Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Dhaubhadel, Sangeeta

Affiliation

Agriculture and Agri-Food Canada

2nd Supervisor

Bernards, Mark

Co-Supervisor

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.

Summary for Lay Audience

Plants make molecules called specialized metabolites that they use in their own protection from external stresses, like extreme weather conditions, diseases, and pests. Soybean is a legume, a family of plants that make unique specialized metabolites called isoflavonoids. In addition to protecting plants from external threats, isoflavonoids play a role in allowing soybean to form symbiotic relationships with species of bacteria called rhizobia to obtain nitrogen from the air. Nitrogen fertilizers are unsustainable and cause environmental destruction. Climate change creates harsher weather conditions and may allow for more/new diseases to attack crops where they were previously not a problem. For this reason, understanding the intricate machinery behind how, when, and why isoflavonoids are made will likely prove to be useful for creating crops that require less nitrogen fertilizer and have increased disease protection. To this extent, I explored when, and how isoflavonoids are made by studying an enzyme called arogenate dehydratase (ADT). ADT makes phenylalanine, the starting molecule used to make isoflavonoids. Phenylalanine is also a building block for proteins. How plants divide available phenylalanine between these two different outputs is unknown, but critical to our understanding of plant function. Furthermore, ADTs are found mostly in the chloroplast, but some may also be found in the cytosol. It has been shown that a cytosolic bi-functional ADT directly interacts with the machinery that makes isoflavonoids. Thus, it is likely that cytosolic bi-functional ADTs direct phenylalanine to isoflavonoid production, while the others make phenylalanine for proteins. In my project I confirmed which GmADT genes make functional ADT proteins. I then identified which ADT genes could possibly make cytosolic versions of the protein. I concluded that one ADT, GmADT12A, likely directs phenylalanine to isoflavonoid production in soybean. This knowledge serves as an important step towards building a sustainable future.

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