"Chitin-specific" peroxidases in plants

The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega (Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.     

Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH

Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.     

Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R₀ plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.     

Fractionation and Characterization of Two Forms of Peroxidase from Oryza Sativa

Abstract

Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.     

Identification and characterisation of Ca-pectate binding peroxidases inArabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca²⁺ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca²⁺-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca²⁺-pectate but not to Ca²⁺-alginate, a polyuronate gel similar to Ca²⁺-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca²⁺-pectate and Ca²⁺-alginate was also assessed. It appeared that 3 of them exhibited a Ca²⁺-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca²⁺-alginate was much weaker than to Ca²⁺-pectate, confirming the specificity of the interaction with the pectic structure.     

Acetylation degree of chitin in the protective response of wheat plants

Influences on the acetylation degree of chitin manifested by proteins from cultural filtrates of strains of the fungus Septoria nodorum different in aggressiveness and of extracts from leaves of the susceptible (Triticum aestivum) and resistant (Triticum timopheevii) wheat plants infected with these strains were studied. Chitin deacetylase was found among the extracellular proteins of the fungus. Its activity was higher in the aggressive strain of the fungus than in the non-aggressive one, and this suggested that this enzyme could play an important role in the further formation of compatible relationship of the pathogens with the plants. Protein extracts from the susceptible wheat seedlings infected with the septoriosis agent also contained a component decreasing the acetylation degree of chitin. Protein extracts from the resistant wheat seedlings increased the chitin acetylation degree. It is supposed that this can be a pattern of the plant counteracting the action of chitin deacetylases of the pathogen.     

Use of chitin for controlling plant plant-parasitic nematodes

Summary Bean and tomato seedlings, treated with different amounts of ammonia and chitin, and inoculated with the root-knot nematode,Meloidogyne javanica, were reared in two consecutive 35-day growth of shoot and root and the condition of the root systems of both plant species and degree of infection byM. javanica were decreased by increasing amounts of ammonia. Chitin caused a relatively small reduction in gall formation but almost no changes in fresh shoot weights. The effect of chitin on plant growth and nematode attack were also compared in irradiated and non-irradiated soil. In the first cycle galling index of the chitin treated plant was similar to that of untreated plants maintained in the irradiated soil conditions, while in the non-irradiated soil, chitin treatment reduced galling index. In the second cycle, chitin treatment reduced galling index in both irradiated and non-irradiated soils, especially in the latter, where galling index greatly decreased compared with the non-treated plants. Differences in fresh shoot weight between nematode-infected and nematode-free plants amended with chitin were greater under non-irradiated than irradiated conditions, especially in the second cycle. In non-irradiated soil, a higher level of chitinolytic microorganisms, particularly actinomycetes, was found in the second cycle. Contribution from the Agricultural Research Organization (ARO), Bet Dagan, Israel. No. 1520-E, 1985 series.     

The Peroxidase Gene Family in Plants: A Phylogenetic Overview

The 73 class III peroxidase genes in Arabidopsis thaliana were used for surveying the evolutionary relationships among peroxidases in the plant kingdom. In Arabidopsis, the 73 genes were clustered in robust similarity groups. Comparison to peroxidases from other angiosperms showed that the diversity observed in Arabidopsis preceded the radiation of dicots, whereas some clusters were absent from grasses. Grasses contained some unique peroxidase clusters not seen in dicot plants. We found peroxidases in other major groups of land plants but not in algae. This might indicate that the class III peroxidase gene family appeared with the colonization of land by plants. The present survey may be used as a rational basis for further investigating the functional roles of class III peroxidases.     

Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides,Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.     

Isolation and localization by histoimmunology of isoperoxidases specific for male flowers of the dioecious species Mercurialis annua L.

