Endogenous Nitric Oxide Content Research Articles

Combined hydrogen peroxide and nitric oxide priming modulate salt stress tolerance in acclimated and non-acclimated oilseed rape (Brassica napus L.) plants

We examined the combined effects of hydrogen peroxide (H2O2) and nitric oxide (NO) on the responses of oilseed rape(Brassica napus L.) plants to salt stress under acclimated and non-acclimated conditions. The results of the shoot and root dry weight traits together with the measurement of malondialdehyde (MDA) indicated that salt acclimation with a low concentration of NaCl (50 mM) could not alleviate the inhibitory effect of high salinity (200 mM NaCl). Under acclimated conditions, seed priming with H2O2 or NO resulted in effective protection against salt stress, however, maximum amelioration of salt stress was found by the combined treatments of H2O2 + NO. Interestingly, in the salt-exposed non-acclimated plants, only seed priming with H2O2 + NO was effective in improving salt tolerance. Pretreatment with H2O2 + NO tended to limit Na translocation into photosynthetic organs to prevent salt damages. Additionally, a large increase in salicylic acid contentwas correlated with phenylalanine ammonia lyase activation and flavonoid biosynthesis was observed when oilseed rape plants exposed to salinity in the presence of H2O2+NO. Interestingly, in this study, endogenous NO content of H2O2–primed plants exhibited a significant increase under non-saline conditions, indicating that H2O2 influences NO accumulation. In addition, oilseed rape plants primed with H2O2 + NO exhibited lower MDA and H2O2 content, contributing to the better induction of antioxidative enzyme activities. Higher levels of antioxidant enzyme activities maintained the integrity of cell membranes, resulting in better plant growth under salt stress. Taken together, our results revealed that oilseed rape plants pretreated with H2O2 + NO exhibited more effective tolerance to salt stress than plants that were pretreated with H2O2 or NO alone.

Main nitric oxide (NO) hallmarks to relieve arsenic stress in higher plants

Arsenic (As) is a toxic metalloid that adversely affects plant growth, and poses severe risks to human health. It induces disturbance to many physiological and metabolic pathways such as nutrient, water and redox imbalance, abnormal photosynthesis and ATP synthesis and loss of membrane integrity. Nitric oxide (NO) is a free radical molecule endogenously generated in plant cells which has signalling properties. Under As-stress, the endogenous NO metabolism is significantly affected in a clear connection with the metabolism of reactive oxygen species (ROS) triggering nitro-oxidative stress. However, the exogenous NO application provides beneficial effects under As-stress conditions which can relieve oxidative damages by stimulating the antioxidant systems, regulation of the expression of the transporter and other defence-related genes, modification of root cell wall composition or the biosynthesis of enriched sulfur compounds such phytochelatins (PCs). This review aims to provide up-to-date information on the key NO hallmarks to relieve As-stress in higher plants. Furthermore, it will be analyzed the diverse genetic engineering techniques to increase the endogenous NO content which could open new biotechnological applications, especially in crops under arsenic stress.

Nitric Oxide and Its Interaction with Hydrogen Peroxide Enhance Plant Tolerance to Low Temperatures by Improving the Efficiency of the Calvin Cycle and the Ascorbate–Glutathione Cycle in Cucumber Seedlings

