Identification of aminotransferases involved in plant phenylalanine biosynthesis

Phenylalanine (Phe) is an important amino acid that is used for protein biosynthesis and precursor for various plant natural products. Phe is synthesized from prephenate, a product of the shikimate pathway, via two alternative pathways that include arogenate or phenylpyruvate as intermediates. Although the arogenate pathway plays a predominant role in Phe biosynthesis in plants, a gene encoding prephenate aminotransferase (PPA-AT), which converts prephenate to arogenate, has not been identified. Here, PPA-ATs from Arabidopsis ( AtPPA-AT) and petunia (PhPPA-AT) were identified using bioinformatic analysis and comparative genomic approaches. Biochemical characterization of AtPPA-AT and PhPPA-AT enzymes indicated that PPA-ATs exhibited high affinity for prephenate and did not use phenylpyruvate or 4-hydroxyphenylpyruvate as amino acceptors. Downregulation of PhPPA-AT in petunia results in significant decrease of Phe levels, suggesting that Phe is mainly synthesized via the arogenate route in plants. However, Phe level was reduced only by 20% in the PhPPA-AT RNAi line, raising a question whether the remaining PPA-AT is enough to direct carbon flux toward the arogenate route and synthesize up to 80% of Phe, or whether flux is redirected through the alternative phenylpyruvate route for Phe biosynthesis. To test the possibility of an alternative route for plant Phe biosynthesis, RNAi transgenic petunia plants in which both the PhADT1 and PhPPA-AT genes were simultaneously downregulated in petunia petals were generated. While in PhADT1-RNAi line the levels of Phe and Phe-derived volatiles were reduced by as much as 80%, those in the PhADT1xPhPPA-AT RNAi line were rescued to wild-type levels, indicating the involvement of an alternative Phe biosynthetic pathway. We have further isolated petunia phenylpyruvate aminotransferase ( PhPPY-AT), which complements the Escherichia coli (E. coli) Phe auxotrophic mutant. Transient downregulation of PhPPY-AT and feeding experiment of 15N-Tyr, together with cytosolic localization of PhPPY-AT, showed that microbial-like phenylpyrvute pathway operates for plant Phe formation in the cytosol and its contribution increases when the arogenate pathway is limiting. To elucidate the regulatory mechanisms involved in Phe biosynthetic pathways in plants, we generated site-directed mutations in the putative allosteric regulatory region of the PhADT1, which relaxed feedback sensitivity by Phe. This mutation resulted in perturbation in the levels of Phe and Phe-derived volatiles, suggesting that unknown regulatory mechanism(s) control carbon flux toward aromatic amino acid biosynthesis based on Phe level in plastids, which need to be further elucidated. Taken together, our research enhances knowledge on biochemical and genetic pathways of Phe biosynthesis and its regulation, which can elucidate the mechanisms of biosynthesis in Phe-derived secondary metabolites