Functional Characterization of Arogenate Dehydratase Isoforms in Soybean
Abstract
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.