Localization of nitric-oxide synthase in plant peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.     

Peroxisomal NADP-Dependent Isocitrate Dehydrogenase. Characterization and Activity Regulation during Natural Senescence

The peroxisomal localization and characterization of NADP-dependent isocitrate dehydrogenase (perICDH) in young and senescent pea (Pisum sativum) leaves was studied by subcellular fractionation, kinetic analysis, immunoblotting, and immunoelectron microscopy. The subunit molecular mass for perICDH determined by immunoblotting was 46 kD. By isoelectric focusing (IEF) of the peroxisomal matrix fraction, the NADP-ICDH activity was resolved into four isoforms, perICDH-1 to perICDH-4, with isoelectric points (pIs) of 6.0, 5.6, 5.4, and 5.2, respectively. The kinetic properties of the NADP-ICDH in peroxisomes from young and senescent pea leaves were analyzed. The maximum initial velocity was the same in peroxisomes from young and senescent leaves, while the Michaelis constant value in senescent leaf peroxisomes was 11-fold lower than in young leaf peroxisomes. The protein levels of NADP-ICDH in peroxisomes were not altered during senescence. The kinetic behavior of this enzyme suggests a possible fine control of enzymatic activity by modulation of its Michaelis constant during the natural senescence of pea leaves. After embedding, electron microscopy immunogold labeling of NADP-ICDH confirmed that this enzyme was localized in the peroxisomal matrix. Peroxisomal NADP-ICDH represents an alternative dehydrogenase in these cell organelles and may be the main system for the reduction of NADP to NADPH for its re-utilization in the peroxisomal metabolism.     

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.     

Mechanical Stress Elicits Nitric Oxide Formation and DNA Fragmentation in Arabidopsis thaliana

The effect of mechanical stress (centrifugation) on the inductionof nitric oxide (NO) formation and DNA fragmentation was investigatedin leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifugedleaves from wild-type and nitrate reductase (NR)nia1, nia2 doublemutant, defective in the assimilation of nitrate, were labelledwith 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualizein vivo NO production. After these treatments, DNA fragmentationwas detected by the terminal deoxynucleotidyl transferase-mediateddUTP nick end in situ labelling (TUNEL) method. Exposure toan NO-releasing compound, sodium nitroprusside (SNP) mimickedthe cell response to centrifugation (20 g). The involvementof endogenous NO as a signal in mechanical stress and in DNAfragmentation was confirmed by inhibition of NO production usinga nitric oxide synthase (NOS) inhibitor viz. N^G-monomethyl-L -arginine (L -NMMA). These results indicate that NOS-likeactivity was present in A. thaliana leaves and was increasedby mechanical stress. The effect of leaf-wounding on nitricoxide production was identical to that of centrifugation. Experimentswith A. thaliana NR mutant also showed that NO bursts were inducedby mechanical and wounding stresses and that NO was not a by-productof NR activity. A positive and significant correlation betweenNO production and DNA fragmentation was recorded for both centrifugedand non-centrifuged cells. Our results suggest that factorsother than NO contribute to DNA damage and cell death, and furthermore,that an inducible form of NOS is present in A. thaliana. Copyright2001 Annals of Botany Company Arabidopsis thaliana, cell death, DNA fragmentation, NO, plant stress, wounding     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Peroxisomal xanthine oxidoreductase: characterization of the enzyme from pea (Pisum sativum L.) leaves

The presence and properties of the enzyme xanthine oxidoreductase (XOR) in peroxisomes from pea (Pisum sativum L.) leaves were studied using biochemical and immunological methods. The activity analysis showed that, in leaf peroxisomes, the superoxide-generating XOR form, xanthine oxidase (XOD), was predominant over the xanthine dehydrogenase form (XDH), with a XDH/XOD ratio of 0.5. However, in crude extracts of pea leaves, the XDH form was more abundant, with a XDH/XOD ratio of 1.6. The native molecular mass of the peroxisomal XOR determined by polyacrylamide gel electrophoresis was 290kDa. Using western blot assays, we identified an immunoreactive band of 59kDa that was not affected by the reducing reagent DTT or endogenous proteases. The analysis of pea leaves by electron microscopy immunogold labeling with affinity-purified antibodies against rat XOD confirmed that this enzyme was localized in the matrix of peroxisomes, as well as in chloroplasts and cytosol. In pea plants subjected to abiotic stress by cadmium, the activity of the peroxisomal XOR was reduced, whereas its protein level expression increased. The results confirmed that leaf peroxisomes contain XOR, and suggest that this peroxisomal metalloflavoprotein enzyme is involved in the mechanism of response of pea plants to abiotic stress by heavy metals.     