The present study was aimed to assess the effect of nitric oxide (NO) and the interaction of NO with hydrogen peroxide (H2O2) on plant tolerance to low temperatures in cucumber seedlings. Exogenous NO significantly increased the endogenous NO content, initial and total activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO carboxylation rate (Vc,max), RuBP regeneration rate (Jmax) and the transcript levels of related genes in cucumber seedlings under low temperatures (11 °C/7 °C); however, the effect of NO was blocked by PTIO (NO scavenger). In addition, the SNP treatment significantly improved the contents of glucose, fructose, sucrose, starch, and the activities of sucrose phosphate synthase (SPS), acid invertase (AI), sucrose synthase (SS), as well as the expression levels of SUCROSE PHOSPHATE SYNTHASE 1&2 (SPS1, SPS2), SUCROSE TRANSPORTER 1&2 (SUT2, SUT4), β-starch hydrolase (BAM), and invertase gene (INVERTASE) in cucumber leaves under low temperatures, and the positive effect of NO was impaired by PTIO. Furthermore, we found that the H2O2, induced by NO, participated in NO-induced elevation of ascorbic acid (AsA), glutathione (GSH), and increased activities of related enzymes in the AsA-GSH cycle at low temperatures. However, the positive effect of NO was blocked by l-NAME (NOS inhibitor), PTIO, DPI (inhibitor of NADPH oxidase), and DMTU (reactive oxygen species scavenger). Taken together, our findings indicate that NO increased the low temperature tolerance of cucumber seedlings via H2O2 by improving the efficiency of the Calvin cycle, which in turn increased the carbohydrates content and accelerated the AsA-GSH cycle to enhance ROS scavenging.

Iron homeostasis regulates maturation of tomato (climacteric) and capsicum (non-climacteric) fruits

Endogenous iron quickly interchanges its oxidation states (Fe III and Fe II), which modulate production of reactive oxygen species (ROS) and nitric oxide (NO), and also serve as co-factor for various enzymes. A sharp increase in Fe(III) content in mature fruits of tomato (climacteric) is a unique observation from the present work. Iron homeostasis in non-climacteric capsicum fruits, however, accompanies a balancing of Fe(III) and Fe(II) levels during turning and ripe stages. Heme oxygenase-1 (HO-1), which mobilizes toxic heme and produces Fe(II), apparently regulates iron homeostasis in tomato. Nearly static HO-1 activity in capsicum indicates that HO-1 probably does not have a major role in iron homeostasis in non-climacteric fruit maturation. Endogenous NO content is much higher during tomato fruit maturation than in capsicum. ROS production is highest in green (unripe) tomato and capsicum fruits and it sharply declines with fruit maturation. Tomato fruits exhibit very high β-cyanoalanine synthase (β-CAS) activity in turning stage and mature fruits, highlighting its crucial role in maintaining a static level of cyanide (CN) generated as a byproduct of ethylene biosynthesis during fruit maturation. In contrast, β-CAS activity is very low in maturing capsicum fruits. Thus, a coordination between iron signal and, NO and ROS accumulation in climacteric and non-climacteric fruits is apparently perceived by hormonal (ethylene) signals getting altered differentially during fruit ripening.

Full sunlight acclimation mechanisms in Riccia discolor thalli: Assessment at morphological, anatomical, and biochemical levels

Light occupies a central position in regulating development of plants. Either little or excess of light could be harmful for plants. Since bryophytes are shade loving organisms, they must adapt to function in fluctuating light regimes. Therefore, the aim of this study was to investigate acclimatory responses of Riccia discolor thalli grown under full sunlight, and were compared with shade grown thalli (control). Length, width, and fresh mass of thallus were significantly lower (by 27, 41 and 37%, respectively) but endogenous nitric oxide content (by 81%) and nitric oxide synthase like activity (by 58%) were higher in full sunlight grown thalli than shade grown thalli. Number of rhizoids was greater in shade but length and width of rhizoids were higher (by 36 and 25%, respectively) in full sunlight grown thalli. The content of carotenoids was higher (by 34%) in full sunlight grown thalli. In full sunlight grown thalli, chloroplasts exhibited avoidance movement but in shade grown thalli they exhibited accumulation movement. Photosynthetic yields were higher in shade grown thalli. Among energy fluxes, ABS/RC did not vary but DI0/RC was higher (by 12%) in full sunlight grown thalli. Reactive oxygen species and damage were greater in full sunlight grown thalli despite enhanced levels of antioxidants i.e. superoxide dismutase (by 66%) and catalase (by 34%). Overall results suggest that full sunlight acclimation in Riccia discolor thalli occurred at various levels in which endogenous NO plays a positive role.

