Hydroxypyruvate Reductase Activity Research Articles

Chromium(VI) Toxicity and Active Tolerance Mechanisms of Wheat Plant Treated with Plant Growth-Promoting Actinobacteria and Olive Solid Waste.

The present study aimed to assess the potential of plant growth-promoting Actinobacteria and olive solid waste (OSW) in ameliorating some biochemical and molecular parameters of wheat (Triticum aestivum) plants under the toxicity of high chromium levels in the soil. With this aim, a pot experiment was conducted, where the wheat plants were treated with a consortium of four Actinobacterium sp. (Bf treatment) and/or OSW (4% w/w) under two levels of nonstress and chromium stress [400 mg Cr(VI) per kg of soil] to estimate the photosynthetic traits, antioxidant protection machine, and detoxification activity. Both Bf and OSW treatments improved the levels of chlorophyll a (+47-98%), carotenoid (+324-566%), stomatal conductance (+17-18%), chlorophyll fluorescence (+12-28%), and photorespiratory metabolism (including +44-72% in glycolate oxidase activity, +6-72% in hydroxypyruvate reductase activity, and +5-44% in a glycine to serine ratio) in leaves of stressed plants as compared to those in the stressed control, which resulted in higher photosynthesis capacity (+18-40%) in chromium-stressed plants. These results were associated with an enhancement in the content of antioxidant metabolites (+10-117%), of direct reactive oxygen species-detoxifying enzymes (+49-94%), and of enzymatic (+40-261%) and nonenzymatic (+17-175%) components of the ascorbate-glutathione cycle in Bf- and OSW-treated plants under stress. Moreover, increments in the content of phytochelatins (+38-74%) and metallothioneins (+29-41%), as markers of detoxification activity, were recorded in the plants treated with Bf and OSW under chromium toxicity. In conclusion, this study revealed that the application of beneficial Actinobacteria and OSW as biofertilization/supplementation could represent a worthwhile consequence in improving dry matter production and enhancing plant tolerance and adaptability to chromium toxicity.

Galaxolide-contaminated soil and tolerance strategies in soybean plants using biofertilization and selenium nanoparticle supplementation.

The current study aimed to address the response of soybean (Glycine max) plants to biofertilization and selenium supplementation treatments under galaxolide contamination of soil. In this regard, a pot experiment was carried out where the soybean plants were treated with the plant growth-promoting Actinobacteria (Actinobacterium sp.) as a biofertilizer (PGPB treatment) and/or selenium nanoparticles (Se treatment; 25 mg L-1) under two non-polluted and galaxolide-polluted soils (250 mg galaxolide per kg of soil) to assess the modifications in some plant physiological and biochemical traits. Although higher accumulation of oxidative biomarkers, including hydrogen peroxide (+180%), malondialdehyde (+163%), and protein oxidation (+125%), indicating oxidative stress in galaxolide-contaminated plants, an apparent decline in their contents was observed in response to biofertilization/supplementation treatments in contaminated soil, especially. It was mainly related to the higher detoxification of ROS in PGPB- and Se-treated plants under galaxolide-induced oxidative stress, in which the direct ROS-scavenging enzymes (+44 -179%), enzymatic (+34 - 293%) and non-enzymatic (+35 - 98%) components of the ascorbate-glutathione pathway, and antioxidant molecules (+38 - 370%) were more activated than in control plants. In addition, a higher accumulation of detoxification activity markers, including phytochelatins (+32%) and metallothioneins (+79%), were found in the combined treatments (PGPB+Se) under galaxolide contamination. Moreover, combined treatment with PGPB and Se ameliorated the levels of chlorophyll a content (+58%), stomatal conductance (+57%), the maximum efficiency of photosystem II (PSII) (+36%), and photorespiratory metabolism (including +99% in glycolate oxidase and +54% in hydroxypyruvate reductase activity) in leaves under galaxolide contamination, which resulted in higher photosynthesis capacity (+36%) and biomass production (+74%) in galaxolide-stressed plants as compared to control group. In conclusion, the application of beneficial Actinobacteria and selenium nanoparticles as biofertilization/supplementation is expected to be useful for improving plant toleration and adaptation against galaxolide contamination.

Moderate modulation by S-nitrosoglutathione of photorespiratory enzymes in pea (Pisum sativum) leaves, compared to the strong effects of high light.

