NO参与AM真菌与烟草共生过程

以烟草（Nicotiana tabacum,品种CF90NF）为材料,利用分光光度法和荧光显微技术结合药理学实验,探讨在AM真菌摩西球囊霉(Glomus mosseae,G.m)与烟草共生过程中一氧化氮(nitric oxide, NO)的作用。结果表明,烟草侧根中含有一定水平的内源NO,苗期接种G.m 10天后,烟草根系NO含量显著增加,侧根中的NO荧光强度也在接种后10天达到最强;一定浓度的NO供体硝普钠(sodium nitroprusside,SNP)能促进G.m对烟草的侵染,而NO的清除剂2-4,4,5,5-苯-四甲基咪唑-1-氧-3-氧化物( 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxidepotassium salt,cPTIO)可明显减弱侧根和菌丝中的NO的荧光强度,降低AM真菌的侵染率,表明NO参与G.m与烟草的共生过程;在G.m与烟草的共生过程中,烟草根系硝酸还原酶(nitrate reductase,NR)活性与Nia-1的表达量明显升高,且NR的抑制剂钨酸钠(sodium tungstate,Na2WO4)可以降低烟草侧根中的荧光强度,但对菌丝中的NO的荧光强度无明显影响。由此推测,来自根系NR途径的NO参与AM真菌与烟草的共生过程,菌丝中可能存在其他来源的NO。     

Nitric oxide (NO) as an intermediate in the cryptogein-induced hypersensitive response--a critical re-evaluation

A hypersensitive response (HR) was induced in tobacco leaves and cell suspensions by the fungal elicitor cryptogein, and NO production was followed by chemiluminescence and occasionally by diaminofluorescein (DAF)-fluorescence. Results from both methods were at least partly consistent, but kinetics was different. NO emission was not induced by cryptogein in leaves, whereas in cell suspensions some weak NO emission was observed, which was nitrate reductase (NR)-dependent, but not required for cell death. Nitric oxide synthase (NOS) inhibitors did not prevent cell death, but PR-1 expression was weakened. In conclusion, neither NR nor NOS appear obligatory for the cryptogein-induced HR. However, a role for NO was still suggested by the fact that the NO scavenger cPTIO prevented the HR. Unexpectedly, cPTI, the reaction product of cPTIO and NO, also impaired the HR but without scavenging NO. Thus, prevention of the HR by cPTIO is not necessarily indicative for a role of NO. Further, even a 100-fold NO overproduction (over wild type) by a nitrite reductase-deficient mutant did not interfere with the cryptogein-induced HR. Accordingly, the role of NO in the HR should be reconsidered.     

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Q_i‐site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Q_o‐site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate‐grown wild‐type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium‐grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate‐grown plants. Hence the ETC, likely the Q‐cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non‐energy‐conserving electron sink upstream of Complex III.     

Impact of the C-N status on the amino acid profile in tobacco source leaves

This paper investigates the influence of the carbon (C) and nitrogen (N) status on the amino acid profile in tobacco source leaves. Treatments used included growing plants at different light intensities, using an antisense RBCS (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) construct to inhibit Rubisco activity, growing plants on 12 or 0.5 mM nitrate, comparing wild-types with genotypes that have small and large decreases in nitrate reductase (NIA) activity, and sampling plants at different times during the diurnal cycle. This combination of experiments provides information on how amino acid levels respond to several inputs including the C and N status, nitrate, excess light and light-dark transitions. The data set was analysed using principal component analysis, regression analysis and by normalizing the level of each individual amino acid on the total amino acid pool. Most amino acids show a downward trend when the C or the N status is decreased, and rise during day and fall at night during the diurnal cycle. However, individual amino acids often showed deviating responses. Furthermore, no evidence was found for feedback inhibition of minor amino acid synthesis, either within or between pathways, when 18 individual amino acids were supplied to detached leaves. Results indicate that regulation of amino acid metabolism, for example by the C and N status, leads to qualitatively similar responses of many amino acids, but homeostatic mechanisms involving feedback inhibition within or between individual amino acid biosynthesis pathways are not stringent. All of the above inputs affect the level of phenylalanine, an amino acid that is also the substrate for an important sector of secondary metabolism. The levels of glutamate were remarkably constant, indicating that unknown mechanisms stabilize the concentration of this key central amino acid. Analyses of metabolite levels and feeding experiments indicated that 2-oxoglutarate plays an important role in regulating glutamate levels. Glutamate was the most effective inhibitor of NIA activity when 18 individual amino acids were supplied to detached leaves. Feeding glutamate, and other downstream amino acids, led to an increase of glutamine, indicating glutamate exerts feedback regulation on ammonium metabolism.     

