植物细胞一氧化氮信号转导研究进展

一氧化氮(nitric oxide, NO)作为重要的信号分子, 调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程, 并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道, 精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基, 通过金属亚硝基化、巯基亚硝基化和Tyr-硝基化等化学修饰方式, 调节靶蛋白的活性, 并影响cGMP和Ca²⁺信使系统等下游信号途径, 调控相应的生理过程。最新的一些研究结果也显示, MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外, 作为活跃的小分子信号, NO和活性氧相互依赖并相互影响, 共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展, 对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

植物体内一氧化氮合成途径研究进展

一氧化氮(NO)作为一种气体信号分子,在植物生理过程中发挥重要作用,它参与调节植物的生长、发育及对外界环境的应激反应.植物体内主要通过酶催化途径和非酶催化途径合成NO.酶催化途径合成NO的主要酶包括一氧化氮合酶(nitric oxide synthase,NOS)和硝酸还原酶(nitrate reductase,NR),以及在某些植物的特定组织或器官或在特殊环境条件下存在的一氧化氮氧化还原酶(nitric oxide oxidoreductase,Ni-NOR)和黄嘌呤氧化还原酶(xanthine oxidoreductase,XOR).非酶催化合成途径主要是在酸性和还原剂存在条件下将亚硝酸盐还原成NO.该文主要结合研究方法,综述了植物体内NO合成途径的研究进展,为植物体内NO信号的作用机理的深入研究提供信息资料.     

植物细胞一氧化氮信号转导研究进展

一氧化氮（nitric oxide,NO）作为重要的信号分子,调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程,并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道,精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基,通过金属亚硝基化、巯基亚硝基化和Tyr．硝基化等化学修饰方式,调节靶蛋白的活性,并影响cGMP和Ca2＋信使系统等下游信号途径,调控相应的生理过程。最新的一些研究结果也显示,MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外,作为活跃的小分子信号,NO和活性氧相互依赖并相互影响,共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展,对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

Formation and possible roles of nitric oxide in plant roots

Nitric oxide has been reported to act as a signalling molecule in different plant tissues and to participate in a variety of physiological processes. It is produced by different enzymes and sources. The root-specific plasma membrane-bound enzymes forming NO from the substrates nitrate and nitrite are of particular interest because roots serve as interfaces between plants and the soil. The co-ordinated activity of the root-specific plasma membrane-bound nitrate reductase (PM-NR) and nitrite:NO reductase (NI-NOR) suggests that NO might also be involved in root signalling and development. The rate of enzymatic production of this NO depends largely on the environmental conditions, mainly the availability of nitrate and oxygen and it is proposed that this NO plays a role during anoxia as an indicator of the external nitrate availability and in regulating symbiotic interactions at the root surface.     

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.     

Nitric oxide detoxification in the rhizobia-legume symbiosis

NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes). These observations have led to the question of how nodules overcome the toxicity of NO. On the bacterial side, one candidate for NO detoxification in nodules is the respiratory Nor (NO reductase) that catalyses the reduction of NO to nitrous oxide. In addition, rhizobial fHbs (flavohaemoglobins) and single-domain Hbs which dioxygenate NO to form nitrate are candidates to detoxify NO under free-living and symbiotic conditions. On the plant side, sHbs (symbiotic Hbs) (Lb) and nsHbs (non-symbiotic Hbs) have been proposed to play important roles as modulators of NO levels in the rhizobia-legume symbiosis. In the present review, current knowledge of NO detoxification by legume-associated endosymbiotic bacteria is summarized.     

Nitric oxide reduces hydrogen peroxide accumulation involved in water stress-induced subcellular anti-oxidant defense in maize plants

Nitric oxide （NO） is a bioactive molecule involved in many biological events, and has been reported as pro-oxidant as well as anti-oxidant in plants. In the present study, the sources of NO production under water stress, the role of NO in water stress-induced hydrogen peroxide （H2O2） accumulation and subcellular activities of anti-oxidant enzymes in leaves of maize （Zea mays L.） plants were investigated. Water stress induced defense increases in the generation of NO in maize mesphyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. Water stress-induced defense increases in the production of NO were blocked by pretreatments with inhibitors of NOS and nitrate reductase （NR）, suggesting that NO is produced from NOS and NR in leaves of maize plants exposed to water stress. Water stress also induced increases in the activities of the chloroplastic and cytosolic anti-oxidant enzymes superoxide dismutase （SOD）, ascorbate peroxidase （APX）, and glutathione reductase （GR）, and the increases in the activities of anti-oxidant enzymes were reduced by pretreatments with inhibitors of NOS and NR. Exogenous NO increases the activities of water stress-induced subcellular anti-oxidant enzymes, which decreases accumulation of H2O2. Our results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is, at least in part, due to the induction of a subcellular anti-oxidant defense.     

Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.     

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.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

Nitric oxide synthase I mediates osteoclast activity in vitro and in vivo

Bone resorption is responsible for the morbidity associated with a number of inflammatory diseases such as rheumatoid arthritis, orthopedic implant osteolysis, periodontitis and aural cholesteatoma. Previous studies have established nitric oxide (NO) as a potentially important mediator of bone resorption. NO is a unique intercellular and intracellular signaling molecule involved in many physiologic and pathologic pathways. NO is generated from L-arginine by the enzyme nitric oxide synthase (NOS). There are three known isoforms of NOS with distinct cellular distributions. In this study, we have used mice with targeted deletions in each of these isoforms to establish a role for these enzymes in the regulation of bone resorption in vivo and in vitro. In a murine model of particle induced osteolysis, NOS I-/- mice demonstrated a significantly reduced osteoclast response. In vitro, osteoclasts derived from NOS I-/- mice were larger than wild type controls but demonstrated decreased resorption. Although NOS I has been demonstrated in osteoblasts and osteocytes as a mediator of adaptive bone remodeling, it has not previously been identified in osteoclasts. These results demonstrate a critical role for NOS I in inflammatory bone resorption and osteoclast function in vitro.     

Oxidation of hydroxylamines to NO by plant cells

At least theoretically, plants may synthesize nitric oxide (NO) either by reduction of N in higher oxidations states, or by oxidation of more reduced N-compounds. The well established reductive pathway uses nitrite as a substrate, produced by cytosolic nitrate reductase. The only oxidative pathway described so far comprises nitric oxide synthase (NOS)-like activity, where guanidino-N from L-arginine is oxidized to NO. In our previous paper we have demonstrated yet another form of oxidative NO formation, whereby hydroxylamine (HA), but also the AOX-inhibitor salicylhydroxamate (SHAM) is oxidized to NO by tobacco suspension cells. Oxidation of HA to NO was also demonstrated in vitro by using ROS producing enzymes. Apparently superoxide radicals and/or hydrogen peroxide served as oxidants. Another unexpected observation pointed to a special role for superoxide dismutase in oxidative NO formation.Key words: hydroxylamine, nitric oxide, oxidative NO formation, reactive oxygen species, salicyl hydroxamate, superoxide dismutase     

Nitric oxide-induced salt stress tolerance in plants: ROS metabolism,signaling, and molecular interactions

Nitric oxide (NO), a non-charged, small, gaseous free-radical, is a signaling molecule in all plant cells. Several studies have proposed multifarious physiological roles for NO, from seed germination to plant maturation and senescence. Nitric oxide is thought to act as an antioxidant, quenching ROS during oxidative stress and reducing lipid peroxidation. NO also mediates photosynthesis and stomatal conductance and regulates programmed cell death, thus providing tolerance to abiotic stress. In mitochondria, NO participates in the electron transport pathway. Nitric oxide synthase and nitrate reductase are the key enzymes involved in NO-biosynthesis in aerobic plants, but non-enzymatic pathways have been reported as well. Nitric oxide can interact with a broad range of molecules, leading to the modification of protein activity, GSH biosynthesis, S-nitrosylation, peroxynitrite formation, proline accumulation, etc., to sustain stress tolerance. In addition to these interactions, NO interacts with fatty acids to form nitro-fatty acids as signals for antioxidant defense. Polyamines and NO interact positively to increase polyamine content and activity. A large number of genes are reprogrammed by NO; among these genes, proline metabolism genes are upregulated. Exogenous NO application is also shown to be involved in salinity tolerance and/or resistance via growth promotion, reversing oxidative damage and maintaining ion homeostasis. This review highlights NO-mediated salinity-stress tolerance in plants, including NO biosynthesis, regulation, and signaling. Nitric oxide-mediated ROS metabolism, antioxidant defense, and gene expression and the interactions of NO with other bioactive molecules are also discussed. We conclude the review with a discussion of unsolved issues and suggestions for future research.     

