Localization Of Peroxidase Activity Research Articles

Potential role for Trichoderma harzianum, Clonostachys rosea and salicylic acid to induce resistance of date palm roots to Bayoud disease (Fusarium oxysporum f. sp. albedinis)

Bayoud disease caused by Fusarium oxysporum f. sp. albedinis (FOA) poses a major problem to date palm (Phoenix dactylifera L.) in Northern Africa. To solve this issue, biocontrol agents and chemicals inducers were used to trigger defense reactions. The present study indicates the direct effect of two antagonistic fungi, Trichoderma harzianum (TH) and Clonostachys rosea (CR), and salicylic acid (SA) application (1 mM, 1.5 mM) in date palm against FOA. Date palm plants were infected by FOA, inoculated with TH or CR, 3 days earlier, or immersed at two SA concentrations (1 mM, 1,5 mM), daily for 3 days. Plants were harvested and examined 3 days after FOA inoculation with pathogen. FOA alone treated plants were led to strong induction of phenylalanine ammonia-lyase (PAL) and peroxidase (POX), all biocontrol treatments showed an increase in chitinase (CHI) and glucanase (GLU) activi-ties. SA 1mM treated plants also showed high levels of lipoxygenase (LOX), SA 1,5 mM treated plants were increased in POX. Histochemical localization of POX activity and lignin content showed that high POX activity was already observed in plants treated with TH and CR. These findings support are useful in understanding the role of two antagonists and inducer abiotic to trigger defense related enzymes in imparting resistance to date palm against FOA.

Cytochemical localization of peroxidase activity in early developmental stages of the moss Ceratodon purpureus. Light-microscopic observations

Peroxidase activity was localized in the cell walls and minute cytoplasm granules of swollen and germinating spores and in the several-celled protonema of the moss <i>Ceratodon purpureus</i> kept in darkness. Kinetin in a concentration of 100 μM inhibited the protonema development and also depressed the activity of this enzyme.

Cytochemical localization of peroxidase activity in the protonema of the moss Ceratodon purpureus during differentiation of gametophore buds induced by kinetin

An increase in the activity of peroxidases under the influence of kinetin has been demonstrated in the protonema of the moss <i>Ceratodon purpureus</i>. It was connected above all with initiation and acceleration of the differentiation process of gametophore buds which showed a high activity of these enzymes. The increase in peroxidase activity was also noted in the degenerating intercalary cells which produced the buds. The colour reaction characteristic for peroxidases was best noticeable in the minute spherical granules (peroxysomes), nucleoli and cell walls.

Peroxidase activity develops in the micropylar endosperm of tomato seeds prior to radicle protrusion.

Peroxidase activity developed specifically in the micropylar region of the endosperm of imbibed tomato seeds prior to radicle emergence. The activity was first detected approximately 24 h after the start of imibibition (6 h before radicle emergence) and increased markedly thereafter. In the lateral portion of the endosperm, peroxidase activity was undetectable for the first 2 d after the start of imbibition. Although the activity in the lateral endosperm became detectable 3 d after imbibition, the extent of the development of the activity was slight. The localization of peroxidase activity in the micropylar endosperm 2 d after the start of imbibition was confirmed by tissue printing analyses. When the endosperm tissues were wounded, there was an enhancement of the enzyme activity at the wounded region. H2O2 was formed at the expense of NADH only in the presence of Mn2+ and dinitrophenol by the extract from the micropylar endosperm in which peroxidase activity was present. The presence of H2O2 in the micropylar portion of the endosperm was shown histochemically. The possible functions of the peroxidases that develop in the endosperm of tomato seeds are discussed.

