NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hypersensitive response (HR) during plant-pathogen interactions. Here we demonstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Arabidopsis cells independently of reactive oxygen species (ROS). We also provide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of PCD of mammalian cells: NO induced chromatin condensation and caspase-like activity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, blocked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-permeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cell death or potentiate NO-induced cell death. This suggests that cGMP synthesis is required but not sufficient for NO-induced cell death in Arabidopsis. In-gel protein kinase assays showed that NO activates a potential mitogen-activated protein kinase (MAPK), although a specific inhibitor of mammalian MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Effects of exogenous nitric oxide donor on antioxidant metabolism in wheat leaves under aluminum stress

With the enhancement of aluminum stress, the content of chlorophyll in wheat seedlings (Triticum aestivum L.) decreased dramatically. At 0.2 mM AlCl₃, the chlorophyll content halved. The aluminum-induced decrease in chlorophyll content could be alleviated by exogenous nitric oxide donor, sodium nitroprusside (SNP) in a dose-dependent manner. Treatment with SNP dramatically promoted the activities of superoxide dismutase, catalase, ascorbate peroxidase and increased the proline content, whereas it decreased hydrogen peroxide and malondialdehyde and maintained the level of soluble protein as compared with water controls. Therefore, NO donor enhanced the antioxidant capacity in wheat seedlings under aluminum stress.     

外源-氧化氮供体硝普钠对黑麦草幼苗耐碱性的影响

采用营养液砂培方法,研究外源一氧化氮(NO)供体硝普钠(SNP)对NaHCO3胁迫下黑麦草幼苗生长、活性氧代谢和渗透溶质积累的影响.结果表明:60 μmol·L-1 SNP能够缓解100 mmol·L-1NaHCO3胁迫对黑麦草幼苗生长的抑制作用,减缓NaHCO3胁迫导致的叶片O2产生速率、H2O2和丙二醛(MDA)含量的增加,提高NaHCO3胁迫下幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、质膜H+-ATP酶的活性和谷胱甘肽(GSH)、可溶性糖、可溶性蛋白质和脯氨酸的含量及K+/Na+比,降低过氧化氢酶(CAT)活性和抗坏血酸(AsA)含量,对游离氨基酸含量影响不大.上述结果表明,NO可能通过激活抗氧化系统活性、促进渗透溶质积累和改善Na+、K+平衡等缓解碱胁迫对幼苗的伤害,从而提高黑麦草的耐碱性.     

Serotonin binds to purified neuronal nitric oxide synthase: A possible explanation for ROS production induced by 5HT in the presence of nNOS

Serotonin (5HT) was shown to induce in vitro the production of ROS in the presence of neuronal nitric oxide synthase (nNOS) in addition to the basal NO^? formation. With the aim of understanding this mechanism, this study investigated the potential binding of 5HT to nNOS. By using [³H]5HT, it is reported here that 5HT binds to nNOS, but only when the enzyme is active and in a superoxide-dependent manner. This binding is prevented by DPI but not by L-NAME. The formation of 5HT-nNOS complex was shown to be very well correlated with the production of ROS by 5HT in the presence of nNOS. A mechanism involving nNOS only in its initial step is proposed to explain both the formation of 5HT-nNOS complex and the production of ROS observed in the presence of nNOS and 5HT.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

一氧化氮对不同耐旱性杨树幼苗抗旱性的影响

以乡土树种小青杨(Populus pseudo-simonii Kitag)和速生品种欧美杨107杨(Populus × euramericana cv."74/76")幼苗为试验材料,在水分胁迫条件下研究不同浓度的外源NO供体硝普纳(sodium nitroprusside, SNP)对2种杨树抗旱性的影响.结果表明,经不同浓度SNP处理后,2种杨树叶片水势对干旱胁迫的敏感程度下降,气孔导度降低,蒸腾作用减弱,叶片含水量升高,保水能力增强.同时,SNP处理能提高杨树叶片可溶性糖和可溶性蛋白含量,降低杨树叶片相对电导率,使杨树在干旱条件下的抗脱水能力提高,减轻了干旱胁迫对细胞膜的伤害.从品种间作用效果看,SNP对107杨的抗旱性提高的幅度高于小青杨,表明SNP更有利于提高速生杨的抗旱能力.     

