Polyamines and Nitric Oxide Link in Regulation of Dormancy Removal and Germination of Apple (Malus domestica Borkh.) Embryos

Polyamines (PAs) belong to plant growth regulators and in complex with classical phytohormones take part in regulation of seed dormancy and germination. Although the impact of reactive oxygen (ROS) and nitrogen (RNS) species on seed germination is well described, the cross talk of PAs with ROS/RNS has never been analyzed. Due to the close connection of PAs and ethylene biosynthetic pathways to arginine (Arg)-dependent NO biosynthesis we investigated production of nitric oxide (NO), peroxynitrite (ONOO^?) and the level of O ₂ ^?? or H₂O₂ in apple embryos, germination of which was PA regulated. PAs: putrescine (Put) and spermidine (Spd) in contrast to spermine (Spm) stimulated germination of apple embryos. Among amino acids, stimulation of germination was observed in Arg and ornithine (Orn) only. Dormancy removal of embryos by PAs was associated with increased accumulation of H₂O₂ and O ₂ ^?? in embryonic axes. At the same stage of completion of sensu stricto germination the stimulatory effect of PAs (Put and Spd) and amino acids, mainly Arg and Orn, was accompanied by enhanced NO and ONOO^? production in embryonic axis. The beneficial effect of PAs (Put and Spd) and their precursors on germination of apple embryos was removed by NO scavenging, suggesting a crucial role of NO in termination of embryo germination and radicle growth. Moreover, activity of polyamine oxidase in embryo axes was greatly enhanced by embryo fumigation with NO. Our data demonstrate the interplay of RNS/ROS with PAs and point to NO action as an integrator of endogenous signals activating germination.     

Nitric oxide contributes to copper tolerance by influencing ROS metabolism in Arabidopsis

Key message

Nitric oxide improves copper tolerance via modulation of superoxide and hydrogen peroxide levels. This reflects the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.

Abstract

Copper (Cu) excess causes toxicity and one probable consequence of this is the disturbance of cell redox state maintenance, inter alia, by reactive oxygen- (ROS) and nitrogen species (RNS). The objective of this paper was to examine the role of nitric oxide (NO) in Cu stress tolerance and its relationship with ROS in Arabidopsis. In agar-grown seedlings, concentration-dependent Cu accumulation was observed. The 5 μM Cu resulted in reduced cell viability in the NO overproducing nox1 and gsnor1-3 root tips compared to the wild-type (WT). In contrast, 25 and 50 μM Cu caused higher viability in these mutants, while in the NO-lacking nia1nia2 lower viability was detected than in the WT. The exogenous NO donor enhanced cell viability and scavenging endogenous NO decreased it in Cu-exposed WT seedlings. Besides, SNP in nia1nia2 roots led to the improvement of viability. The ascorbic acid-deficient mutants (vtc2-1, vtc2-3) possessing slightly elevated ROS levels proved to be Cu sensitive, while miox4 showing decreased ROS production was more tolerant to Cu than the WT. In nox1 and gsnor1-3, Cu did not induce superoxide formation, and H₂O₂ accumulation occurred only in the case of NO deficiency. Based on these, under mild stress NO intensifies cell injury, while in the case of severe Cu excess it contributes to better viability. ROS were found to be responsible for aggravation of Cu-induced damage. NO alleviates acute Cu stress via modulation of O ₂ ^·? and H₂O₂ levels reflecting the necessity of a well-coordinated interplay between NO and ROS during stress tolerance.     

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.     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide     

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.     

Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H₂O₂ causes salt resistance via the action of plasma membrane H⁺-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H₂O₂ mediating salt resistance.Key Words: hydrogen peroxide, nitric oxide, signaling molecule, salt resistanceUnder salinity conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol.^1,2 Control of Na movement across the plasma membrane (PM) and tonoplast in order to maintain a low Na concentration in the cytoplasm is a key factor of cellular adaptation to salt stress.^3,4 Na transport across the PM is dependent on the electrochemical gradient created by the PM H⁺-ATPase.^5,6 It has been proven that the activity of the PM H⁺-ATPase is a key index of plant adaptation to salt stress.⁷ Therefore, the regulation of expression of the PM H⁺-ATPase may represent an important cellular mechanism for salt resistance. In contrast to our understanding of the regulation of PM H⁺-ATPase by other factors, the roles of NO and H₂O₂ act as signals under salt stress have been less known.Previous studies have shown that both NO and H₂O₂ function as stress signals in plants, mediating a range of resistance mechanisms in plants under stress conditions.^8–10 We have previously shown that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H⁺-ATPase activity in calluses from reed.¹¹ Although NO acts as a signal molecule under salt stress and induces salt resistance by increasing PM H⁺-ATPase activity, our research results also indicated NO can not activate purified PM H⁺-ATPase activity, at least in vitro. Subsequently, we set out to find the other signal molecules and factors between NO and PM H⁺-ATPase activity. Since our studies have indicated that NO can not induce salt resistance directly, what roles dose it play in salt resistance in tolerant cells under salt stress? We initially hypothesized ABA or H₂O₂ might be downstream signal molecules to regulate the activity of PM H⁺-ATPase. Further results indicated H₂O₂ content increased greatly under salt stress. Since H₂O₂ might be the candidate downstream signal molecule, we tested PM H⁺-ATPase activity and K to Na ratio in calluses by adding H₂O₂. The results suggested that H₂O₂ inducing an increased PM H⁺-ATPase activity resulted in an increased K to Na ratio. Summing up this new assay that allows us to speculate NO maybe regulate the H₂O₂ generation.Since H₂O₂ is involved in downstream signal molecule of NO, PM NADPH oxidase, the main source of H₂O₂ production, might be the regulated target of NO. We took a pharmacological approach to examine the speculation. The results indicated that PM NADPH oxidase is required for H₂O₂ accumulation and PM NADPH oxidase activity could attribute to NO in calluses under salt stress. These results also raised another question regarding what concentrations of NO can induce such effects. In our experiments, NO content was induced 1.6 times higher than the control values under salt treatment. We speculated there exists an effective balance point in NO signal system similar to previous reports by Delledonne et al.¹² in disease resistance.Further research work is required to decipher the mechanism through which NO and H₂O₂ acts and how K and Na elements uptake might be connected with salt resistance. We would like to propose a simple testable model that accounts for the results reported in this paper (Fig. 1). According to our model, H₂O₂ rather than NO is the major signaling molecular that mediated directly PM H⁺-ATPase under salt stress. Normally, NO generated from nitric oxide synthase (NOS) acts as a signal molecule to regulate other mechanisms. Under salt stress, accumulated NO activates PM NADPH oxidase activity. Then, a number of H₂O₂ is produced from PM NADPH oxidase. The PM H⁺-ATPase is activated greatly by the accumulated H₂O₂. Eventually, the transmembrane electrochemical gradient is created and K to Na ratio increases. The model we have proposed here is testable and should provide further insights into salt resistance mechanism regulated by NO and H₂O₂ signal molecules.Open in a separate windowFigure 1Hypothetical model for the potential function of NO and H₂O₂ as signaling molecules in inducing salt resistance. Salt stress activates a signal transduction cascade that leads to the increased activity of PM H⁺-ATPase, whose expression produces salt resistance. NO is generated by NOS, and H₂O₂ is produced by NADPH oxidase attributed to NO. The activity of PM H⁺-ATPase is regulated by H₂O₂ directly under salt stress. The model is based on the recent results in calluses from P. euphratica¹² and those previously reported on the NO function in reed.¹¹Research on roles of NO and H₂O₂ under stress conditions in plant is advancing rapidly. Further analysis of salt resistance mechanism with novel technology will certainly increase our knowledge in this field.     