When plants are exposed to water stress, photosynthesis is downregulated due to enhanced reactive oxygen species (ROS) and nitric oxide (NO). In contrast, photorespiratory metabolism protected photosynthesis and sustained yield. Modulation of photorespiration by ROS was established, but the effect of NO on photorespiratory metabolism was unclear. We, therefore, examined the impact of externally added NO by using S-nitrosoglutathione (GSNO), a natural NO donor, in leaf discs of pea (Pisum sativum) under dark or light: moderate or high light (HL). Maximum NO accumulation with GSNO was under high light. The presence of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, prevented the increase in NO, confirming the release of NO in leaves. The increase in S-nitrosothiols and tyrosine-nitrated proteins on exposure to GSNO confirmed the nitrosative stress in leaves. However, the changes by GSNO in the activities and transcripts of five photorespiratory enzymes: glycolate oxidase, hydroxypyruvate reductase, catalase, glycerate kinase, and phosphoglycolate phosphatase activities were marginal. The changes in photorespiratory enzymes caused by GSNO were much less than those with HL. Since GSNO caused only mild oxidative stress, we felt that the key modulator of photorespiration might be ROS, but not NO.

Potassium (K+) Starvation-Induced Oxidative Stress Triggers a General Boost of Antioxidant and NADPH-Generating Systems in the Halophyte Cakile maritima.

Potassium (K+) is an essential macro-element for plant growth and development given its implication in major processes such as photosynthesis, osmoregulation, protein synthesis, and enzyme function. Using 30-day-old Cakile maritima plants as halophyte model grown under K+ deprivation for 15 days, it was analyzed at the biochemical level to determine the metabolism of reactive oxygen species (ROS), key photorespiratory enzymes, and the main NADPH-generating systems. K+ starvation-induced oxidative stress was noticed by high malondialdehyde (MDA) content associated with an increase of superoxide radical (O2•−) in leaves from K+-deficient plants. K+ shortage led to an overall increase in the activity of hydroxypyruvate reductase (HPR) and glycolate oxidase (GOX), as well as of antioxidant enzymes catalase (CAT), those of the ascorbate-glutathione cycle, peroxidase (POX), and superoxide dismutase (SOD), and the main enzymes involved in the NADPH generation in both leaves and roots. Especially remarkable was the induction of up to seven CuZn-SOD isozymes in leaves due to K+ deficiency. As a whole, data show that the K+ starvation has associated oxidative stress that boosts a biochemical response leading to a general increase of the antioxidant and NADPH-generating systems that allow the survival of the halophyte Cakile maritima.

Identification and Characterization of Genes Encoding the Hydroxypyruvate Reductases in Chlamydomonas Reveal Their Distinct Roles in Photorespiration.

Photorespiration plays an important role in maintaining normal physiological metabolism in higher plants and other oxygenic organisms, such as algae. The unicellular eukaryotic organism Chlamydomonas is reported to have a photorespiration system different from that in higher plants, and only two out of nine genes encoding photorespiratory enzymes have been experimentally characterized. Hydroxypyruvate reductase (HPR), which is responsible for the conversion of hydroxypyruvate into glycerate, is poorly understood and not yet explored in Chlamydomonas. To identify the candidate genes encoding hydroxypyruvate reductases in Chlamydomonas (CrHPR) and uncover their elusive functions, we performed sequence comparison, enzyme activity measurement, subcellular localization, and analysis of knockout/knockdown strains. Together, we identify five proteins to be good candidates for CrHPRs, all of which are detected with the activity of hydroxypyruvate reductase. CrHPR1, a nicotinamide adenine dinucleotide (NADH)-dependent enzyme in mitochondria, may function as the major component of photorespiration. Its deletion causes severe photorespiratory defects. CrHPR2 takes part in the cytosolic bypass of photorespiration as the compensatory pathway of CrHPR1 for the reduction of hydroxypyruvate. CrHPR4, with NADH as the cofactor, may participate in photorespiration by acting as the chloroplastidial glyoxylate reductase in glycolate-quinone oxidoreductase system. Therefore, the results reveal that CrHPRs are far more complex than previously recognized and provide a greatly expanded knowledge base for studies to understand how CrHPRs perform their functions in photorespiration. These will facilitate both modification of photorespiration and genetic engineering for crop improvement by synthetic biology.

