Influence of environmental factors on the generation of nitric oxide in the roots of etiolated pea seedlings

The article studies the nitric oxide (NO) levels in the roots of etiolated seedlings of garden peas (Pisum sativum L.) using the DAF-2DA fluorescent probe and fluorescent microscopy. Cross sections of roots of 100-150 microm (the site of a root which is 10-15 mm from the apex) are analyzed. It is shown that the level of NO in the roots after 24 h increased by more than a factor of 2 in the versions with NaNO2 and sodium nitroprusside. At feeding the seedlings with KNO3, a peak in the accumulation of NO in the roots (twofold increase) was observed after 30 min. Fertilizing seedlings with L-arginine (2 mM) increased the intensity of the fluorescence of the root sections by more than a factor of 2. The inoculation of seedlings of rhizobia (Rhizobium leguminosarum by. viceae) contributed to the reduction of NO on the background of the control (H20) and sodium nitroprusside and nitrogen compounds. Scavengers of NO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), hemoglobin) and inhibitors of nitrate reductase and animal NO synthase (sodium tungstate and aminoguanidine hydrochloride) reduced the level of NO in the roots. The results are discussed in relation to the role of NO in plants under the influence of biotic and abiotic factors.     

Initiation of nitric oxide (NO) synthesis in roots of etiolated seedlings of pea (Pisum sativum L.) under the influence of nitrogen-containing compounds

The level of nitric oxide (NO) in roots of 2-day-old etiolated pea (Pisum sativum L.) seedlings was investigated by fluorescence microscopy using the fluorescent probe 4,5-diaminofluorescein diacetate. Segments representing transversal (cross) cuts of the roots having thickness of 100 to 150 μm (a segment of the root located 10 to 15 mm from the apex) were analyzed. A substantial concentration of NO in the roots was registered when the seedlings were grown in water (control). Addition of 4 mM sodium nitroprusside, 20 mM KNO₃, 2 mM NaNO₂, 2 mM L-arginine into the growth medium increased NO concentration with respect to the control by 1.7- to 2.3-fold. Inhibitors of animal NO-synthase — 1 mM Nω-nitro-L-arginine methyl ester hydrochloride and 1 mM aminoguanidine hydrochloride — reduced the intensity of fluorescence in the root segments in the presence of all the studied compounds. In medium with KNO₃, the inhibitor of nitrate reductase ?150 μM sodium tungstate -lowered the fluorescence intensity by 60%. Scavengers of nitric oxide — 100 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 4 μM hemoglobin — lowered NO concentration in all the studied variants. Potassium ferrocyanide (4 mM) as the inactive analog of sodium nitroprusside inhibited generation of NO. These results are discussed regarding possible pathways of NO synthesis in plants.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.     

Nitric oxide affecting root growth,lignification and related enzymes in soybean seedlings

This study analyzed the involvement of nitric oxide (NO) in the root lignification of soybean seedlings. To this end, changes in root cell viability; phenylalanine ammonia-lyase (PAL) and soluble and cell wall bound peroxidase (POD) activities and lignin and hydrogen peroxide (H₂O₂) contents of soybean roots treated with the NO-donor sodium nitroprusside (SNP) and its relationships with root growth were evaluated. Seedlings were cultivated in a nutrient solution supplemented with 5 to 1,000 μM SNP for 24 h. At an extremely low concentration (5 μM), SNP induced root growth and increased lignification and activities of related enzymes (PAL and cell wall-bound POD). At a high concentration (1,000 μM), SNP reduced root growth and lignification (PAL activity and H₂O₂ and lignin contents) and caused a loss of cell viability. Application of potassium ferrocyanide (an analog of SNP that cannot release NO) and PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoleline-1-oxyl-3-oxide, a scavenger of NO) revealed that the inhibitory/stimulatory effects on root lignification may be due to NO itself. These results indicate that NO, depending on its concentration, may act as a stress factor, due to its toxic action, or as a signal molecule, inducing soybean root growth and lignification.     

