Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O₂-resistant photosynthesis. The apparent K_m for H₂O₂ was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.     

Selection and characterization of tobacco plants with novel o(2)-resistant photosynthesis

Plants were obtained with novel O₂-resistant photosynthetic characteristics. At low CO₂ (250-350 μL CO₂ L⁻¹) and 30°C when O₂ was increased from 1% to 21% to 42%, the ratio of net CO₂ uptake in O₂-resistant whole plants or leaf discs compared to wild type increased progressively, and this was not related to stomatal opening. Dihaploid plantlets regenerated from anther culture were initially screened and selected for O₂-resistant growth in 42% O₂/160 μL CO₂ L⁻¹ and 0.18% of the plantlets showed O₂-resistant photosynthesis. About 30% of the progeny (6 of 19 plants) of the first selfing of a fertile plant derived from a resistant dihaploid plant had O₂-resistant photosynthesis, and after a second selfing this increased to 50% (6 of 12 plants). In 21% O₂ and low CO₂, net photosynthesis of the resistant plants was about 15% greater on a leaf area basis than wild type. Net photosynthesis was compared in leaf discs at 30 and 38°C in 21% O₂, and at the higher temperature O₂-resistant plants showed still greater photosynthesis than wild type. The results suggest that the O₂-resistant photosynthesis described here is associated with a decreased stoichiometry of CO₂ release under conditions of rapid photorespiration. This view was supported by the finding that leaves of O₂-resistant plants averaged 40% greater catalase activity than wild type.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Chitin-graft-poly(2-methyl-2-oxazoline) enhanced solubility and activity of catalase in organic solvent

Catalase from bovine liver was lyophilized from an aqueous solution containing chitin-graft-poly(2-methyl-2-oxazoline) (3), which was synthesized by the reaction of 52% deacetylated chitin (1) with living poly(2-methyl-2-oxazoline) (2). The rate of consumption of H₂O₂ in chloroform catalyzed by the lyophilized catalase with 3 was enhanced more than 10 times that by catalase without 3. The dispersibility and solubility of lyophilized catalase with 3 in chloroform were improved in comparison with catalase itself.     

Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity

Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2). The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress. The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity. H2O2 resistance correlates well with catalase activity. An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance. These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity.     

Effect of chromate on photosynthesis in Cr(VI)-resistant Chlorella

Chromate-resistant Chlorella spp. isolated from effluents of electroplating industry could grow in the presence of 30 μM K₂Cr₂O₇. Since photosynthesis is sensitive to oxidative stress, chromate toxicity to photosynthesis was examined in this algal isolate. Chromate [Cr(VI)] up to 100 μM was found to stimulate photosynthesis, while 90% inhibition was found, when the cells were incubated with 1 mM Cr(VI) for 4 h. Photosystem (PS) II was inhibited by 80% and PSI by 40% after such Cr(VI) treatment. Thermoluminescence studies on cells treated with 1 mM Cr(VI) for 4 h showed that S₂Q_A ^? recombination peak (Q) was shifted to higher temperature, whereas S₂/S₃Q_B ^? recombination peak (B) was shifted to lower temperature. These shifts indicated alga stress response in order to overcome an excitation stress resulting from the inhibition of photosynthesis by Cr(VI). The nontreated Chlorella cells kept in the dark showed periodicity of four for the Q peak (4–8°C) and B peak (34–38°C) after exposure to series of single, turnover, saturating flashes. This periodicity was lost in Cr(VI)-treated cells. Higher concentrations of Cr(VI) inhibited mainly the electron flow in the electron transport chain, inactivated oxygen evolving complex, and affected also Calvin cycle enzymes in the Cr(VI)-resistant isolates of Chlorella.     

Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light

Transgenic tobacco deficient in either Cat1 (Cat1AS), Cat2 (Cat2AS), or both (CatGH) was generated through sense and antisense technology. Cat1AS, Cat2AS, and CatGH plants showed no visible phenotype when grown at low light (100 µmol m⁻² sec⁻¹. Under these conditions, deficiency in Cat1 and/or Cat2 did not lead to constitutive pathogenesis-related (PR-1) expression and did not potentiate PR-1 induction by exogenous salicylic acid. This demonstrates that catalase suppression per se is not a sufficient signal for PR-1 induction. In Cat1-deficient plants exposed to higher light intensities (250–1000 µmol m⁻² sec⁻¹), PR-1 expression was induced without pathogenic challenge and multiplication of Pseudomonas syringae pv. syringae was repressed. Yet, it is unlikely that Cat1 deficiency is mimicking the mode of action of salicylic acid in tobacco, because, concurrent with PR-1 induction, Cat1 deficiency at high light provoked severe leaf damage, characterized by white necrotic lesions. Taken together, these results do not support the model that catalase inactivation is the key route by which salicylic acid induces PR defense responses in healthy tissue. However, because catalase deficiency is potentially lethal to leaves, catalase inactivation by salicylic acid could be of importance in the establishment of hypersensitive responses.     

