Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies

The cellular location of three peroxidase isoenzymes (PRX) in mature leaf tissue of Petunia and their affinity for Concanavalin A-Sepharose were investigated. The isoenzymes PRXa, PRXb and PRXc were identified by their positions in starch-gel zymograms. The fast-moving anodic and cathodic peroxidase bands, the isoenzymes PRXa and PRXc respectively, were the most active peroxidases in extracellular extracts. The molecular forms of PRXa showed a tissue-specific distribution between midrib and remaining leaf tissue. An intermediate-moving anodic peroxidase band, the isoenzyme PRXb, was the most active peroxidase released after extraction of isolated mesophyll protoplasts. Small amounts of the peroxidase isoenzymes were present in cell-wall-bound fractions. Incubation of a crude protein fraction with Concanavalin A-Sepharose showed that the isoenzyme PRXb bound more firmly to Concanavalin A-Sepharose than the isoenzymes PRXa and PRXc, of which only one molecular form bound partly. The results are discussed with respect to a possible function of one of the peroxidase isoenzymes, and a possible role of oligosaccharide chains in determining the cellular location of plant peroxidases is suggested.Abbreviations Con A Concanavalin A - PRX peroxidase (isoenzyme)     

Modulation of expression of SOD isoenzymes in mud crab (Scylla serrata): effects of inhibitors,salinity and season

Presence of several isoenzymes of superoxide dismutase (SOD) were demonstrated in tissues (abdominal muscle: 7 number, hepatopancreas: 13 number and gills: 7 number) of mud crabs (Scylla serrata) by employing specific staining of the enzyme in native-PAGE. SOD isoenzymes in tissues of mud crab were found to be thermolabile. The intensity of a major SOD band in tissues of crabs was reduced by the treatment of H₂O₂ or chloroform:ethanol. KCN treatment resulted in splitting of that major SOD band into two or more distinct bands. SDS treatment resulted in disruption of SOD bands. A sex-specific SOD isoenzyme band of higher molecular weight was observed in gills and muscle in winter and summer seasons, respectively. The observed different SOD isoenzyme pattern in tissues at altered salinities and seasons suggests separate tissue-specific antioxidant adaptation strategies of crabs against abiotic factors.     

苦参和苦豆子的过氧化物酶同工酶谱分析和比较

采用聚丙烯酰胺凝胶电泳技术分析和比较了5个不同产地的苦参和1种苦豆子的过氧化物酶同工酶活性。结果表明：不同产地苦参品种和苦豆子的过氧化物酶同工酶谱迁移率在0.14～0.28之间,共6条酶谱带。聚类分析表明,苦参和苦豆子的亲缘关系较远,而不同产地的苦参种质也存在一定的差异,可分为3类：甘肃成县栽培的苦参与甘肃岷县栽培的苦参为一类,河南卢氏野生苦参单独为一类,河北承德野生苦参和甘肃成县野生苦参为一类。     

山楂叶螨危害对海棠叶片POD的影响

以人工接种的方法研究了山楂叶螨(Tetranychus viennensis Zacher)危害初期(9d内)对海棠(Malus zumi)叶片过氧化物酶(POD)的影响。结果表明,随危害时间延长和虫口密度增大,POD活性迅速升高,30头／叶危害9d和60头／叶危害6d时均约达对照的190％;PAGE分析结果显示叶螨危害使海棠叶片产生2种新的POD同工酶,受害植株的未接虫叶片的POD活性同时升高,并出现与接虫叶片相同的PAGE谱带,揭示叶螨危害对海棠叶片POD有系统诱导性。POD的变化可能是受害植株对叶螨危害的一种应激反应。     

Infrared spectroscopic studies of carbonyl horseradish peroxidases.

