Inhibition of electron transfer through the cytochrome b-c 1 complex by nitric oxide in a photodenitrifier,Rhodopseudomonas sphaeroides forma sp. denitrificans

The effects of nitric oxide (NO) on electron transfer were studied with a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans. NO inhibited the oxidation of cytochrome c induced by continuous illumination in intact cells. NO inhibited the re-reduction of cytochrome c, the slow phase of the carotenoid bandshift, and the oxidation of cytochrome b after a flash illumination, suggesting that NO inhibited the photosynthetic cyclic electron transfer through the cytochrome b-c ₁ region. NO also inhibited the nitrite (NO ₂ ^- ) and NO reductions with succinate as the electron donor in intact cells, but did not inhibit the NO ₂ ^- and NO reductions in chromatophore membranes with ascorbate and phenazine methosulfate as the electron donors. NO reversibly inhibited the ubiquinol: cytochrome c oxidoreductase of the membranes, suggesting that NO inhibited the electron transfer through the cytochrome b-c ₁ region and that the cytochrome b-c ₁ complex also was involved in the electron transport in both NO ₂ ^- and NO reductions. The catalytic site of NO reduction was distinct from the inhibitory site of NO.Abbreviations UHDBT 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole - UHNQ 3-undecyl-2-hydroxy-1,4-naphthoquinone - MOPS 3-(N-morpholino)propane-sulfonic acid - PMS phenazine methosulfate - DCIP 2,6-dichlorophenol indophenol - DDC diethyl-dithiocarbamate     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Nitric oxide is induced by wounding and influences jasmonic acid signaling in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Nitric oxide (NO) has been associated with plant defense responses during microbial attack, and with induction and/or regulation of programmed cell death. Here, we addressed whether NO participates in wound responses in Arabidopsis thaliana (L.) Heynh.. Real-time imaging by confocal laser-scanning microscopy in conjunction with the NO-selective fluorescence indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) uncovered a strong NO burst after wounding or after treatment with JA. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive responses. Furthermore, we were able to detect NO in plants (here induced by wounding) by means of electron paramagnetic resonance measurements using diethyldithiocarbamate as a spin trap. When plants were treated with NO, Northern analyses revealed that NO strongly induces key enzymes of jasmonic acid (JA) biosynthesis such as allene oxide synthase (AOS) and lipoxygenase (LOX2). On the other hand, wound-induced AOS gene expression was independent of NO. Furthermore, JA-responsive genes such as defensin (PDF1.2) were not induced, and NO induction of JA-biosynthesis enzymes did not result in elevated levels of JA. However, treatment with NO resulted in accumulation of salicylic acid (SA). In transgenic NahG plants (impaired in SA accumulation and/or signaling), NO did induce JA production and expression of JA-responsive genes. Altogether, the presented data demonstrate that wounding in Arabidopsis induces a fast accumulation of NO, and that NO may be involved in JA-associated defense responses and adjustments.Abbreviations AOS Allene oxide synthase - cPTIO Carboxy-2-phenyl-4,4,5,5-tetramethylimidazolinone-3-oxide-1-oxyl - DAF-2 DA 4,5-Diaminofluorescein diacetate - DETC Diethyldithiocarbamate - EPR Electron paramagnetic resonance - iNOS Inducible nitric oxide synthase - JA Jasmonic acid - JIP Jasmonic acid-induced protein - LOX2 Lipoxygenase 2 - NO Nitric oxide - OPR3 12-Oxophytodienoate reductase - PDF1.2 Plant defensin - ROS Reactive oxygen species - SA Salicylic acid - SNP Sodium nitroprusside     

Nitric Oxide is Involved in the <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>-induced Lateral Root Formation in Tomato

