Nitric oxide counteracts the senescence of rice leaves induced by abscisic acid

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by ABA. Senescence of rice leaves was determined by the decrease of protein content. ABA treatment resulted in (1) induction of leaf senescence, (2) increase in H2O2 and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase). All these ABA effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-alpha-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA + NaNO2] were effective in reducing ABA-induced leaf senescence. PBN prevented ABA-induced increase in the contents of H2O2 and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on ABA-promoted senescence, ABA-increased H2O2 content and lipid peroxidation, ABA-decreased GSH and AsA, and ABA-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of ABA-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H2O2.     

盐及干旱胁迫对油菜抗氧化系统和RbohC、RbohF基因表达的影响

以白菜型油菜‘陇油6号’和‘天油2号’为试验材料,经MAPK抑制剂U0126、H₂O₂清除剂DMTU、NADPH氧化酶抑制剂DPI和IMD预处理后再分别进行盐胁迫、PEG-6000模拟干旱胁迫,研究其对两种油菜幼苗活性氧、抗氧化酶活性和RbohC、RbohF基因表达的影响.结果表明: 盐胁迫和PEG-6000模拟干旱胁迫下,两种白菜型油菜中H₂O₂积累量上升,O₂^-·积累量下降,抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT、抗坏血酸过氧化物酶APX和谷胱甘肽还原酶GR)活性和RbohC、RbohF基因表达均升高.与单独胁迫处理相比,两种油菜O₂^-·积累、抗氧化酶活性和RbohC、RbohF基因的表达量均明显降低,经DMTU、DPI和IMD预处理后再分别进行盐和干旱胁迫,H₂O₂积累量下降,但U0126预处理后再进行胁迫处理,H₂O₂积累量上升.说明NADPH氧化酶、MAP激酶级联途径、H₂O₂参与了盐、干旱胁迫下活性氧产生、抗氧化酶活性变化和RbohC、RbohF基因表达的调控.     

盐及干旱胁迫对油菜抗氧化系统和RbohC、RbohF基因表达的影响

以白菜型油菜‘陇油6号’和‘天油2号’为试验材料,经MAPK抑制剂U0126、H_2O_2清除剂DMTU、NADPH氧化酶抑制剂DPI和IMD预处理后再分别进行盐胁迫、PEG-6000模拟干旱胁迫,研究其对两种油菜幼苗活性氧、抗氧化酶活性和RbohC、RbohF基因表达的影响.结果表明:盐胁迫和PEG-6000模拟干旱胁迫下,两种白菜型油菜中H_2O_2积累量上升,O_2^-·积累量下降,抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT、抗坏血酸过氧化物酶APX和谷胱甘肽还原酶GR)活性和RbohC、RbohF基因表达均升高.与单独胁迫处理相比,两种油菜O_2^-·积累、抗氧化酶活性和RbohC、RbohF基因的表达量均明显降低,经DMTU、DPI和IMD预处理后再分别进行盐和干旱胁迫,H_2O_2积累量下降,但U0126预处理后再进行胁迫处理,H_2O_2积累量上升.说明NADPH氧化酶、MAP激酶级联途径、H_2O_2参与了盐、干旱胁迫下活性氧产生、抗氧化酶活性变化和RbohC、RbohF基因表达的调控.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