Phosphomimetic T335D Mutation of Hydroxypyruvate Reductase 1 Modifies Cofactor Specificity and Impacts Arabidopsis Growth in Air.

Photorespiration is an essential process in oxygenic photosynthetic organisms triggered by the oxygenase activity of Rubisco. In peroxisomes, photorespiratory HYDROXYPYRUVATE REDUCTASE1 (HPR1) catalyzes the conversion of hydroxypyruvate to glycerate together with the oxidation of a pyridine nucleotide cofactor. HPR1 regulation remains poorly understood; however, HPR1 phosphorylation at T335 has been reported. By comparing the kinetic properties of phosphomimetic (T335D), nonphosphorylatable (T335A), and wild-type recombinant Arabidopsis (Arabidopsis thaliana) HPR1, it was found that HPR1-T335D exhibits reduced NADH-dependent hydroxypyruvate reductase activity while showing improved NADPH-dependent activity. Complementation of the Arabidopsis hpr1-1 mutant by either wild-type HPR1 or HPR1-T335A fully complemented the photorespiratory growth phenotype of hpr1-1 in ambient air, whereas HPR1-T335D-containing hpr1-1 plants remained smaller and had lower photosynthetic CO2 assimilation rates. Metabolite analyses indicated that these phenotypes were associated with subtle perturbations in the photorespiratory cycle of HPR1-T335D-complemented hpr1-1 rosettes compared to all other HPR1-containing lines. Therefore, T335 phosphorylation may play a role in the regulation of HPR1 activity in planta, although it was not required for growth under ambient air controlled conditions. Furthermore, improved NADP-dependent HPR1 activities in peroxisomes could not compensate for the reduced NADH-dependent HPR1 activity.

Characterization of Arabidopsis thaliana Hydroxyphenylpyruvate Reductases in the Tyrosine Conversion Pathway.

Tyrosine serves as a precursor to several types of plant natural products of medicinal or nutritional interests. Hydroxyphenylpyruvate reductase (HPPR), which catalyzes the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), has been shown to be the key enzyme in the biosynthesis of rosmarinic acid (RA) from tyrosine and, so far, HPPR activity has been reported only from the RA-accumulating plants. Here, we show that HPPR homologs are widely distributed in land plants. In Arabidopsis thaliana, which does not accumulate RA at detectable level, two homologs (HPPR2 and HPPR3) are functional in reducing pHPP. Phylogenetic analysis placed HPPR2 and HPPR3 in two separate groups within the HPPR clade, and HPPR2 and HPPR3 are distinct from HPR1, a peroxisomal hydroxypyruvate reductase (HPR). In vitro characterization of the recombinant proteins revealed that HPPR2 has both HPR and HPPR activities, whereas HPPR3 has a strong preference for pHPP, and both enzymes are localized in the cytosol. Arabidopsis mutants defective in either HPPR2 or HPPR3 contained lower amounts of pHPL and were impaired in conversion of tyrosine to pHPL. Furthermore, a loss-of-function mutation in tyrosine aminotransferase (TAT) also reduced the pHPL accumulation in plants. Our data demonstrate that in Arabidopsis HPPR2 and HPPR3, together with TAT1, constitute to a probably conserved biosynthetic pathway from tyrosine to pHPL, from which some specialized metabolites, such as RA, can be generated in specific groups of plants. Our finding may have broad implications for the origins of tyrosine-derived specialized metabolites in general.

Paracoccus simplex sp. nov., a New Methylamine-Utilizing Facultative Methylotroph

A facultative methylotroph, strain F5Т, which uses methylamine and a broad spectrum of polycarbon substrates as carbon and energy sources, was isolated from silt of a freshwater lake in the southern Moscow region. The cells were gram-negative, coccoid, non-spore-forming, nonmotile, colorless, reproducing by binary fission and possessing a capsule. The organism was mesophilic, neutrophilic, not halophilic, oxidase- and catalase-positive, and capable of nitrate reduction to nitrite. Methylamine was oxidized by amine dehydrogenase and via the icl- serine pathway of С1 metabolism, as was indicated by activities of hydroxypyruvate reductase and serine-glyoxylate aminotransferase and by the absence of hexulosephosphate synthase and ribulose bisphosphate carboxylase. Predominant fatty acids were C18:1ω7c (72.3%) and C16:0 (11.6%). Phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine were the dominant phospholipids. The G + C DNA content was 65.8 mol % (Tm). Q10 was the dominant ubiquinone. Strain F5T exhibited high similarity of the 16S rRNA gene sequences with Paracoccus strains: P. aminovorans JCM7685T = VKM B-2140Т (98.0%), P. huijuniae FLN-7Т (97.9%), and P. limosus NB88T (97.5%). However, the level of DNA-DNA relatedness between the strains F5Т and P. aminovoransТ was only 21 ± 3%. Based on the data obtained, strain F5Т was identified as a new Paracoccus species with proposed name Paracoccus simplex sp. nov. (= VKM B-3226T = CCUG 71989T).

