Azide-dependent nitric oxide emission from the water fern Azolla pinnata

Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (N O ₂ ^? ) the amino acid L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN₃). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.     

Nitrate reductase from <Emphasis Type="Italic">Triticum aestivum</Emphasis> leaves: Regulation of activity and possible role in production of nitric oxide

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation. In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR. The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule. Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue.     

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ

At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.     

Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

Increase in gastric pH reduces hypotensive effect of oral sodium nitrite in rats

The new pathway nitrate-nitrite-nitric oxide (NO) has emerged as a physiological alternative to the classical enzymatic pathway for NO formation from l-arginine. Nitrate is converted to nitrite by commensal bacteria in the oral cavity and the nitrite formed is then swallowed and reduced to NO under the acidic conditions of the stomach. In this study, we tested the hypothesis that increases in gastric pH caused by omeprazole could decrease the hypotensive effect of oral sodium nitrite. We assessed the effects of omeprazole treatment on the acute hypotensive effects produced by sodium nitrite in normotensive and L-NAME-hypertensive free-moving rats. In addition, we assessed the changes in gastric pH and plasma levels of nitrite, NO(x) (nitrate+nitrite), and S-nitrosothiols caused by treatments. We found that the increases in gastric pH induced by omeprazole significantly reduced the hypotensive effects of sodium nitrite in both normotensive and L-NAME-hypertensive rats. This effect of omeprazole was associated with no significant differences in plasma nitrite, NO(x), or S-nitrosothiol levels. Our results suggest that part of the hypotensive effects of oral sodium nitrite may be due to its conversion to NO in the acidified environment of the stomach. The increase in gastric pH induced by treatment with omeprazole blunts part of the beneficial cardiovascular effects of dietary nitrate and nitrite.     

Protective effect of supplemental low intensity white light on ultraviolet-B exposure-induced impairment in cyanobacterium <Emphasis Type="Italic">Spirulina platensis</Emphasis>: formation of air vacuoles as a possible protective measure

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.     

Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria.

Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.     

Modulation of nitrate reductase activity by photosynthetic electron transport chain and nitric oxide balance in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta)

Nitrate reductase (NR), a key enzyme in nitrogen metabolism, has been implicated in the production of nitric oxide (NO) in plants. The effect of photosynthetic electron transport chain inhibitors and NO scavengers or donors on NR activity of Gracilaria chilensis was studied under experimental laboratory conditions. Effective quantum yield (Φ _PSII) and NR activity were significantly diminished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, two photosynthetic electron flux inhibitors of photosystem (PS) II and PSI, respectively, but not by diphenyleneiodonium, a NADPH oxidase inhibitor, indicating a direct dependence of NR activity on the PSII and PSI electron flux. Nitrate reductase activity was sensitive to a decrease or increase of NO levels when NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO donor (sodium nitroprusside) were added. Moreover, the addition of 8Br-cGMP, a secondary signal molecule, stimulated NR activity. These results evidence a modulation of the photosynthetic electron transport chain and NO balance on G. chilensis NR activity. This association could be linked to the crucial tight modulation of nitrogen assimilation and carbon metabolism to guarantee nitrite incorporation into organic compounds and to avoid toxicity by nitrite, reactive oxygen species, or nitric oxide in the cells. Nitric oxide showed to be an important signaling molecule regulating NR activity and cGMP could participate as secondary messenger on this regulation by phosphorylation and desphosphorylation processes.     

Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.)

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.     

Oral nitrite ameliorates dextran sulfate sodium-induced acute experimental colitis in mice

Inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the intestinal tract with excessive production of cytokines, adhesion molecules, and reactive oxygen species. Although nitric oxide (NO) is reported to be involved in the onset and progression of IBDs, it remains controversial as to whether NO is toxic or protective in experimental colitis. We investigated the effects of oral nitrite as a NO donor on dextran sulfate sodium (DSS)-induced acute colitis in mice. Mice were fed DSS in their drinking water with or without nitrite for up to 7 days. The severity of colitis was assessed by disease activity index (DAI) observed over the experimental period, as well as by the other parameters, including colon lengths, hematocrit levels, and histological scores at day 7. DSS treatment induced severe colitis by day 7 with exacerbation in DAI and histological scores. We first observed a significant decrease in colonic nitrite levels and increase in colonic TNF-α expression at day 3 after DSS treatment, followed by increased colonic myeloperoxidase (MPO) activity and increased colonic expressions of both inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) at day 7. Oral nitrite supplementation to colitis mice reversed colonic nitrite levels and TNF-α expression to that of normal control mice at day 3, resulting in the reduction of MPO activity as well as iNOS and HO-1 expressions in colonic tissues with clinical and histological improvements at day 7. These results suggest that oral nitrite inhibits inflammatory process of DSS-induced experimental colitis by supplying nitrite-derived NO instead of impaired colonic NOS activity.     

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

Background and Aims

Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum).

Methods

NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings.

Key Results

Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N₂ flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate.

