Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Oxygen stress and adaptation of a semi-aquatic plant: rice ( Oryza sativa)

One of the major abiotic stresses that affects plant growth and development is anoxia or hypoxia. Rice is a semi-aquatic plant bestowed with the capability of overcoming oxygen limitation for a considerable period of time. For instance, it can withstand submergence stress either by inherent metabolic adaptations (resistant type), or by keeping its leaves above the water surface by continuously elongating the stem (avoiding type). In the former case, an interplay of several metabolic pathways engaged in anaerobic fermentation keeps the submerged plant alive for a certain period of time. In the latter type, also known as deepwater rice, continuous stem elongation brought about by a series of reactions in planta enables the shoot to remain above the water surface and thus maintain respiration and photosynthesis. However, the earliest event, i.e., sensing the oxygen level that brings about all the changes, has not been clearly understood. This paper intends to evaluate the metabolic adaptations of rice plants to oxygen constraints. Electronic Publication     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Plasmid maintenance and gene expression of a recombinant culture under aerobic and anaerobic conditions

Summary The stability and gene expression of a batch culture ofEscherichia coli, strain C600, carrying the plasmid pKN401 under both aerobic and anaerobic conditions were studied. It was observed that the plasmid was stable under both conditions and during a step change in the environment in which oxygen and nitrogen were supplied alternately. In addition, the -lactamase specific activity was sensitive to the availability of oxygen, with higher activities and a strong dependence on optical density observed under anaerobic conditions.     

Properties of nitrate reductase from Fusarium oxysporum 11dn1 fungi grown under aerobic and anaerobic conditions

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.     

Role of two 2-oxoglutarate:ferredoxin oxidoreductases in Hydrogenobacter thermophilus under aerobic and anaerobic conditions

Hydrogenobacter thermophilus TK-6 is a thermophilic, hydrogen-oxidizing bacterium that fixes carbon dioxide as a sole carbon source via the reductive tricarboxylic acid cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes in the pathway. Strain TK-6 has at least two isozymes of OGOR, namely For and Kor. These OGORs showed different expression patterns under aerobic conditions than under anaerobic conditions. In this work, we developed a homologous recombination method for Hydrogenobacter, and constructed a For mutant and a Kor mutant. Observation of phenotypes of the mutant strains showed that Kor was essential for anaerobic growth and that For activity supported robust aerobic growth of the organism.     

Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions

The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.     

Swarming characteristics of Proteus mirabilis under anaerobic and aerobic conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.     

Effects of abscisic acid on ozone tolerance of rice (Oryza sativa L.) seedlings

Ozone is one of the major gaseous pollutants detrimental to crop growthand metabolism. The objective of this research was to study how ABA amelioratesthe effects of ozone on rice seedlings. Seedlings of two rice cultivars withdifferent sensitivities to ozone (Tainung 67, tolerant; and Taichung Native 1,sensitive) were treated with 400 ppb of ozone or ABA and 400ppb of ozone to determine their effect on growth, stomatalmovement, chlorophyll characteristics, and the activity of antioxidant enzymes.Activities of the enzymes SOD, APOD, GR and POD were significantly higher inthesensitive cultivar, TN 1, than in the tolerant cultivar, TNG 67. Seedlings ofthe sensitive cultivar pretreated with ABA (10 M) weresignificantly more tolerant of ozone than control seedlings. Pretreatment withABA effectively reduced stomatal conductance and the degree of injury. Abscisicacid also increased ascorbate peroxidase and glutathione reductase activity.Ozone increased peroxidase activity in sensitive seedlings, but ABA decreasedperoxidase activity. The sensitive cultivar had a higher density of stomata onits leaves than the tolerant cultivar. The results suggest that ABA inducedtolerance to ozone may be more associated with its effects on stomatal movementthan on the modulation of antioxidant enzyme activity.     

Genome-wide survey of the RIP domain family in Oryza sativa and their expression profiles under various abiotic and biotic stresses

Ribosome-inactivating proteins (RIPs) are N-glycosidases that inhibit protein synthesis by depurinating rRNA. Despite their identification more than 25 years ago, little is known about their biological functions. Here, we report a genome-wide identification of the RIP family in rice based on the complete genome sequence analysis. Our data show that rice genome encodes at least 31 members of this family and they all belong to type 1 RIP genes. This family might have evolved in parallel to species evolution and genome-wide duplications represent the major mechanism for this family expansion. Subsequently, we analyzed their expression under biotic (bacteria and fungus infection), abiotic (cold, drought and salinity) and the phytohormone ABA treatment. These data showed that some members of this family were expressed in various tissues with differentiated expression abundances whereas several members showed no expression under normal growth conditions or various environmental stresses. On the other hand, the expression of many RIP members was regulated by various abiotic and biotic stresses. All these data suggested that specific members of the RIP family in rice might play important roles in biotic and abiotic stress-related biological processes and function as a regulator of various environmental cues and hormone signaling. They may be potentially useful in improving plant tolerance to various abiotic and biotic stresses by over-expressing or suppressing these genes.     

Effect of hypoxia on the antioxidative enzymes in aerobically grown rice (Oryza sativa) seedlings

The development of the antioxidative system is repressed in rice ( Oryza sativa L. cv. Yamabiko) seedlings germinated under water, but the system is synthesized within 24 h after exposure of the seedlings to air. In order to see whether this regulation of the antioxidative system is reversible, the changes were investigated after submergence of aerobically grown seedlings under water. Low survivability, no morphological change and a slight increase in activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) were observed 24 h after the submergence. Activities of some antioxidative enzymes were decreased, but they were still higher than those in seedlings germinated under water. Superoxide dismutase (SOD; EC 1.15.1.1) in mitochondria, in addition to cytosolic forms of SOD, showed no significant changes, whereas plastidic SOD decreased drastically, which reflected the changes in the development of the corresponding organelles. Thus, the regulation of the antioxidative system was, in part, reversible. In contrast, catalase (EC 1.11.1.6) activity alone was increased by the shift from air to submergence.     

Reduction of soluble and insoluble iron forms by membrane fractions of Shewanella oneidensis grown under aerobic and anaerobic conditions

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of Shewanella oneidensis MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O(2), fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe(3+), ferrozine-Fe(3+), and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe(3+) was greater than the reduction of citrate-Fe(3+). With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe(3+) as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.     

Divergent roles of RpoS in Escherichia coli under aerobic and anaerobic conditions

Escherichia coli exhibited different levels of rpoS expression and general stress resistance under aerobiosis and anaerobiosis. Expression measured using reporter gene fusions and protein levels was lower under anaerobic conditions. Consistent with earlier findings, rpoS mutants were selected in aerobic nutrient-limited cultures but rpoS mutants were not enriched under anaerobiosis. This result suggested that, despite its decreased level, RpoS had a function under anaerobic conditions not essential under aerobiosis. Competition experiments between rpoS(+) and rpoS bacteria confirmed the advantage conferred by RpoS under anaerobiosis. In contrast, stress resistance assays suggested RpoS made a greater contribution to general stress resistance under aerobiosis than anaerobiosis. These results indicate a significant, but different role of RpoS in aerobic and anaerobic environments.