Effect of Pichia membranaefaciens on ROS metabolism and postharvest disease control in citrus fruit

The yeast Pichia membranaefaciens has antagonistic effects against a wide range of phytopathogenic fungi that cause postharvest fruit decay. This work evaluated the effects of P. membranaefaciens on the reactive oxygen species (ROS) metabolism and disease control in harvested citrus fruit (Citrus sinensis L. Osbeck). The lesion diameter caused by Penicillium italicum and Penicillium digitatum on citrus fruit was remarkably reduced when the fruit was point-inoculated or dipped in a suspension of P. membranaefaciens at 1 × 10⁸ CFU mL⁻¹. The application of P. membranaefaciens on citrus fruit enhanced the activity of superoxide dismutase, ascorbate peroxidase, and glutathione reductase, as well as the levels of hydrogen peroxide, the superoxide anion and glutathione, but inhibited the decreasing ascorbic acid content. Furthermore, catalase activity was decreased by the same treatment. These results indicated that yeast treatment induced the synthesis of antioxidant enzymes which might have antagonistic effects against postharvest green and blue mold infection in citrus fruit.     

NaCl和PEG6000胁迫下枣组培苗中ZjAPX的表达

为了探索枣树遭遇盐、干旱胁迫时抗坏血酸过氧化物酶基因(ZjAPX)在抗氧化系统中的响应,以枣树组织培养植株为试材,进行了NaCl和PEG6000(模拟干旱)胁迫,观察受胁迫后植株外观形态变化,并从植株叶片中提取RNA进行反转录。根据ZjAPX的cDNA序列设计特异性引物,以反转录产物为模板,对ZjAPX基因在胁迫条件下的表达情况进行了定量分析。结果表明,ZjAPX基因受NaCl和PEG6000诱导表达,而且在一定浓度和时间范围内,随着浓度的增大和胁迫时间的延长表达量增高,说明ZjAPX基因参与了植株抵抗盐和干旱带来的伤害。     

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。     

Overproduction of reactive oxygen species involved in the pathogenicity of Fusarium in potato tubers

Differences in virulence between Fusarium sulphureum and Fusarium sambucinum were compared. Changes in reactive oxygen species production and metabolism in inoculated slices of potato tubers were also compared. The result showed that Fusarium infection induced significant production of ROS, lipid peroxidation and loss of cell membrane integrity, but low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Compared to F. sambucinum, F. sulphureum led larger lesion diameters on potato tubers and slices. It resulted in more superoxide anion ($O_2^{-}$) and earlier peak of hydrogen peroxide (H₂O₂), but lower activity of catalase (CAT) and APX, and accompanied with higher malondialdehyde (MDA) content and lower cell membrane integrity. These findings suggested that overproduction of ROS involved in the pathogenicity of Fusarium in potato tubers.     

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。     

枣树抗坏血酸过氧化物酶基因ZjAPX生物信息学分析及植物表达载体的构建

利用生物信息学软件对已获得的枣树抗坏血酸过氧化物酶基因cDNA序列ZjAPX进行了同源性及功能位点等多项参数分析。结果表明,此序列为全长753 bp的开放读码框(ORF),编码251氨基酸,分子量为62.55 kDa,理论等电点为5.10,为一个膜外蛋白;序列保守性分析表明,该序列具有典型的铁氧化还原蛋白结合区域,属于一个典型的植物亚铁血红素过氧化物酶家族蛋白;氨基酸序列相似性分析表明,该蛋白与已知的其他植物抗坏血酸过氧化物酶APX具有极高的同源性,与可可树APX和陆地棉APX同源性均为93%,命名为ZjAPX(DDBJ/EMBL/GenBank注册号:AB608053)。用Swiss Model程序(http://au.expasy.org/tools/)推测了ZjAPX基因编码蛋白的三维结构。以ZjAPX的cDNA序列为模板,PCR扩增后,经限制性内切酶消化,与PEZR(K)-LNY载体进行重组连接,测序结果显示,其植物表达载体构建成功。此结果为研究枣树抗坏血酸过氧化物酶基因在植物体内的生物学功能以及可能的活性调节方式奠定了基础。     

Hydrogen peroxide pretreatment alters the activity of antioxidant enzymes and protects chloroplast ultrastructure in heat-stressed cucumber leaves

