The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells

Abstract

Besides the cytochrome c pathway, plant mitochondria have an alternative respiratory pathway that is comprised of a single homodimeric protein, alternative oxidase (AOX). Transgenic cultured tobacco cells with altered levels of AOX were used to test the hypothesis that the alternative pathway in plant mitochondria functions as a mechanism to decrease the formation of reactive oxygen species (ROS) produced during respiratory electron transport. Using the ROS-sensitive probe 2,7-dichlorof luorescein diacetate, we found that antisense suppression of AOX resulted in cells with a significantly higher level of ROS compared with wild-type cells, whereas the overexpression of AOX resulted in cells with lower ROS abundance. Laser-scanning confocal microscopy showed that the difference in ROS abundance among wild-type and AOX transgenic cells was caused by changes in mitochondrial-specific ROS formation. Mitochondrial ROS production was exacerbated by the use of antimycin A, which inhibited normal cytochrome electron transport. In addition, cells overexpressing AOX were found to have consistently lower expression of genes encoding ROSscavenging enzymes, including the superoxide dismutase genes SodA and SodB, as well as glutathione peroxidase. Also, the abundance of mRNAs encoding salicylic acid-binding catalase and a pathogenesis-related protein were significantly higher in cells deficient in AOX. These results are evidence that AOX plays a role in lowering mitochondrial ROS formation in plant cells.

Metal Ion availability in mitochondria

“…The ferroxidase center in mitochondrial ferritin contains a diiron carboxylate center (Levi et al 2001). A related site is present in the alternative oxidase (AOX) found in the mitochondrial respiratory chain of plants and some fungi (Maxwell et al 1999).…”

Evaluation of community respiratory mechanisms with oxygen isotopes: A case study in Lake Kinneret

“…In particular, Maxwell et al (1999) demonstrated the engagement of this mechanism as a response to the formation of reactive oxygen species that resulted in oxidative stress. They suggested that the role of AOX was to decrease this stress.…”

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

“…Intracellular production of ROS was measured using 2 0 ,7 0 -dichlorofluorescein diacetate (H 2 DCF-DA) (Maxwell et al 1999). Dichlorofluorescein (DCF) fluorescence was measured using a microplate spectrofluorometer (Molecular Devices Spectra Max Gemini).…”

“…Excessive mitochondrial ROS formation is seen as an indicator of mitochondrial dysfunction (Raha and Robinson 2000) and is a key trigger of programmed cell death (Lam et al 2001). Using transgenic tobacco cells, Maxwell et al (1999) showed that cells lacking AOX had increased mitochondrial-derived ROS formation and an upregulation of key genes involved in antioxidant defence, when compared with wild-type cells. More recently, these same AOX antisense cells have been shown to have a greater susceptibility for mitochondrial-dependent and mitochondrial-independent cell death .…”