Microscale vertical profiles of n(2) fixation, photosynthesis, o(2), chlorophyll a, and light in a cyanobacterial assemblage

Profiles of N(2) fixation, O(2) production (gross photosynthesis), O(2) concentration, chlorophyll a concentration, and photon fluence rates were measured with 50-mum resolution in colonies of the heterocyst-forming cyanobacterium Nostoc parmelioides. Microelectrode measurements were made after 20 h of incubation under N(2) gas. Colonies were frozen, and 50-mum sections were prepared by using a freezing microtome and analyzed for N enrichment and chlorophyll a concentration. Colonies exhibited steep spatial gradients in rates of gross photosynthesis, O(2) concentration, and irradiance, with the highest values generally occurring at the surface. O(2) concentration, photosynthesis, and irradiance all showed positive correlations, but chlorophyll a concentrations varied independently of photosynthesis and irradiance. Forty-four percent of the variation in N incorporation was explained by gross photosynthesis (a positive correlation) when incorporation of N was expressed per unit of biomass (chlorophyll a).     

Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts

The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO₂- dependent O₂ evolution.

Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO₂ fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H₂O₂.

It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H₂O₂ under the best experimental conditions for CO₂ fixation.

     

Exogenous H(2)O(2) and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum

Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.     

Reversible o(2) inhibition of nitrogenase activity in attached soybean nodules

Various forms of stress result in decreased O₂ permeability or decreased capacity to consume O₂ in legume root nodules. These changes alter the nodule interior O₂ concentration (O_i). To determine the relationship between O_i and nitrogenase activity in attached soybean (Glycine max) nodules, we controlled O_i by varying external pO₂ while monitoring internal H₂ concentration (H_i) with microelectrodes. O_i was monitored by noninvasive leghemoglobin spectrophotometry (nodule oximetry). After each step-change in O_i, H_i approached a new steady state, with a time constant averaging 23 s. The rate of H₂ production by nitrogenase was calculated as the product of H_i, nodule surface area, and nodule H₂ permeability. H₂ permeability was estimated from O₂ permeability (measured by nodule oximetry) by assuming diffusion through air-filled pores; support for this assumption is presented. O_i was nearly optimal for nitrogenase activity (H₂ production) between 15 and 150 nm. A 1- to 2-min exposure to elevated external pO₂ (40-100 kPa) reduced H_i to zero, but nitrogenase activity recovered quickly under air, often in <20 min. This rapid recovery contrasts with previous reports of much slower recovery with longer exposures to elevated pO₂. The mechanism of nitrogenase inhibition may differ between brief and prolonged O₂ exposures.     

The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity

Tobacco plants (Nicotiana tabacum L.) transformed with an inverted cDNA encoding ribulose 5-phosphate kinase (phosphoribulokinase,PRK; EC 2.7.1.19) were employed to study the in vivo relationship between photosynthetic electron transport and the partitioning of electron transport products to major carbon metabolism sinks under conditions of elevated ATP concentrations and limited ribulose 1,5-bisphosphate (RuBP) regeneration. Simultaneous measurements of room temperature chlorophyll fluorescence and CO₂ gas exchange were conducted on intact leaves. Under ambient CO₂ concentrations and light intensities above those at which the plants were grown, transformants with only 5% of PRK activity showed down-regulation of PS II activity and electron transport in response to a decrease in net carbon assimilation when compared to wild-type. This was manifested as a decline in the efficiency of PS II electron transport (_{PS II}), an increase in dissipation of excess absorbed light in the antennae of PS II and a decline in: total linear electron transport (J₁), electron transport dedicated to carbon assimilation (J_A) and electron transport allocated to photorespiration (J_L). The transformants showed no alteration in the Rubisco specificity factor measured in vitro and calculated in vivo but had a relatively smaller ratio of RuBP oxygenation to carboxylation rates (v_o/v_c), due to a higher CO₂ concentration at the carboxylation site (C_c). The relationship between _{PS II} and _{CO 2}was similar in transformants and wild-type under photorespiratory conditions demonstrating no change in the intrinsic relationship between PS II function and carbon assimilation, however, a novel result of this study is that this similar relationship occurred at different values of quantum flux, J₁, J_A, J_L and v_o/v_c in the transformant. For both wild-type and transformants, an assessment was made of the possible presence of a third major sink for electron transport products, beside RuBP oxygenation and carboxylation, the data provided no evidence for such a sink.Abbreviations C_c CO₂ concentration at the site of carboxylation - C_i intercellular CO₂ concentration - g_m mesophyll conductance to CO₂ - J₁ total linear electron flow - J_A linear electron flow allocated to CO₂ assimilation - J_c linear electron flow supporting carbon reduction and oxidation cycles - J_L linear electron flow allocated to photorespiration (RuBP oxygenation and fixation of released photorespiratory CO₂) - PRK phosphoribulokinase - q_P, q_N coefficients for photochemical and non-photochemical quenching of fluorescence respectively - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase - S Rubisco specificity to CO₂/O₂ - v_c, v_o rates of RuBP carboxylation and RuBP oxygenation, respectively - _{CO 2} relative quantum yield of CO₂ assimilation - _C maximum _{CO 2} under non-photorespiratory conditions - _exc the efficiency of excitation capture by open PS II centres - _{PS II} relative quantum yield of PS II electron transport     

Isolation of a lipopolysaccharide (LPS)-resistant mutant, with defective LPS binding, of cultured macrophage-like cells.

Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.     

An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.