Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Contribution of insulin to the ascorbate recycling system in the chicken liver

The effect of insulin on the ascorbate recycling system in the chicken liver was examined. First, insulin was injected subcutaneously into the chicken, and liver glutathione‐dependent dehydroascorbate reductase (GSH‐DHAR) activity was determined. Insulin increased liver GSH‐DHAR activity, but did not affect plasma and liver ascorbate concentration. Dehydroascorbate increased plasma and liver ascorbate levels, and liver GSH‐DHAR activity. However, distinct changes in plasma insulin level were not observed by dehydroascorbate injection. In addition, reduction of external dehydroascorbate in cultured chicken hepatocytes could not be observed in an insulin‐deprived culture, although the cells reduced external dehydroascorbate in a serum‐free culture with insulin. We concluded that insulin affects the ascorbate recycling system as an essential factor in the chicken liver.     

Differential levels of antioxidants in paraquat-resistant and -susceptible Erigeron canadensis biotypes in Korea

To characterize the biochemical differences in paraquat-resistant and -susceptible biotypes of Erigeron canadensis L. collected from Korea, we investigated the constitutive levels of various antioxidants such as antioxidant enzymes and low molecular weight antioxidants in leaves, as well as after paraquat treatment. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, and catalase were higher in the paraquat-resistant biotype than in the paraquat-susceptible biotype. Reduced ascorbic acid content was higher in the resistant biotype, but the content of reduced glutathione was higher in the susceptible biotype. These results indicate that one of the paraquat-resistant mechanisms in E. canadensis in the present study might be related to protecting the activities of antioxidant enzymes, such as superoxide dismutase, peroxidase ascorbate peroxidase, and catalase, as well as the contents of low molecular weight antioxidants such as ascorbate and glutathione.     

胁迫对菜用大豆种子抗坏血酸-谷胱甘肽循环的影响

采用蛭石栽培,在100 mmol/L NaCl 胁迫下,对耐盐性不同的两个菜用大豆［Glycine max (L.)Merr.］品种种子的过氧化氢（H2O2）含量及抗坏血酸-谷胱甘肽（AsA-GSH）循环进行了研究。结果显示,NaCl胁迫显著增加了菜用大豆种子的H2O2含量,但耐盐品种 绿领特早的增幅低于盐敏感品种理想高产95-1。NaCl胁迫期间,绿领特早种子中抗坏血酸过氧化物酶（APX）、脱氢抗坏血酸还原酶（DHAR）、谷胱甘肽还原酶（GR）活性,AsA、GSH含量以及AsA/DHA值和GSH/GSSG值的增幅高于同期的理想高产95-1,或降幅低于同期的理想高产95-1; 脱氢抗坏血酸（DHA）、氧化型谷胱甘肽（GSSG）含量的增幅低于同期的理想高产95-1。表明 绿领特早种子在胁迫期间能够保持较高的AsA-GSH循环效率,可有效地抑制H2O2的积累,这可能是其耐盐性较强的重要原因之一。     

马铃薯抗坏血酸含量及其代谢相关酶活性关系的研究

 为探讨马铃薯不同器官中抗坏血酸(AsA) 含量及其代谢相关酶活性关系, 研究了马铃薯幼叶、功能叶、老叶、茎和块茎中AsA和其氧化态脱氢抗坏血酸(DHA) 的含量与L - 半乳糖- 1, 4 - 内酯脱氢酶( GalLDH) 、脱氢抗坏血酸还原酶(DHAR) 、谷胱甘肽还原酶( GR ) 、抗坏血酸过氧化物酶(APX) 、抗坏血酸氧化酶(AO) 和单脱氢抗坏血酸还原酶(MDHAR) 等6种酶活性之间的相关性。结果表明, 马铃薯AsA在幼叶和块茎中含量很高。叶片和茎的抗坏血酸库(AsA与DHA之和) 水平与GalLDH活性显著相关, 而AsA含量与DHAR活性显著相关, DHA含量与APX活性显著相关。说明在马铃薯幼叶中高含量的AsA可能由于GalLDH和DHAR的高活性; 而块茎中AsA的积累, 主要来自于叶片的运输和DHAR催化的DHA再生。     