Localization of hydrogen peroxide production in Zinnia elegans L. stems

Abstract Hydrogen peroxide is required for a variety of physiological processes associated with plant cell wall biosynthesis. Several recent studies have suggested that starch-KI could be used as an effective histochemical stain for studying the spatial and temporal regulation of H2O2 production during plant development. In this study, we found that detection of H2O2 in Zinnia elegans stems using starch-KI staining was highly dependent upon plant age, developmental status, and the manner in which the tissue was handled prior to staining. An alternative staining technique based on cerium perhydroxide deposition clearly showed that most of the H2O2 detected by starch-KI resulted from wounding at the cut surfaces of stem sections. However, with cerium staining, substantial H2O2 production was apparent well below the cut surface in collenchyma cell walls where previous work had demonstrated localization of peroxidase activity. Co-localization of H2O2 and peroxidase in collenchyma tissues may suggest an involvement in crosslinking of the pectins and hemicelluloses that predominate the walls of these cells. Cerium deposition also suggested that H2O2 production in xylem vessel walls may lag slightly behind secondary cell wall thickening.

Localization of Peroxidase Activity in Tegumental, Muscle, and Parenchymal Cells of the Lung Fluke Paragonimus miyazakii (Digenea: Troglotrematidae)

The distribution and localization of peroxidase activity were examined in adults of Paragonimus miyazakii in sections stained with 3,3'-diaminobenzidine in the presence of hydrogen peroxide. Peroxidase activity was detected in tegumental cell mitochondria. With cell development, the reaction intensity became greater. Activity extended from the inner membrane (undifferentiated stage) to the intermembrane space (differentiating stage) and then to the matrix (differentiated stage). Mitochondria with the most intense reaction were densely distributed in the outer syncytial region. Strong activity was also present in mitochondria of muscle cells and in those of Type II parenchymal cells. Intermembrane spaces were the predominant site of this activity. Characteristically, both 10 mM azide and 1 mM cyanide were mildly effective in the inhibition of mitochondrial peroxidase activity in each cell type.

Localization of peroxidase activity in the retina and the retinal pigment epithelium of the syrian golden hamster ( Mesocricetus auratus)

1. 1. Endogenous peroxidase was localized by electron microscopy within the retinal tissue of the golden Syrian hamster. 2. 2. Peroxidase activity was found in the apical parts of rod outer segments, in the cytoplasm and phagosomes of the retinal pigment epithelium (RPE), and extracellularly between the basal infoldings of the RPE. 3. 3. No reaction product was detected when either H 2O 2 or tetramethyl benzidine were omitted from the reaction medium. 4. 4. The same was true when the tissue was osmicated before the incubation in the reaction medium or treated with 100 mM sodium azide. 5. 5. These results indicate that the peroxidative activity was enzymatically mediated.

Simultaneous detection of membrane markers with monoclonal antibodies and peroxidatic activities in leukaemia: ultrastructural analysis using a new method of fixation preserving the platelet peroxidase.

Simultaneous detection of specific surface markers by immunogold and intracellular peroxidase activity was determined ultrastructurally in normal and leukaemic progenitors of platelets, erythrocytes and granulocytes. A new method of fixation was employed to preserve platelet peroxidase activity. Monoclonal antibodies to platelet glycoproteins labelled exclusively platelet peroxidase (PPO) positive cells, i.e. platelets, megakaryocytes and promegakaryoblasts (PMKB). In acute megakaryoblastic leukaemia, most PMKB possessed both markers while a few PMKB identified by PPO did not bind monoclonal antibodies. This result suggests that PPO appears earlier in maturation than platelet glycoproteins. Although all glycoproteins (GP) displayed fewer sites in PMKB than platelets, GP Ib was often observed in more mature megakaryocytes. Surface (glycophorin A) and intracytoplasmic markers including ferritin, intra-mitrochondrial iron and diffuse peroxidase activity due to haemoglobin of erythroid progenitors, appeared simultaneously. The number of glycophorin A sites increased with maturation. In leukaemia involving PMKB and proerythroblasts, the surface markers were coincident with the localization of peroxidase activity; glycophorin A was always absent from blasts which exhibited PPO activity localized in endoplasmic reticulum. Platelet glycoproteins were never expressed in any other cell lineage. The myeloid surface antigen was present on normal late neutrophilic promyelocytes after the cessation of myeloperoxidase synthesis. In some cases of M1 and M2 AML (FAB classification), labelling was identical to normal cells while in others the antigen appeared earlier than normal. Our findings show that the surface phenotype of blasts from non-lymphoid leukaemia and the intracellular peroxidase activity of a given cell type can be simultaneously demonstrated and analysed by electron microscopy.