Infrared difference spectra, FeIIICO vs. FeIII of horseradish peroxidase isoenzymes A2 and C were recorded from 2000 to 1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibit two CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH is lowered the low-frequency band for each isoenzyme decreases in intensity with a concommitant appearance and increase in intensity of a band at 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conform to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the infrared results was simplified by the observation that a linear relationship exists between the redox potential, Em7, for the FeIII/FeII system vs. the infrared CO stretching frequency, vCO, for cytochrome a3, hemoglobin, myoglobin, and cytochrome P-450 cam with substrate. This relationship suggests that the primary force altering vCO in these heme proteins is a variation in electron density at the heme iron and not direct protein interactions with the CO ligand. The horseradish peroxidase infrared bands in the 1930-cm-1 region correlate well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration are consistent with horseradish peroxidase having a single CO binding site which can hold in two geometries, one of which contains an amino acid moiety capable of forming a hydrogen bond to the carbonyl oxygen.     

Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

Summary Hairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.     

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H₂O₂ content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.     

Cytoplasmic effects on activity of individual anionic isoenzymes of peroxidase in crosses between two genotypes of flax (Linum usitatissimum L.)

Four anionic peroxidase isoenzymes in main stem tissues of flax (Linum usitatissimum L.) were separated electrophoretically on acrylamide gels and their individual activities measured spectrophotometrically in the gels. Activities were expressed in terms of areas of optical density peaks corresponding to the locations of the isoenzymes. Four isoenzymes were assayed for activity in this way in two inbred genotypes and their F₁, F₂, and first backcross progeny. Crosses were made in all combinations between the two parental genotypes and their reciprocal F₁ hybrids to produce 16 progenies of the generations above. Isoenzyme separations and assays for activity were carried out on individual plants of each of the 16 progenies. A model estimating cytoplasmic effects across segregating generations as differences between all progeny of the one compared to the other F₁ reciprocal hybrid, either in a male or a female direction, was fitted to the activity data by weighted least-squares procedures. Cytoplasmic effects transmitted through male gametes from the F₁ reciprocals were demonstrable for three of the four anionic peroxidase isoenzymes.Supported in part by the National Research Council of Canada.     

Isozymes variability among <Emphasis Type="Italic">Fusarium udum</Emphasis> resistant cultivars of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) (Millsp)

The present study was carried out to select the different pigeonpea cultivars for resistance against wilt caused by Fusarium udum and to assess the genetic variability among the resistant and susceptible cultivars. These cultivars were screened by root dip inoculation and classified into resistant (ICP 8863 and 9145), moderately resistant (ICP 11681 and Selection-1), susceptible (ICP 7118, TRG-1 and LRG-30) and highly susceptible cultivars (ICP-2376 and LRG-41). The peroxidase activity (PEO) in both leaf and root tissues of four pigeonpea cultivars (ICP 8863, Selection-1, ICP 2376 and LRG-30) were determined at 1^st, 4^th and 7^th day after inoculation (DAI) in healthy and F. udum infected tissues. Higher PEO activity in both leaf and root was observed and at 4^th DAI in susceptible cultivars. In native-PAGE analysis of isozymes, the induction of specific leaf peroxidase band (Em=0.17) and two root peroxidase bands (Em=0.24 and 0.55) were observed in ICP 8863 after inoculation. Significant differences were observed in the leaf phosphatase and esterase banding profiles of all the cultivars. The presence of leaf phosphatase band at Em of 0.04 was observed only in ICP 8863 and 11681. The leaf esterase band (Em=0.3) was well expressed in ICP 8863 when compared to other cultivars. The significance of peroxidase in plant defense mechanism and utility of biochemical markers in breeding programmes are discussed. Part of M.Sc. (Ag) thesis of the first author and approved by the Acharya N.G. Ranga Agricultural University during March 2002.     

Effect of gamma irradiation on peroxidase isoenzymes during suberization of wounded potato tubers

A comparison of peroxidase isoenzymes in skin, cortex and pith tissues of the potato tuber by thin-layer isoelectric focusing in Sephadex revealed major differences in the isoenzyme patterns. Wounding induced several-fold increases in the peroxidase activity which were correlated with the increased amounts of specific isoenzymes. The anodic and cathodic forms with high activity, normally present in large amounts in skin, were found to be preferentially synthesized in suberizing tissues, suggesting a functional role for peroxidase in the suberization process. Cycloheximide treatment prevented the rapid increase in the content and activity of these specific isoenzymes, which indicated that the increase in peroxidase is due to a de novo synthesis of the enzyme. Suberization is not inhibited by gamma irradiation at sprout-inhibiting dose levels.     

The Subcellular Localization of Isoperoxidases in Capsicum annuum Leaves and their Different Expression in Vegetative and Flowered Plants

The subcellular localization of leaf peroxidases (EC 1.11.1.7)and their expression in vegetative and flowered plants has beenstudied in Capsicum annuum (var. annuum) in order to assesswhether the expression of new peroxidase isoenzymes can characterizethe floral state which determines the beginning of reproductivedevelopment. The results showed that floral development is accompaniedby a significant increase in the level of soluble (non-sedimentable)leaf peroxidase, independently of leaf position along the internodes,and therefore independently of the leaf age. An analysis ofthe leaf peroxidase isoenzyme patterns along the internodesfor vegetative and flowered plants shows that the increase inperoxidase activity is due to a general increase in the activityof all the pre-existing peroxidase isoenzymes, although isoenzymeB₂ and, especially, isoenzyme A₁ showed a distinctive and majorincrease in activity. These two isoenzymes are mainly ionically-boundto cell walls, probably in equilibrium with the same isoenzymesmoving freely in the cell-wall free spaces. The differs fromother peroxidase isoenzymes, such as isoperoxidase B₆, whichis mainly located in the covalently-bound cell-wall fractionand in mesophyll vacuoles. These results are discussed in thelight of a possible role of cell wall peroxidases as markersof the floral state in Capsicum annuum morphogenesis.Copyright1993, 1999 Academic Press Capsicum, floral state, leaf peroxidases, subcellular localization, vegetative state     

Peroxidase and α-Amylase Activities in Relation to Germination of Dormant and Nondormant Wheat

Germination capacity, and α-amylase production in relation to the peroxidase and isoperoxidase activities in the grains of three varieties of wheat have been analysed and compared. A high percentage of germination and α-amylase producation at 25°C are associated with low peroxidase activity of the isolated embryo. This correlation is lacking when the intact grain is considered. A 2-day treatment at 4°C which further increases the percentage germination and enhances α-amylase synthesis, lowers the activity of peroxidase in the embryos. A general decrease in activity of all the isoenzymes is observed. Based on the above data and on differences in the activity of the most cathodic isoperoxidasic bands, a hypothesis is put forward which suggests that a sufficiently low peroxidase activity and a minimum auxin level of the embryo are responsible for the onset of germination.     

Biochemical and ecological characterization of two peroxidase isoenzymes from the mangrove, Rhizophora mangle

This study examines phenolic peroxidase (POX) in Rhizophora mangle L. leaves in order to assess its role in phenolic manipulation and H₂O₂ scavenging. Sun-exposed and understorey leaves experiencing varying degrees of nutrient stress were analysed from an oligotrophic cay off the coast of Belize. POX activity was unaffected by growth environment, but increased throughout leaf development and persisted through senescence and after abscission. Histochemical analyses indicated POX activity throughout leaf tissues, especially in the apoplast. Phenolics were similarly broadly distributed. Two isoenzymes of POX were partially characterized with pIs of 4.1 and 6.3 and masses of 65.5 and 54.3 kDa, respectively. The larger, more acidic isoenzyme showed especially high heat stability, showing no reduced activity after 24 h at 60 °C. Rhizophora mangle POX oxidized quercetin preferentially, and, to a lesser extent, coniferyl alcohol, caffeic acid, chlorogenic acid, and p-coumaric acid. It did not oxidize ascorbate, but ascorbate could act as a secondary electron donor in the presence of a phenolic substrate and H₂O₂. However, because quercetin and other aglycones were not present in R. mangle leaves, and because POX showed no activity with the most abundant leaf flavonoid, rutin, it was concluded that detoxification of H₂O₂ is secondary to the other roles of POX in manipulation of phenolics.     

Thermal activation of peroxidase from tobacco leaf mesophyll cell walls.