Salicylic acid-induced nitric oxide enhances arsenic toxicity tolerance in maize plants by upregulating the ascorbate-glutathione cycle and glyoxalase system

The role of nitric oxide (NO) in salicylic acid (SA)-induced tolerance to arsenic (As) stress in maize plants is not reported in the literature. Before starting As stress (AsS) treatments, SA (0.5 mM) was sprayed to the foliage of maize plants. Thereafter, AsV (0.1 mM as sodium hydrogen arsenate heptahydrate) stress (AsS) was initiated and during the stress period, sodium nitroprusside (SNP 0.1 mM), a NO donor, was sprayed individually or in combination with SA. Furthermore, cPTIO (0.1 mM) was also applied as a NO scavenger during the stress period. Arsenic stress led to significant reductions in plant growth, photosynthesis, water relation parameters and endogenous NO content, but it increased hydrogen peroxide, malondialdehyde, electrolyte leakage, methylglyoxal, proline, the activities of major antioxidant enzymes, and leaf and root As content. The combined treatment of SA+SNP was more effective to reverse oxidative stress related parameters and reduce the As content in both leaves and roots, with a concomitant increase in antioxidant defense system, the ascorbate-glutathione (AsA-GSH) cycle-related enzymes, glyoxalase system enzymes, plant growth, and photosynthetic traits. The beneficial effects of SA were completely abolished with cPTIO supply by blocking the NO synthesis in AsS-maize plants, indicating that NO effectively participated in SA-improved tolerance to AsS in maize plants.

The role of nitrate reductase in brassinosteroid-induced endogenous nitric oxide generation to improve cadmium stress tolerance of pepper plants by upregulating the ascorbate-glutathione cycle

A study was performed to assess if nitrate reductase (NR) participated in brassinosteroid (BR)-induced cadmium (Cd) stress tolerance primarily by accelerating the ascorbate-glutathione (AsA-GSH) cycle. Prior to initiating Cd stress (CdS), the pepper plants were sprayed with 0.5μM 24-epibrassinolide (EBR) every other day for 10 days. Thereafter the seedlings were subjected to control or CdS (0.1mM CdCl2) for four weeks. Cadmium stress decreased the plant growth related attributes, water relations as well as the activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but enhanced proline content, leaf Cd2+ content, oxidative stress-related traits, activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the activities of antioxidant defence system-related enzymes as well as NR activity and endogenous nitric oxide content. EBR reduced leaf Cd2+ content and oxidative stress-related parameters, enhanced plant growth, regulated water relations, and led to further increases in proline content, AsA-GSH cycle-related enzymes' activities, antioxidant defence system-related enzymes as well as NR activity and endogenous nitric oxide content. The EBR and the inhibitor of NR (tungstate) reversed the positive effects of EBR by reducing NO content, showing that NR could be a potential contributor of EBR-induced generation of NO which plays an effective role in tolerance to CdS in pepper plants by accelerating the AsA-GSH cycle and antioxidant enzymes.

Nitric oxide is involved in the brassinolide-induced adventitious root development in cucumber

BackgroundBrassinolide (BR), as a new type of plant hormones, is involved in the processes of plant growth and stress response. Previous studies have reported the roles of BR in regulating plant developmental processes and also response tolerance to abiotic stresses in plants. The main purpose of our study was to explore whether nitric oxide (NO) plays a role in the process of BR-induced adventitious root formation in cucumber (Cucumis sativus L.).ResultsExogenous application of 1 μM BR significantly promoted adventitious rooting, while high concentrations of BR (2–8 μM) effectively inhibited adventitious rooting. NO donor (S-nitroso-N-acerylpenicillamine, SNAP) promoted the occurrence of adventitious roots. Simultaneously, BR and SNAP applied together significantly promoted adventitious rooting and the combined effect was superior to the application of BR or SNAP alone. Moreover, NO scavenger (c-PTIO) and inhibitors (L-NAME and Tungstate) inhibited the positive effects of BR on adventitious rooting. BR at 1 μM also increased endogenous NO content, NO synthase (NOS-like) and Nitrate reductase (NR) activities, while BRz (a specific BR biosynthesis inhibitor) decreased these effects. In addition, the relative expression level of NR was up-regulated by BR and SNAP, whereas BRz down-regulated it. The application of NO inhibitor (Tungstate) in BR also inhibited the up-regulation of NR.ConclusionBR promoted the formation of adventitious roots by inducing the production of endogenous NO in cucumber.