Behavior of the Inner Wall Layer of the GerminatingFuligo SepticaSpore: Evidence of Peroxidase Activity

The spore wall of Fuligo septica is composed of an exterior electron-dense, spined layer and an interior more electron-transparent, fibrous layer. The fibrous material in the interior layer is often tightly packed. During germination, the exterior layer splits, but the inner layer does not. A remnant of this inner layer surrounds the newly released protoplast if the remnant is not left behind. The remnant appears to be derived from the fibrous wall material. However, swarm cells appear to be free of structural wall components. Cytochemical tests suggest peroxidase activity in the inner wall layer. Initial localization of peroxidase activity near the split in the outer wall may indicate peroxidase plays a role in the germination process. Light microscopic observations of Fuligo septica (L.) Wiggers show that during germination the spore wall ruptures to form a wedge-shaped split through which the protoplast emerges (11). Electron micrographs of this species indicate that the spore wall is composed of two layers: an outer electron-dense layer with spines and an inner, relatively electron-transparent layer (3). A two-layered wall has been observed in other Myxomycetes such as Arcyria cinerea (Bull.) Pers. (17) and Stemonitis virginiensis Rex (18). In these two species the inner layer is elastic and remains intact longer than the outer layer. However, in F. septica there are no published studies dealing with the behavior of the inner layer. In the present study, we examined the inner wall layer and compared its behavior with that of other Myxomycetes.

THE CYTOCHEMICAL LOCALIZATION OF PEROXIDASE IN TOMATO FRUIT CELLS

The localization of peroxidase activity a t the ultrastructural level in tomato fruit (Lycopersicon esculentum Mill cv. Walters) has been accomplished using the diaminobenzidine histochemical reaction. In immature tomato fruit the peroxidase activity was most prominent at the inner surface of the tonoplast, although some cell wall activity was observed. However, in mature tomato fruit most of the peroxidase activity was localized in the cell wall. No activity was found in organelles or in the cytoplasm of either immature or mature fruit. The significance of these observations is discussed in relation to the physiological role of the enzyme in fruit ripening.

Localization of peroxidase activity in tracheal epithelium.

The activity of endogenous peroxidase was demonstrated in light ciliated cells, mature goblet cells, and nonciliated serous cells, but not in basal cells, intermediate cells, dark ciliated cells or immature goblet cells of the upper tracheal epithelium of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The peroxidase activity ws the most intense in the epithelium of the first tracheal ring and gradually diminished at lower tracheal levels, becoming extremely faint in the vicinity of the middle of the trachea. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum (r-ER) including the nuclear envelope, some vesicles and saccules of the Golgi complex, secretory granules, and small apical vesicles. It is believed that the basal cells were transformed into the intermediate cells, and some of the intermediate cells were turned into the ciliated cells, while other intermediate cells were differentiated into the goblet cells. The dark ciliated cells proved to be the immature ciliated cells immediately after the transformation from the intermediate cells. In these cells, peroxidase is not yet produced.

Peroxidase activity in root hairs of cress (Lepidium sativum L.). Cytochemical localization and radioactive labelling of wall bound peroxidase

The ultrastructural localization of peroxidase activity in young, growing root hairs of cress (Lepidium sativum L.) after assay with 3,3′-diaminobenzidine is reported. Prominent peroxidase activity has been found in the dictyosomes and the associated vesicles, in ribosomes on ER-cisternae, as well as in the cell wall. On the basis of both ultrastructural and cytochemical evidence it is proposed that peroxidase in root hairs is synthesized on the ER- and within dictyosome cisternae, packaged and transported in secretory vesicles and extruded into the cell wall particularity at the tip region of a root hair. The kinetic of Golgi apparatus mediated peroxidase secretion was monitored by measuring the55Fe protoheme content of primary cell walls. Peroxidase secretion seems to be enhanced during stress incubation in destilled water. Secretory activity in root hairs is 20 times higher than in cells of the root body.

