Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

Expression analysis of the two ferrochelatase genes in Arabidopsis in different tissues and under stress conditions reveals their different roles in haem biosynthesis

The Arabidopsis thaliana genome has two genes (AtFC-I and AtFC-II), encoding ferrochelatase, the terminal enzyme of haem biosynthesis. The roles of the two enzymes in the synthesis of haem for different haemoproteins was investigated using reporter gene analysis. A 1.41 kb fragment from the 5' upstream region of the AtFC-II gene was fused to the luciferase gene, and then introduced into tobacco plants, followed by luciferase activity measurements. AtFC-II-LUCwas expressed in all aerial parts of the plant, and was highest in flowers, but it was not expressed in roots. It was unaffected by viral infection, and considerably reduced by wounding or oxidative stress. Similarly, a 1.76 kb region of the AtFC-I promoter was fused to the uidAgene encoding -glucuronidase. AtFC-I-GUS was expressed in all tissues of the plant, but was higher in roots and flowers than in leaves or stems. It was induced by sucrose, wounding and oxidative stress and, most markedly, by plants undergoing the hypersensitive response to TMV infection. Levels of endogenous ferrochelatase activity were increased in pea chloroplasts isolated from wounded leaves, indicating that the induction in promoter activity is likely to result in increased haem biosynthetic potential. Salicylic acid, but not methyl-jasmonate was able to replace the stress treatment in induction of AtFC-I expression, suggesting that the requirement for haem synthesis is part of the defence response. The implications of the results for the different roles of the two ferrochelatases in haem biosynthesis are discussed.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity

Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species. Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized. Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine (Vitis vinifera) suspension cells, and the mutant-plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1. Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.     

Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H₂O₂-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.     

Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress

Salt stress is one of the major abiotic stresses in agriculture worldwide, especially in the Mediterranean area. We report here a proteomic approach to investigate the salt stress-responsive proteins in grapevine (Vitis vinifera). Two-dimensional electrophoresis (2-DE) was used to analyze the proteome of the salt-tolerant Tunisian grapevine cultivar Razegui, subjected to a supply of 100mm NaCl over 15d. Analysis of 2-DE gels derived from stressed plants revealed more than 800 reproducibly detected protein spots, with 48 proteins displaying a differential expression pattern, including 32 up-regulated, 9 down-regulated and 7 new protein spots induced after salt treatment. The presence of stress-responsive proteins in the different plant organs suggests that salt spreads systemically. Edman degradation analysis and database searching aided us in identifying a major protein GP. Database analysis revealed that this peptide has a 98% sequence similarity with a pathogenesis-related (PR) protein 10 (V. vinifera). A full-length cDNA encoding the GP protein was isolated from grapevine salt-stressed leaves and sequenced. The predicted protein contained 158 amino acids and showed 98% identity with PR10 protein of of V. vinifera (accession no. Cac16165).     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Antioxidant enzyme alterations in experimental and clinical diabetes

Previous studies from our laboratory have demonstrated the presence of complex alterations in the activities of antioxidant enzymes in various tissues of rats with streptozotocin (STZ)-induced diabetes. In the present investigation, it is shown that rats made diabetic with alloxan (ALX), an agent differing from STZ both chemically and in its mechanism of diabetogenesis, show virtually identical tissue antioxidant enzyme changes which, as is the case with STZ, are preventable by insulin treatment. The finding that the patterns of antioxidant enzyme alterations in chemically-induced diabetes are independent of the diabetogenic agent used and the presence of similar abnormalities in tissues of spontaneously diabetic (BB) Wistar rats (particularly when diabetic control is less than optimal) suggest that the changes observed are a characteristic feature of the uncontrolled diabetic state and that these may be responsible for (or predispose to) the development of secondary complications in clinical diabetes. Comparative studies involving red cells of diabetic rats and human diabetics revealed a number of common changes, namely an increase in glutathione reductase activity, a decreased susceptibility to oxidative glutathione depletion (which was related to the presence of hyperglycemia) and an increased production of malondialdehyde (an indirect index of lipid peroxidation) in response to in vitro challenge with hydrogen peroxide. In the diabetic patients, the extent of this increase in susceptibility of red cell lipids to oxidation paralleled the severity of diabetic complications. Our results suggest that increased (or uncontrolled) oxidative activity may play an important role in the pathogenesis of complications associated with the chronic diabetic state.This work was supported by grants from the British Columbia Health Care Research Foundation and the Canadian Diabetes Association.     

Oxidant-induced cell-cycle delay in Saccharomyces cerevisiae: the involvement of the SWI6 transcription factor

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a cell-cycle delay that may provide a mechanism to overcome oxidative stress. Strains sensitive to LoaOOH from the genome-wide deletion collection were screened to identify deletants in which the cell-cycle delay phenotype was reduced. Forty-seven deletants were identified that were unable to mount the normal delay response, implicating the product of the deleted gene in the oxidant-mediated cell-cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to its role in cell-cycle progression through Start. The swi6 deletant strain was delayed on entry into the cell cycle in the absence of an oxidant, and oxidant addition caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in oxidant sensing leading to cell division delay. Micro-array studies identified genes whose expression in response to LoaOOH depended on SWI6. The screening identified 77 genes that were upregulated in the wild-type strain and concurrently downregulated in the swi6 deletant treated with LoaOOH. These data show that functions such as heat shock response, and glucose transport are involved in the response.