Interactive role of exogenous 24 Epibrassinolide and endogenous NO in Brassica juncea L. under salinity stress: Evidence for NR-dependent NO biosynthesis

The present study unravels origin of nitric oxide (NO) and the interaction between 24-Epibrassinolide (EBL) and nitrate reductase (NR) for NO production in Indian mustard (Brassica juncea L.) under salinity stress. Two independent experiments were performed to check whether (i) Nitrate reductase or Nitric oxide synthase takes part in the biosynthesis of endogenous NO and (ii) EBL has any regulatory effect on NR-dependent NO biosynthesis in the alleviation of salinity stress. Results revealed that NR-inhibitor tungstate significantly (P ≤ 0.05) decreased the NR activity and endogenous NO content, while NOS inhibitor l-NAME did not influence NO biosynthesis and plant growth. Under salinity stress, inhibition in NR activity decreased the activities of antioxidant enzymes, increased H2O2, MDA, protein carbonyl content and caused DNA damage, implying that antioxidant defense might be related to NO signal. EBL supplementation enhanced the NR activity but did not influence NOS activity, suggesting that NR was involved in endogenous NO production. EBL supplementation alleviated the inhibitory effects of salinity stress and improved the plant growth by enhancing nutrients, photosynthetic pigments, compatible osmolytes, and performance of AsA-GSH cycle. It also decreased the superoxide ion accumulation, leaf epidermal damages, cell death, DNA damage, and ABA content. Comet assay revealed significant (P ≤ 0.05) enhancement in tail length and olive tail moment, while flow cytometry did not showed any significant (P ≤ 0.05) changes in genome size and ploidy level under salinity stress. Moreover, EBL supplementation increased the G6PDH activity and S-nitrosothiol content which further boosted the antioxidant responses under salinity stress. Taken together, these results suggested that NO production in mustard occurred in NR-dependent manner and EBL in association with endogenous NO activates the antioxidant system to counter salinity stress.

Nitric oxide is involved in the regulation of melatonin-induced antioxidant responses in Catharanthus roseus roots under cadmium stress

Nitric oxide (NO) and melatonin are two biostimulant molecules in plants that not only modulate the growth and development of plants but also confer enhanced tolerance to abiotic stresses. Therefore, in this study, the interactive effects of melatonin and nitric oxide (NO) on seedlings of Catharanthus roseus (L.) G. Don was evaluated under both control and conditions of stress due to Cd. Our results show that both melatonin and sodium nitroprusside (SNP, as an NO donor) significantly improved seedling growth, which was associated with the enhanced concentration of photosynthetic pigments in both the control plants and under Cd-stress conditions. Impacts of both melatonin and SNP were more pronounced in the Cd-stressed plants than control seedlings. The Cd stress increased H2O2 and lipid peroxidation levels in roots. Melatonin, as well as SNP, increased endogenous NO concentration in roots. Both melatonin and SNP enhanced the concentration of proline and the activities of antioxidant enzymes (SOD, POD, APX, CAT) and lowered H2O2 and lipid peroxidation levels in roots of C. roseus plants under Cd stress. These melatonin-induced responses in the roots were suppressed by 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO), but inhibition of melatonin biosynthesis by p–chlorophenylalanine, could not reverse the protective effects conferred by NO. These outcomes suggest that NO, as a downstream signaling molecule, is implicated in the melatonin-promoted antioxidant responses in roots of C. roseus plants.