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.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Stress responses of wheat leaves to dehydration: Participation of endogenous NO and effect of sodium nitroprusside

Dynamics of endogenous NO in the leaves of 7-day-old seedlings of spring wheat (Triticum aestivum L., cv. Debyut) and the effect of exogenous NO donor (sodium nitroprusside, SNP) on the development of oxidative stress and activity of antioxidant enzymes in the leaves under water deficiency were investigated. Quick and phasic accumulation of NO in the leaves was observed under growing dehydration (0–3 h) and subsequent rehydration (0–3 h), which points to identical response of NO signal system to opposite changes in the water status of plants. A decrease in relative turgidity of tissues brought about accumulation of H₂O₂ and MDA therein. Protective effect of NO donor infiltrated in the leaves was associated with an elevation of ascorbate peroxidase and catalase activities and suppression of lipid peroxidation upon dehydration. Pretreatment with SNP (50–250 μM) induced the elevation of NO level in the leaves both before action of the stress agent and in the beginning (0–30 min) of dehydration. The obtained results suggest that brief increase in endogenous NO is necessary for triggering protective and adaptive responses in wheat plants during the development of water deficiency.     

Level nitric oxide (NO) and growth of roots of etiolated pea seedlings

Data regarding the interrelation of nitric oxide (NO) content in roots of 3-day-old etiolated pea seedlings and their growth under different concentrations of N-containing compounds were obtained. The concentration of exogenous compounds (sodium nitroprusside SNP, KNO₃, NaNO₂, L-arginine) rendering an inhibiting effect on the growth of roots were established, and the NO content in roots was determined at these concentration. It was shown that the inhibition of growth and highest NO content in the roots was determined with SNP (4 mM) and NaNO₂ (2 mM) during 24 h exposition of seedlings. This dependence was not established in combinations with KNO₃ (20 mM) and L-arginine (4 mM). We established that a NO scavenger, hemoglobin (4 μM), fully or partially removed the toxic effect of SNP, nitrate, and nitrite on growth. The effect of NO on the growth and the participation of N-containing compounds in generation of NO in roots of pea seedlings is discussed.     

Induction of heat resistance in wheat seedlings by exogenous calcium,hydrogen peroxide,and nitric oxide donor: functional interaction of signal mediators

Functional interactions of calcium ions, hydrogen peroxide, and nitric oxide as signal mediators in root cells of wheat (Triticum aestivum L.) seedlings upon induction of their heat resistance was studied with use of inhibitor-based analysis. Treatment of the seedlings with hydrogen peroxide or a combination of calcium chloride with ionophore A23187 significantly increased their content of nitric oxide, which peaked 0.5–1 h after the start of the treatment. CaCl₂ or exogenous NO donor (sodium nitroprusside, SNP) transitorily increased the hydrogen peroxide level in the roots. Seedlings pretreatments with calcium chelator (EGTA), blocker of Ca²⁺ channels (LaCl₃), inhibitor of phospholipase C (neomycin), or antagonist of cyclic adenosine-5'-diphosphatribose formation (nicotinamide) more or less prevented the rise in the nitric oxide content in roots caused by exogenous H₂O₂; the SNP-induced rise in hydrogen peroxide was also damped down. However, the seedlings pretreatment with antioxidants ionol or dimethylthiourea did not hinder the increase in the NO level, which was caused by exogenous Ca²⁺. The inhibitors of NO synthase (N^G-nitro-L-arginine methyl ester, L-NAME) or nitrate reductase (sodium tungstate) did not interfere in the accumulation of H₂O₂ in root tissues stimulated by exogenous calcium. Calcium antagonists diminished the seedlings heat resistance increased by hydrogen peroxide or SNP. Antioxidants and inhibitors of NO synthase or nitrate reductase weakened the calcium-stimulated enhancement in the seedlings heat resistance. It was concluded that calcium may activate NO- and H₂O₂-generating enzymatic systems as well as participate in the transduction of signals of these mediators into genetic apparatus and in the formation of physiological reactions underlying the enhanced heat resistance.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Effects of the nitric oxide donor sodium nitroprusside on antioxidant enzymes in wheat seedling roots under nickel stress