Energy transduction efficiencies in nitrogenous oxide respirations of Azospirillum brasilense Sp7

For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H⁺-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth yields (Y _s ^max -values) were essentially the same with O₂ or N₂O but were 1/3 and 2/3 lower with NO ₂ ^- or NO ₃ ^- , respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N₂-fixing conditions drastically reduced the Y _s ^max -values in the N₂O and O₂-respiring cells. In the H⁺-translocation measurements, the /oxidant ratios were 5.6 for O₂→H₂O, 2.5–2.8 for NO ₃ ^- →NO ₂ ^- , 2.2 for NO ₂ ^- →N₂O and 3.1 for N₂O→N₂ respirations when the cells were preincubated with valinomycin and K⁺. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP⁺) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H⁺-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore, nitrate and nitrite respirations were accompanied by a H⁺-uptake . Any significant H⁺-translocation could not be detected in N₂O- and O₂-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal molar growth yield determinations and the absence of any H⁺-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of respiration. The low H⁺-extrusion values for N₂O-respiration compared to O₂-respiration in cells treated with valinomycin plus TPMP⁺ are, however, not in accord with such an interpretation.     

Hydrogen peroxide- and nitric oxide-induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants

We tested whether pre-treatments of roots with H₂O₂ (10 mM for 8 h) or sodium nitroprusside (SNP; 100 μM for 48 h), a donor of NO, could induce prime antioxidant defense responses in the leaves of citrus plants grown in the absence or presence of 150 mM NaCl for 16 d. Both root pre-treatments increased leaf superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, and induced related-isoform(s) expression under non-NaCl-stress conditions. When followed by salinity, certain enzymatic activities also exhibited an up-regulation in response to H₂O₂ or SNP pre-exposure. An NaCl-stress-provoked decrease in the ascorbate redox state was partially prevented by both pre-treatments, whereas the glutathione redox state under normal and NaCl-stress conditions was increased by SNP. Real-time imaging of NO production was found in vascular tissues and epidermal cells. Furthermore, NaCl-induced inhibition in OH scavenging activity and promotion of OH-mediated DNA strand cleavage was partially prevented by SNP. Moreover, NaCl-dependent protein oxidation (carbonylation) was totally reversed by both pre-treatments as revealed by quantitative assay and protein blotting analysis. These results provide strong evidence that H₂O₂ and NO elicit long-lasting systemic primer-like antioxidant activity in citrus plants under physiological and NaCl-stress conditions.     

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.     

Effects of nitric oxide and Fe supply on recovery of Fe deficiency induced chlorosis in peanut plants

The effects of nitric oxide (NO) and/or iron (Fe) supplied to Fe deficient plants have been investigated in peanut (Arachis hypogaea L.) grown in Hoagland nutrient solution with or without Fe. Two weeks after Fe deprivation, recovery was induced by addition of 250 μM sodium nitroprusside (SNP, a NO donor) and/or 50 μM Fe (Fe-EDTA) to the Fe deprived (-Fe) nutrient solution. Activities of antioxidant enzymes, leaf chlorophyll (Chl), and active Fe content decreased, whereas activities of H⁺-ATPase, ferric-chelate reductase (FCR), nitrate reductase, and nitric oxide synthase and NO production increased in Fe deficient plants, consequently an Fe chlorosis symptom appeared obviously. In contrast, these symptoms disappeared gradually after two weeks with NO and/or Fe supply, which caused an increases in leaf Chl and active Fe content, especially following by co-treatment with NO and Fe to values found in Fe sufficient plants. Increased activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and decreased accumulation of reactive oxygen species (H₂O₂, O ₂ ^?? ) and malondialdehyde enhanced the ability of resistance to oxidative stress. Supplied NO alone had the obvious effect on increased NO production and on activity of H⁺-ATPase and FCR, whereas root length and root/shoot ratio were most effectively increased by Fe supplied alone. Co-treatment with NO and Fe did the best effects on recovery peanut chlorosis symptoms by significantly increased Chl and available Fe content and adjusted distribution of Fe and other mineral elements (Ca, Mg, and Zn) in both leaves and roots.     