To elucidate the physiological mechanism of heat stress mitigated by exogenous hydrogen peroxide (H₂O₂) further, seedlings of Cucumis sativus cv Lvfeng no. 6 were subjected to two temperatures (42/38 and 25/18 °C) after pretreatment with 1.5 mM H₂O₂. We investigated whether exogenous H₂O₂ could protect chloroplast ultrastructure under heat stress (42/38 °C) and whether the protective effect was associated with the regulation of antioxidant enzymes. The heat condition disintegrated the membranes of 71.4% chloroplasts in the leaf cells and resulted in the elevated levels of malondialdehyde (MDA) and endogenous H₂O₂. When H₂O₂ pretreatment was combined with the heat stress, the abnormal chloroplasts occurred at a rate of 33.3%, and the contents of MDA and endogenous H₂O₂ were decreased. Heat stress and exogenous H₂O₂ both increased the activities of antioxidant enzymes such as glutathione peroxidase (GSH-Px, EC 1.11.1.9), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reducatase (MDHAR, EC 1.6.5.4), and glutathione reductase (GR, EC 1.6.4.2). The combined effect of heat stress and H₂O₂ pretreatment led to higher activity of antioxidant enzymes including superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), GSH-Px, DHAR, MDHAR and GR in comparison to the heat treatment alone. We propose that exogenous H₂O₂ increases antioxidant enzyme activities in cucumber leaves, decreases lipid peroxidation, and thus protects the ultrastructure of chloroplasts under heat stress.     

Purification and Characterisation of Ascorbate Oxidase from Flour and Immature Wheat Kernels

White flour from wheat was shown to contain basic-ascorbate oxidase (AOX) enzymes (pI 7·6–9·6) and acidic-AOX enzymes (pI 5·1–6·6) in a ratio of 0·4:1, based on chromatography data. Immature wheat kernels (two weeks post-anthesis) contained about 12 times more AOX activity (units/g dry weight) than flour from mature grain, and the ratio of basic- to acidic-AOX was 5:1. Acidic-AOX was purified 90-fold from flour by hydrophobic interaction, gel filtration and anion exchange chromatography. Basic-AOX was purified 20 000-fold from immature wheat by hydrophobic interaction, anion exchange, cation exchange and gel filtration chromatography in a yield of 5%. The acid-AOX had a M of 140 k, was optimally active at pH 6·3 and 40 °C, and was stable in the pH range 5–9 and at 30 °C for 0·5 h at pH 6·2. The Km values were 0·26 m for L-ascorbic acid and 0·93 m for D-iso ascorbic acid. The basic-AOX had a M of 139 k and subunit M of 72 k. The enzyme was optimally active at pH 6·2 and 50 °C, and was stable in the pH range 5–9 and at 40 °C for 0·5 h at pH 6·2. The Km values were 0·30 m for L-ascorbic acid and 0·53 m for D-iso ascorbic acid. The absorption spectrum of basic-AOX had absorption maxima at 280 nm and 607 nm of similar magnitude to those measured in AOX fromCucurbita species (squash). This indicates that wheat AOX contains protein-bound copper similar to other plant AOX.     

Induction of disease resistance and ROS metabolism in navel oranges by chitosan

The objective of this work was to evaluate how disease resistance and reactive oxygen species (ROS) metabolism in harvested navel oranges (Citrus sinensis L. Osbeck) may be affected by chitosan. Fresh navel oranges were treated with 2% chitosan or 0.5% glacial acetic acid (control) solution for 1 min, and some were inoculated with Penicillium italicum and Penicillium digitatum. Then, the fruit were stored at 20 °C and 85–95% RH. Treatment with 2% chitosan significantly reduced the disease incidence and the lesion diameter compared with control fruit. This treatment effectively enhanced the activities of peroxidase (POD) and superoxide dismutase (SOD), and levels of glutathione (GSH) and hydrogen peroxide (H₂O₂), inhibited the activities of catalase (CAT) and the decreases of ascorbate (AsA) content during navel orange fruits storage. Ascorbate peroxidase (APX) activity in the navel orange fruit was induced slightly by the chitosan treatment during 14–21 days storage. However, glutathione reductase (GR) activity in the fruit was not enhanced by the chitosan treatment. These results indicated that chitosan treatment could induce the navel orange fruit disease resistance by regulating the H₂O₂ levels, antioxidant enzyme and ascorbate–glutathione cycle.     

水稻类病斑突变系的培育与研究

利用γ射线辐照水稻品种嘉浙B的种子,从处理后代中获得了稳定的类病斑突变体嘉浙DB。为了阐明该突变体性状形成的遗传和生物学基础,开展了突变基因定位和类病斑特征及叶绿体相关特性的研究。结果表明,嘉浙DB是一种典型的Sekiguchi类病斑突变体,在无病原菌侵染和外界胁迫的情况下,叶片上可自发形成棕黄色病斑,病斑的面积随着植株的生长不断扩大;在病斑扩展阶段,突变体叶片细胞中叶绿体结构紊乱,提早降解,光合作用效率显著下降;嘉浙DB的类病斑性状属隐性性状,很可能受LOC_Os12g16720的1个突变等位基因控制,其第1 425位核苷酸G缺失可导致移码突变从而编码一个截断的蛋白;突变体中清除活性氧的4个叶绿体抗坏血酸过氧化物酶(APX)的基因转录水平在白天光合作用下均显著高于野生型,而在夜间则无显著差异。叶绿体的结构紊乱及细胞内的高活性氧含量可能是引发突变体叶片细胞凋亡的原因。本研究为从叶绿体的角度阐述Sekiguchi类病斑发生的分子机制提供了依据。