Nitric oxide： orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response

Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations, the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex Ⅳ) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of “metabolic hypoxia”-a situation in which, although oxygen is available, the cell is unable to utilise it.     

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant‐adaptive response to hypoxia is not known. Here, we investigated whether ethylene‐induced aerenchyma requires hypoxia‐induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia‐induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide potassium salt) and ethylene biosynthetic inhibitor CoCl₂ both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium‐dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca‐CAMK, inositol 1,4,5‐trisphosphate 5‐phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.     

施氮量对小麦叶片硝酸还原酶活性、一氧化氮含量和气体交换的影响

研究了不同施氮量对冬小麦分蘖到抽穗期叶片硝酸还原酶(NR)活性、一氧化氮(NO)含量、气体交换参数和籽粒产量的影响.结果表明:叶片光合速率（P_n）、蒸腾速率（T_r）、瞬时水分利用效率（IWUE）和产量均随施氮量的增加呈先升高后降低的趋势,在180 kg·hm^-2氮处理时达到最高.随施氮量的增加,叶片NR活性提高; 在分蘖期和拔节期,叶片NR活性与NO含量呈显著线性相关（R²≥0.68,n=15）,NO含量和气孔导度（G_s）呈显著正二次相关（R²≥0.43,n=15）;低氮处理下,NR活性较低使叶片NO含量维持在较低水平,促进气孔开放,高氮处理下,NR活性较高使叶片NO含量增加,诱导气孔关闭;在抽穗期叶片NR活性和NO含量无显著相关关系,虽然NO含量和G_s也呈显著正二次相关（R²≥0.36,n=15）,但不能通过施氮提高NR活性来影响叶片NO含量,进而调节叶片气孔行为.合理施氮使小麦叶片NO含量维持在较低水平,可提高叶片G_s、T_r和IWUE,增强作物抗旱能力,促进光合作用,提高小麦产量.     

Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation

Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO₂ or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO₂ was removed in continuous light, and was reactivated when CO₂ was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots.     

Nitric oxide oscillations in Paracoccus denitrificans: the effects of environmental factors and of segregating nitrite reductase and nitric oxide reductase into separate cells

Nitric oxide is a denitrification intermediate which is produced from nitrite and then further converted via nitrous oxide to nitrogen. Here, the effect of low concentrations of the protonophore carbonylcyanide m-chlorophenylhydrazone on the time courses for dissolved gases was examined. While NO was found to oscillate, N(2)O only increased gradually as the reduction of nitrite progressed. The frequency and shape of protonophore-induced NO oscillations were influenced by temperature and the concentration of electron donor N,N,N',N'-tetramethyl-p-phenylene diamine (TMPD) in a manner compatible with the observed differential effects on the two involved enzyme activities. We demonstrated the existence of a pH interval, where [NO] oscillates even without uncoupler addition. Occurrence of nitric oxide oscillations in mixtures of a nitrite reductase mutant with a nitric oxide reductase mutant suggests that they cannot be due to a competition of the enzymes for redox equivalents from one common respiratory chain.     

Seasonal characteristics of nitric oxide emission from a typical Chinese rice–wheat rotation during the non-waterlogged period

Nitric oxide emissions from a typical rice–wheat rotation system in southeastern China were continuously measured with an automatic system in 1996–1997. The seasonal pattern of the NO emissions was characterized for the non‐waterlogged period of a rotation cycle. Nitric oxide emissions during the period from March through June were 3.9–6.3 folds for the fertilized plots and 1.6 folds for the unfertilized plot larger than those from November through December. Nitric oxide emissions were not detectable during the winter period from January through February. Amendment of synthetic fertilizer N significantly enhanced the NO emission by a factor of 6.5, but the enhancement was significantly mitigated by 25% through substituting ca. 16% of the synthetic fertilizer N with organic N from fermented crop residues or by 21% through deep tillage. The NO–N emission factor, defined as the amount of NO–N released per unit of synthetic fertilizer N input, was determined to be 0.025 kg NO–N kg ^?1 of N applied for the non‐waterlogged period, which was reduced by 32% through substituting part of the synthetic N fertilizer with fermented crop residues or by 24% through deep tillage. In addition, the NO emission factor, defined as the amount of NO–N emitted from unit unfertilized area per day, was observed to be ca. 3.8 g N ha ^?1 d ^?1 . Approximately 0.55 Tg N yr ^?1 was likely released as NO from Chinese cultivated lands.     