Melatonin alleviates heat-induced damage of tomato seedlings by balancing redox homeostasis and modulating polyamine and nitric oxide biosynthesis

BackgroundMelatonin is a pleiotropic signaling molecule that plays multifarious roles in plants stress tolerance. The polyamine (PAs) metabolic pathway has been suggested to eliminate the effects of environmental stresses. However, the underlying mechanism of how melatonin and PAs function together under heat stress largely remains unknown. In this study, we investigated the potential role of melatonin in regulating PAs and nitric oxide (NO) biosynthesis, and counterbalancing oxidative damage induced by heat stress in tomato seedlings.ResultsHeat stress enhanced the overproduction of reactive oxygen species (ROS) and damaged inherent defense system, thus reduced plant growth. However, pretreatment with 100 μM melatonin (7 days) followed by exposure to heat stress (24 h) effectively reduced the oxidative stress by controlling the overaccumulation of superoxide (O2•−) and hydrogen peroxide (H2O2), lowering the lipid peroxidation content (as inferred based on malondialdehyde content) and less membrane injury index (MII). This was associated with increased the enzymatic and non-enzymatic antioxidants activities by regulating their related gene expression and modulating the ascorbate–glutathione cycle. The presence of melatonin induced respiratory burst oxidase (RBOH), heat shock transcription factors A2 (HsfA2), heat shock protein 90 (HSP90), and delta 1-pyrroline-5-carboxylate synthetase (P5CS) gene expression, which helped detoxify excess ROS via the hydrogen peroxide-mediated signaling pathway. In addition, heat stress boosted the endogenous levels of putrescine, spermidine and spermine, and increased the PAs contents, indicating higher metabolic gene expression. Moreover, melatonin-pretreated seedlings had further increased PAs levels and upregulated transcript abundance, which coincided with suppression of catabolic-related genes expression. Under heat stress, exogenous melatonin increased endogenous NO content along with nitrate reductase- and NO synthase-related activities, and expression of their related genes were also elevated.ConclusionsMelatonin pretreatment positively increased the heat tolerance of tomato seedlings by improving their antioxidant defense mechanism, inducing ascorbate–glutathione cycle, and reprogramming the PAs metabolic and NO biosynthesis pathways. These attributes facilitated the scavenging of excess ROS and increased stability of the cellular membrane, which mitigated heat-induced oxidative stress.

Nitric oxide ameliorates salinity tolerance in Pyrodwarf pear (Pyrus communis) rootstocks by regulating polyamine content

Nitric oxide (NO), an endogenous signaling molecule, is involved in various physiological processes and stress responses in plants. In the present research, Pyrodwarf pear (Pyrus communis) rootstocks were grown by nutrient solution to investigate the effects of sodium nitroprusside (SNP) application as an NO donor at 0, 0.1, 0.5, and 1 mM levels on plant stress tolerance, content of main polyamines, physiological reactions, and activity of CAT and APX enzymes under NaCl stress condition at 0, 50, 100, and 150 mM concentrations. Exogenous NO significantly increased endogenous NO content of leaves, height of the plants and relative water content (RWC) of leaves while it decreased malondialdehyde (MDA) content. NaCl stress increased the content of putrescine (Put) and spermine (Spm), and exogenous NO resulted in a further increase the content of Spm in plant leaves under NaCl stress. Pyrodwarf pear rootstocks significantly showed higher content of proline and antioxidant activity under NaCl stress, and NO treatment further increased the content of proline and antioxidant activity in plants exposed to NaCl stress. The activity of antioxidant enzymes including catalase (CAT) and peroxidase (POD) increased in plants under NaCl stress, and exogenous NO further induced those antioxidant enzyme activities. Exogenous NO resulted in the enhancement of Spm content and plants with higher Spm content exhibited high levels of antioxidant activity and proline content under NaCl stress, indicating the beneficial effects of exogenous NO under salinity conditions. NO improved the tolerance of Pyrodwarf pear rootstocks under NaCl stress by regulating the content and the ratio of polyamines.