Conclusions

The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein.Key words: Nitric oxide, nitrate, nitrate reductase, post-translational regulation, tomato, Solanum lycocarpum     

Gene Expression of Lupeol Synthase and Biosynthesis of Nitric Oxide in Cell Suspension Cultures of Betula platyphylla in Response to a Phomopsis Elicitor

The current work aimed to characterize the generation of nitric oxide (NO) and gene expression of lupeol synthase (LUS) in Betula platyphylla cells exposed to a Phomopsis elicitor. The effects of nitrate reductase (NR) and NO synthase (NOS), the two key enzymes responsible for endogenous NO biosynthesis in plants, were also investigated. NO production in B. platyphylla cell cultures exhibited a biphasic pattern, reaching the Wrst plateau within 1.0–10 h of exposure to the Phomopsis elicitor. LUS gene expression was found to increase abruptly 10 h after Phomopsis induction, reaching its highest level (18.08) at 24 h. The maximum levels of NOS and NR activities in elicitor-treated cells were found to be 1.7-fold and 6.9-fold those of untreated cells, respectively. Pharmacological experiments showed that Phomopsis elicitor-induced NO production and LUS gene expression level were significantly suppressed by the NOS inhibitor N^G-nitro-l-Arg methyl ester (l-NAME), the NR inhibitor sodium azide (NaN₃), and the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). NaNO₂ and l-arginine (the substrates that produce NO via NR and NOS) and NO donor sodium nitroprusside (SNP) were found to increase both NO production and LUS gene expression. These results suggest that the increase in LUS gene expression due to fungal elicitor-induced NO may involve the NR and NOS biosynthetic pathways.     

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.     

Nitrite-dependent nitric oxide production pathway: implications for involvement of active nitrogen species in photoinhibition in vivo

Air pollution studies have shown that nitric oxide (NO), a gaseous free radical, is a potent photosynthetic inhibitor that reduces CO2 uptake activity in leaves. It is now recognized that NO is not only an air pollutant but also an endogenously produced metabolite, which may play a role in regulating plant cell functions. Although many studies have suggested the presence of mammalian-type NO synthase (NOS) in plants, the source of NO is still not clear. There has been a number of studies indicating that plant cells possess a nitrite-dependent NO production pathway which can be distinguished from the NOS-mediated reaction. Nitrate reductase (NR) has been recently found to be capable of producing NO through one-electron reduction of nitrite using NAD(P)H as an electron donor. This review focuses on current understanding of the mechanism for the nitrite-dependent NO production in plants. Impacts of NO produced by NR on photosynthesis are discussed in association with photo-oxidative stress in leaves.     

Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana

Wild type (WT), and nitrate reductase (NR)- and nitrite-reductase (NiR)-deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate-grown WT cells was very low in air, increased slightly after addition of nitrite (200 microM), but strongly under anoxia. Importantly, even completely NR-free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR-free cells. In contrast, NiR-deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.     

Influences of some internal and external conditions on the induction of nitrate reductase in rice seedlings

Induction of nitrate reductase (NR) in 7-day-old rice seedlingswas depressed when endosperms were removed. NR activity in theseedlings from which endosperms were removed (deendospermedseedlings), reached a maximum 6 hr after supplying NaNO₃ andthen gradually decreased. That in intact seedlings continuedto increase for 12 hr, and then decreased fairly rapidly. Sucrose(30 mM concentration) supplied exogenously to deendospermedseedlings raised NR activity to the level of the intact seedlings.Macronutrients added exogenously did not show such an effect. NR activity in deendospermed seedlings placed in the dark wasextremely low. However, in the presence of exogenous sucrose,the activity was raised to the same level as that in the lightin the absence of exogenous sucrose. This suggests that sucrosesubstitutes for light in the induction of NR in deendospermedseedlings. Protein inhibitors suppressed NR induction when theplants were fed continuously with nitrate solution containingthe inhibitor. In cases where the plant roots were immersedin inhibitor solutions for 2 hr before transfer to nitrate solution,only chloramphenicol promoted and the others inhibited NR induction.NR induction was also suppressed by respiratory inhibitors,of which sodium azide was very potent. (Received August 25, 1976; )     

Nitric oxide homeostasis in Salmonella typhimurium: roles of respiratory nitrate reductase and flavohemoglobin

Nitric oxide (NO) is generated in biological systems primarily via the activity of NO synthases and nitrate and nitrite reductases. Here we show that Salmonella enterica serovar Typhimurium (S. typhimurium) grown anaerobically with nitrate is capable of generating polarographically detectable NO after nitrite (NO(2)(-)) addition. NO accumulation is sensitive to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Neither an fnr mutant nor an fnr hmp double mutant produces NO, indicating the involvement in NO evolution from NO(2)(-) of protein(s) positively regulated by FNR. Contrary to previous findings in Escherichia coli, we demonstrate that neither the periplasmic nitrite reductase (NrfA) nor the cytoplasmic nitrite reductase (NirB) is involved in NO production in S. typhimurium. However, mutant cells lacking the membrane-bound nitrate reductase, NarGHI, and membranes derived from these cells are unable to produce NO, demonstrating that, in wild-type S. typhimurium, this enzyme is responsible for NO production. Membrane terminal oxidases cannot account for the NO levels measured. The nitrate reductase inhibitor, azide, abrogates NO evolution by Salmonella, and production of NO occurs only in the absence from the assays of nitrate; both features reveal a marked similarity between the NO-generating activities of this bacterium and plants. Unlike the situation in E. coli, an S. typhimurium hmp mutant produces NO both aerobically and anaerobically. Under aerobic conditions, when a functional flavohemoglobin is present, no NO is detectable. We propose a homeostatic mechanism in S. typhimurium, in which NO produced from NO(2)(-) by nitrate reductase derepresses Hmp expression (via FNR and NsrR) and NorV expression (via NorR) and thus limits NO toxicity.