Proteomic analysis of soybean seedling leaf under waterlogging stress in a time-dependent manner

Leaf is sensitive to environmental changes and exhibits specific responses to abiotic stress. To identify the response mechanism in soybean leaf under waterlogging stress, a gel-free/label-free proteomic technique combined with polyethylene glycol fractionation was used. Attenuated photosynthesis by waterlogging stress in the leaf of soybean seedlings was indicated from proteomic results. Defensive mechanisms such as reactive oxygen species (ROS) scavenging was also recognized. Cluster analysis revealed that proteins that exhibit characteristic dynamics in response to waterlogging were mainly related to photosynthesis. Among the identified photorespiration-related proteins, the protein abundance and enzyme activity of hydroxypyruvate reductase were transiently increased in control plants, but were clearly decreased in response to waterlogging stress. These results suggest that waterlogging directly impairs photosynthesis and photorespiration. Furthermore, hydroxypyruvate reductase may be a critical enzyme controlling the rate of photorespiration.

Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration

BackgroundProtein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. MethodsWe studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches. ResultsProteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. ConclusionThese findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. General significanceThis is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H2O2 contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H2O2 contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.

Metabolism of methanol and glucose in Angulomicrobium tetraedrale

Enzymes were assayed by routine methods [3‐5] in the extracts of cells grown on methanol or glucose. Autotrophic growth was studied in an H 2 : CO 2 : O 2 (75 : 15 : 10) atmosphere. The vitamin requirements of the strain was determined by growing it on methanol in a medium containing various vitamins (Sigma) at concentrations of 20 µ g/l instead of yeast extract. As can be seen from the table, A. tetraedrale possesses a complete set of enzymes necessary for the oxidation of methanol to CO 2 (the dehydrogenases of methanol, formaldehyde, and formate). The high activity of formaldehyde and formate dehydrogenases in the presence of NAD + indicates that the first step of oxidation of e 1 -substrates produces reduced pyridine nucleotides. The absence of serine‐glyoxylate aminotransferase and 3-hexulose-6-phosphate synthase suggests that the serine and ribulose monophosphate pathways are not involved in e 1 -metabolism. The higher activity of hydroxypyruvate reductase in glucose-grown cells may indicate that this enzyme is not involved in methanol metabolism. The high activity of phosphoribulokinase and ribulose 1,5-bisphosphate carboxylase shows that methanol is assimilated at the level of CO 2 via the ribulose bisphosphate (RuBP) cycle. Transketolase and transaldolase are shown to be involved in the conversion of glyceraldehyde-3-phosphate to xylulose-5phosphate, resulting in the regeneration of the primary CO 2 acceptor ribulose-1,5-bisphosphate. The cells grown on methanol or glucose were found to contain glycerate kinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphate aldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase, and 6-phosphofructokinase (ATP). Therefore, carbohydrates in this bacterium are metabolized via the glycolytic, pentose phosphate, and the Entner‐ Doudoroff pathways. The cells grown on glucose contained active glucokinase (ATP), pyruvate dehydrogenase, citrate synthase, 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase (NADP + ), and malate dehydrogenase. The decreased activity of some enzymes of the tricarboxylic acid (TCA) cycle in the methanolgrown cells suggests that the role of this cycle in methylotrophs is predominantly biosynthetic. The activities of the key enzymes of the glyoxylate cycle (isocitrate lyase and malate synthase) in the methanol-grown cells were also lower than in the glucose-grown cells. was assimilated with the involvement of glycine dehydrogenase (NADPH), glutamate dehydrogenase (NAD(P)H), and the glutamate cycle enzymes glutamate synthase and glutamine synthetase. The anaplerotic fixation of CO 2 required pyruvate carboxylase, PEP carboxylase, and PEP carboxykinase. The activity of PEP carboxylase was higher during the methylotrophic growth of the bacterium. Thus, A. tetraedrale assimilates methanol by oxidizing it to CO 2 via formaldehyde and formate, followed by the fixation of the CO 2 in the RuBP pathway. Hence, this bacterium may be considered a facultative autotroph with elements of methylotrophic metabolism. This suggestion is confirmed by the ability of A. tetraedrale to grow in an atmosphere of CO 2 + H 2 + O 2 . The growth of this bacterium was found to be stimulated by biotin. The key enzymes of the autotrophic fixation of CO 2 (phosphoribulokinase and ribulose bisphosphate carboxylase) were induced during bacterial growth on methanol.

Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses.

Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. The NADP-malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP-malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses.

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and 0-12, growing in the temperature range from 30 to 61 degrees C with an optimum at 55 degrees C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes, membrane-bound methane monooxygenase, and phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulose phosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55 degrees C that in cells grown at 37 degrees C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH4+ assimilation occurred through reductive amination of alpha-ketoglutarate and via the glutamate cycle. The relationship between the physiological-biochemical peculiarities of the isolates and their thermophilic nature is discussed.

Changes in some enzymes of microbodies and plastid development in excised radish cotyledons: Effect of narciclasine

Narciclasine (NCS), isolated from mucilage of Narcissus bulb, showed inhibitory effects on growth and plastid development of excised radish cotyledons. NCS (0.1 mumol/L) started to show inhibitory effects on isocitrate lyase and hydroxypyruvate reductase activities after 24 h incubation in light. When NCS concentration was increased to 10 mumol/L, the activities of both enzymes are completely inhibited. From ultrastructural studies, NCS markedly prevented the degradation of protein bodies and lipid bodies, as well as chloroplast formation of excised radish cotyledons. There was only little degradation of protein and lipid bodies, and almost no chloroplast formation in the excised radish cotyledon treated with 1 mumol/L NCS. Therefore, our results provide clear evidence that NCS inhibited the transition of glyoxysomes and peroxisomes, and chloroplast development.

Growth of mycobacteria on carbon monoxide and methanol.

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.

Utilization of methane and carbon dioxide by symbiotrophic bacteria in gills of Mytilidae (Bathymodiolus) from the Rainbow and Logachev hydrothermal fields on the Mid-Atlantic Ridge

Bivalve mollusks Bathymodiolus asoricus and Bathymodiolus puteoserpentis collected from the Rainbow and Logachev hydrothermal fields during dives of Mir 1 and Mir 2 deep-sea manned submersibles were studied. Rates of methane oxidation and carbon dioxide assimilation in mussel gill tissue were determined by radiolabel analysis. During oxidation of 14Ch4, radiocarbon was detected in significant quantities not only in carbon dioxide but also in dissolved organic matter, most notably 14C-formate and 14C-acetate, occurring in a 2:1 ratio. Activities of hexulose-phosphate synthase, phosphoribulokinase, and ribulose 1,5-bisphosphate carboxylase were shown in the soluble fraction of gill tissue cells. At the same time, no activity of hydroxypyruvate reductase--the key enzyme of the serine pathway of C1-assimilation--was detected. The results of PCR amplification using genetic probes for membrane-bound methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF) attest to the presence of the genes of these enzymes in the total DNA extracted from gill samples. However, no appropriate PCR responses were obtained with the mmoX primer system, which is a marker for soluble methane monooxygenase. All samples studied showed amplification with primers for the genera Methylobacter and Methylosphaera. At the same time, no genes specific to the genera Methylomonas, Methylococcus, Methylomicrobium, or Methylosinus and Methylocystis were detected. Electron microscopic examinations revealed the presence of two groups of endosymbiotic bacteria in the mussel gill tissue. The first group was represented by large cells possessing a complex system of cytoplasmic membranes, typical of methanotrophs of morphotype I. The other type of endosymbionts, having much smaller cells and lacking intracellular membrane structures, is likely to be constituted by sulfur bacteria.

Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase

Barley ( Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)H-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate ( K i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-1 in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon.

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2).

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.