Salicylic acid reduces the accumulation of aluminum in Panax notoginsen root cell wall pectin via the NO signaling pathway

Salicylic acid (SA) and nitric oxide (NO) are key signal molecules involved in the reduction of Al accumulation in many plants. The purpose of this study was to investigate whether they could reduce the root content of Al and/or its binding to the root cell wall (CW) fractions in Panax notoginseng, and to investigate the corresponding regulatory mechanisms. Effects of Al treatments on the endogenous SA and NO contents of P. notoginseng were detected. Exogenous SA and paclobutrazol (PAC, a SA synthesis inhibitor) treatments were conducted to test the effects on endogenous NO content; exogenous sodium nitroprusside (SNP, a NO doner) and 2-phenyl-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxyde (cPTIO, a NO synthesis inhibitor) treatments were conducted to test the effects on endogenous SA content. Pectin methyltransferase (PMT) and pectin methylesterase (PME) activity and the gene expression, pectin methylesterification degree (PMD), pectin content, and the accumulation of Al in roots were also studied under the treatments described above. Al stress induced a significant increase of the activity and expression of phenylalanine ammonia-lyase (PAL), and promoted the significant increase of endogenous SA content of P. notoginseng roots. The Al promoted accumulation of endogenous NO could be enhanced by exogenous SA treatment, but was reduced by PAC. Accumulation of endogenous NO that promoted by Al could be enhanced by exogenous SA treatment, but was reduced by PAC treatment. However, SNP or cPTIO treatments had no significant effect on the Al-induced endogenous SA content of P. notoginseng. This showed that NO acted downstream of SA signaling of P. notoginseng under Al stress. Al stress can increase the activity and genes expression of PME in the roots of P. notoginseng, reduce PMD, increase CW pectin content, and thus, the binding capacity of CW pectin to Al was enhanced in P. notoginseng roots. Exogenous SA or SNP both reduced the Al-binding capacity of root CW pectin by decreasing the pectin content, and increased the PMD by inhibiting the genes expression and activity of PME. The effects of PAC or cPTIO on the above-mentioned indicators under Al stress were opposite to that of exogenous SA or SNP treatments. The Al stress induced accumulation of pectin and the reduction of PMD in the root CW of P. notoginseng could be reversed by the treatments of exogenous SA or SNP, then the Al contents in root CW pectin were reduced. Al stress activated the endogenous SA and NO signaling pathways in P. notoginseng, and NO acted downstream of SA. It showed that the Al-activated NO-SA signaling pathway contributed in the reduction of Al binding to the root under Al stress.

Exogenous nitric oxide donor and arginine provide protection againstshort-term drought stress in wheat seedlings.

Nitric oxide (NO) is an important plant signaling molecule that has a vital role in abiotic stress tolerance. In the present study, we assesseddrought-induced (15 and 30% PEG, polyethylene glycol) damage in wheat (Triticum aestivum L. cv. Prodip) seedlings and mitigation by the synergistic effect of exogenous Arg (0.5mM l-Arginine) and an NO donor (0.5mM sodium nitroprusside, SNP). Drought stress sharply decreased the leaf relative water content (RWC) but markedly increased the proline (Pro) content in wheat seedlings. Drought stress caused overproduction of reactive oxygen species (ROS) and methylglyoxal (MG) due to the inefficiency of antioxidant enzymes, theglyoxalase system, and theascorbate-glutathione pool. However, supplementation with theNO donor and Arg enhanced the antioxidant defense system (both non-enzymatic and enzymatic components) in drought-stressed seedlings. Application of theNO donor and Arg also enhanced the glyoxalase system and reduced the MG content by increasing the activities of the glyoxalase system enzymes (Gly I and Gly II), which restored the leaf RWC and further increased the Pro content under drought stress conditions. Exogenous NO donor and Arg application enhanced the endogenous NO content, which positively regulated the antioxidant system and reduced ROS production. Thus, the present study reveals the crucial roles of Arg and NO in enhancing drought stress tolerance in wheat seedlings by upgrading their water status and reducing oxidative stress and MG toxicity.

Distribution of Endogenous NO Regulates Early Gravitropic Response and PIN2 Localization in Arabidopsis Roots.

