Characterizing the Functions of the Alternative Oxidase and Type II NAD(P)H dehydrogenase Gene Families in Arabidopsis thaliana

The mitochondrial electron transport chain (mETC) plays a central role in the bioenergetics of all eukaryotic cells. However, the structure of the mETC varies somewhat between plants and higher animals. Plants have a more branched mETC, providing alternative pathways for electron flow, including type II NAD(P)H dehydrogenases (ND) and alternative oxidases (AOX). Recent studies suggest that these “alternative respiratory pathways” may help to minimize the production of reactive oxygen species (ROS) by the mETC. To better understand the connections between mETC structure and ROS production in plants, we transformed Arabidopsis thaliana with several RNAi vectors designed to silence all AOX genes (5 total) or all external ND genes (NDout) (4 total) in the Arabidopsis genome. Real time RT-PCR analyses demonstrated significantly reduced levels of target AOX and NDout transcripts in several transgenic lines. In vivo respiratory assays of AOX-silenced lines showed significantly reduced AOX capacity (~40% reduction) compared to wild-type. Likewise, we saw a decrease in external calcium-dependent NADH oxidation in mitochondria isolated from NDout-silenced lines. Hydrogen peroxide production and lipid peroxidation levels were also measured in AOX- and NDout-silenced lines. The strongest effects were seen in an AOX-silenced line, which showed a 72% increase in leaf H2O2 content in leaf tissue, a 54% increases in H2O2 production in isolated mitochondria, and a 45% increase in total lipid peroxidation. These results suggest that the AOX gene family may play an important role in controlling in vivo mitochondrial ROS production in plants.