胞外H2O2及NADPH氧化酶参与了铜胁迫对植物细胞死亡的诱导

以烟草悬浮细胞BY-2(Nicotiana tabacum L.cv.Bright Yellow-2)为材料,探讨了在铜离子胁迫下植物细胞死亡发生过程中胞外H₂O₂及NADPH氧化酶所扮演的角色。实验结果表明,随着外源CuCl₂浓度的上升(从0~700 μmol·L^-1),细胞死亡水平不断上升,且胞外H₂O₂的水平也不断增加。在300 μmol·L^-1的CuCl₂诱导细胞死亡的过程中,加入H₂O₂清除剂N-N-二甲基硫脲(DMTU)降低了胞外CuCl₂胁迫下H₂O₂含量增加的同时也降低了细胞死亡水平的上升,这一观察表明了铜离子胁迫所导致的细胞死亡的发生和胞外H₂O₂的增加有关。进一步的研究表明,300 μmol·L^-1 CuCl₂的胁迫导致了NADPH氧化酶活性的显著性上升,而加入NADPH氧化酶的抑制剂（二亚苯基碘,DPI,)则降低了CuCl₂胁迫所导致的细胞死亡和胞外H₂O₂含量的上升。上述结果表明,胞外H₂O₂和NADPH氧化酶参与了CuCl₂对植物细胞死亡的诱导作用。     

香菇菌丝后熟转色形成的活性氧作用与自噬特征

为了解活性氧(reactive oxygen species,ROS)在香菇菌丝后熟转色形成中的作用及其自噬细胞学特征,以香菇工厂化菌株KS11为研究材料,分析其在菌丝后熟转色过程中4个时间点(30、45、60、75 d)的活性氧含量(ROS)、丙二醛(MDA)含量、NADPH氧化酶浓度、抗氧化酶活性以及外源活性氧和DPI对其影响的表型试验,利用透射电镜观察该过程菌丝细胞自噬特征变化,并运用实时荧光定量PCR对自噬基因Atg8的表达水平进行比较分析。结果表明：(1) H₂O₂作为主要的活性氧因子在菌丝后熟转色形成中呈现显著动态变化,后熟转色过程中不断升高,并在转色中第60天呈高峰值。(2) NADPH氧化酶浓度与H₂O₂含量变化呈紧密正相关。(3)外源施加一定浓度H₂O₂显著促进香菇菌丝后熟转色,且DPI作为NADPH氧化酶抑制剂显著抑制了香菇菌丝后熟转色的发生。(4)香菇菌丝后熟转色过程中,细胞自噬特征逐渐增强,并在转色中后期最显著。上述结果表明以H₂O₂为主的活性氧在香菇菌丝后熟转色过程中起重要作用,并且可能参与了香菇菌丝后熟转色过程中的自噬进程。     

水杨酸诱发的丹参悬浮培养细胞内H2O2迸发与其培养基碱化的关系

水杨酸(salicylic acid,SA)处理可诱导丹参悬浮培养细胞内H2O2产生及其培养基碱化。利用NADPH氧化酶抑制剂咪唑(imidazole,IMD)、H2O2淬灭剂二甲基硫脲(dimethylthiourea,DMTU)、质膜H+-ATPase抑制剂钒酸钠(Na3VO4)及激活剂壳梭孢菌素(fusicoccin,FC)处理丹参悬浮培养细胞,探讨SA诱导的H2O2迸发与培养基碱化之间的关系。结果表明,H2O2可促发培养基碱化,IMD和DMTU抑制SA诱发的培养基碱化,说明H2O2参与SA诱发的培养基碱化过程;SA抑制质膜H+-ATPase活性,Na3VO4引发培养基碱化并使H2O2迸发时间提前,FC处理逆转了SA诱导的培养基碱化及H2O2迸发,说明质膜H+-ATPase调控培养基pH值变化,培养基碱化促进了H2O2产生。因此,丹参悬浮培养细胞内H2O2水平与其培养基碱化程度之间相互关联、共同作用,协同响应SA的诱导。     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H₂O₂ release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H₂O₂ release. H₂O₂ was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H₂O₂ production. H₂O₂ release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with -glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H₂O₂ generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H₂O₂ release in insect mitochondria supplemented with -glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H₂O₂ production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

