Stable Form of Ascorbate Peroxidase from the Red Alga Galdieria partita Similar to Both Chloroplastic and Cytosolic Isoforms of Higher Plants

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, K _m values, k _cat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.     

植物维生素C过氧化物酶研究进展

维生素C过氧化物酶(ascorbate peroxidase,APX)是植物体内的重要酶系,是植物AsA-GSH氧化还原途径的重要组分,是清除H2O2(特别是叶绿体中的H2O2)的关键酶.本文综述了维生素C过氧化物酶表达调控方面的研究进展,包括逆境(干旱胁迫、空气污染、微量元素缺乏、离子胁迫、过度光强、照射以及盐胁迫等)与APX的表达调控、植物细胞程序性死亡(PCD)与APX的表达调控、植物生长发育与APX的表达调控、植物进化与APX表达调控等.植物体内的APX基因包括基质和类囊体两类,不同的APX基因序列存在一定差异,本文还综述了这两类APX基因在植物方面的分离和克隆进展情况,同时对APX基因的遗传转化进行了简要回顾,最后指出了APX今后的研究方向.     

Cloning and Expression of One Chloroplastic Ascorbate Peroxidase Gene from Nelumbo nucifera

A novel ascorbate peroxidase (APX) cDNA was obtained from Nelumbo nucifera (Elian). The phylogenetic analysis indicated that N. nucifera APX grouped together with chloroplastic APX of high plants. The recombinant protein expressed by PET-30a vector showed APX activity (0.04?mM ascorbate min(-1) mg(-1) protein). The APX mRNA was expressed in young leaves, roots, terminal buds, and leafstalks. Synergistic expression of N. nucifera APX and MnSOD mRNA was indicated in the short-term response to mechanical wounding.     

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。     

Cytosolic Ascorbate Peroxidase in Seedlings and Leaves of Maize (Zea mays)

Ascorbate peroxidase (APX) was purified to homogeneity frommaize (Zea mays L. cv.) coleoptiles. APX was a monomer witha molecular mass of 28 kDa, as determined by gel nitration andSDS-polyacrylamide gel electrophoresis. It contained one protohememoiety per molecule, with the oxidized form giving a Soret peakat 403 nm with small peaks at 502 and 638 nm, and the reducedform giving peaks at 435 and 556 nm. The enzyme was not inactivatedby depletion of ascorbate. Cell fractionation and immunohistochemicalstudies using polyclonal antibodies raised against maize APXrevealed that the enzyme was not located in the chloroplastsof green leaves. It was abundant in the cytoplasm but not inthe vacuoles of cells in the coleoptile, mesocotyl and youngleaves of seedlings. In mature green leaves, small amounts ofthe enzyme were distributed in vascular systems, in particularin the companion cells. The N-terminal amino acid sequence ofmaize APX exhibited high homology to pea cytosolic APX, spinachAPX and Arabidopsis APX, but not to APX from tea chloroplasts. (Received February 15, 1993; Accepted May 6, 1993)     

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。     

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.     

毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。     

Ascorbate Peroxidase in Tea Leaves: Occurrence of Two Isozymes and the Differences in Their Enzymatic and Molecular Properties

Two isozymes of ascorbate (AsA) peroxidase were found in tealeaves, and one of them (AsA peroxidase II) was purified tohomogeneity, as judged by polyacrylamide gel electrophoresis.AsA peroxidase II is a monomer with a molecular weight of 34,000and contains protoheme, but it is not a glycoprotein. The enzymeshowed a Soret peak at 409 run and at 420 nm when oxidized andreduced, respectively, with an a-band at 556 nm. The oxidizedenzyme showed two small peaks at 478 nm and 530 nm. The peakat 478 nm disappeared when the enzyme was inactivated by depletionof AsA or by the addition of cyanide. Antibody raised againstAsA peroxidase II from tea did not cross-react with guaiacolperoxidase from spinach, and antibody against the guaiacol peroxidasedid not with AsA peroxidases from tea leaf. The amino acid compositionand amino acid sequence of the amino-terminal region of AsAperoxidase II were determined. Little homology in terms of aminoacid sequence was found between AsA peroxidase II and variousguaiacol peroxidases. The enzymatic and molecular propertiesof the two isozymes showed distinct differences with respectto molecular weight, sensitivity to AsA-depletion, specificityfor the electron donor, and other enzymatic properties. (Received April 13, 1989; Accepted July 25, 1989)     

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.     