Cell wall-bound peroxidase isolated from tobacco leaf mesophyll cell walls was found to consist of two isoenzymes (P1 and P2). Each exists in the form of a single subunit with the same molecular weight (35 000) as determined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Both isoenzymes exhibited maximum activities at 70 degrees. P1 increased to 265% and P2 to 140% of the activities assayed at 27 degrees; below this temperature the two isoenzymes had the same specific activity. On hydrolysis, P1 showed a carbohydrate content of 26.22% when the monosaccharides were analyzed by gas liquid chromatography; P2 gave 21.45%.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.     

Peroxidase Isoenzymes in Development of Tall and Dwarf Cultivars of Rice

Four cultivars of rice, Hansraj (tall), Jamuna, Padma and Sabarmati(all dwarf) were analysed for variation in isoperoxidase patternsduring development by horizontal starch-gell electrophoresis.The samples were taken at weekly intervals, starting with soakedseeds (12 h) until the post panicle stage. There is a distinctpattern of peroxidase isoenzymes in the tall and dwarf cultivarsstudied. Some isoenzymes appear at a particular developmentalstage while others disappear while some remain fairly constantonce they are activated. Hansraj showed more qualitative fluctuationsin peroxidase than did the three dwarf varieties. A few isoperoxidaseswere specific to some varieties such as bands A₃ and A₇ forHansraj and band A₄ for Sabarmati. These specific bands of peroxidasemay have some role to play in development of Hansraj (tall)and Sabarmati (with aroma). Besides this, greater fluctuationsin peroxidase activity in the tall variety may be due to loosecontrol in the regulation of peroxidase activity. The significanceof controlling elements of enzyme activity is discussed.     

The Tonoplast Localization of Two Basic Isoperoxidases of High pI in Lupinus

The ultrastructural localization of peroxidase (EC 1.11.1.7) activity in cambial initial and xylem parenchyma cells of etiolated Lupinus albus hypocotyls (cv. Multolupa) revealed that, unlike phloem tissues, most of the enzymatic activity is located as dark electron-dense deposits on the tonoplast. Subcellular fractionation studies of plasmolyzed hypocotyls revealed that this enzymatic activity is apparently due to two basic peroxidase isoenzymes of isoelectric points 9.5 and 9.7 for B₃ and B₄, respectively. These isoenzymes are found mainly in young (5-day old) seedlings, and are probably involved, as indicated by other authors, in the metabolism of quinolizidine alkaloids in Lupinus species.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

Summary A method for the localization of pyruvate kinase isoenzymes type L, M₂ and M₁ in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M₁ was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M₁. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M₂ was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M₂ was detected in high concentrations in the granulation tissue of type M₂ was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M₂ in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

几种植物组织及其原生质体过氧化物酶同工酶的比较研究

对马尾松幼苗子叶和胚轴、甘蔗、小麦、烟草、黄花菜等幼叶及其原生质体的过氧化物酶同工酶(POI)分别作比较研究。观察到凡经纤维素酶处理的各植物组织POI酶带数均多于未经纤维素酶处理的组织的酶带数;除个别例外,后者一般又比无壁原生质体的酶带数多。此种差异随植株生长年龄而增大,表明植物组织内大部分POI主要存于质外体中。     

Localization and General Properties of Developing Peach Seed Coat and Endosperm Peroxidase Isoenzymes

Changes of soluble and ionically bound peroxidase and indoleacetic acid (IAA) oxidase activities were followed during peach seed development. Soluble peroxidase activity was located mainly in the embryo plus endosperm tissue, whereas wall ionically bound activities were found predominantly in the integument tissue. The different peroxidase isoenzymes present in the extracts were characterized by polyacrylamide gel electrophoresis and isoelectric focusing; the main soluble isoenzyme of embryo plus endosperm tissue was an anionic isoperoxidase of R _F 0.07. Basic ionically bound isoenzymes were located only in the integument tissue, but two soluble anionic isoenzymes of R _F 0.23 and 0.51 were also present in this tissue. In parallel, peroxidase protein content was estimated specifically using polyclonal antibodies. The kinetic data and the changes of seed IAA oxidase activity during fruit development suggested that basic peroxidase isoenzymes from ionically bound extracts of integument might be involved in IAA degradation. Received September 11, 1997; accepted October 21, 1997