过氧化氢缓解裸燕麦幼苗低温胁迫的主成分和隶属函数分析

为探明信号分子过氧化氢（H₂O₂）提高裸燕麦幼苗耐冷性的作用,以‘定莜6号’沙培幼苗为材料,在3叶期喷施10 μmol·L^-1 H₂O₂ 12 h后于8℃/5℃（昼/夜）条件下低温胁迫,以喷蒸馏水（H₂O）为对照,分别在低温处理的0、1、2、3、4、5 d取幼苗叶片测定超氧阴离子（O₂^-·）、H₂O₂、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）、抗坏血酸（AsA）、谷胱甘肽（GSH）、可溶性糖（SS）、脯氨酸（Pro）、可溶性蛋白质（SP）和热稳定性蛋白质（HSP）13项与耐冷性有关的生理指标及低温处理5 d后植株株高和生物量增量,采用主成分和隶属函数分析综合评价H₂O₂对裸燕麦幼苗耐冷性的影响。结果表明,与对照相比,喷施H₂O₂显著提高了低温胁迫下裸燕麦幼苗株高和生物量增量,降低了裸燕麦幼苗叶片O₂^-·和MDA及低温胁迫2~5 d的H₂O₂含量,促进低温胁迫期间裸燕麦幼苗叶片SOD、CAT、POD和APX活性提高及AsA、GSH、SS、Pro、SP和HSP积累。主成分分析13项生理指标离差标准化数据,提取的前4个主成分累积方差贡献率达85.6%;隶属函数综合评价4个主成分得分值显示,喷施H₂O₂显著提高了低温胁迫0~5 d的综合评价值。表明喷施H₂O₂能够通过调控生理生化代谢提高裸燕麦幼苗的耐冷性。     

拟南芥金属蛋白酶FtSH4通过生长素与活性氧调控叶片衰老

植物金属蛋白酶Ft SH基因家族在拟南芥(Arabidopsis thaliana)中有12个成员,目前各基因的功能还不清楚。该文利用细胞生物学和遗传学方法初步分析了拟南芥FtSH4在叶片衰老中的功能。ftsh4-4突变体叶片中H_2O_2含量及细胞死亡率增加,叶绿素含量降低;此外,突变体中过氧化物酶基因表达上调,过氧化物酶活性增加,出现早衰表型。外源抗氧化剂As A、内源和外源生长素能够通过降低ftsh4-4体内H_2O_2含量、过氧化物酶基因的表达及过氧化物酶活性,恢复ftsh4-4叶片的衰老表型。ftsh4-4突变体中生长素响应因子基因ARF2和ARF7上调表达,外源生长素和抗氧化剂能够降低ARF2和ARF7的表达,并且ARF2突变能够降低ftsh4-4的H_2O_2含量并恢复其早衰表型。以上结果表明,FtSH4基因通过生长素与活性氧在调控植物叶片衰老中起重要作用。     

外源ABA、NO和H2O2对葱莲花瓣及叶片表皮气孔开闭的影响

以葱莲(Zephyranthes candida)为材料,研究不同浓度外源脱落酸、硝普钠(sodium nitroprusside,SNP)及过氧化氢对花瓣和叶片表皮气孔开闭的影响,以期为三者在切花保鲜中的应用提供新的依据。实验结果表明,10~1000 μmol/L脱落酸和硝普钠均能不同程度地引起花瓣和叶片表皮气孔关闭,且花瓣气孔较叶片气孔有更高的敏感性。过氧化氢对叶片表皮气孔开闭的影响大于对花瓣气孔的影响,花瓣表皮的气孔孔径仅在1000 μmol/L处理时变化显著。这说明在外源信号物质延缓切花衰老的过程中,花瓣表皮气孔的运动也可能起到了一定的作用。适当外源信号物质处理能诱导花瓣表皮气孔关闭,从而使花瓣的蒸腾作用减小,维持植物体内水势,延缓切花衰老。     