In vitro effect of nanosilver on gene expression of superoxide dismutases and nitric oxide synthases in chicken sertoli cells

To evaluate effects of different concentrations of nanosilver colloid on the cell culture of Sertoli cells, the proportion of lipid peroxidation, antioxidant capacity, nitric oxide (NO) production and genes expression of superoxide dismutases (SOD1 and SOD2) and nitric oxide synthases (eNOS and iNOS) were measured. Sertoli cells were incubated at concentrations of 25, 75 and 125 ppm nanosilver for 48 h. There was progressive lipid peroxidation in treatments according to increasing of nanosilver. Lipid peroxidation, as indicated by malondialdehyde levels, was significantly elevated by the highest concentration of silver colloid (125 ppm), although antioxidant capacity, as measured by ferric ion reduction, was unaffected. Nitrite, as an index of NO production was reduced only in 125 ppm of nanosilver. Expression of SOD1 gene was reduced in nanosilver-treated cells at all concentrations, whereas expression of SOD2 gene was reduced only in cells treated with 125 ppm nanosilver. Expression of iNOS gene was progressively increased with higher concentrations of nanosilver. Expression of eNOS gene was also increased in 125 ppm of nanosilver. In conclusion, toxic effects of nanosilver could be due to high lipid peroxidation and suppression of antioxidant mechanisms via reduced expression of SOD genes and increased expression of NOS genes.     

一氧化氮对水分胁迫下玉米种子萌发及幼苗生理特性的影响

以0.4mol/L的甘露醇(M)模拟水分胁迫状况,研究了外源一氧化氮(NO)供体硝普钠(SNP)对水分胁迫下玉米种子萌发、幼苗生长和生理特性的影响。结果表明:(1)水分胁迫下,玉米种子萌发和幼苗生长受到抑制,叶片丙二醛(MDA)含量、质膜相对透性、脯氨酸含量均显著增加;(2)SNP能显著提高水分胁迫下玉米种子的发芽率、发芽势、发芽指数和活力指数,增加玉米幼苗的根长、茎长、根重和整株干重,抑制水分胁迫下玉米幼苗叶片MDA含量的上升,降低叶片质膜相对透性,降低脯氨酸含量。其中以100μmol/L和200μmol/LSNP对水分胁迫的缓解效果最佳。     

Involvement of endogenous nitric oxide in myeloperoxidase mediated benzo(a)pyrene induced polymorphonuclear leukocytes injury

The present study was undertaken to investigate the involvement of nitric oxide in the augmentation of benzo(a)pyrene induced cellular injury in polymorphonuclear leukocytes (PMNs). Polymorphs were isolated from the blood collected from Wistar rats treated with and without benzo(a)pyrene (50mg/kg, i.p.) through cardiac puncture. Catalase, superoxide dismutase (SOD), glutathione-s-transferase (GST), myeloperoxidase (MPO) and nitrite content were estimated in PMNs using standard procedures. Inducible nitric oxide synthase (iNOS) and cytochrome P-4501A1 (CYP1A1) expression in PMNs were also analyzed in presence or absence of nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG, 5mM) and L-N^G nitro L-arginine methyl ester (L-NAME, 1mM). A significant augmentation was observed in the nitrite content, activities of superoxide dismutase, MPO and GST and the expressions of iNOS and CYP1A1, however, catalase activity was attenuated in PMNs of benzo(a)pyrene treated rats as compared with their respective controls. AG and L-NAME resulted in a significant attenuation in nitrite content, MPO activity and iNOS expression; however, no significant alteration was observed in CYP1A1 expression. CYP1A1 inhibitor alpha-naphthoflavone inhibited the expression of iNOS in PMNs of benzo(a)pyrene treated animals significantly. The results obtained thus suggest that CYP1A1 induces iNOS expression leading to the generation of endogenous nitric oxide (NO) that could be responsible for the augmentation of myeloperoxidase-mediated benzo(a)pyrene-induced injury in PMNs.     

一氧化氮对大鼠背根神经节神经元膜电位的作用

目的：观察一氧化氮对大鼠背根神经节神经元的作用及有关离子机制,并探讨一氧化氮在痛觉信息传递过程中的作用。方法：在分离的大鼠背根神经节标本上,应用细胞内记录技术,给予灌流一氧化氮供体硝普钠,观察硝普钠诱导的神经元膜反应。结果：大部分神经元对硝普钠敏感（79／102,77．45％）,滴加硝普钠（10～100mmol／L）后可引起浓度依赖性的超极化反应,剩余神经元没有反应。硝普钠（100mmol／L）可使神经元膜电导由（21．06±1．94）nS增加到（23．08±0．92）nS。L-NAME（非选择性一氧化氮合酶抑制剂,1mmol／L）、CdCl2（非选择性钙通道阻断剂,0．1mmol／L）、无Na^＋平衡盐液对硝普钠引起超极化反应无明显影响。四乙基碘化铵（非选择性钾通道阻断剂,10mmol／L）明显抑制硝普钠引起的超极化反应。结论：硝普钠在大鼠背根神经节神经元上可引起浓度依赖的超极化反应,且此超极化反应是钾电导介导的。     

