Functional Characterization of Arogenate Dehydratase Isoforms in Soybean

Phenylalanine flux is partitioned between phenylpropanoid and protein synthesis. The mechanisms behind the metabolic channeling of phenylalanine are largely unknown. Arogenate dehydratase (ADT) enzymes, which catalyze the last and rate-limiting step in the synthesis of phenylalanine in plants, have been shown to interact with the isoflavonoid metabolon in the cytosol. Cytosolic phenylalanine, however, can only be synthesized through prephenate dehydratase (PDT) activity. In this study, putative soybean ADTs (GmADTs) were characterized for their ADT and PDT activity. This was done using complementation assays with two different knockout yeast strains, aro8aro9 and pha2, which lack prephenate aminotransferase and PDT activity, respectively. Additionally, GmADTs with alternate transcripts that exclude the transit peptide were identified through qRT- PCR. It was determined that, of 8 putative GmADTs, GmADT11B had the most ADT and PDT activity. GmADT12B and GmADT12C were found to have some ADT activity but to a lesser degree. The remaining 5 GmADTs had the least ADT activity, if any. Some PDT activity was detected in GmADT12A and GmADT13A, while none was detected in the remaining 5 GmADTs. Furthermore, it was determined that GmADT12B and GmADT11A contain alternate transcripts that exclude the sequence for the transit peptide. If these GmADTs have a cytosolic isoform, they are likely involved in directing phenylalanine flux to phenylpropanoid synthesis. These findings provide insight into possible mechanisms of regulation controlling specialized metabolite synthesis in plants

Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon