH function. The pdh1mutants lackedPDHtranscript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence thatMtPDH1is responsible for the PDH activity detected inM. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity ofpdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism.
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Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta
l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure–function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.
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- PAR ID:
- 10309340
- Date Published:
- Journal Name:
- The Plant Journal
- ISSN:
- 0960-7412
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1111/tpj.15597