High-resolution and automated image analysis of individual roots demonstrated that endogenous nitric oxide (NO) contribute significantly to gravitropism of Arabidopsis roots. Lowering of endogenous NO concentrations strongly reduced and even reversed gravitropism, resulting in upward bending, without affecting root growth rate. Notably, the asymmetric accumulation of NO along the upper and lower sides of roots correlated with a positive gravitropic response. Detection of NO by the specific DAF-FM DA fluorescent probe revealed that NO was higher at the lower side of horizontally-oriented roots returning to initial values 2 h after the onset of gravistimulation. We demonstrate that NO promotes plasma membrane re-localization of PIN2 in epidermal cells, which is required during the early root gravitropic response. The dynamic and asymmetric localization of both auxin and NO is critical to regulate auxin polar transport during gravitropism. Our results collectively suggest that, although auxin and NO crosstalk occurs at different levels of regulation, they converge in the regulation of PIN2 membrane trafficking in gravistimulated roots, supporting the notion that a temporally and spatially coordinated network of signal molecules could participate in the early phases of auxin polar transport during gravitropism.

Alterations in HO-1 expression, heme oxygenase activity and endogenous NO homeostasis modulate antioxidant responses of Brassica nigra against nano silver toxicity

Silver nanoparticles (AgNPs) are one of the most widely-used nanomaterials, which are toxic and can cause physiological disorders in plants. The aim of the present study was to investigate whether a possible signaling link between heme oxygenase (HO) and nitric oxide (NO) is implicated in alleviating the toxicity of AgNPs as well as AgNO3. The results showed that exposure to 400 mg L−1 of AgNPs or AgNO3 reduced the chlorophyll content and the growth parameters in Brassica nigra. Data on Ag accumulation as well as the evaluation of lipid peroxidation and the H2O2 content in roots and shoots revealed that AgNP exerted more toxicity than AgNO3. Applying AgNP and AgNO3, respectively, increased HO transcripts by 87.5 and 37.3% and elevated the endogenous NO content 51.8 and 28.5%. The application of both hematin (as an inducer of HO) and sodium nitroprusside (SNP, as a NO donor) reversed the chlorosis and improved plant growth under AgNP and Ag+ ions stresses. Hematin decreased Ag accumulation in plants, indicating that this compound triggered an avoidance mechanism. Hematin and SNP enhanced the activities of antioxidant enzymes and proline accumulation, in parallel to increasing HO transcripts and NO release levels in the roots. ZnPPIX, as the inhibitor of HO, and cPTIO, as the specific NO scavenger, differentially blocked these effects. These findings revealed for the first time that HO might confer an increased tolerance to AgNP by activating the antioxidant systems, which was partially mediated by NO signal.

Nitric oxide synthase-dependent nitric oxide production enhances chilling tolerance of walnut shoots in vitro via involvement chlorophyll fluorescence and other physiological parameter levels

Abstract The role of nitric oxide (NO) in response to chilling stress in walnut (Juglans regia L.) seedlings was investigated. Walnut shoots were treated in vitro with sodium nitroprusside (SNP), NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), NO synthase (NOS) inhibitor N-nitro-L-Arg-methyl ester (L-NAME), and nitrate reductase (NR) inhibitor sodium tungstate. Their effects on chilling tolerance, the contents of soluble sugar, proline, and total phenol, reduced glutathione (GSH) and redox state (GSH/GSSG), and the activities of NOS and NR and NO level were analyzed. The results showed that low temperatures at 4 °C induced electrolyte leakage, lipid peroxidation and photosynthetic efficiency suppression, which were dramatically alleviated by exogenous application of SNP. The levels of soluble sugar, proline, total phenol, GSH and GSH/GSSG increased evidently in the presence of SNP under chilling stress. The above protective effect of SNP could be reversed by NO scavenger PTIO or NOS inhibitor. However, NR inhibitors did not affect the protective effect of SNP. Moreover, measurements of NOS activity and NO production showed that both NOS activity and endogenous NO content increased markedly under chilling stress. The NO production was markedly reduced by NOS inhibitors, but not affected by NR inhibitors in the presence or absence of chilling stress. Taken together, these results suggest that NOS-dependent NO production may confer chilling tolerance in walnut shoots in vitro by enhancing the levels of soluble sugar, proline, total phenol, GSH and GSH/GSSG to alleviate electrolyte leakage, lipid peroxidation and photosynthetic efficiency suppression induced by chilling stress.