Nitric oxide promoted rhizome induction in Cymbidium shoot buds under magnesium deficiency

Cymbidium shoot buds grown under Mg²⁺ deficiency without naphthalene acetic acid (NAA) showed knotted appearance. Ultrastructure of the cortical cells showed a progressive disorganization and disintegration of chloroplast membranes. The growth of shoots was resumed with the addition of 10 μM NAA. Specific NO scavenger, cPTIO induced deformation in shoot growth in 80 % of cultures. In longitudinal sections of shoots treated with cPTIO, depositions of densely stained particles in cells were observed. These inhibitory responses of cPTIO were ameliorated by 10 μM NAA. The NO donor, sodium nitroprusside (SNP), treated shoot buds displayed rapid senescence followed by necrosis of leaves. Ultrastructure of cortical cells at this stage revealed the endocytosis of mitochondria along with membrane bound cytoplasmic inclusions in the vacuole. A sharp increase in H₂O₂ generation was observed with a little change in the activity of antioxidant glutathione disulfide (GSSG), suggesting NO mediated oxidative stress. Surprisingly, after 4 weeks these necrotic shoots were converted into a globular, embryo like shoot tip with numerous structures termed here as ‘neomorph’ in its base. Neomorphs were different from protocorm like bodies both anatomically and morphologically. Ultrastructure of the rhizome tip exhibited numerous amyloplast and round mitochondria. At this stage, the generation of high rate of H₂O₂ was masked by GSSG, and the generation of GSSG was proportional with the concentrations of SNP, and not observed in the control (without SNP). The neomorphs were further sub-cultured to medium with different concentrations of SNP or cPTIO. After 4 weeks of culture, only the neomorphs sub-cultured on medium with SNP developed into shoots and approximately ten shoots were observed to emerge from the axils of each rhizome. Ultrastructure of cells of regenerating green neomorphs showed different shapes of mitochondria and chloroplasts and presence of active dictyosomes. The obtained shoots subjected to the acclimatization in polyhouse, expressed good growth with 85 % survival. Therefore it is reasonable to suggest that the process of de-differentiation and re-differentiation leading to rhizome formation under the condition of Mg²⁺ deficiency is NO mediated.     

Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin

The aim of this work was to assess the capacities of some ^·NO-donors to release ^·NO, and consequently NOx in aerobic medium, or to give peroxynitrite. The method was based on the differential reactivity of serotonin (5-HT) with either NO_x or peroxynitrite, leading in phosphate-buffered solutions to 4-nitroso- and 4-nitro-5-HT formation, respectively. Yields and formation rates of 5-HT derivatives with ^·NO-donor were compared to those obtained with authentic ^·NO or peroxynitrite in similar conditions. Aside from the capacity of diazenium diolates (SPER/NO and DEA/NO) to release ^·NO spontaneously, converting 5-HT exclusively to 4-nitroso-5-HT, all other ^·NO donors must undergo redox reactions to produce ^·NO. S-nitrosoglutathione (GSNO) and sodium nitroprus-side (SNP) modified 5-HT only in the presence of Cu²⁺, GSNO yielding 6 times more 4-nitroso-5-HT than SNP. Furthermore, in the presence of Cu⁺, the yield of ^·NO-release from GSNO was 45%. The molsidomine metabolite (SIN-1), which was presumed to release both ^·NO and O2/·- at pH 7.4, reacted with 5-HT differently, depending on the presence of reductant or oxidant. Under aerobic conditions, SIN-1 acted predominantly as a 5-HT oxidant and also as a poor ^·NO and peroxynitrite donor (15% yield of ^·NO-release and 14 % yield of peroxynitrite formation). The strong oxidant Cu²⁺, even in the presence of air oxygen, accelerated oxidation and increased ^·NO release from SIN-1 up to 86%. Only a small part of SIN-1 gave simultaneously ^·NO and O2/·- able to link together to give peroxynitrite, but other oxidants could enhance ^·NO release from SIN-1.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 NO和H₂O₂是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H₂O₂在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H₂O₂的变化, 以及外源NO和H₂O₂诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L^–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H₂O₂含量。施加0.25 mmol·L^–1外源NO供体亚硝基铁氰化钠(Na₂[Fe(CN)₅NO]·2H₂O, sodium nitroprusside, SNP)和0.1 mmol·L^–1H₂O₂, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L^–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C₁₄H₁₆N₂O₄·K, carboxy-PTIO, cPTIO)和150 U·mL^–1 H₂O₂清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H₂O₂的积累, 而外源H₂O₂对根尖NO的含量无显著影响。这表明NO和H₂O₂是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H₂O₂的形成, 进而诱导大豆根尖及RBCs的耐铝反应。     