Azospirillum spp. is a well known plant-growth-promoting rhizobacterium. Azospirillum-inoculated plants have shown to display enhanced lateral root and root hair development. These promoting effects have been attributed mainly to the production of hormone-like substances. Nitric oxide (NO) has recently been described to act as a signal molecule in the hormonal cascade leading to root formation. However, data on the possible role of NO in free-living diazotrophs associated to plant roots, is unavailable. In this work, NO production by Azospirillum brasilense Sp245 was detected by electron paramagnetic resonance (6.4 nmol. g^–1 of bacteria) and confirmed by the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA). The observed green fluorescence was significantly diminished by the addition of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Azospirillum-inoculated and noninoculated tomato (Lycopersicon esculentum L.) roots were incubated with DAF-2 DA and examined by epifluorescence microscopy. Azospirillum-inoculated roots displayed higher fluorescence intensity which was located mainly at the vascular tissues and subepidermal cells of roots. The Azospirillum-mediated induction of lateral root formation (LRF) appears to be NO-dependent since it was completely blocked by treatment with cPTIO, whereas the addition of the NO donor sodium nitroprusside partially reverted the inhibitory effect of cPTIO. Overall, the results strongly support the participation of NO in the Azospirillum-promoted LRF in tomato seedlings.     

Nitric oxide plays a central role in determining lateral root development in tomato

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological processes in plants, most of them involving cross-talk with traditional phytohormones. Auxin is the main hormone that regulates root system architecture. In this communication we report that NO promotes lateral root (LR) development, an auxin-dependent process. Application of the NO donor sodium nitroprusside (SNP) to tomato (Lycopersicon esculentum Mill.) seedlings induced LR emergence and elongation in a dose-dependent manner, while primary root (PR) growth was diminished. The effect is specific for NO since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) blocked the action of SNP. Depletion of endogenous NO with CPTIO resulted in the complete abolition of LR emergence and a 40% increase in PR length, confirming a physiological role for NO in the regulation of root system growth and development. Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed that the NO signal was specifically located in LR primordia during all stages of their development. In another set of experiments, SNP was able to promote LR development in auxin-depleted seedlings treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Moreover, it was found that LR formation induced by the synthetic auxin 1-naphthylacetic acid (NAA) was prevented by CPTIO in a dose-dependent manner. All together, these results suggest a novel role for NO in the regulation of LR development, probably operating in the auxin signaling transduction pathway.Abbreviations CPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - DAF-2 DA 4,5-Diaminofluorescein diacetate - LR Lateral root - NAA 1-Naphthylacetic acid - NO Nitric oxide - NPA N-1-Naphthylphthalamic acid - PR Primary root - SNP Sodium nitroprusside     

Decomposition of N-nitrosamines, and concomitant release of nitric oxide by Fenton reagent under physiological conditions

N-Nitrosodimethylamine (NDMA) in phosphate buffer was rapidly decomposed by Fenton reagent composed of H₂O₂, and Fe(II) ion. Electron spin resonance (ESR) studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that characteristic four line 1:2:2:1 ESR signals due to the DMPO-OH adduct formed on treatment of DMPO with Fenton reagent disappeared in the presence of NDMA, and N-nitrosodiethylamine (NDEA), suggesting the interaction of the N-nitrosamines with Fenton reagent. Treatment of the N-nitrosamines with Fenton reagent generated nitric oxide (NO) as estimated by ESR technique using cysteine–Fe(II), and N-methyl- -glucaminedithiocarbamate (MGD)–Fe(II) complexes. Characteristic 3, and single line signals due to 2 cysteine–Fe(II)–NO, and 2 cysteine–Fe(II)–2 NO complexes, respectively, and three line signals due to MGD–Fe(II)–NO were observed. Considerable amount of NO were liberated as determined by NO₂⁻, the final oxidation product of NO formed by reaction with dissolved oxygen in the aqueous medium. Spontaneous release of a small amount of NO from the N-nitrosamines was observed only on incubation in neutral buffers. Above results indicate that the N-nitrosamines were decomposed accompanying concomitant release of NO on contact with reactive oxygen species.     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

