Expression of NO scavenging hemoglobin is involved in the timing of bolting in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Plants contain three classes of hemoglobin genes of which two, class 1 and class 2, have a structure similar to classical vertebrate globins. We investigated the effect of silencing the class 1 non-symbiotic hemoglobin gene, GLB1, and the effect of overexpression of GLB1 or the class 2 non-symbiotic hemoglobin gene, GLB2, in Arabidopsis thaliana. Lines with GLB1 silencing had a significant delay of bolting and after bolting, shoots reverted to the rosette vegetative phase by formation of aerial rosettes at lateral meristems. Lines with overexpression of GLB1 or GLB2 bolted earlier than wild type plants. By germinating the lines in a medium containing the nitric oxide (NO) donor, sodium nitroprusside (SNP), it was demonstrated that both GLB1 and GLB2 promote bolting by antagonizing the effect of NO, suggesting that non-symbiotic plant hemoglobin controls bolting by scavenging the floral transition signal molecule, NO. So far, NO scavenging has only been demonstrated for class 1 non-symbiotic hemoglobins. A direct assay in Arabidopsis leaf cells shows that GLB1 as well as the class 2 non-symbiotic hemoglobin, GLB2, scavenge NO in vivo. NO has also been demonstrated to be a growth stimulating signal with an optimum at low concentrations. It was observed that overexpression of either GLB1 or GLB2 shifts the optimum for NO growth stimulation to a higher concentration. In conclusion, we have found that expression of NO scavenging plant hemoglobin is involved in the control of bolting in Arabidopsis.     

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development

Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from l-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.     

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 and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

S-nitrosylation: An emerging post-translational protein modification in plants

Increasing evidences support the assumption that nitric oxide (NO) acts as a physiological mediator in plants. Understanding its pleiotropic effects requires a deep analysis of the molecular mechanisms underlying its mode of action. In the recent years, efforts have been made in the identification of plant proteins modified by NO at the post-translational level, notably by S-nitrosylation. This reversible process involves the formation of a covalent bond between NO and reactive cysteine residues. This research has now born fruits and numerous proteins regulated by S-nitrosylation have been identified and characterized. This review describes the basic principle of S-nitrosylation as well as the Biotin Switch Technique and its recent adaptations allowing the identification of S-nitrosylated proteins in physiological contexts. The impact of S-nitrosylation on the structure/function of selected proteins is further 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.     

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.     

植物一氧化氮生物学的研究进展

一氧化氮(NO)是植物中的一种关键的信号分子.在植物中,NO的潜在来源包括一氧化氮合成酶、硝酸还原酶、黄嘌呤氧化还原酶和非酶促途径.NO能促进植物生长,延缓叶片、花和果实衰老,促进休眠和需光种子的萌发,能与植物激素相互作用调节气孔运动,诱导程序性细胞死亡和防御相关基因的表达,并在逆境中作为一种抗氧化剂起作用.NO的细胞内信号反应包括环鸟苷酸、环腺苷二磷酸核糖的产生和细胞质Ca2 浓度的增加,其信号转导途径及其生物化学和细胞学本质还不十分清楚.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron–dithiocarbamate complexes

The free radical, nitric oxide (√NO), is responsible for a myriad of physiological functions. The ability to verify and study √NO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of √NO at relevant cellular sites. With this in mind, several iron-chelates (Fe²⁺(L)₂) have been developed, which have provided a useful tool for the study and identification of √NO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of √NO detection is dependent on the Fe²⁺(L)₂ complex. The development of more efficient and stable Fe²⁺(L)₂ chelates may help to better understand the role of √NO in vivo. In this paper, we present data comparing several proline derived iron–dithiocarbamate complexes with the more commonly used spin traps for √NO, Fe²⁺-di(N-methyl-D-glutamine-dithiocarbamate) (Fe²⁺(MGD)₂) and Fe²⁺-di(N-(dithiocarboxy)sarcosine) (Fe²⁺(DTCS)₂). We evaluate the apparent rate constant (k_app) for the reaction of √NO with these Fe²⁺(L)₂ complexes and the stability of the corresponding Fe²⁺(NO)(L)₂ in presence of NOS I.     

Effect of l-Arginine Analogs and a Calcium Chelator on Nitric Oxide (NO˙) Production by Leishmania sp.

Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes were grown in the presence of l-arginine analogs such as Nω-nitro-l-arginine methyl ester (l-NAME), NG-nitro-l-arginine (l-NNA) and d-arginine (an inactive l-arginine isomer), besides an intracellular calcium chelator [ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid; EGTA] to verify the importance of l-arginine metabolism and the cofactors for these parasites. The parasite's growth curve was followed up and the culture supernatants were used to assay nitric oxide (NO˙) production by the Griess reaction. The results showed a significant effect of l-arginine analogs on NO˙ production by all Leishmania species studied, especially l-NAME, an irreversible inhibitor of the constitutive nitric oxide synthase (cNOS). When L. amazonensis promastigotes were pre-incubated with l-NAME, the infection range of the murine macrophages was lowered to 61% in 24?h and 19% after 48?h. These data demonstrated that the parasite NO˙ pathway is important to the establishment of the infection.     

植物一氧化氮(NO)研究进展

一氧化氮（NO）是植物的重要生物活性分子,它参与植物生长发育的许多过程,如种子萌发、下胚轴伸长、叶扩展、根生长、侧根形成、细胞凋亡以及植物抗逆反应等。大量的证据表明,植物可以通过与动物NO合酶类似的酶产生NO。此外,植物还可通过硝酸还原酶产生NO。NO在植物中的信号传递途径仍不十分清楚,植物有可能采用与动物相类似的机制。由于植物的大多数生长发育现象都受到植物激素的调节和控制,NO与植物激素之间的关系也受到越来越多的关注。通过激素起作用可能是植物内源NO作用的机理之一。     

Nitric oxide in legume-rhizobium symbiosis

Nitric oxide (NO) is a gaseous signaling molecule with a broad spectrum of regulatory functions in plant growth and development. NO has been found to be involved in various pathogenic or symbiotic plant-microbe interactions. During the last decade, increasing evidence of the occurrence of NO during legume-rhizobium symbioses has been reported, from early steps of plant-bacteria interaction, to the nitrogen-fixing step in mature nodules. This review focuses on recent advances on NO production and function in nitrogen-fixing symbiosis. First, the potential plant and bacterial sources of NO, including NO synthase-like, nitrate reductase or electron transfer chains of both partners, are presented. Then responses of plant and bacterial cells to the presence of NO are presented in the context of the N₂-fixing symbiosis. Finally, the roles of NO as either a regulatory signal of development, or a toxic compound with inhibitory effects on nitrogen fixation, or an intermediate involved in energy metabolism, during symbiosis establishment and nodule functioning are discussed.     

Involvement of nitric oxide in water stress-induced responses of cucumber roots

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors.A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ. Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.     

Bioactivity-based analysis and chemical characterization of anti-inflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated RAW264.7 cells

Inflammation is not only a self-defense response of the innate immune system, but also the pathogenesis mechanism of multiple diseases such as arthritis, neurodegeneration, and cancer. Curcuma zedoaria Roscoe (Zingiberaceae), an indigenous plant of India, has been used traditionally in Ayurveda and folk medicine. As part of our ongoing efforts to screen traditional medicinal plants exhibiting pharmacological potential and to characterize the compounds involved, we examined the anti-inflammatory effects of the MeOH extract of C. zedoaria rhizomes using lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells and found that MeOH extract inhibited the synthesis of nitric oxide (NO) in a dose-dependent manner (IC₅₀: 23.44 ± 0.77 μg/mL). In our efforts to characterize the compounds responsible for these anti-inflammatory effects, bioactivity-guided fractionation of the MeOH extract and chemical investigation of its active hexane-soluble fraction led to the successful isolation of five sesquiterpenes (1–5), the structures of which were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among them, curcuzedoalide (5) exhibited potent inhibitory effects on NO synthesis (IC₅₀: 12.21 ± 1.67 μM) and also suppressed pre-inflammatory protein expression of iNOS and COX-2. Curcuzedoalide (5) was thus determined to be a contributor to the anti-inflammatory effect of C. zedoaria rhizomes and could be a potential candidate for therapeutic applications.     