Nitric oxide synthesis and signalling in plants

As with all organisms, plants must respond to a plethora of external environmental cues. Individual plant cells must also perceive and respond to a wide range of internal signals. It is now well-accepted that nitric oxide (NO) is a component of the repertoire of signals that a plant uses to both thrive and survive. Recent experimental data have shown, or at least implicated, the involvement of NO in reproductive processes, control of development and in the regulation of physiological responses such as stomatal closure. However, although studies concerning NO synthesis and signalling in animals are well-advanced, in plants there are still fundamental questions concerning how NO is produced and used that need to be answered. For example, there is a range of potential NO-generating enzymes in plants, but no obvious plant nitric oxide synthase (NOS) homolog has yet been identified. Some studies have shown the importance of NOS-like enzymes in mediating NO responses in plants, while other studies suggest that the enzyme nitrate reductase (NR) is more important. Still, more published work suggests the involvement of completely different enzymes in plant NO synthesis. Similarly, it is not always clear how NO mediates its responses. Although it appears that in plants, as in animals, NO can lead to an increase in the signal cGMP which leads to altered ion channel activity and gene expression, it is not understood how this actually occurs.
NO is a relatively reactive compound, and it is not always easy to study. Furthermore, its biological activity needs to be considered in conjunction with that of other compounds such as reactive oxygen species (ROS) which can have a profound effect on both its accumulation and function. In this paper, we will review the present understanding of how NO is produced in plants, how it is removed when its signal is no longer required and how it may be both perceived and acted upon.     

Nitric oxide is formed in Medicago truncatula-Sinorhizobium meliloti functional nodules

Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.     

Are there protective enzymatic pathways to regulate high local nitric oxide (NO) concentrations in cells under stress conditions?

This paper examines, from a chemical perspective, the hypothesis of the existence of protective enzymes whose role would be to regulate the high local nitric oxide (NO) concentrations that are released in NO-generating cells in situations of response to oxidative stress. These enzymes should play the role, with respect to NO, either of a reductase or of a dismutase. The energetics of the intervening transformations is herein presented, along with a review of pertinent literature. An attempt is made in order to describe the physiognomy of such enzymes, in relation with the literature data. Experimental investigation is needed to further evaluate the validity of such a hypothesis.     

真菌异化硝酸盐还原机理的研究进展

真菌异化硝酸盐还原途径的发现打破了反硝化仅存在于原核细胞这一传统观念。真菌异化硝酸盐还原途径是在环境中氧供给受限的情况下发生的, 包括反硝化和氨的发酵。硝酸盐能诱导产生反硝化作用的酶, 其中, 硝酸盐还原酶与亚硝酸还原酶位于线粒体中, 它们所催化的酶促反应能偶联呼吸链ATP合成酶合成ATP, 同时产生NO。与参与反硝化作用前两个酶不同, 真菌NO还原酶能以NADH为直接电子供体将NO还原为N2O, 在NAD+的再生和自由基NO的脱毒中起着重要作用。氨发酵则将硝酸盐还原成NH4+, 同时偶联乙酸的生成和底物水平磷酸化。此文从参与该过程的关键酶、关键酶的表达调节、真菌与细菌异化硝酸盐还原的比较等角度综述了真菌异化硝酸盐还原的最新研究进展。     

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O₂^?*), to form peroxynitrite (ONOO^?) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.     

Nitric oxide signaling in yeast

As a cellular signaling molecule, nitric oxide (NO) is widely conserved from microorganisms, such as bacteria, yeasts, and fungi, to higher eukaryotes including plants and mammals. NO is mainly produced by NO synthase (NOS) or nitrite reductase (NIR) activity. There are several NO detoxification systems, including NO dioxygenase (NOD) and S-nitrosoglutathione reductase (GSNOR). NO homeostasis based on the balance between NO synthesis and degradation is important for the regulation of its physiological functions because an excess level of NO causes nitrosative stress due to the high reactivity of NO and NO-derived compounds. In yeast, NO may be involved in stress responses, but NO and its signaling have been poorly understood due to the lack of mammalian NOS orthologs in the genome. Even though the activities of NOS and NIR have been observed in yeast cells, the gene encoding NOS and the NO production mechanism catalyzed by NIR remain unclear. On the other hand, yeast cells employ NOD and GSNOR to maintain an intracellular redox balance following endogenous NO production, exogenous NO treatment, or environmental stresses. This article reviews NO metabolism (synthesis, degradation) and its regulation in yeast. The physiological roles of NO in yeast, including the oxidative stress response, are also discussed here. Such investigations into NO signaling are essential for understanding the NO-dependent genetic and physiological modulations. In addition to being responsible for the pathology and pharmacology of various degenerative diseases, NO signaling may be a potential target for the construction and engineering of industrial yeast strains.     

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both beta- and gamma-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a "functional group" defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.