苹果L-半乳糖-1-磷酸磷酸酶cDNA克隆及其表达

 【目的】了解苹果果实L-半乳糖-1-磷酸磷酸酶(L-galactose-1-phosphate phosphatase, GPP)基因的特性,探索苹果GPP基因表达特性及其与抗坏血酸(ascorbic acid,AsA)水平的关系。【方法】通过RT-PCR从苹果果实中克隆GPP 全长 cDNA,分析其序列特征,进行原核表达,制备GPP特异抗体,分析GPP mRNA及其蛋白表达水平与苹果不同组织AsA的关系。【结果】从‘嘎啦’苹果果实中克隆的GPP cDNA(GenBank登录号为 FJ752240)包含的最大开放阅读框(open reading frame,ORF)为813 bp,编码270个氨基酸残基,预测分子量为29 kD,该基因与其它植物报道的GPP基因具有较高的相似性,但与肌醇-1-磷酸磷酸酶基因差异较大。构建的pET-32a(＋)-GPP载体在大肠杆菌BL21(E. coli BL21)中异源表达后,获得主要以包涵体存在约50 kD的融合蛋白GPP-His(His约21 kD)。以该蛋白制备抗体,与重组蛋白的Western杂交表明该抗体能与GPP发生特异反应。对苹果可溶性蛋白杂交显示,苹果体内GPP蛋白约33 kD。在苹果不同组织中,GPP mRNA与蛋白质的相对表达水平与AsA含量存在明显的一致性。【结论】以单体形式存在的苹果GPP蛋白具有翻译后修饰特性,且该基因的表达在苹果AsA合成调控中可能起重要作用。     

Continuous Spectrophotometric Assay and Properties of Ascorbic Acid Oxidising Factors in Wheat

A continuous spectrophotometric assay was developed to measure ascorbic acid oxidation in crude Na₂SO₄ extracts of flour. The rate of ascorbic acid oxidation in flour extracts measured using this method was similar to the rate in flour-water suspensions and 2–4 fold less than the rate in dough measured using an indophenol-xylene extraction method. Flour extracts appeared to contain two ascorbic acid oxidising factors; one with optimal activity at pH 6·3 and 30 °C and the other with optimal activity at pH 10 and 40 °C. The pH 6·3 factor had properties similar to those of ascorbate oxidase (EC 1·10·3·3) in its pH and temperature stability, strong inhibition by NaN₃, KCN and diethyldithiocarbamate, inactivation by proteases, and greater stereospecificity towards -ascorbic acid than -isoascorbic acid. The pH 6·3 factor was most concentrated in the pollard milling fraction of wheat and was lowest in flour. The pH 10 factor had several properties indicating non-enzymic oxidation of ascorbic acid; it was not inactivated by proteases, it was inhibited poorly or not at all by the above ascorbate oxidase inhibitors, and it had low specificity for stereoisomers of ascorbic acid.     

Changes in antioxidative system and membrane damage of lettuce in response to salinity and boron toxicity

Individual and combined effects of salinity and B toxicity on growth, the major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX) activities, ascorbic acid, proline, and H₂O₂ accumulation, and stomatal resistance (SR), malondialdehyde (MDA), membrane permeability (MP) and the concentrations of sodium (Na), chloride (Cl) and boron (B) of lettuce were investigated. Boron toxicity and salinity reduced growth of lettuce plants. Under B toxicity, B concentration of the plants was increased, but in the presence of NaCl, the concentration of B was significantly reduced. Sodium and Cl concentrations were increased in B + NaCl and NaCl treatments. Membrane damage was more pronounced in NaCl and B + NaCl treatments. Stomatal resistance of the plants was significantly increased by salinity treatments. The accumulation of proline and ascorbic acid was the highest in the B + NaCl treatment. In general, stress conditions significantly increased H₂O₂ and antioxidant enzyme (SOD, CAT and APX) activities. The present results indicate that stomatal closure is an important response of lettuce against NaCl and B + NaCl stress. Furthermore NaCl and B + NaCl toxicity-induced oxidative stress in lettuce resulting in lipid peroxidation and membrane damage. Increased antioxidant enzyme activities and also accumulation of ascorbic acid and proline are involved in order to overcome B- and NaCl-induced oxidative stress.