Renata Santos

Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis

In nitrogen‐poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium–legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen‐fixing ability was severely impaired. More strikingly, most SOD‐deficient bacteria did not reach the differentiation stage of nitrogen‐fixing bacteroids. The SOD‐deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.

Reactive oxygen species, nitric oxide and glutathione: a key role in the establishment of the legume-Rhizobium symbiosis?

Reactive oxygen species are generated in the first steps of the Rhizobium–legume symbiosis. Superoxide radicals and hydrogen peroxide have been detected in infection threads and there is also evidence of the presence of nitric oxide in young alfalfa nodules. Moreover, rhizobial mutants, with a reduced antioxidant defense, exhibit an impaired capacity to nodulate. The oxidative burst generated in response to symbiotic infection can be consistent with rhizobia being initially perceived as invaders by the plant; in this framework, it may be correlated with the existence of abortive infections. However, the burst appears to be also involved in the expression of early nodulins associated with successful infections. Thus, in parallel to its involvement in defense processes, a positive role for the oxidative burst (including nitric oxide) in the establishment of the symbiotic interaction can also be proposed. The burst could trigger the expression of plant and/or bacterial genes which are essential for the nodulation process. In this framework, glutathione and homoglutathione could be key intermediates for gene expression, via the modification of the redox balance. Thus, the oxidative burst may have a dual role in the establishment of the symbiosis.

Glutathione-dependent redox status of frataxin-deficient cells in a yeast model of Friedreich's ataxia

Friedreichs ataxia is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulphur cluster defects and high sensitivity to oxidative stress. Glutathione is a major protective agent against oxidative damage and glutathione-related systems participate in maintaining the cellular thiol/disulfide status and the reduced environment of the cell. Here, we present the first detailed biochemical study of the glutathione-dependent redox status of wild-type and frataxin-deficient cells in a yeast model of the disease. There were five times less total glutathione (GSH+GSSG) in frataxin-deficient cells, imbalanced GSH/GSSG pools and higher glutathione peroxidase activity. The pentose phosphate pathway was stimulated in frataxin-deficient cells, glucose-6-phosphate dehydrogenase activity was three times higher than in wild-type cells and this was coupled to a defect in the NADPH/NADP(+) pool. Moreover, analysis of gene expression confirms the adaptative response of mutant cells to stress conditions and we bring evidence for a strong relation between the glutathione-dependent redox status of the cells and iron homeostasis. Dynamic studies show that intracellular glutathione levels reflect an adaptation of cells to iron stress conditions, and allow to distinguish constitutive stress observed in frataxin-deficient cells from the acute response of wild-type cells. In conclusion, our findings provide evidence for an impairment of glutathione homeostasis in a yeast model of Friedreichs ataxia and identify glutathione as a valuable indicator of the redox status of frataxin-deficient cells.

Essential Role of Superoxide Dismutase on the Pathogenicity of Erwinia chrysanthemi Strain 3937

The sodA gene from Erwinia chrysanthemi strain 3937 was cloned by functional complementation of an Escherichia coli sodA sodB mutant and sequenced. We identified a 639-bp open reading frame, which encodes a protein that is 85% identical to the E. coli manganese-containing superoxide dismutase MnSOD. Promoter elements of this gene were identified by transcriptional mapping experiments. We constructed an E. chrysanthemi deltasodA mutant by reverse genetics. The deltasodA mutation resulted in the absence of a cytoplasmic SOD, which displays the same characteristics as those of MnSOD. The deltasodA mutant was more sensitive to paraquat than the wild-type strain. This mutant could macerate potato tubers, similar to the wild-type strain. In contrast, when inoculated on African violets, the mutant produced, at most, only small necrotic lesions. If the inoculum was supplemented with the superoxide anion-scavenging metalloporphyrin MnTMPyP or purified SOD and catalase, the deltasodA mutant was able to macerate the inoculated zone. Generation of superoxide anion by African violet leaves inoculated with E. chrysanthemi was demonstrated with nitroblue tetrazolium as an indicator. Therefore, at the onset of infection, E. chrysanthemi cells encounter an oxidative environment and require active protective systems against oxidative damages such as MnSOD to overcome these types of conditions.

Candida albicans lacking the frataxin homologue: a relevant yeast model for studying the role of frataxin.

We cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans. CaYFH1 was expressed in Δyfh1 Saccharomyces cerevisiae cells, where it compensated for all the phenotypes tested except for the lack of cytochromes. Double ΔCayfh1/ΔCayfh1 mutant had severe defective growth, accumulated iron in their mitochondria, lacked aconitase and succinate dehydrogenase activity and had defective respiration. The reductive, siderophore and haem uptake systems were constitutively induced and the cells excreted flavins, thus behaving like iron‐deprived wild‐type cells. Mutant cells accumulated reactive oxygen species and were hypersensitive to oxidative stress, but not to iron. Cytochromes were less abundant in mutants than in wild‐type cells, but this did not result from defective haem synthesis. The low cytochrome concentration in mutant cells was comparable to that of iron‐deprived wild‐type cells. Mitochondrial iron was still available for haem synthesis in ΔCayfh1/ΔCayfh1 cells, in contrast to S. cerevisaeΔyfh1 cells. CaYFH1 transcription was strongly induced by iron, which is consistent with a role of CaYfh1 in iron storage. Iron also regulated transcription of CaHEM14 (encoding protoporphyrinogen oxidase) but not that of CaHEM15 (encoding ferrochelatase). There are thus profound differences between S. cerevisiae and C. albicans in terms of haem synthesis, cytochrome turnover and the role of frataxin in these processes.

Zinc suppresses the iron-accumulation phenotype of Saccharomyces cerevisiae lacking the yeast frataxin homologue (Yfh1).

Analysis of Saccharomyces cerevisiae cell transcriptome revealed that iron deprivation/supplementation affects genes other than those of the iron regulon (controlled by Aft proteins). Several genes regulated by zinc (induced by zinc deprivation) were induced by iron. Cells lacking the yeast frataxin homologue Yfh1 accumulate large amounts of iron in their mitochondria. We have shown that the zinc metabolism of these cells is also impaired: zinc uptake and zinc accumulation were both much lower in Delta yfh1 cells than in wild-type cells. Excess zinc in the growth medium also influenced the phenotypes of Delta yfh1 cells. It prevented the accumulation of iron in the mitochondria of Delta yfh1 cells and increased the growth rate of these cells and their resistance to oxidative stress. However, zinc did not restore the deficiency of Fe-S and haem proteins of Delta yfh1 cells. Zinc inhibited mitochondrial respiration and protected Yah1p, the mitochondrial ferredoxin. These results suggest that zinc nutrition may be important in the aetiology of Friedreichs ataxia.

Characteristics of Protoporphyrinogen Oxidase

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the oxidative O2-dependent aromatization of the colorless protoporphyrinogen IX to the highly conjugated protoporphyrin IX, the precursor of both hemes and chlorophylls (Fig. 1). It is the final enzyme in the common branch of the heme and chlorophyll biosyn-thetic pathways in plants (Fig. 2).