Functional Characterisation of Alternative Oxidase Protein Isoproteins in <scp> <i>Arabidopsis thaliana</i> </scp>

The Alternative Oxidase (AOX) is encoded by a small gene family in plants. While being one of the most intensively studied plant mitochondrial proteins, it is primarily only one isoform, AOX1a, that is well studied. We investigated the sub-and neo-functionalisation of AOX isoforms in Arabidopsis thaliana by constructing over-expressing lines for all five AOX isoforms in an aox1a knock-out mutant line, where no AOX protein can be detected. In Arabidopsis thaliana, knock-out mutants for aox1a are unable to support germination on antimycin A, despite the presence of four functional AOX genes. Sub-functionalisation was observed for AOX1a in that its 3 kb promoter region supports germination on antimycin A when driving the expression of other AOX isoforms, indicating that it is a promoter-based trait, rather than a specific function of the AOX1a isoprotein. Further evidence of sub-functionalisation was evident as AOX1d and AOX1a tagged with GFP enhanced epidermal expression but not when the other isoforms were used. Arabidopsis AOX1c represents an example of neo-functionalisation as aox1c knock-out had a slightly retarded growth phenotype, while mis-expression of AOX1c, using either the CaMV 35S promoter or the 3 kb of the AOX1c promoter, resulted in enhanced growth. This was even more evident under high-light conditions, where greater tolerance to high light was observed compared to wild type (Col-0) plants. These examples indicate that functional analysis of all AOX isoforms is crucial to gain a full understanding of the role of AOX in plant metabolism and growth.