Nitric oxide synthase activity and the level of nitrates/nitrites in brain regions during spontaneous morphine withdrawal in rats

Activity of nitric oxide synthase (NOS) and concentrations of nitrate/nitrites (NO _x ^? ) were measured in brain regions of rats during spontaneous morphine withdrawal, which was modeled in male Wistar rats. The animals were injected with the increasing intraperitoneal doses (10–100 mg/kg, twice a day) of morphine hydrochloride for 6 days. Thirty six hours after the last injection the severity of the spontaneous morphine withdrawal syndrome was determined by specific autonomic and locomotor indices The withdrawal was accompanied by the increase of both NOS activity and NO _x ^? levels in the midbrain and hippocampus, the decrease of these parameters in striatum and hypothalamus, and lack of changes in cerebral cortex and brain stem. In cerebellum NOS activity decreased whereas NO _x ^? concentrations remained unchanged. In the cerebral cortex, striatum, midbrain, and cerebellum activity of NOS and NO _x ^? concentrations correlated with the withdrawal syndrome severity and also with the specific signs of abstinence.     

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite

Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation. Received: 8 May 2000 / Accepted: 24 August 2000     

Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes

The indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 10⁴ cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co-culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti-CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H-2^d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H-2^b B10BR; H-2^k) nonadherent lymphocytes, which were stimulated with conA or anti-CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co-cultured with SEA-reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti-CD3, proliferated well by the addition of L-arginine homologue, N^G-monomethyl-L-arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti-CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of N^G-monomethyl-L-arginine.     

一氧化氮促进体外培养大鼠脑室下区神经干细胞的分化

目的：探讨一氧化氮（NO）对新生大鼠体外培养的神经干细胞（NSCs）分化的作用。方法：采用常规方法分离新生大鼠脑室下区（SVZ）组织,进行NSCs体外培养。用DETA／NO作为NO供体,用L-NAME作为一氧化氮合酶（NOS）抑制剂。免疫荧光法检测NSCs标志物-巢蛋白（nestin）、神经元标志物-8Ⅲ型微管蛋白（Tuj-1）和星型胶质细胞标志物-胶质原纤维酸性蛋白（GFAP）的表达,还检测了神经元型NOS的表达。用Greiss还原法检测培养液中总NO的浓度。结果：培养的神经球均为nestin阳性、BIdu阳性和nNOS阳性。NSCs和40μmol／L、50μmol／L、60μmol／LDEFA／N0共培养5d,实验组培养液中N0浓度较对照组显著增高（P〈0．01）,相应实验组分化的神经元数和星型胶质细胞数较对照组明显增加（P〈0．01和P〈0．05）。NSCs和100μmol／L、150μmol／L、200μmol／LL-NAME共培养5d,实验组培养液中NO浓度较对照组降低（P〈0．05）,相应实验组分化的神经元数和星型胶质细胞数也较对照组减少（P〈0．05）。结论：NO能直接促进大鼠SVZ体外培养的NSCs分化。     

Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes

* Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components. * NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues. * Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin. * Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.     

Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide

Endothelial cell injury is a critical event in tissue damage accompanying inflammation, in which both inflammatory cytokines and reactive oxygen species may play pivotal roles, although the exact mechanism has not yet been clarified. We found that combined stimulation with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) induced both cytotoxicity to murine vascular endothelial cell line F-2 and an increase in nitric oxide (NO). Therefore, in the present study, the implication of NO in cytotoxicity was examined. A potent iNOS-specific inhibitor ONO-1714 completely blocked both cytokine-induced cytotoxicity and NO production. NO scavengers such as carboxy-PTIO and hemoglobin blocked cytotoxicity. Moreover, exogenous NO from NOC 18 also caused cytotoxicity. These results together demonstrated that cytotoxicity of IFN-gamma and TNF-alpha for endothelial cell F-2 was mediated by NO, suggesting a pathogenic role of cytokine-induced NO production in endothelial damage under inflammatory conditions.