Endogenous Nitric Oxide Content in the Cellular Layers of the Retina

Nitric oxide is a universal molecule which regulates numerous functions in the body. In the retina, nitric oxide plays a regulatory role modulating synaptic transmission between photoreceptors and bipolar cells; it also has a toxic role and is a necessary inducer of the development of apoptosis in the outer nuclear layer and the ganglion cell layer. We report here studies of the endogenous nitric oxide concentration in vivo in the cellar layers of the retina. Nitric oxide concentrations were determined by two independent methods – using a spin trap and electron paramagnetic resonance (EPR) and an NO-sensitive microelectrode, to obtain in situ measurements in the live isolated frog retina. Significant nitric oxide concentrations were seen only in the ganglion cell layer (~0.25 μM) and the inner segments of photoreceptors (~0.6 μM). NO synthase was present in the same locations; the activity and kinetic parameters of this enzyme were also determined.

Heme Oxygenase-1 Delays Gibberellin-Induced Programmed Cell Death of Rice Aleurone Layers Subjected to Drought Stress by Interacting with Nitric Oxide.

Cereal aleurone layers undergo a gibberellin (GA)-regulated process of programmed cell death (PCD) following germination. Heme oxygenase-1 (HO-1) is known as a rate-liming enzyme in the degradation of heme to biliverdin IXα, carbon monoxide (CO), and free iron ions (Fe2+). It is a critical component in plant development and adaptation to environment stresses. Our previous studies confirmed that HO-1 inducer hematin (Ht) promotes the germination of rice seeds in drought (20% polyethylene glycol-6000, PEG) conditions, but the corresponding effects of HO-1 on the alleviation of germination-triggered PCD in GA-treated rice aleurone layers remain unknown. The present study has determined that GA co-treated with PEG results in lower HO-1 transcript levels and HO activity, which in turn results in the development of vacuoles in aleurone cells, followed by PCD. The pharmacology approach illustrated that up- or down-regulated HO-1 gene expression and HO activity delayed or accelerated GA-induced PCD. Furthermore, the application of the HO-1 inducer Ht and nitric oxide (NO) donor sodium nitroprusside (SNP) not only activated HO-1 gene expression, HO activity, and endogenous NO content, but also blocked GA-induced rapid vacuolation and accelerated aleurone layers PCD under drought stress. However, both HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) and NO scavenger 2-(4-carboxyphenyl0-4, 4,5,5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) reserved the effects of Ht and SNP on rice aleurone layer PCD under drought stress by down-regulating endogenous HO-1 and NO, respectively. The inducible effects of Ht and SNP on HO-1 gene expression, HO activity, and NO content were blocked by cPTIO. Together, these results clearly suggest that HO-1 is involved in the alleviation of GA-induced PCD of drought-triggered rice aleurone layers by associating with NO.

Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons

Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29 kDa) of HO-1. NaCl-modulated HO-1 activity correlates with endogenous NO content in the cotyledons. Increased NO accumulation by hemin treatment also correlates with enhanced activity of HO-1 in both control and NaCl stress conditions. Present work indicates that NO positively modulates HO-1 activity in sunflower seedling cotyledons. NaCl stress tends to antagonize NO action on HO-1 activity. NO (from sodium nitroprusside; SNP) is probably positively modulating HO-1 activity by way of its interaction/binding with heme group. Present work also shows enhanced NO accumulation in seedling cotyledons both in the absence or presence of iron in the growth medium, in response to NaCl stress. Thus, a probable link between endogenous NO, NaCl stress and iron-homeostasis by way of modulation of HO-1 activity at early stage of sunflower seedling growth has been proposed.