Ammonium and nitrate as nitrogen sources in two Eriophorum species

Summary We compared ammonium and nitrate nutrition in Eriophorum scheuchzeri and E. vaginatum, two Alaskan sedges that are native to high- and low-fertility sites, respectively. When grown in solution culture, the two species were similar in their kinetics of NH _inf4 ^sup+ NO _inf3 ^sup- absorption: at nitrogen concentrations below 50 M, net NH _inf4 ^sup+ and NO _inf3 ^sup- were absorbed at similar rates, but at higher concentrations, net uptake of NO _inf3 ^sup- was significantly faster than that of NH _inf4 ^sup+ . The two species also showed similar abilities to assimilate NO _inf3 ^sup- . Growth of E. vaginatum under NO _inf3 ^sup- nutrition was only slightly less than that under NH _inf4 ^sup+ . The observed similarities between these species from contrasting edaphic habitats indicate that factors other than tissue-specific rates of nitrogen acquisition and assimilation may underlie local adaptation to soil N fertility. Moreover, the capacity of these species to exploit NO _inf3 ^sup- as a N source supports the view that NO _inf3 ^sup- availability may be significant even in wet, acidic, arctic soils.     

叶施NO对NaCl胁迫下红砂幼苗叶片和根系中氮代谢酶及营养物质的影响

以当年生红砂(Reaumuria soongorica)幼苗为材料,采用盆栽实验,考察叶面喷施不同浓度(0、0.01、0.10、0.25、0.50、1.00 mmol·L^-1)NO供体硝普钠 (SNP) 对NaCl(300 mmol·L^-1)胁迫下红砂根、叶中可溶性蛋白、游离氨基酸和硝态氮含量,以及谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、硝酸还原酶(NR)活性的影响,并采用主成分分析和隶属函数法筛选NO对NaCl胁迫缓解效应的氮代谢指标和最佳NO浓度,以探讨外源NO对NaCl 胁迫下红砂缓解效应的氮代谢响应机制。结果表明：(1)在300 mmol·L^-1 NaCl胁迫处理下,红砂幼苗根、叶中可溶性蛋白、硝态氮含量以及GS、GOGAT、NR活性均比对照显著下降。(2)外源NO能显著提高盐胁迫下红砂叶、根中GS、GOGAT、NR活性和硝态氮含量,增加根中可溶性蛋白和游离氨基酸含量。(3)NR和GOGAT活性可用于评价NO对NaCl胁迫下红砂幼苗的缓解作用,外源NO(SNP)对红砂幼苗在NaCl胁迫下的缓解效果强弱表现为0.25 mmol·L^-1> 0.50 mmol·L^-1> 0.10 mmol·L^-1> 1.00 mmol·L^-1> 0.01 mmol·L^-1。研究发现,300 mmol·L^-1 NaCl胁迫显著抑制了红砂幼苗氮代谢,外源NO(SNP)有助于提高盐胁迫下红砂NR活性,加快硝态氮转化为铵态氮,促进红砂叶片和根中GS/GOGAT对转化物的同化,从而增强红砂幼苗的耐盐性,并以0.25 mmol·L^-1SNP处理时缓解作用最佳;NR和GOGAT活性可作为NO缓解盐胁迫的评价指标。     

Trypanosoma cruzi: Effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice

Leukotrienes are important mediators of inflammatory responses. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide (NO) and iNOS expression in cardiac tissue of mice infected with Trypanosoma cruzi, the agent of Chagas’ disease. NO is a key mediator of parasite killing in mice experimentally infected with T. cruzi, and previous studies have suggested that leukotrienes, such as LTB₄, induces NO synthesis in T. cruzi-infected macrophages and plays a relevant role in the killing of parasite in a NO-dependent manner. We therefore investigated whether leukotrienes would have a similar role in vivo in controlling the parasite burden by regulating NO activity. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased NO and IL-6 production in the plasma with a concomitant decrease in the expression of iNOS in the cardiac tissue on day 12 after T. cruzi infection. These findings indicate that endogenous leukotrienes are important regulators of NO activity in the heart and therefore influence the cardiac parasite burden without exerting a direct action on IL-6 production in the acute phase of infection with T. cruzi.     

NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Transgenic alfalfa (Medicago sativa L.) root cultures expressing sense and antisense barley (Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000-g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7–8 and a strong affinity to NADH and NADPH (K _m 3 M for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35±4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.Abbreviation Hb Hemoglobin     

Ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions increase levels of nitric oxide and matrix metalloproteinase-9 without a direct association between them

Inflammatory and infectious conditions were simulated in cultures of ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells, using interferon-gamma (IFN-γ, 100 units/ml) and lipopolysaccharide (LPS, 0.5 μg/ml) co-treatment for 24 h, to investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA and the production of NO. Aminoguanidine (AG, 1 mM; an NOS inhibitor) along with IFN-γ and LPS, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 100 μM; an NO donor) and/or (±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4, 100 μM; an NO donor), were also added to analyze the possible association of NO with matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Co-treatment of cells with IFN-γ and LPS increased iNOS mRNA expression, NO production, MMP-9 mRNA expression, and 105 kDa MMP-9 production. Additional treatment with the NOS inhibitor AG inhibited NO production, but did not down-regulate the expression of MMP-9 mRNA or 105 kDa MMP-9. The NO donors SNAP and NOR4 did not affect the expression of MMP-9 mRNA, 105 kDa MMP-9 or TIMP-1 mRNA. These results suggest that ras/myc SFME cells respond to infectious and inflammatory conditions and can enhance malignancy as cancer cells due to their increased levels of NO and MMP-9 production, but that NO is not directly associated with MMP-9 in these cells. H. Yamaguchi and Y. Kidachi contributed equally to this work     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

The production and utilization of nitric oxide by a new,denitrifying strain of Pseudomonas aeruginosa

When a new strain of Pseudomonas aeruginosa was grown aerobically and then transferred to anaerobic conditions, cells reduced NO ₃ ^– quantitatively to NO ₂ ^– in NO ₃ ^– -respiration. In the absence of nitrate, NO ₂ ^– was immediately reduced to NO or N₂O but not to N₂ indicating that NO ₂ ^– -reductase but not N₂O-reductase was active. The formation of the products NO or N₂O depended on the pH in the medium and the concentration of NO ₂ ^– present. When P. aeruginosa was grown anaerobically for at least three davs N₂O-reductase was also active. Such cells reduced NO to N₂ via N₂O. The new strain generated a H⁺-gradient and grew by reducing N₂O to N₂ but not by converting NO to N₂O. For comparison, Azospirillum brasilense Sp7 showed the same pattern of NO-reduction. In contrast, Paracoccus denitrificans formed 3.5 H⁺/NO during the reduction of NO to N₂O in oxidant pulse experiments but could not grow in the presence of NO. Thus the NO-reduction pattern in P. denitrificans on one side and P. aeruginosa and A. brasilense on the other was very different. The mechanistic implications of such differences are discussed.     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species

A double-mutant cell line, which was unable to grow in a medium with NO ₃ ^- as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179–183) supplemented with 50 mM chlorate and 229 M 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO ₃ ^- medium and then by transfer to the NO ₃ ^- medium supplemented with 229 M 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO ₃ ^- medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.Abbreviations B5 medium of Gamborg et al. (1968) - MS medium of Murashage and Skoog (1962) - 5MT 5-thethyltryptophan     

Significance of gaseous NO for ammonia oxidation by Nitrosomonas eutropha

Nitrification by the obligately lithoautotrophic ammonia oxidizer Nitrosomonas eutropha was significantly inhibited when nitric oxide was removed from the culture medium by means of intensive aeration and turbulence. Nearly complete recovery of ammonia oxidation could be achieved by adding 100 ppm NO to the supplied air. Inhibition of ammonia oxidation occurred also upon addition of the NO binding agens 2,3-Dimercapto-1-propane-sulfonic acid (DMPS). Recovery of ammonia oxidation occurred within 3 h in the presence of 100 ppm NO and within 76 h in the absence of externally added NO. In co-cultures of N. eutropha and the NO detoxifying bacterium Pseudomonas PS88, hardly any nitrification was detectable and release of NO was extremely low when the heterotroph was provided with an organic substrate. When cells of Pseudomonas PS88 were added to a mixotrophically nitrifying culture of N. eutropha the release of NO decreased drastically upon the addition and ammonia oxidation ceased. These results confirm for the first time the significance of NO in the course of ammonia oxidation by N. eutropha.     

Nitric oxide decreases ammonium release in tadpoles of the clawed frog, <Emphasis Type="Italic">Xenopus laevis</Emphasis>, Daudin

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or l-arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nω-methyl-l-arginine (l-NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.