Performance of wave function and density functional methods for water hydrogen bond spin–spin coupling constants

Spin–spin coupling constants in water monomer and dimer have been calculated using several wave function and density functional-based methods. CCSD, MCSCF, and SOPPA wave functions methods yield similar results, specially when an additive approach is used with the MCSCF. Several functionals have been used to analyze their performance with the Jacob’s ladder and a set of functionals with different HF exchange were tested. Functionals with large HF exchange appropriately predict ¹ J _{O H} , ² J _{H H} and ^2h J _{O O} couplings, while ^1h J _{O H} is better calculated with functionals that include a reduced fraction of HF exchange. Accurate functionals for ¹ J _{O H} and ² J _{H H} have been tested in a tetramer water model. The hydrogen bond effects on these intramolecular couplings are additive when they are calculated by SOPPA(CCSD) wave function and DFT methods.

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Graphical Abstract Evaluation of the additive effect of the hydrogen bond on spin-spin coupling constants of water using WF and DFT methods.

     

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

Background and Aims

Nitric oxide (NO) has been demonstrated to stimulate the activity of nitrate reductase (NR) in plant roots supplied with a low level of nitrate, and to affect proteins differently, depending on the ratio of NO to the level of protein. Nitrate has been suggested to regulate the level of NO in plants. This present study examined interactive effects of NO and nitrate level on NR activity in roots of tomato (Solanum lycocarpum).

Methods

NR activity, mRNA level of NR gene and concentration of NR protein in roots fed with 0·5 mm or 5 mm nitrate and treated with the NO donors, sodium nitroprusside (SNP) and diethylamine NONOate sodium (NONOate), and the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), were measured in 25-d-old seedlings.

Key Results

Addition of SNP and NONOate enhanced but cPTIO decreased NR activity in the roots fed with 0·5 mm nitrate. The opposite was true for the roots fed with 5 mm nitrate. However, the mRNA level of the NR gene and the protein concentration of NR enzyme in the roots were not affected by SNP treatment, irrespective of nitrate pre-treatment. Nevertheless, a low rate of NO gas increased while cPTIO decreased the NR activities of the enzyme extracts from the roots at both nitrate levels. Increasing the rate of NO gas further increased NR activity in the enzyme extracts of the roots fed with 0·5 mm nitrate but decreased it when 5 mm nitrate was supplied. Interestingly, the stimulative effect of NO gas on NR activity could be reversed by NO removal through N₂ flushing in the enzyme extracts from the roots fed with 0·5 mm nitrate but not from those with 5 mm nitrate.

Conclusions

The effects of NO on NR activity in tomato roots depend on levels of nitrate supply, and probably result from direct interactions between NO and NR protein.Key words: Nitric oxide, nitrate, nitrate reductase, post-translational regulation, tomato, Solanum lycocarpum     

Formation of inorganic protonic-acid polymer via inorganic-organic hybridization: Synthesis and characterization of polymerizable olefinic organosilyl derivatives of mono-lacunary Dawson polyoxometalate

A novel polymerizable organosilyl-modified Dawson-type polyoxometalate (POM) [α₂-P₂W₁₇O₆₁{CH₂C(CH₃)COO(CH₂)₃Si}₂O]⁶⁻ (1) was synthesized as both salt (Me₂NH₂-1) and H⁺ form (H-1). They were characterized with complete elemental analysis, thermogravimetric and differential thermal analysis (TG/DTA), FTIR, (¹H, ¹³C, ²⁹Si, ³¹P and ¹⁸³W) NMR and n-butylamine titration method. H-1 was immobilized to a polymer network through free radical copolymerization with methyl methacrylate (MMA). The acidities of H-1 and hybrid copolymer (H-1-co-MMA) were evaluated using the Hammett indicators (dicinnamalacetone and benzalacetophenone; pK_a values of the protonated indicators are −3.0 and −5.6, respectively). The pK_a value of H-1 was estimated as that between −3.0 and −5.6 in CH₃CN solution and H-1 was immobilized in H-1-co-MMA with the original acidity being retained. Glass transition point (T_g) and molecular weight distribution of H-1-co-MMA were affected by the used amount of H-1 because of the cross-linking effect of H-1.