植物抗坏血酸过氧化物酶的表达调控以及对非生物胁迫的耐受作用

抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)属于I型血红素过氧化物酶, 它催化H2O2依赖的L-抗坏血酸氧化作用, 对抗坏血酸表现出高度的专一性。植物APX基因家族由4个亚家族组成, 分别为细胞质、叶绿体、线粒体和过氧化物酶体基因亚家族, 每个亚家族中又含有不同的APX同工酶。作为植物抗坏血酸-谷胱甘肽循环中的一个关键组分, APX在细胞H₂O₂代谢过程中起着至关重要的作用。研究表明植物APX是氧化还原信号系统中调节细胞水平H₂O₂非常重要的一种酶, APX同工酶的表达机制非常复杂, 细胞质APX受多种信号调节表达, 两种叶绿体APX通过选择性剪接进行组织特异性调节。通过调控产生的APX可调节细胞中的氧化还原信号, 进而提高植物对非生物胁迫的耐受性。文章综述了植物APX的催化机制、表达调控机理以及响应植物非生物逆境胁迫的重要作用。     

植物抗坏血酸过氧化物酶研究进展(综述)

本文从酶的作用机制、酶学特征、分子生物学等方面综述植物抗坏血酸过氧化物酶（APX）的研究进展。     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

植物抗坏血酸过氧化物酶的作用机制、酶学及分子特性

介绍叶绿体中H2O2的产生和清除,抗坏血酸过氧化物酶(APX)的酶学和分子特性,APX同工酶在植物体内的分布和功能及其相互之间的区别,APX与细胞色素C过氧化酶(CPX)和谷胱苷肽过氧化物酶(GPX)等一些在不同生物中的H2O2清除酶的异同之处,以及有关APX基因工程的研究进展.     

Inactivation Mechanism of Ascorbate Peroxidase at Low Concentrations of Ascorbate; Hydrogen Peroxide Decomposes Compound I of Ascorbate Peroxidase

One of the characteristic properties of ascorbate peroxidase(APX), which distinguishes it from guaiacol peroxidase, Cytc peroxidase and glutathione peroxidase, is the rapid inactivationof the enzyme under conditions where an electron donor is absent.When thylakoid-bound APX (tAPX) in 100 µM ascorbate wasdiluted 500-fold with an ascorbate-depleted medium, the enzymaticactivity was lost with half time of about 15 s. The inactivationof tAPX was suppressed under anaerobic conditions and also bythe addition of catalase, but it was unaffected by the additionof superoxide dismutase. These observations suggest that hydrogenperoxide at nanomolar levels, produced by autooxidation of ascorbateat lower than micromolar levels, might participate in the inactivationof tAPX. The participation of hydrogen peroxide was confirmedby the inactivation of tAPX upon incubation with hydrogen peroxideunder anaerobic conditions. In the absence of ascorbate, theheme of the two-electron-oxidized intermediate of tAPX (designatedCompound I) is decomposed by hydrogen peroxide. Thus, the instabilityof Compound I to hydrogen peroxide is responsible for the inactivationof APX when ascorbate is not available for Compound I and theenzyme cannot turnover. (Received October 16, 1995; Accepted February 21, 1996)     

Overexpression of Superoxide Dismutase Protects Plants from Oxidative Stress (Induction of Ascorbate Peroxidase in Superoxide Dismutase-Overexpressing Plants)

Photosynthesis of leaf discs from transgenic tobacco plants (Nicotiana tabacum) that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD+) was protected from oxidative stress caused by exposure to high light intensity and low temperature. Under the same conditions, leaf discs of plants that did not express the pea SOD isoform (SOD-) had substantially lower photosynthetic rates. Young plants of both genotypes were more sensitive to oxidative stress than mature plants, but SOD+ plants retained higher photosynthetic rates than SOD- plants at all developmental stages tested. Not surprisingly, SOD+ plants had approximately 3-fold higher SOD specific activity than SOD- plants. However, SOD+ plants also exhibited a 3- to 4-fold increase in ascorbate peroxidase (APX) specific activity and had a corresponding increase in levels of APX mRNA. Dehydroascorbate reductase and glutathione reductase specific activities were the same in both SOD+ and SOD- plants. These results indicate that transgenic tobacco plants that overexpress pea Cu/Zn SOD II can compensate for the increased levels of SOD with increased expression of the H2O2-scavenging enzyme APX. Therefore, the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection in SOD+ plants could result not only from increased SOD levels but from the combined increases in SOD and APX activity.     

植物中的抗坏血酸氧化酶

文章简要介绍植物中抗坏血酸氧化酶的结构、功能、基因的表达调控及其参与的生理反应研究进展。     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

过量表达水稻细胞质型OsAPXs基因提高转基因烟草的耐盐性

为了验证水稻(Oryza sativa L.)细胞质型APXs与细胞耐盐性的关系,实验分别将OsAPXa和OsAPXb（基因登录号：D45423、AB053297）转化到烟草(Nictiana tabacum,N.plum)植株中。Southern结果表明,二基因分别整合到烟草的基因组;Northern分析表明,外源基因在转基因烟草中得到高效表达;在碳酸盐逆境下,T2代转基因植株与野生型对照相比,其APX活性呈现显著的提高,T₂代品系的H₂O₂含量和叶片受害程度显著低于野生型;T₂代品系分别在含有10 mmol·L^-1 NaHCO₃、5 mmol·L^-1 Na₂CO₃的MS培养基上生长,根的生长受到抑制,叶片产生黄化;野生型烟草则难以存活。水稻细胞质型OsAPXs基因的过量表达提高了转基因烟草的耐盐性,揭示出OsAPXa和OsAPXb在碳酸盐逆境应答过程中发挥着重要的作用。     

cDNA Cloning of Thylakoid-Bound Ascorbate Peroxidase in Pumpkin and Its Characterization

A cDNA for thylakoid-bound ascorbate peroxidase of pumpkin wascloned and characterized. Thylakoid-bound ascorbate peroxidasehad a high similarity to cytosolic ascorbate peroxidases, andthe precursor contained a transit peptide to chloroplasts atits ammo-terminus and a putative membrane-spanning region atits carboxy-terminus. (Received February 23, 1996; Accepted March 25, 1996)