Cytochemical Localization of Peroxidase Activity in the Miracidium of Schistosoma mansoni

Distribution of peroxidase activity in the mitochondria of the miracidium of the blood fluke, Schistosoma mansoni, was investigated cytochemically using the diaminobenzidine (DAB) technique. Re-action product was localized in the mitochondria of this larvae stage at pH 7.4 and 9.7. The reaction was peroxide-dependent and insensitive to either potassium cyanide, sodium azide, or 3-amino-1,2,4-triazole at the concentrations used. The reaction was inactivated by heat and by pretreatment with methanol-nitro-ferricyanide, and inhibitor of peroxidase. A perioxide-independent reaction was also observed in the mitochondria. This latter reaction was sensitive to potassium cyanide and sodium azide. It is hypothesized that the peroxidase either may act where peroxide is an electron acceptor in a flavoprotein-linked system or may be a vestige of a more primitive pathway. No peroxidase activity was observed in the mitochondria of other stages of the life cycle of the worm.

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha.

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorphia has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop growth on or methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H2O2 dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H2O2 dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H2O2 was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H2O2 for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.

The fine structural localization of peroxidase activity in digestive organs of rats and mice

An endogenous peroxidase activity is demonstrated in acinar cells of the salivary gland and epithelial cells of the colonic crypt of normal rats and mice using electron microscopic histochemistry. The main site of the enzymatic activity is cisternae of the rough endoplasmic reticulum including those of the nuclear envelope, while the intensity of the activity is greatly variable among cell types. Some vesicular and cisternal elements of the Golgi apparatus and secretory granules exhibit the reaction, but it is not consistent in all cells with the peroxidase-positive endoplasmic reticulum. It is very interesting that the peroxidase activity is positive in the rough endoplasmic reticulum-Golgi complex-secretory granule system (EGG system) of the cells located at the beginning and the end of the digestive tract. This suggests a peroxidase-dependent anti-infectious mechanism.

Localization of peroxidase activity in the developing submandibular gland of normal and isoproterenol-treated rats.

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.

Ultrastructural localization of peroxidase activity in Gomori-positive glia.

Ultrastructural localization of peroxidase activity in Gomori-positive glia.

Cytochemical localization of peroxidase activity in root cells

The distribution of peroxidase in the apical 3 mm of pea roots has been investigated using the histochemical method employing 3,3-diaminobenzidine as a substrate. At the tissue level the enzyme is localized predominately in the root cap, epidermis, inner cortical cells, endodermis, phloem and maturing xylem. At the subcellular level peroxidase is found mainly in the intercellular regions of the cortex cell walls and in the cytoplasm and vacuoles of the steler cells. Root microbodies, unlike those of leaves, do not appear to be able to oxidize this substrate. The significance of these observations is discussed in relation to the validity of the technique and the proposed roles of the enzyme in cellular metabolism.

Toxoplasma gondii: Localization of peroxidase activity

Localization of peroxidase activity in Toxoplasma gondii was studied using the method of Graham and Karnovsky (1966). Pcroxidase reaction was confined to mitochondria and no other organelles such as the polar ring, conoid, micronemes, micropyle and submembranous fibrils were found to have positive reactions.

Ultrastructural localization of peroxidase activity in human basophil leukocytes.

The cytoplasmic granules of the human basophil leukocyte have been shown to exhibit peroxidase activity both at the light and electron microscopic levels when exposed to the 3,3’- diaminobenzidine tetrahydrochloride peroxidase procedure. Reactivity is diminished when exogenous peroxide is omitted from the incubation medium and is completely abolished when catalase is included in the peroxide-free medium