4,5-diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5-Diaminofluoroscein diacetate, DAF-2 DA. Following DAF-2 loading, ganglia showed cell-specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME prevented much of the initial development of DAF-2 fluorescence, whereas the inactive isomer D-NAME had no effect. Washout of preincubated L-NAME caused increased cell-specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L-arginine also resulted in an increase of DAF-2 fluorescence in a cell-specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF-2 fluorescence increases in most cells. We therefore presume that the cell-specific pattern of DAF-2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF-2 staining pattern and previously published studies of NOS activity are discussed.     

Natural Senescence of Pea Leaves (An Activated Oxygen-Mediated Function for Peroxisomes)

We studied the activated oxygen metabolism of peroxisomes in naturally and dark-induced senescent leaves of pea (Pisum sativum L.). Peroxisomes were purified from three different types of senescent leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. The activities of the O2-- and H2O2-producing enzymes were enhanced by natural senescence. Senescence also produced an increase in the generation of active oxygen species (O2- and H2O2) in leaf peroxisomes and in the activities of two glyoxylate-cycle marker enzymes. A new fraction of peroxisomes was detected at an advanced stage of dark-induced senescence. Electron microscopy revealed that this new peroxisomal fraction varied in size and electron density. During senescence, the constitutive Mn-superoxide dismutase (SOD) activity of peroxisomes increased and two new CuZn-SODs were induced, one of which cross-reacted with an antibody against glyoxysomal CuZn- SOD. This fact and the presence of glyoxylate-cycle enzymes support the idea that foliar senescence is associated with the transition of peroxisomes into glyoxysomes. Our results indicate that natural senescence causes the same changes in peroxisome-activated oxygen metabolism as dark-induced senescence, and reinforce the hypothesis of an effective role of peroxisomes and their activated oxygen metabolism in this stage of the life cycle.     

Methods of nitric oxide detection in plants: A commentary

Over the last decade nitric oxide (NO) has been shown to influence a range of processes in plants. However, when, where and even if NO production occurs is controversial in several physiological scenarios in plants. This arises from a series of causes: (a) doubts have arisen over the specificity of widely used 4,5-diaminofluorescein diacetate (DAF-2DA)/4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) dyes for NO, (b) no plant nitric oxide synthase (NOS) has been cloned, so that the validity of using mammalian NOS inhibitors to demonstrate that NO is being measured is debatable, (c) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) needs to be used with caution, and (d) some discrepancies between assays for in planta measurements and another based on sampling NO from the gas phase have been reported. This review will outline some commonly used methods to determine NO, attempt to reconcile differing results obtained by different laboratories and suggest appropriate approaches to unequivocally demonstrate the production of NO.     

Nitric oxide generation in Vicia faba phloem cells reveals them to be sensitive detectors as well as possible systemic transducers of stress signals

Vascular tissue was recently shown to be capable of producing nitric oxide (NO), but the production sites and sources were not precisely determined. Here, NO synthesis was analysed in the phloem of Vicia faba in response to stress- and pathogen defence-related compounds. The chemical stimuli were added to shallow paradermal cortical cuts in the main veins of leaves attached to intact plants. NO production in the bare-lying phloem area was visualized by real-time confocal laser scanning microscopy using the NO-specific fluorochrome 4,5-diaminofluorescein diacetate (DAF-2 DA). Abundant NO generation in companion cells was induced by 500 microm salicylic acid (SA) and 10 microm hydrogen peroxide (H(2)O(2)), but the fungal elicitor chitooctaose was much less effective. Phloem NO production was found to be dependent on Ca(2+) and mitochondrial electron transport and pharmacological approaches found evidence for activity of a plant NO synthase but not a nitrate reductase. DAF fluorescence increased most strongly in companion cells and was occasionally observed in phloem parenchyma cells. Significantly, accumulation of NO in sieve elements could be demonstrated. These findings suggest that the phloem perceives and produces stress-related signals and that one mechanism of distal signalling involves the production and transport of NO in the phloem.     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.     

A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle.

We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.     

Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O2*(-)) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals O2*(-). Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP(+) can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO*), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).     

Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging

We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptor proteins (SNAREs), and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and rat lung exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated 1) whether this trafficking block affects subcellular localization and function of endothelial nitric oxide (NO) synthase (eNOS) and 2) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced "megalocytosis" of pulmonary arterial endothelial cells (PAEC) was accompanied by a loss of eNOS from the plasma membrane, with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogeneous compartments and partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN, and ER Tracker, but not Lyso Tracker. Hypoxia and senescence also produced enlarged PAEC, with dysfunctional Golgi and loss of eNOS from the plasma membrane, with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO with 4,5-diaminofluorescein diacetate (DAF-2DA) as probe showed a marked loss of caveolar NO after MCTP, hypoxia, and senescence. Although ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased DAF-2DA fluorescence in MCTP-treated and senescent PAEC, suggesting localization of eNOS in an aberrant cytoplasmic compartment that was readily discharged by Ca(2+)-induced exocytosis. Thus monocrotaline, hypoxia, and senescence produce a Golgi blockade in PAEC, leading to sequestration of eNOS away from its functional caveolar location and providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension, despite sustained eNOS protein levels.     

Retinal Cell Death Induced by TRPV1 Activation Involves NMDA Signaling and Upregulation of Nitric Oxide Synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.     

Nitric oxide production inhibited by xenobiotic compounds in the protozoan Paramecium primaurelia

The notable increase in agricultural and industrial activities over the last decades has caused a considerable increase in anthropogenic waste and, consequently, the presence of pollutants in both water and sediments. For this reason, there is great interest in identifying alternative models and bioassays complying with the 3Rs strategy (aimed at Reducing, Refining and Replacing tests on vertebrate organisms in toxicological studies). Protozoa seem to be well suited to this strategy and it is widely accepted that assays with protozoa are relevant to the study of environmental modifications due to the presence of xenobiotic compounds.Recently, we detected the presence of nitric oxide synthase (NOS)-related NADPH-diaphorase activity and neuronal NOS-related molecules, immunologically recognized by the anti-rat brain NOS antibody, in a single-cell freshwater eukaryote, Paramecium primaurelia. In this work we have looked for the basal NO production in living cells of P. primaurelia using the specific fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) and measuring the intracellular NO levels with image analysis. The NO production was sensitive to compounds modulating NOS activity such as: S-methyl-tiocitrulline, an NOS activity inhibitor, l-NAME, an analogue of arginine that inhibits NO production, arginine, an NOS substrate, or sodium nitroprusside, an NO donor. The NO production in P. primaurelia was also shown to be sensitive to μM concentrations of heavy metals (HgCl₂ and CdCl₂), or μM concentrations of pesticides (diazinon and AFD 25), thus representing a potential biomarker for environmental biomonitoring. The possible involvement of cellular Ca²⁺ concentration, assayed by the fluorescent probe chlortetracycline hydrochloride, in NO production was examined after xenobiotic exposure.     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signalmolecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show thatexogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin torapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number ofLRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturatedconcentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-IX (ZnPPIX) were added. Interestingly, depletion of endogenousNO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor N~G-nitro-L-arginine methyl ester (L-NAME),led to the complete abolition ofLR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, theinduction of LR development by 200 umol/L sodium nitroprusside (SNP),an NO donor, was not affected by the presenceor absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopywith the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NOrelease compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especiallyafter 36 h treatment. The LRP were found to have similar morphology in control, SNP-and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstreamof CO signaling, which has also been shown to occur in animals.     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signal molecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show that exogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin to rapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number of LRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturated concentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-Ⅸ (ZnPPIX) were added. Interestingly, depletion of endogenous NO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME), led to the complete abolition of LR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, the or absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopy with the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NO release compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especially after 36 h treatment. The LRP were found to have similar morphology in control, SNP- and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstream of CO signaling, which has also been shown to occur in animals.     

4,5‐diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5‐Diaminofluoroscein diacetate, DAF‐2 DA. Following DAF‐2 loading, ganglia showed cell‐specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L ‐NAME prevented much of the initial development of DAF‐2 fluorescence, whereas the inactive isomer D ‐NAME had no effect. Washout of preincubated L ‐NAME caused increased cell‐specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L ‐arginine also resulted in an increase of DAF‐2 fluorescence in a cell‐specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF‐2 fluorescence increases in most cells. We therefore presume that the cell‐specific pattern of DAF‐2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF‐2 staining pattern and previously published studies of NOS activity are discussed. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 361–369, 2002