Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes

* Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components. * NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues. * Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin. * Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.     

Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings

In this study, we examined the regulation by putrescine, spermidine and spermine of nitric oxide (NO) biosynthesis in Arabidopsis thaliana seedlings. Using a fluorimetric method employing the cell-impermeable NO-binding dye diaminorhodamine-4M (DAR-4M), we observed that the polyamines (PAs) spermidine and spermine greatly increased NO release in the seedlings, whereas arginine and putrescine had little or no effect. Spermine, the most active PA, stimulated NO release with no apparent lag phase. The response was quenched by addition of 2-aminoethyl-2-thiopseudourea (AET), an inhibitor of the animal nitric oxide synthase (NOS) and plant NO biosynthesis, and by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO), an NO scavenger. By fluorescence microscopy, using the cell-permeable NO-binding dye diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM), we observed that PAs induced NO biosynthesis in specific tissues in Arabidopsis seedlings. Spermine and spermidine increased NO biosynthesis in the elongation zone of the Arabidopsis root tip and in primary leaves, especially in the veins and trichomes, while in cotyledons little or no effect of PAs beyond the endogenous levels of NO-induced fluorescence was observed. We conclude that PAs induce NO biosynthesis in plants.     

NO signalling in cytokinin-induced programmed cell death

Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and N^G.-monomethyl- l -arginine ( l -NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.     

外源一氧化氮对镉胁迫下玉米幼苗根生长及氧化伤害的影响

以玉米幼苗为材料,通过在镉处理的同时补充外源一氧化氮(NO)供体硝普钠(SNP)及其类似物[K3Fe(CN)6]、以及NO消除剂,分析NO对植物耐镉性的影响,探讨NO在植物逆境胁迫响应中的作用及其机理。结果显示:添加20μmol·L-1 SNP能显著降低镉引发的玉米幼苗根生长抑制及根尖内源镉的积累,减少电解质的渗漏以及超氧化物自由基(O2.-)和过氧化氢(H2O2)的上升幅度,抑制超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性的增加,进一步提高镉胁迫下谷胱甘肽还原酶(GR)的活性。SNP的上述效应可被NO消除剂2-(4-羧基-2-苯基)-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)所逆转,而SNP类似物K3Fe(CN)6的应用对上述反应几乎无影响,说明该反应具有NO特异性。研究表明,外源NO能够显著缓解镉胁迫对玉米幼苗生长造成的伤害,该缓解作用主要是通过降低植株体内内源镉积累和减轻镉诱发的氧化伤害来实现的。     

Cytokinin biosynthesis in cultured rootless tobacco plants

Biosynthesis of cytokinin in shoots was examined by growing rootless tobacco (Nicotiana tabacum) plants in vitro. The rootless plants were originated by culturing tobacco callus on a high cytokinin-low auxin medium to induce the formation of plantlets which were then grown on medium without exogenous cytokinin and auxin. The rootless plants supplied with [(14)C]adenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N(6)-(Delta(2)-isopentenyl)adenine, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine. The total amount of these four major cytokinins was estimated to be present at a concentration of 14 to 23 nanomoles per kilogram of rootless plant. These data indicate that adenine serves as a precursor of the purine moiety of cytokinin molecules and that the cytokinin biosynthetic sites are also located in the shoot in addition to the presumed root sites.     

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.     

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N⁶-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.     

Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production

The capacity of 12 cytokines to induce NO2- or H2O2 release from murine peritoneal macrophages was tested by using resident macrophages, or macrophages elicited with periodate, casein, or thioglycollate broth. Elevated H2O2 release in response to PMA was observed in resident macrophages after a 48-h incubation with IFN-gamma, TNF-alpha, TNF-beta, or CSF-GM. Of these, only IFN-gamma induced substantial NO2- secretion during the culture period. The cytokines inactive in both assays under the conditions tested were IL-1 beta, IL-2, IL-3, IL-4, IFN-alpha, IFN-beta, CSF-M, and transforming growth factor-beta 1. Incubation of macrophages with IFN-gamma for 48 h in the presence of LPS inhibited H2O2 production but augmented NO2- release, whereas incubation in the presence of the arginine analog NG-monomethylarginine inhibited NO2- release but not H2O2 production. Although neither TNF-alpha nor TNF-beta induced NO2- synthesis on its own, addition of either cytokine together with IFN-gamma increased macrophage NO2- production up to six-fold over that in macrophages treated with IFN-gamma alone. Moreover, IFN-alpha or IFN-beta in combination with LPS could also induce NO2- production in macrophages, as was previously reported for IFN-gamma plus LPS. These data suggest that: 1) tested as a sole agent, IFN-gamma was the only one of the 12 cytokines capable of inducing both NO2- and H2O2 release; 2) the pathways leading to secretion of H2O2 and NO2- are independent; 3) either IFN-gamma and TNF-alpha/beta or IFN-alpha/beta/gamma and LPS can interact synergistically to induce NO2- release.     

Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus) cultured cells

Programmed cell death (PCD) plays a vital role in plant development and is involved in defence mechanisms against biotic and abiotic stresses. Different forms of PCD have been described in plants on the basis of the cell organelle first involved. In sycamore ( Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin (FC) induces cell death. However, only a fraction of the dead cells shows the typical hallmarks of animal apoptosis, including cell shrinkage, chromatin condensation, DNA fragmentation and release of cytochrome c from the mitochondrion. In this work, we show that the scavenging of nitric oxide (NO), produced in the presence of FC, by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and rutin inhibits cell death without affecting DNA fragmentation and cytochrome c release. In addition, we show that FC induces a massive depolymerization of actin filaments that is prevented by the NO scavengers. Finally, the addition of actin-depolymerizing drugs induces PCD in control cells and overcomes the inhibiting effect of cPTIO on FC-induced cell death. Vice versa, the addition of actin-stabilizing drugs to FC-treated cells partially inhibits the phytotoxin-induced PCD. These results suggest that besides an apoptotic-like form of PCD involving the release of cytochrome c , FC induces at least another form of cell death, likely mediated by NO and independent of cytochrome c release, and they make it tempting to speculate that changes in actin cytoskeleton are involved in this form of PCD.     

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.     

A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation

Cytokinin is an adenine derivative plant hormone that generally regulates plant cell division and differentiation in conjunction with auxin. We report that a major cue for the negative regulation of sulfur acquisition is executed by cytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor in Arabidopsis root. We constructed a green fluorescent protein (GFP) reporter system that generally displays the expression of the high-affinity sulfate transporter SULTR1;2 in Arabidopsis roots. GFP under the control of SULTR1;2 promoter showed typical sulfur responses that correlate with the changes in SULTR1;2 mRNA levels; accumulation of GFP was induced by sulfur limitation (-S), but was repressed in the presence of reduced sulfur compounds. Among the plant hormones tested, cytokinin significantly downregulated the expression of SULTR1;2. SULTR1;1 conducting sulfate uptake in sultr1;2 mutant was similarly downregulated by cytokinin. Downregulation of SULTR1;1 and SULTR1;2 by cytokinin correlated with the decrease in sulfate uptake activities in roots. The effect of cytokinin on sulfate uptake was moderated in the cre1-1 mutant, providing genetic evidence for involvement of CRE1/WOL/AHK4 in the negative regulation of high-affinity sulfate transporters. These data demonstrated the physiological importance of the cytokinin-dependent regulatory pathway in acquisition of sulfate in roots. Our results suggested that two different modes of regulation, represented as the -S induction and the cytokinin-dependent repression of sulfate transporters, independently control the uptake of sulfate in Arabidopsis roots.     

Impact of nitrate on the structure and function of bacterial biofilm communities in pipelines used for injection of seawater into oil fields

We studied the impact of NO(3)(-) on the bacterial community composition, diversity, and function in in situ industrial, anaerobic biofilms by combining microsensor profiling, (15)N and (35)S labeling, and 16S rRNA gene-based fingerprinting. Biofilms were grown on carbon steel coupons within a system designed to treat seawater for injection into an oil field for pressurized oil recovery. NO(3)(-) was added to the seawater in an attempt to prevent bacterial H(2)S generation and microbially influenced corrosion in the field. Microprofiling of nitrogen compounds and redox potential inside the biofilms showed that the zone of highest metabolic activity was located close to the metal surface, correlating with a high bacterial abundance in this zone. Upon addition, NO(3)(-) was mainly reduced to NO(2)(-). In biofilms grown in the absence of NO(3)(-), redox potentials of <-450 mV at the metal surface suggested the release of Fe(2+). NO(3)(-) addition to previously untreated biofilms induced a decline (65%) in bacterial species richness, with Methylophaga- and Colwellia-related sequences having the highest number of obtained clones in the clone library. In contrast, no changes in community composition and potential NO(3)(-) reduction occurred upon subsequent withdrawal of NO(3)(-). Active sulfate reduction was below detection levels in all biofilms, but S isotope fractionation analysis of sulfide deposits suggested that it must have occurred either at low rates or episodically. Scanning electron microscopy revealed that pitting corrosion occurred on all coupons, independent of the treatment. However, uniform corrosion was clearly mitigated by NO(3)(-) addition.     

N9-Substituted N⁶-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N(6)-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2-C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N(6)-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N(6)-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by (1)H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ≥ 50 μM.     

An internal carbon source for improving biological nutrient removal

This study investigates the potential of mechanically disintegrated surplus activated sludge (SAS) to be used as an internal carbon source for biological nutrient removal (BNR) using two laboratory tests. In the phosphorus release test, the addition of disintegrated sludge as a carbon source was able to enhance phosphate (PO(4)-P) release by 14.9 mg l(-1) PO(4)-P when compared with acetate (7.9 mg l(-1) PO(4)-P), considering the 4.3 mg l(-1) PO(4)-P released in the control vessel, without carbon addition. Similarly, in the denitrification test, the nitrate (NO(3)-N) consumption rate was improved after the addition of disintegrated sludge (14.9 mg NO(3)-Ng(-1)VSS h(-1)) compared with acetate (7.0 mg NO(3)-Ng(-1)VSS h(-1)), taking in consideration the rate obtained in the control vessel (6.9 mg NO(3)-Ng(-1)VSS h(-1)). Two to five minutes of SAS disintegration time in the deflaker (2300-6200 kJ kg(-1) total solids) is recommended for this application.     

Dynamic compression influences interleukin-1beta-induced nitric oxide and prostaglandin E2 release by articular chondrocytes via alterations in iNOS and COX-2 expression

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.