Changes of peroxidase,esterase isozyme activities and some cell inclusions in regenerated vascular tissues after girdling inBroussonetia papyrifera (L.) Vent

The isozyme zymogram of peroxidase and esterase, and some cell inclusion contents were changed with the differentiation of regenerated vascular stem tissues after girdling inBroussnetia papyrifera (L.). Vent. Presence or disappearance of some peroxidase and esterase isozyme bands was related to wounding. Some isoperoxidase bands disappeared at the time of vascular tissue formation, but some esterase isozyme bands appeared in phloem or cambial regions as sieve-like elements or mature xylem were formed. The inclusion grains progressively disappeared with the formation of callus and initiation of vascular meristems. The cell inclusions reappeared during the formation of regenrated vascular tissues. Histochemical study indicated that the inclusion grains could be a complex compound of a protein mass encircling polysaccharide in the center with a proteinous nucleus.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Immunolocalization of a plant glutathione peroxidase-like protein

We report on the localization of GPXle-1, a plant glutathione peroxidase (GPX)-like protein, in the internode of Lycopersicon esculentum Mill. GPXle-1 was detected in the cytoplasm near to the plasma membrane of trichomes, in the wall of collenchyma and in both the cytoplasm and wall of vascular tissues. GPXle-1 was not found in the epidermis or parenchyma. After mechanical stimulation, a change in its cell distribution was recorded. In stimulated plants, GPXle-1 was detected throughout the cytoplasm in the epidermis, collenchyma and cortical parenchyma.Abbreviations GPX Glutathione peroxidase - ROS Reactive oxygen species     

Ethylene effect on extensin and peroxidase distribution in the subapical region of pea epicotyls

In dark grown pea (Pisum sativum) seedlings ethylene causes the triple response in which elongation growth is inhibited, radial growth is promoted, and orientation of shoots to gravity is altered. The distribution of extensin and peroxidase activity in pea epicotyls upon ethylene treatment was studied by tissue printing on nitrocellulose paper. It was found that the localization of extensin and peroxidase activity changes after 72 and 96 hours of ethylene treatment. In untreated plants, peroxidase activity is detected only in the vascular bundles. Nonetheless, after 72 and 96 hours of ethylene treatment peroxidase activity is hardly detected in the vascular system but present in the epidermal and cortical cells. Extensin increases in the epidermal and cortical cells upon ethylene treatment but it also appears in the vascular system when peroxidase activity is no longer detected.     

Diffusion barrier to horseradish peroxidase in the vascular stria of the guinea pig

Summary Guinea pigs were given horseradish peroxidase intracardially and its diffusion in the vascular stria was studied. The Peroxidase spred freely among the intermediate cells and the marginal cells, but was never found to have passed zones occupied by tight junctions. It is concluded that the zones of tight junctions bordering the vascular stria represent a diffusion barrier to horseradish peroxidase.     

Identification and characterization of VPO1, a new animal heme-containing peroxidase

Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K_m for H₂O₂ of 1.5 mM. When co-expressed in cells, VPO1 can use H₂O₂ produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Vascular reactions to horseradish peroxidase in the guinea pig

Summary Albino guinea pigs were given intradermal injections of the protein tracer horseradish peroxidase. In a 0.1 mM concentration the tracer did not increase vascular permeability to Evans blue-labelled plasma proteins. In a 1 mM concentration, however, the peroxidase induced a local vascular leakage. This leakage was almost totally inhibited by pretreating the animals with acetylsalicylic acid, while antihistamine had only a weak inhibitory effect. We therefore believe that prostaglandins are important mediators in this HRP-induced vascular reaction.     

A quantitative histochemical study of the microvasculature of irradiated skin

Short-term X-ray damage to the microvasculature of the skin of newborn rats has been quantitated using Horseradish Peroxidase as a tracer. Image analysis of thick sections on which peroxidase was demonstrated histochemically revealed a radioinduced increase in vascular volume coupled with a decrease in vascular length and an altered frequency distribution of blood vessel calibers which resulted in early telangiectasia. The results afforded by direct counting of peroxidase positive macrophagic cells and microphotometric evaluation of peroxidase present in the connective tissue indicate a progressive increase in capillary permeability as a function of dose and time post-irradiation. The accuracy with which the affected region of blood vessels coincided with the area exposed to the beam favours the hypothesis of direct damage to the vessel wall as a major cause of radioinduced lesion.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Immune-complex trapping in the splenic ellipsoids of rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss), immunised with horseradish peroxidase, were given horseradish peroxidase intravenously, and the trapping of antigen in the spleen was followed 1, 24, and 48 h after injection. After 1 h, the localisation of horseradish peroxidase indicated that the antigen had been extensively trapped in the walls of the splenic ellipsoids. The colocalisation of horseradish peroxidase with rainbow trout immunoglobulin M and complement factor 3 was shown with a double immunofluorescence technique and suggested that horseradish peroxidase was trapped in the form of immune complexes. After 24 and 48 h, very little horseradish peroxidase was detected in the ellipsoids, and horseradish peroxidase was mainly found in association with large cells with prominent cytoplasmic extensions. In nonimmunised fish given horseradish peroxidase intravenously, antigen was not detected in ellipsoids. Thus, the observed difference between immunised and nonimmunised trout suggests a specific role for the splenic ellipsoids in rapid immune-complex trapping and invites speculation on its significance in a secondary immune response.     

