IRKUTSK, RUSSIA
Institute of Plant Physiology and Biochemistry
Russian Academy of Sciences

NOVOSIBIRSK, RUSSIA
Institute of Cytology and Genetics
Russian Academy of Sciences

Differential expression of mitochondrial genes under redox state changes caused by respiratory inhibitor antimycine A

— Tarasenko, VI; Garnik, EY; Kobzev, VF; Konstantinov, YM

Regulation of gene expression by redox conditions is considered to be one of the key regulatory mechanisms in most of living organisms. We have previously demonstrated the existence of redox control of RNA synthesis in organello in maize mitochondria (Konstantinov et al., Biochem. Mol. Biol. Intern. 36:319–326, 1995). Significant activation of transcription under oxidizing conditions and its repression under reducing conditions can indicate the existence of a special regulatory mechanism in plant mitochondria. Another group has independently shown dependence of RNA synthesis in potato mitochondria on the redox state of the electron transport chain (Wilson et al., Eur J. Biochem. 242:81–85, 1996). Both groups have used an approach allowing detection of total RNA synthesis level, so it is unknown if this process could be specific for transcripts of certain mitochondrial genes.

In this investigation we studied changes in the transcript levels of a few mitochondrial genes in maize seedlings treated with respiratory inhibitor antimycine A. This compound blocks electron transport at the level of respiratory complex III causing overreduction of the electron transport chain. This state could in turn lead to an increase in the generation of reactive oxygen species (ROS) by the respiratory chain. Changes of both the mitochondria redox state and an elevated ROS level could serve as signals for modulation of gene expression. Plant mitochondria contain an alternative oxidase which directly transfers electrons from ubiquinone to oxygen, lowering the extent of respiratory chain overreduction and ROS formation. To inhibit alternative oxidase activity we used salicylhydroxamic acid (SHAM).

The intact seedlings were soaked in water solutions containing antimycine A, SHAM or both compounds during time periods of 2, 4, 6, 10 and 24 hours. Plant material was frozen in liquid nitrogen and immediately subjected to RNA isolation with use of the phenol extraction method. These RNA samples were electrophoresed in agarose gel containing formaldehyde and hybridized with PCR-labelled DNA probes for maize mitochondrial genes atp9, cox1, cox3, cob, rps13 and rrn26.

Figure 1 shows the results of the experiments. The treatment of seedlings with antimycine A alone led to gradual increase of atp9, cox1, cox3 and cob gene transcript levels followed by decrease up to the level lower than in the control. The highest amounts of transcripts were detected after 4 h following treatment for cox1, cox3 and cob, and after 10 h following treatment for the atp9 gene. Two peaks of transcript accumulation (after both 4 h and 10 h) were observed for the rps13 gene.

The treatment with SHAM decreased the atp9 transcript level during the first 6 h, after which partial recovery of transcript content was observed. Fluctuations of transcript level, with maximum at 24 h, were characteristic of rps13 gene expression. The expression of other genes tested was unaffected by SHAM treatment.

The treatment with antimycine A and SHAM led to rapid decrease of atp9, cox1, cox3 and cob transcript level (after 2 and 4 h). Contrary to this, rps13 transcript amount decreased rapidly after 2 h of treatment, but rose again to control level after 4 h and remained stable until the end of the treatment.

Thus, we have found that the redox state of the mitochondrial respiratory chain affects transcript levels of mitochondrial genes and these changes are gene-specific. The expression patterns were quite similar for cox1, cox3 and cob genes, which might reflect localization of proteins encoded by these genes in the same respiratory complex. The variations of the atp9 transcript level were not identical, but in some extent they resemble ones detected for the aforementioned genes. On the other hand, the expression pattern of the rps13 gene encoding ribosomal protein was very different in most cases. In general, our results point to the involvement of the redox state of the respiratory chain and/or ROS level in regulation of mitochondrial gene expression in vivo.

 

Fig. 1. Northern blot analysis of expression of mitochondrial atp9, cox1, cox3, cob, rps13 and rrn26 genes under redox state changes caused by treatment of seedlings by antimycine A; treatment duration 2, 4, 6, 10 and 24 hours.


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