H2O2参与水杨酸诱导丹参培养细胞中丹酚酸B合成的信号转导

过氧化氢(Hydrogen peroxide,H2O2)为活性氧(Reactive oxygen species,ROS)的一种,存在于许多生物体系中并介导植物中多种生理和生化过程。为了探讨H2O2作为信号分子在水杨酸(Salicylic acid,SA)诱导丹参培养细胞合成丹酚酸B(Salvianolic acid B,Sal B)过程中的作用,分别考察了SA和H2O2、过氧化氢酶(Catalase,CAT)、二甲基硫脲(2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,DMTU)及咪唑(Imidazole,IMD)对苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)和酪氨酸氨基转移酶(Tyrosine aminotransferase,TAT)的活性及Sal B含量的影响。结果表明,SA处理可有效地诱导丹参培养细胞中H2O2产生、PAL和TAT活性升高以及Sal B合成积累量的增加;外源施加10～30 mmol/L H2O2也可以有效促进PAL、TAT活性升高和Sal B合成积累量的增加;用H2O2的清除剂CAT处理发现,CAT对SA或外源H2O2诱导的Sal B合成积累具有消除作用,说明H2O2可能作为SA诱导Sal B积累过程中的上游信号分子起作用;用H2O2淬灭剂DMTU处理,可以有效抑制SA对Sal B合成的促进作用;用质膜烟酰胺腺嘌呤二核苷酸(Nicotinamide vadenine dinucleotide phosphate,NADPH)氧化酶(H2O2来源的主要酶)抑制剂IMD处理,可以抑制Sal B的合成,但这种抑制效果可以部分被外源施加的SA削弱,说明通过HADPH氧化酶产生的H2O2受阻时,SA诱导的Sal B合成积累也会受到抑制。表明H2O2是介导SA诱导丹参培养细胞中Sal B合成积累的信号分子。     

A novel high-throughput genetic screen for stress-responsive mutants of Arabidopsis thaliana reveals new loci involving stress responses

Activation sequence-1 (as-1) cognate promoter elements are widespread in the promoters of plant defense-related genes as well as in plant pathogen promoters, and may play important roles in the activation of defense-related genes. The as-1-type elements are highly responsive to multiple stress stimuli such as jasmonic acid (JA), salicylic acid (SA), H(2)O(2), xenobiotics and heavy metals, and therefore provide a unique opportunity for identifying additional signaling components and cross-talk points in the various signaling networks. A single as-1-type cis-element-driven GUS reporter Arabidopsis line responsive to JA, SA, H(2)O(2), xenobiotics and heavy metals was constructed for mutagenesis. A large-scale T-DNA mutagenesis has been conducted in the reporter background, and an efficient high-throughput mutant screen was established for isolating mutants with altered responses to the stress chemicals. A number of mutants with altered stress responses were obtained, some of which appear to identify new components in the as-1-based signal transduction pathways. We characterized a mutant (Delta8L4) with a T-DNA insertion in the coding sequence of the gene At4g24275. The as-1-regulated gene expression and GUS reporter gene expression were altered in the Delta8L4 mutant, but there was no change in the expression of genes lacking as-1 elements in their promoters. The phenotype observed with the Delta8L4 mutant was further verified using RNAi plants for At4g24275 (8L4-RNAi), suggesting the feasibility of use of this high-throughput mutant screening in isolating stress-signaling mutants.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

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。     

Dimerumic acid protected oxidative stress-induced cytotoxicity in isolated rat hepatocytes

Dimerumic acid (DMA) is contained in Monascus anka and Monascus pilosus fermented products. The purpose of this study was to evaluate the effect of DMA against salicylic acid (SA)- and tert-butylhydroperoxide (t-BHP)-induced oxidative stress and cytotoxicity in the liver, using rat liver microsomes and isolated rat hepatocytes. DMA was extracted from monascus-garlic-fermented extract using M. pilosus. In rat liver microsomes, 1 microM DMA decreased SA-induced lipid peroxidation but did not affect the production of the oxidative metabolite of SA via CYP. In isolated rat hepatocytes, 1 microM DMA decreased SA-induced lipid peroxidation and chemiluminescence (CL) generation and the intracellular glutathione-reduced form/oxidized form (GSH/GSSG) ratio in the presence of 1 microM DMA was higher than that without DMA; however, 100 microM DMA suppressed the leakage of lactate dehydrogenase (LDH). On the other hand, t-BHP-induced lipid peroxidation, CL generation, and LDH leakage were prevented by 100 microM DMA. Thus, DMA showed an antioxidative effect in hepatocytes and protected against hepatotoxicity by suppressing oxidative stress without affecting CYP enzymes.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Contribution of macrophage migration inhibitory factor to extracellular signal-regulated kinase activation by oxidative stress in cardiomyocytes

In response to oxidative stress, the pathogenesis of a number of cardiovascular events and several genes are stimulated by extracellular signal-regulated kinases (ERK1/2). Biphasic (early, 10 min; and delayed, 120 min) ERK1/2 activation by H(2)O(2), a reactive oxygen species, was observed in cultured neonatal rat cardiomyocytes. We investigated the hypothesis that the delayed activation of ERK1/2 depends on a factor secreted by oxidative stress (FSO). The delayed activation was inhibited by calphostin C, a protein kinase C inhibitor. Conditioned medium (CM) obtained from cells stimulated with H(2)O(2) induced rapid and monophasic ERK1/2 activation, which was not inhibited by calphostin C. In contrast, calphostin C-pretreated CM did not activate ERK1/2. Macrophage migration inhibitory factor (MIF) was one of the candidate FSOs activating ERK1/2. The existence of MIF in CM, the recombinant MIF-stimulated ERK1/2 rapid activation, and anti-MIF neutralizing antibody-induced inhibition of the delayed activation implied that MIF could be the FSO. Pretreatment of cardiomyocytes with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor did not suppress the MIF secretion, although it prevented the ERK1/2 activation by H(2)O(2). These results indicate that MIF is secreted from cardiomyocytes as a result of oxidative stress and activates ERK1/2 through a MEK1/2-dependent mechanism, although the secretion is not regulated by ERK1/2 but by protein kinase C.