The mitochondria of higher plants have two electron transport pathways: cytochrome pathway and alternative pathway (Vanlerberghe et al. 1997). The alternative oxidase (AOX), which is the terminal oxidase of the alternative pathway, branches at the ubiquinone pool and bypasses two of the three proton translocation sites, cytochrome bc₁ and cytochrome c oxidase (COX). In this way, the alternative pathway is uncoupled with ATP synthesis, whereas the cytochrome pathway is coupled with ATP synthesis by a proton gradient as the motive force during electron flow. In dicots, the expressions of AOX genes are induced by many stresses, including inhibition of the cytochrome pathway (Vanlerberghe et al. 1997). In Arabidopsis thaliana, for example, expression of AOX1a gene is induced by treatment with antimycin A and sodium azide (NaN₃), both of which inhibit the functions of COX (Saisho et al. 2001). In monocots, there is not yet any evidence that expression of AOX gene is induced by treatment with inhibitors of respiration. In this report, we investigated the expression of AOX1a gene in rice by NaN₃ treatment.

Five-day-old rice seedlings grown under constant light at 28C, were treated with 100 microM NaN₃. Rice leaves were collected just before the treatment started (0 hour), and at 2, 4, 8 and 12 hours after the treatment. Total RNA was extracted from each sample. Northern hybridization was performed using specific probes for AOX1a gene and ATP2 gene. ATP2 gene encodes the F_oF₁-ATP synthase Beta-subunit in rice. NaN₃ increased AOX1a mRNA (Fig. 1). Our result agrees with the findings on Arabidopsis thaliana (Saisho et al. 2001). The amount of AOX1a transcript rapidly increased and reached its maximum 8 hours after treatment with 100 microM NaN₃. On the other hand, the steady-state mRNA level of ATP2 gene hardly changed 12 hours after treatment. These results suggest that expression of rice AOX1a gene is induced to consume the electrons transferred to the ubiquinone pool, because the functions of COX are inhibited by NaN₃ treatment. On the other hand, NaN₃ inhibits apoplastic Cu-Zn SOD and peroxidases (Ogawa et al. 1997), as well as COX. AOX may compensate for the inhibition of activities of apoplastic Cu-Zn SOD and peroxidases. Further analysis is necessary to evaluate the functions of AOX.

References

Vanlerberghe, G. C. and L. McIntosh, 1997. Alternative oxidase: From gene to function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 703-734.

Saisho, D., M. Nakazono, N. Tsutsumi and A. Hirai, 2001. ATP synthesis inhibitors as well as respiratory inhibitors increase steady-state level of alternative oxidase mRNA in Arabidopsis thaliana. J. Plant Physiol. 158: 241-245.

Ogawa, K., S. Kanematsu and K. Asada, 1997. Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification. Plant Cell Physiol. 38: 1118-1126.