朱砂叶螨刺吸胁迫对玉米防御信号分子的诱导作用

【目的】探明朱砂叶螨Tetranychus cinnabarinus持续为害对玉米Zea mays叶片内茉莉酸(jasmonic acid,JA)、水杨酸(salicylic acid,SA)、乙烯(ethylene,ET)、一氧化氮(nitricoxide,NO)、脱落酸(abscisic acid,ABA)和过氧化氢(hydrogen peroxide,H2O2)6个防御信号分子的诱导作用。【方法】室内人工接螨(10,20和30头/叶),采用分光光度法(SP)、高效液相色谱法(HPLC)和酶联免疫法(ELISA),测定了朱砂叶螨持续为害0,24,48,72和96 h后,玉米幼苗叶片内6个信号分子的含量。【结果】朱砂叶螨持续刺吸为害玉米幼苗叶片后,JA,ABA和H2O23个信号分子含量在叶螨刺吸为害24 h内迅速上升,在24 h时达高峰值,叶螨密度为30头/叶时其含量分别为同期未接螨对照的4.13,3.84和3.20倍,24-48 h内迅速下降;此后,ABA和H2O2含量维持在较低水平,而JA含量在48-96 h内又上升至次高峰值。NO含量则在24-48 h内上升较快,48 h时达最高,叶螨密度为30头/叶时其含量为同期未接螨对照的5.09倍。SA和ET含量在96 h内均随刺吸时间的延长而增大,96 h时最高,叶螨密度为30头/叶时其含量分别为同期未接螨对照的5.17和2.99倍。叶螨密度为30头/叶时,6个信号分子含量均显著高于同期未接螨对照(P0.05)。【结论】朱砂叶螨为害对玉米叶片内JA,SA,ET,NO,ABA和H2O2均具有诱导作用,且6个信号分子在叶螨持续为害玉米叶片后循序被诱导。     

油菜RbohB基因的克隆及逆境响应表达分析

利用RACE技术从‘陇油6号’油菜中克隆得到一个新的RbohB基因,全长2 694 bp,开放阅读框2 541 bp,编码846个氨基酸。实时荧光定量PCR分析表明RbohB基因表达受低温、高盐、H₂O₂诱导,MAPKK抑制剂U0126预处理12 h再经低温、高盐、H₂O₂诱导,与单独处理结果相比,RbohB基因表达明显降低,表明该基因在油菜适应低温、高盐、H₂O₂胁迫过程中发挥作用,U0126对该基因的转录有抑制作用。NADPH氧化酶活性受H₂O₂处理的诱导,U0126预处理12 h再经H₂O₂处理,与单独H₂O₂处理结果相比,NADPH氧化酶活性明显降低,表明MAPKK抑制剂U0126对该酶活性有抑制作用。     

ABA诱导玉米叶质外体H2O2积累的机制

通过组织化学染色和电镜观察并结合酶活性分析表明,ABA可通过诱导玉米（Zea mays L、）叶片质膜NADPH氧化酶、细胞壁POD及质外体PAO活性的升高,使其质外体产生H2O2;其中质膜NADPH氧化酶起主要作用。     

24-表油菜素内酯对干旱胁迫下辣椒幼苗叶片抗氧化酶系统及耐旱相关基因表达的影响

报道了干旱胁迫下外源24-表油菜素内酯（EBR）对辣椒幼苗叶片H₂O₂和MDA含量,抗氧化酶活性,以及耐旱相关基因表达的影响。结果表明,0.1 μmol·L^-1 EBR处理诱导了辣椒幼苗叶片H₂O₂含量的增加,并提高了SOD、APX、CAT、DHAR、MDAR和GR活性;干旱胁迫下,EBR处理显著诱导了辣椒叶片抗氧化酶活性的增加,并抑制了H₂O₂和MDA含量的上升;EBR处理也促进了cAPX和MDAR等抗氧化酶基因的表达,以及WRKY3、WRKY6和MYB等转录因子的表达。由此认为,适宜浓度的外源EBR可能是通过信号分子H₂O₂调控辣椒叶片中WRKY和MYB等转录因子的表达以调控相关耐旱基因表达,增强细胞的抗氧化酶活性,减轻干旱造成的膜质过氧化伤害,从而增强了辣椒幼苗的耐旱性。