Physiologia Plantarum 129: 225–232. 2007 Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317 Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain Andra ´ s Szarka a, *, Nele Horemans b , Zita Kova ´ cs a , Pa ´ l Gro ´f c , Miklo ´ s Mayer a and Ga ´ bor Ba ´ nhegyi d,e a Department of Biochemistry and Food Technology, Budapest University of Technology and Economics, Budapest, Hungary b Department of Biology, University of Antwerp, Antwerp, Belgium c Department of Biophysics and Radiation Biology, Semmelweis University Budapest, Budapest, Hungary d Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Semmelweis University Budapest, Budapest, Hungary e Department of Pathophysiology, Experimental Medicine and Public Health, University of Siena, Siena, Italy Correspondence *Corresponding author, e-mail: szarka@mail.bme.hu Received 3 April 2006; revised 17 July 2006 doi: 10.1111/j.1399-3054.2006.00810.x The reduction of dehydroascorbate (DHA) was investigated in plant mitochondria. Mitochondria isolated from Bright Yellow-2 tobacco cells were incubated with 1 mM of DHA, and the ascorbate generation was followed by high-performance liquid chromatography. Mitochondria showed clear ability to reduce DHA and to maintain a significant level of ascorbate. Ascorbate generation could be stimulated by the respiratory substrate succinate. The complex I substrate malate and the complex I inhibitor rotenone had no effect on the ascorbate generation from DHA. Similarly, the complex III inhibitor antimycin A, the alternative oxidase inhibitor salicylhydroxamic acid, and the uncoupling agent 2,4-dinitrophenol were ineffective on mitochondrial ascorbate generation both in the absence and in the presence of succinate. However, the competitive succinate dehydrogenase inhibitor malonate almost completely abolished the succinate-dependent increase in ascorbate pro- duction. The complex IV inhibitor KCN strongly stimulated ascorbate accumulation. These results together suggest that the mitochondrial respiratory chain of plant cells – presumably complex II – plays important role in the regeneration of ascorbate from its oxidized form, DHA. Introduction Ascorbate is one of the most important water-soluble antioxidants for plant and animal cells (Ba ´nhegyi et al. 1997, May and Asard 2004). The mitochondrion has a central role in ascorbate metabolism, especially in plants. Plant mitochondria are responsible not only for the synthesis of ascorbate by L-galactono-g-lactone dehydrogenase but also for the regeneration of ascorbate from its oxidized forms (Bartoli et al. 2000, Chew et al. 2003, Jimenez et al. 1997, Yabuta et al. 2000). The recycling is extremely important, since the fully oxidized dehydroascorbic acid has a short half-life and would be lost unless it is reduced back. Mitochondrial dehydroascorbate (DHA) reduction can be caused by two main mechanisms. Electrons are provided by small electron carriers, such as glutathione (GSH) or lipoic acid, or by the respiratory electron transfer chain. Both processes have been elucidated in detail in animal mitochondria (Li et al. 2001, Li et al. 2002). DHA is taken up by both plant and animal mitochon- dria, which suggests that the organelle might be re- sponsible for the regeneration of DHA produced locally Abbreviations – BY-2, Bright Yellow-2; DHA, dehydroascorbate; EPR, electron paramagnetic resonance; ESR, electron spin resonance; GLUT, glucose transporter; GSH, glutathione; HPLC, high-performance liquid chromatography; MOPS, 4-morpholine- propanesulfonic acid; MDA, monodehydroascorbate; NADH, nicotinamide adenine dinucleotide; SHAM, salicylhydroxamic acid. Physiol. Plant. 129, 2007 225