Treatment of Arabidopsis (Arabidopsis thaliana) alternative oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compared with ecotype Columbia (Col-0), as evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments, and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, and sucrose and starch metabolism changed, indicating that effects were not confined to mitochondria, where the AOX1a protein is located. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed that transcripts typically induced upon stress treatment or involved in antioxidant defense systems, especially chloroplast-located antioxidant defense components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signaling pathways that regulate these components. Taken together, these results indicate that aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal redox balance in the cell.

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper: manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu 2+ , Zn 2+ , and Co 2+ was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.

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