Ascorbate Oxidation: UV Absorbance of Ascorbate and ESR Spectroscopy of the Ascorbyl Radical as Assays for Iron

Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example. the concentration of iron can be determined by the metal-dependent rate of ascorbate oxidation in near-neutral solutions. The lower limit of detection of iron, as Fe(III)EDTA. by monitoring the decrease in absorbance at 265 nm of ascorbate is about 200 nM. However, by measuring the concentration of the ascorbyl radical by ESR spectroscopy the lower limit is about 10nM.

Using these assays, I have shown that the typical microliter laboratory syringe can introduce significant iron into solutions. Thus. for studies involving iron, these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.     

Ascorbate Oxidation: UV Absorbance of Ascorbate and ESR Spectroscopy of the Ascorbyl Radical as Assays for Iron

Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example. the concentration of iron can be determined by the metal-dependent rate of ascorbate oxidation in near-neutral solutions. The lower limit of detection of iron, as Fe(III)EDTA. by monitoring the decrease in absorbance at 265 nm of ascorbate is about 200 nM. However, by measuring the concentration of the ascorbyl radical by ESR spectroscopy the lower limit is about 10nM.

Using these assays, I have shown that the typical microliter laboratory syringe can introduce significant iron into solutions. Thus. for studies involving iron, these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.     

Study on Peroxidase Isozymes in Citrus and Fortunella

The acidic peroxidase isozymes in petals of 106 Citrus and Fortunella species and cultivars were studied using polyacrylamide gel electrophoresis. The results (Fig. 1; Tables 1 and 2) suggest that these isozymes be of the monomeric nature coded by a single locus. Five alleles were identified and designated as A, B, C, D and F respectively. No genetic difference in peroxidase isozymes was detected among the mandarins orignated from China, Japan and Indian. Man Ju may be a hybrid between Ponkan and Ougan. Ponkan may have not been involved in the origin of Jiao Gan. Zheng Jiang Jin Ju should be an infraspecific hybrid of C. reticulata. C. sinensis has a close relationship with some Chinese mandarins. Changshou Kumquat and Calamondin are shown to be intergeneric hybrids between Citrus andFortunella.     

非水介质中辣根过氧化物酶（HRP）荧光活性测定法

建立了一种分析ＨＲＰ催化活力的新方法。该方法基于单体（底物）、聚合物（产物）的荧光发射光谱不重叠,使用荧光光谱仪,通过测量底物荧光淬灭来检测ＨＲＰ在非水介质中（二氧六环－水、乙醇－水、丙酮－水体系）催化酚类、芳香胺类物质聚合的活力。此方法迅速、简便,结果是定量并可重复的,并能定量地计算底物转化率。     

Immunological Study of Carbonic Anhydrase in C3 and C4 Plants Using Antibodies to Maize Cytosolic and Spinach Chloroplastic Carbonic Anhydrase

Carbonic anhydrase (CA) was purified to homogeneity from maizeleaves, and antibodies to the purified protein raised usingrabbits. Western blot analyses using antibodies to the purifiedmaize leaf CA indicated cross-reactivity with leaf extractsfrom a variety of C₃ and C₄ monocotyledonous and dicotyledonousplants. However maize leaf CA antibodies quantitatively titratedCA activity only in leaf extracts of maize and sorghum. Pretitrationof crude leaf extracts with maize CA antibodies and activitymeasurements, together with Western blot analyses of the resultantsamples, indicated that there are isozymic forms of CA in plantleaf tissues in both C₃ and C₄ plants which are similar in sizebut which differ antigenically from the maize leaf CA. (Received September 7, 1989; Accepted February 23, 1990)     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

Substrate Specificities of Peroxidase Isozymes in the Developing Pea Seedling

The peroxidase activities of developing pea seedlings were determinedwith several substrates including three phenolic compounds,eugenol, caffeic acid and ferulic acid, which are possible precursorsin the biosynthesis of lignin. Column chromatography of thereaction products of peroxidase with caffeic and ferulic acidsindicates the formation of larger molecular weight complexesof these substrates. The peroxidase isozymes of peas were shownto be heterogenous both in molecular weight and in substratespecificity. Apparent K_m determinations of two isolated isozymesindicate differences in affinities for various substrates. Starchgel zymograms with two different substrates also indicate largedifferences in staining intensities of the different isozymes.The observed pattern of changes in peroxidase level in the developingpea seedling differed according to substrate. For example, whencaffeic acid is the hydrogen donor, a large increase in activitywas observed in the 6th to 8th day of germination. This peakof activity was not observed with other substrates. Pisum sativum, peroxidase isozymes, substrate specificity     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)