Interactions between iron nutrition and Verticillium wilt resistance in tomato

The relationship between Fe nutritional status and Verticillium wilt disease in tomatoes possessing single gene resistance to Race 1 of Verticillium dahliae was investigated using hydroponic culture media. Iron limiting conditions increased the sensitivity of resistant tomatoes to the pathogen as expressed by wilting and chlorosis. Distance of fungal vascular invasion was approximately the same in both Fe replete and Fe limited treatments. Comparison of near-isolines revealed that the magnitude of disease expressed in Fe deficient Pixie II (resistant) was considerably less than that expressed by the susceptible Pixie variety. Infection of tomato did not enhance the severity of low-Fe stress as quantified by root peroxidase activity and chlorophyll content of young leaves.     

Effect of culture temperature on the heterologous expression of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> versatile peroxidase in <Emphasis Type="Italic">Aspergillus</Emphasis> hosts

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L^-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.     

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica

The cDNA encoding for lignin peroxidase of Phanerochaete chrysosporium was expressed in the Pichia methanolica under the control of the alcohol oxidase (AUG1) promoter which was followed by either the lignin peroxidase leader peptide of Phanerochaete chrysosporium or the Saccharomyces cerevisiae alpha-factor signal peptide. Both peptides efficiently directed the secretion of lignin peroxidase from the recombinant yeast cell. The extracellular lignin peroxidase activity in two recombinants was 932 U l(-1) and 1933 U l(-1). The purity of the recombinant product was confirmed by SDS-PAGE.     

Involvement of Lipid Peroxidation in Water Stress-Promoted Senescence of Detached Rice Leaves

Role of lipid peroxidation and antioxidative enzymes (catalase, peroxidase, superoxide dismutase, ascorbate peroxidase and glutathione reductase) in water stress-promoted senescence of detached rice leaves was investigated. The senescence was followed by measuring the decrease in protein content. Increased lipid peroxidation was closely correlated with senescence in water stressed leaves. Decrease in superoxide dismutase activity was evident 8 h after beginning of water stress. However, decreased catalase, peroxidase, and ascorbate peroxidase activity was observed only when senescence was observed. Glutathione reductase was not affected by water stress. Free radical scavengers retarded water stress-enhanced senescence.     

Le phloème deSelaginella willdenowii: histophysiologie comparée

Metaphloem sieve elements ofSelaginella willdenowii are elongated cells with slightly oblique or transverse end walls. Pores are seen on both lateral and end walls, although they are more numerous on the latter. Parenchyma cells exhibiting strong enzyme activities (acid phosphatase, non specific esterase, succinate dehydrogenase, cytochrome oxidase, peroxidase) are present between sieve elements and tracheids in each vascular bundle. A functional association thus appears to exist between these parenchyma cells and the conducting elements.—The occurrence of transverse to slightly oblique end walls in sieve elements seems to characterize the ligulate Lycopsids (as opposed to the aligulateLycopodium where sieve elements possess slanting, very oblique, end walls).

     

Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures

A callus strain with stable ability for vindoline synthesis was selected from many prepared Catharanthus roseus leaf calli to study the regulation of vindoline biosynthesis as well as other indole alkaloids. It was shown that light and plant growth regulators significantly influenced the biosynthesis of vindoline and other alkaloids as well as acidic and basic peroxidase activities. Light promoted vindoline and serpentine biosynthesis, and stimulated plastid development and peroxidase activity. However, 2,4-D suppressed the biosynthesis of all indole alkaloids and peroxidase activity. Our results suggest that light or plant hormones regulate vindoline, serpentine and other alkaloid biosynthesis and accumulation by influencing peroxidase activity and the differentiation status of callus cultures, especially chloroplast development. Some possible relationships between serpentine or vindoline biosynthesis and peroxidase activity are proposed.     

Superoxide dismutase,catalase, and glutathione peroxidase in the swim bladder of the physoclistous fish,Opsanus tau L

Summary The antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase were measured in the rete mirabile and gas gland epithelium area of the swim bladder of the toadfish Opsanus tau. When the concentration of enzyme in the swim bladder was compared with the concentration in other organs (kidney, heart, gills) of the same fish, the swim bladder was found to have the highest concentration of superoxide dismutase but relatively low levels of glutathione peroxidase and catalase.Cytochemical assay for the peroxidatic activity of catalase confirmed that virtually no catalase is present in epithelial cells of the gas gland. A similar assay for peroxidase revealed a cyanide-sensitive peroxidase in the multilamellar bodies of these cells. Most of the catalase and peroxidase in the rete mirabile appears to be confined to the granules of neutrophils and the cytoplasm of erythrocytes. Enzyme activity in the neutrophils is not inhibited by 10^-1 M KCN. Cyanide does appear to inhibit the peroxidase activity in erythrocytes but has little effect on catalase in these cells.Supported by grant No. HL23338 from the National Institutes of Health     

Optimalization of the peroxidase production by tissue cultures of horseradish <Emphasis Type="Italic">in vitro</Emphasis>

Tissue cultures of Armoracia rusticana L., both transformed with Agrobacterium rhizogenes and nontransformed, were screened for peroxidase activity. Most of the derived and tested strains exhibited 20 times higher activity [from 99 to 723 U g⁻¹(d.m.)] than the root of the intact plant [(30 U g⁻¹ (d.m.)]. The highest peroxidase activity was found in tumour culture growing on the medium without growth regulators. The influence of the addition of sugars and heavy metal ions in the medium on peroxidase production was tested. Increase in peroxidase activity was observed after cultivation of horseradish culture with cadmium, cobalt, nickel or lead ions.This work is supported by Grant Agency of Czech Republic Project No. 526/04/0135.