The inhibitory effect of nickel on the growth of wheat (Triticum aestivum L.) seedlings and the alleviation of nickel toxicity by nitric oxide (NO) were investigated. Nickel (Ni) at 100 μM caused striking reduction in seedling growth and significant overproduction of MDA and H₂O₂ in the roots. Supplementation with NO donor sodium nitroprusside (SNP) could significantly reverse the inhibitory effect of nickel in a dose-dependent manner. K₃Fe(CN)₆, a SNP analogue, which does not release NO, had no ameliorative effect on Ni toxicity in wheat.. In addition, application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, could dramatically counteract the stimulatory effects of SNP on the growth of wheat seedling roots under Ni stress, confirming that NO rather than other compounds derived from SNP was responsible for the alleviating effect of Ni toxicity. Further results showed that SNP enhanced the activities of guaiacol peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1..1..5.1..1), glutathione reductase (GR, EC 1.6.4.2), and glutathione S-transferase (GST, EC 2.5.1.18) in wheat seedling roots under nickel stress, while no significant difference in the activity of catalase (CAT, EC 1.11.1.6) in wheat roots supplemented with SNP or without it was observed. These results clearly indicate that NO has a protective role in Ni-induced oxidative damage through modulation of antioxidant enzymes.     

Nitric oxide promotes in vitro organogenesis in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

The effects of nitric oxide (NO) on caulogenesis, shoot organogenesis and rhizogenesis from hypocotyl explants of Linum usitatissimum were investigated. Exogenously supplied NO donors, 5 μM sodium nitroprusside (SNP), 2 μM S-nitroso-N-acetylpenicillamine (SNAP) and 2 μM 3-morpholinosydnonimine (SIN-1), significantly promoted shoot differentiation from the hypocotyl explants of L. usitatissimum excised from its in vitro raised seedlings. Potassium ferrocyanide, a structural analogue of SNP, lacking NO group, did not promote shoot organogenesis. Likewise, products of NO, \textNO₂ ^- {\text{NO}}_{2}^{ - } and \textNO₃ ^- {\text{NO}}_{3}^{ - } supplied as 5 μM NaNO₂ and 5 μM NaNO₃ did not enhance shoot differentiation. Another source of NO, a mixture of sodium nitrite (SN) provided along with ascorbic acid (AsA), also caused significant promotion in the average number of shoots per responding explant. SNP also augmented the rhizogenic response of the microshoots in terms of percentage of responding explants, number of roots per responding explant and average root length. The NO scavengers, 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimideazoline-1-oxyl-3-oxide (cPTIO) or methylene blue (MB), provided along with SNP, SNAP, SIN-1 or SN + AsA, at concentrations equimolar to the optimum concentration of the donors, reversed the promotory influence, thereby, confirming the role of NO in promotion of in vitro morphogenesis. However, NO scavengers individually did not affect the observed morphogenic processes. Morphological and histological studies of hypocotyl segments cultured on BM or BM + SNP for 4, 8 and 12 days demonstrated that SNP enhanced shoot differentiation by inducing a higher number of shoot primordia, each of which develops into a single shoot.     

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1 h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H₂O₂), superoxide (O₂^•−) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.     

