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'-dichlorofluorescein 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 ROS-scavenging 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.

Figure 1

Intracellular ROS abundance in WT and Aox1 transgenic cultured tobacco cells. Intracellular ROS was measured by using H₂DCF-DA as described in Materials and Methods. Measurements were made by using cells 3–4 days after subculture. Results represent the means (± SD) of six to eight separate experiments. AS8, Aox1 antisense; S11, Aox1 overexpresser.

Figure 2

ROS formation in WT and Aox1 transgenic cultured tobacco cells after inhibition of cytochrome c electron transport. Time course of ROS production after addition of antimycin A (5 μM) to intact cells at t = 0. At each time point, an aliquot of cells was removed and incubated with H₂DCF-DA for 30 min before fluorescence measurement. ●, WT; ■, AS8; ▴, S11. Results represent the means (± SD) of four separate experiments.

Figure 3

Intracellular localization of ROS formation. Laser-scanning confocal microscope images of WT and Aox1 transgenic cultured tobacco cells. Cells were double-labeled with H₂DCF-DA and Mitotracker Red. If used, antimycin A (+AA) was added 5 min before the dyes. Magnification for all images was ×350.

Figure 4

Effects of ROS-generating and ROS-scavenging compounds on intracellular ROS levels. (A) Cells were incubated for 4 h in the presence of the ROS-generating compounds antimycin A (AA; 5 μM), H₂O₂ (1 mM), or menadione (Men.; 100 μM) before ROS determination. (B) Effect of common antioxidants on the antimycin A-dependent increase in DCF fluorescence. Cells were preincubated for 1 h with either BHA (100 μM) or flavone (+Flav.; 0.5 mM) before the addition of antimycin A (AA). DCF fluorescence was determined 4 h after the addition of antimycin A. Data are presented as percentages of the absolute values shown in A. Results represent the means (± SD) of three separate experiments.

Figure 5

Expression of genes encoding ROS-scavenging enzymes and PR-1 in WT and Aox1 transgenic cells. RNA gel blot analysis of 25 μg of total RNA isolated from cultured tobacco cells 3–4 days after subculture. (A) Probes for ROS-scavenging transcripts were SodA and SodB from N. plumbaginifolia, GPX from N. tabacum, and SA-Cat from N. tabacum; probes were obtained by RT-PCR. (B) PR-1 from N. tabacum was obtained by RT-PCR. (C) Arabidopsis translation initiation factor elF4A was used to confirm equal loading of RNA.