外源NO对铜胁迫下番茄幼苗根系抗坏血酸谷胱甘肽循环的影响

采用营养液培养方法,研究外源NO对铜胁迫下番茄(Lycopersicon esculentum Mill.)幼苗根系抗坏血酸(AsA)-谷胱甘肽(GSH)循环中抗氧化物质和抗氧化酶系的影响.结果表明：外施适量NO(硝普钠)可提高铜胁迫下番茄幼苗根系AsA、GSH含量和AsA/DHA(氧化型抗坏血酸)、GSH/GSSG(氧化型谷胱甘肽),降低DHA和GSSG含量.添加100 μmol·L^-1 BSO(谷胱甘肽合成酶抑制剂)处理下,外源NO可提高铜胁迫下番茄幼苗根系的AsA含量、AsA/DHA及抗坏血酸酶(AAO)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)比活性,降低DHA、GSH、GSSG含量及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)比活性;添加250 μmol·L^-1 BSO处理下,外源NO提高了铜胁迫下番茄幼苗根系的AsA、GSH、GSSG含量、AsA/DHA及APX和GR比活性,降低了DHA含量及AAO、DHAR和MDHAR比活性.说明外源NO影响了铜胁迫下番茄根系的AsA-GSH代谢循环,并通过调节AsA/DHA、GSH/GSSG的变化来减轻氧化胁迫,从而缓解铜胁迫对番茄根系的伤害.     

外源NO对镉胁迫下番茄活性氧代谢及光合特性的影响

采用水培方法,研究了外源一氧化氮（NO）对镉（Cd）胁迫下番茄幼苗活性氧代谢及光合特性的影响.结果表明：在Cd胁迫下,外施100 μmol·L^-1 硝普钠（SNP）显著增强了番茄超氧化物岐化酶（SOD）、过氧化氢酶（CAT）等抗氧化酶活性,提高了叶片、根系中Ca、Fe等元素含量,提高了叶片叶绿素含量、净光合速率（P_n）、蒸腾速率（T_r）和气孔导度（G_s）,降低了过氧化氢（H₂O₂）、丙二醛（MDA）含量和胞间CO₂浓度（C_i）.但SNP对Cd胁迫下的缓解效应可被牛血红蛋白（Hb,NO的清除剂）消除.在Cd处理液中加入100 μmol·L^-1 NO_x^-（NO的分解产物）或100 μmol·L^-1亚铁氰化钠（SNP的相似物或分解产物）,对Cd胁迫无显著改善.表明外源NO可通过提高活性氧清除能力,维持矿质营养元素平衡,缓解Cd胁迫对番茄幼苗叶片光合机构的破坏,从而维持番茄光合效率.     

Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide

Salinity stress causes ionic stress (mainly from high Na⁺ and Cl^- levels) and osmotic stress (as a result of inhibition of water uptake by roots and amplified water loss from plant tissue), resulting in cell death and inhibition of growth and ultimately adversely reducing crop productivity. In this report, changes in root nitric oxide content, shoot and root biomass, root H₂O₂ content, root lipid peroxidation, root cell death, root caspase-like enzymatic activity, root antioxidant enzymatic activity and root ascorbate and glutathione contents/redox states were investigated in maize (Zea mays L. cv Silverking) after long-term (21 d) salt stress (150 mM NaCl) with or without exogenously applied nitric oxide generated from the nitric oxide donor 2,2′-(Hydroxynitrosohydrazano)bis-ethane. In addition to reduced shoot and root biomass, salt stress increased the nitric oxide and H₂O₂ contents in the maize roots and resulted in elevated lipid peroxidation, caspase-like activity and cell death in the roots. Altered antioxidant enzymatic activities, along with changes in ascorbate and glutathione contents/redox status were observed in the roots in response to salt stress. The detrimental effects of salt stress in the roots were reversed by exogenously applied nitric oxide. These results demonstrate that exogenously applied nitric oxide confers salt stress tolerance in maize by reducing salt stress-induced oxidative stress and caspase-like activity through a process that limits accumulation of reactive oxygen species via enhanced antioxidant enzymatic activity.