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H₂O₂)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Sodium nitroprusside mediates seedling development and attenuation of oxidative stresses in Chinese cabbage

Nitric oxide (NO) has been shown to be involved in diverse physiological processes in microbes, animals and plants. In this study, the involvement of NO in the development and possible roles in oxidative stress protection of Chinese cabbage (Brassica rapa subsp. pekinensis cv. Samrack-ulgari) seedlings were investigated. Exogenous application of sodium nitroprusside (SNP) retarded root elongation, while increasing lateral root formation of Chinese cabbage. Plants showed no signs of external stress due to SNP application in true leaves. Cotyledons of 3-week-old Chinese cabbage plants were found to be highly sensitive to SNP application. Treated cotyledons displayed rapid tissue collapse and associated cell death. Although SNP application reduced root growth under normal growth conditions, it also enhanced methyl viologen (MV)-mediated oxidative stress tolerance. Analysis of SNP application to Chinese cabbage leaf disks, revealed SNP-induced tolerance against oxidative stresses by MV and H₂O₂, and evidence includes prevention of chlorophyll loss, superoxide anion (O₂ ⁻) accumulation and lipid peroxidation. This report supports a role for nitric oxide in modulating early seedling development, programmed cell death and stress tolerance in Chinese cabbage.     

Effects of root and foliar applications of exogenous NO on alleviating cadmium toxicity in lettuce seedlings

The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) toxicity in lettuce seedlings were studied. SNP was added into hydroponic systems or sprayed directly on the leaves of plants grown with and without Cd. Excess supply of Cd (100 μM) caused growth inhibition, dramatically increased Cd accumulation in both leaves and roots, and inhibited the absorption of Ca, Mg, Fe and Cu. Excess Cd also decreased activities of superoxide dismutase peroxidase and catalase in leaves and roots, and increased the accumulation of superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Root or foliar applications of exogenous NO alleviated Cd-induced growth suppression, especially root application of 250 μM SNP and foliar addition of 500 μM SNP. Addition of SNP promoted the chlorophyll synthesis suggesting that the photosynthesis was up-regulated. Exogenous NO increased Cd-decreased activities of antioxidant enzymes and markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation. Moreover, the absorption of Ca, Mg, Fe and Cu was increased, indicating that exogenous NO stimulated H⁺-ATPase activity to promote sequestration or uptake of ions. In addition, exogenous NO also inhibited Cd transfer from roots to shoots, which may indicate that Cd retention in roots induced by NO plays a significant role in Cd tolerance in lettuce seedlings. These data suggest that under Cd stress, exogenous NO improves photosynthesis by increasing chlorophyll synthesis, protects lettuce seedlings against oxidative damage by scavenging ROS, helps to maintain the uptake of nutrient elements, and inhibits Cd transferred to shoots effectively.     

Energy exchange and ultrastructure of plant cells under generation of nitric oxide

The effects of nitric oxide (NO) on oxygen consumption, heat generation, and cell ultrastructure were investigated in the seedlings of wheat (Triticum aestivum L.). The experiments were conducted with the excised roots of 5-day-old seedlings grown in the solution of CaCl₂ (2.5 × 10⁻⁴ M). The source of NO was NaNO₂ (5 × 10⁻³ M) where the roots were incubated. Production of NO was determined by means of EPR, respiration — gasometrically, heat generation — using a microcalorimeter. The results showed that NO was formed in the presence of NaNO₂. This was accompanied by a decrease in the respiration rate by about 30%, which lasted for 5–6 h. Apparently, NO inhibited mitochondrial oxidation because stimulation of oxygen consumption induced by 2,4-DNP was completely removed in the presence of NaNO₂. When the cells were affected by succinic acid in the presence of NaNO₂, respiration was strongly inhibited. The effects of succinic acid and NaNO₂ were negated by ascorbic acid. A decrease in the rate of respiration was accompanied by a reduction in heat generation. Moreover, the efflux of potassium ions to the root incubation medium was stimulated, which may point to changes in ionic membrane permeability. The observed changes in energy exchange were accompanied by disturbances in the cell ultrastructure. Nitric oxide induced a clarification of the mitochondrial matrix and a reduction in the number of cristae. It was concluded that NO excess in plant tissues brings about a deceleration of energy exchange, disturbance of the ultrastructural organization, and cell death.