Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Allison E. McDonald, Ph.D.

Advisor Role

Thesis Advisor

Abstract

Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative pathway for electron transport, allowing plants to maintain respiratory flexibility under environmental stress conditions. While AOX has been extensively studied in angiosperms (flowering plants), little is known about its distribution and conservation in non-angiosperms. Based on gaps identified in the literature, this thesis investigated the distribution of AOX across non-angiosperm plant lineages. Although AOX is present in many green algae and is therefore considered to be an ancient protein that originated before the existence of non-angiosperm plants, it remains unclear whether the protein is uniformly distributed across these groups or whether evolutionary exceptions exist where it is absent. It was hypothesized that AOX is present across all non-angiosperm plant lineages.

To address this question, an integrative approach combining bioinformatic and molecular methodologies was used. Publicly available sequence databases were searched to attempt to identify AOX sequences from ferns, cycads, conifers, ginkgo, lycopods, liverworts, and mosses. Multiple sequence alignments were conducted to assess the conservation of AOX motifs, catalytic residues, and iron-binding sites. Lineage-specific primers were designed and tested on representative non-angiosperm species using reverse-transcriptase polymerase chain reaction (RT-PCR), cloning, transformation, and Sanger sequencing to isolate, amplify, and sequence AOX cDNA.

A total of 222 AOX sequences representing 70 species were retrieved and analyzed from non-angiosperm plants. Conserved AOX iron-binding sites (LETVA, NERMHL, GYLEEA, and RADEAH), catalytic residues (Glutamate and Histidine), and other conserved sequence motifs (CRAMM and PAPIYGH) were identified across all phylogenetic groups examined. Bioinformatic analysis identified in silico evidence of AOX sequences within Ginkophyta, Cycadophyta, and Lycopodiopsida to expand the known phylogenetic distribution of AOX beyond previously reported information in the literature. Molecular analysis successfully amplified putative AOX products from multiple non-angiosperm groups, and subsequent cloning and sequencing confirmed the presence of AOX within the fern Nephrolepis exaltata for the first time. 

Collectively, these findings support the hypothesis that AOX is broadly distributed across non-angiosperm plant lineages and demonstrate that key AOX motifs remain highly conserved throughout plant evolution. This thesis highlights the value of integrating bioinformatics, molecular biology, and taxonomy to investigate the physiological and evolutionary significance of AOX across the Viridiplantae lineage.

Convocation Year

2026

Convocation Season

Fall

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