Ascorbate Biosynthesis in Mitochondria Is Linked to the Electron Transport Chain between Complexes III and IV

Abstract

Ascorbic acid is synthesized from galactono-γ-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-γ-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.

Photosynthesis and State Transitions in Mitochondrial Mutants ofChlamydomonas reinhardtiiAffected in Respiration

“…Therefore, it is tempting to propose that in wild type and in mutants not affected in complex IV, interactions of ascorbate with the photosynthetic electron transport chain participate in non-photochemical PQ reduction and initiate partial state 2 transition. Complex III mutants might even show higher responses because a stimulation of ascorbate synthesis by antimycin A (an inhibitor of Cyt c reduction by complex III) was reported in potato mitochondria (Bartoli et al, 2000). However, to our knowledge, reduction of PQ by ascorbate in thylakoids has not been reported, although chemical reduction of extracted PQ by ascorbate is common routine.…”

“…Reduced Cyt c is then oxidized by complex IV. In potato mitochondria, inhibition of complex IV by cyanide strongly reduces ascorbate synthesis (Bartoli et al, 2000). One can hypothesize that the same pathway occurs in C. reinhardtii and that mitochondrial ascorbate synthesis is specifically inhibited in complex IV mutants.…”

“…The Cyt pathway is inactive in both cases, and we checked that the ATP level in dark-adapted (90 min) cells did not differ significantly (values of 65% to70% of wild type), thus indicating no visible difference in oxidative phosphorylation ability (data not shown). Interestingly, it was found recently that complex IV is specifically involved in ascorbate synthesis in higher plant mitochondria (Bartoli et al, 2000). In plants, the last step of ascorbate biosynthesis consists of the oxidation of l-galactono-lactone by a membrane dehydrogenase that couples l-galactono-lactone oxidation to the reduction of Cyt c (Wheeler et al, 1998).…”

The Response of Carbon Metabolism and Antioxidant Defenses of Alfalfa Nodules to Drought Stress and to the Subsequent Recovery of Plants

“…Ascorbate and dehydroascorbate were measured as described by Bartoli et al (2000) with some modifications (Matamoros et al, 2006) using nodules harvested directly into liquid nitrogen to avoid artifactual oxidation of ascorbate. Extracts were made with 1 M HClO 4 , cleared by centrifugation, neutralized with 1 M K 2 CO 3 to pH 5.6, and centrifuged again.…”

Towards an Analysis of the Rice Mitochondrial Proteome

“…Biochemical investigations have revealed that isolated plant mitochondria share many features with those from animals and fungi. However, plant mitochondria also contain additional components such as nonphosphorylating bypasses of the electron transport chain, specialized metabolite carriers, and enzymes involved in the synthesis of folate, lipoic acid, and vitamin C (Bartoli et al, 2000;Gueguen et al, 2000;Rebeille et al, 1997). Only a few of the nuclear genes encoding proteins that maintain these unique functions have been identified.…”