诱导型一氧化氮合酶抑制剂研发现状

一氧化氮(NO)对炎症性疾病的治疗作用近来引起了广泛的重视。诱导型一氧化合成酶(iNOS)被发现广泛地参与趋炎因子表达和反应性氧化产物(ROS)/反应性氮化产物(RNS)的产生,从而进一步证明了一氧化氮在炎症病理发生发展中的关键作用。由于传统的抗炎药物环氧合酶-2(COX-2)抑制剂被报导有较多副作用,新型抑制炎症药物的研究开发势在必行。本文分别介绍了化学来源、生物来源、植物来原性iNOS抑制剂阻的开发、研究现状,阐述了其在断炎症信息传递通道中的作用。表明了iNOS抑制剂防止炎症损害的相关机理,提出iNOs不仅能在初始阶段影响炎症的发生,也对抑制和终结炎症有作用。最后进一步介绍了用中草药研发iNOs抑制剂的可能性,展望了于中药在该领域内的巨大前景。     

Class-1 hemoglobins,nitrate and NO levels in anoxic maize cell-suspension cultures

Nitric oxide (NO) is a reactive gas involved in many biological processes of animals, plants and microbes. Previous work has demonstrated that NO is formed during hypoxia in alfalfa (Medicago sativa L.) root cultures and that the levels of NO detected are inversely related to the levels of expression of class-1 hemoglobin expressed in the tissue. The objectives of this study were: to examine whether NO is produced in transgenic maize (Zea mays L.) cell-suspension cultures exposed to anoxic growth conditions; to determine whether a similar relationship existed between a class-1 hemoglobin and the amount of NO detected under these conditions; and, to estimate the route of formation and breakdown of NO in the tissue. Maize cell-suspension cultures, transformed to express the sense or antisense strands of barley hemoglobin were used to overexpress or underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with an empty vector was used as a control. Up to 500 nmol NO (g FW)^–1 was detected in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels of NO in the different cell lines under anoxic conditions had an inverse relationship to the level of hemoglobin in the cells. There was no detectable NO produced under aerobic growth conditions. Spectroscopic data demonstrated that recombinant maize hemoglobin reacted with NO to form methemoglobin and NO₃^–. Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension cultures by using ¹⁵NO₃^– and electron paramagnetic resonance spectroscopy, suggesting that NO is formed via nitrate reductase during hypoxia. The results demonstrate that NO is produced in plant tissues grown under low oxygen tensions and suggest that class-1 hemoglobins have a significant function in regulating NO levels.Abbreviations DEANO 2-(N,N-Diethylamino)-diazenolate-2-oxide - EPR Electron paramagnetic resonance - Hb Hemoglobin - MGD N-(Dithiocarbamoyl)-N-methyl-d-glucamine - NOS Nitric oxide synthase - WT Wild type     

Triterpenoid biosynthesis and the transcriptional response elicited by nitric oxide in submerged fermenting Ganoderma lucidum

Ganoderic triterpenoid (GT) is a promising anti-tumour constituent in Ganoderma lucidum. The aim of this study was to investigate induction by and a possible signalling mechanism of nitric oxide (NO) for GT synthesis. Compared to the control, the biomass decreased by 43.5% at 120 h and the GT yield increased by 40.94% at 72 h in the presence of a 5 mM NO donor sodium nitroprusside supplement. The gene expression profiles of G. lucidum in response to NO were investigated by RNA-sequencing. Functional annotation and an enrichment analysis of the differentially expressed genes indicated that NO inhibited mycelial growth probably via the suppression of the glycolysis genes involved in carbohydrate metabolism. NO may function directly as a regulator of gene expression in the mevalonate pathway to induce GT biosynthesis, and the hyper-production of GT in response to NO could also be accomplished by a signalling function involving Ca²⁺ and a reactive oxygen species (ROS) pathway. The results of this study are useful for large-scale GT production and can facilitate further studies on the endogenous signalling pathways involved in the GT biosynthetic pathway.     

NO在植物中的调控作用

一氧化氮(NO)是一种易扩散的生物活性分子,是生物体内重要的信号分子.植物细胞通过NO合酶、硝酸还原酶、或非生化反应途径产生NO.NO参与植物生长发育调控和对生物与非生物环境胁迫的应答反应,大量证据表明NO是植物防御反应中的关键信使,其信号转导机制也受到越来越多的关注.本文主要通过讨论NO的产生、对植物生长周期的影响、在植物代谢中的信号调节以及参与细胞凋亡来阐述NO在植物中的作用.