Specific anodic isoperoxidases of male flowers of the dioecious plant Mercurialis annua were extracted, partially purified, and injected into rabbits. The serum against these specific peroxidases was used after absorption to localize these male isoenzymes during flower development by means of histoimmunology. (Indirect immunoperoxidase method). The peroxidases were characteristic of microsporogenesis and tapetum differentiation. Their synthesis started at an early stage of male organogenesis. They were not observed in other sites of peroxidase activity of male flowers (tunica, endothecium, filament, vascular anatomy) or in female flowers (embryo sac or nucellus). I propose that these isoenzymes constitute an early and specific marker of male organogenesis in higher plants.     

Structural-functional features of plant isoperoxidases

Current data on structural-functional features of plant peroxidases and their involvement in functioning of the pro-/antioxidant system responding to stress factors, especially those of biotic origin, are analyzed. The collection of specific features of individual isoforms allows a plant to withstand an aggressive influence of the environment. Expression of some genes encoding different isoperoxidases is regulated by pathogens (and their metabolites), elicitors, and hormone-like compounds; specific features of this regulation are considered in detail. It is suggested that isoperoxidases interacting with polysaccharides are responsible for a directed deposition of lignin on the cell walls, and this lignin in turn is concurrently an efficient strengthening material and protects the plants against pathogens.     

Sequential release of both basic and acidic isoperoxidases to the media of suspension cultured cells of Capsicum annuum

Summary The establishment of suspension cell cultures from trimmed cotyledons of pepper (Capsicum annuum L.) provides a new experimental system for studying the relationship between release of peroxidase (EC 1.11.1.7) into the free intercellular spaces and plant cell growth. In contrast with several other species, the total peroxidase activity in the medium increased continuously during the post-exponential growth phase of the pepper cell culture, and this was correlated with the growth inhibition of pepper cells cultivated in suspension. The increase in the peroxidase activity in the culture medium was the consequence of a differential release of isoperoxidases, prominently marked by a primary release of basic isoperoxidases, followed by a strong increase in the level of acidic isoperoxidases. Thus, pepper cells cultures constitute a new experimental system for studying the regulation of the sequential release of basic and acidic isoperoxidases, which occurs during the growth cessation of plant cells.     

Elimination of Differences in the Mobility of Flax Isoperoxidases on PAGE by Digestion with alpha-Mannosidase

In the flax (Linum usitatissimum) genotype Stormont cirrus, anodic peroxidases from the genotroph S migrate more slowly on PAGE and SDS-PAGE than the corresponding peroxidases from the genotroph L. When purified isoperoxidases S2 and L2 were digested with α-mannosidase, the difference in mobility was eliminated. Treatment with α-fucosidase and β-xylosidase also altered the mobility of S2 and L2, but affected the sensitivity to the action of endo-β-N-acetylglucosaminidase H of only S2. Our results suggest differences in posttranslational processing of the carbohydrate moiety between S and L isoperoxidases. These differences were also found in other S and L glycoenzymes (anodic acid phosphatases) as well as in the peroxidases of other flax genotypes.     

Isozyme Patterns of Acid Peroxidase in the Genus Stachys

Isozyme patterns of acid peroxidases and their dependence on plant age, cultivation conditions, and tissue type have been investigated by electrophoresis in polyacrylamide gel in four species of the genus Stachys. The most stable peroxidase patterns have been found in the plant roots. Acid peroxidases have been shown to be species-specific, which allows their use in taxonomic studies. Cultivation in vitro and in vivo produces different isozyme patterns in various tissues of plants of various ages.     

Homology of plant peroxidases: Relationships among acidic isoenzymes

Antisera specific for two commercial acidic peroxidases from horseradish ( Amoracea rusticana L.) were used to determine the degree of homology between isoperoxidases from horseradish, turnip ( Brassica rapa L. cv. Purple White Top Globe) and radish ( Raphanus sativus L. cv. Cherry Belle). Ouchterlony agar diffusion, precipitin tests and anticatalaytic assays were used to show that acidic horseradish peroxidases could be distinguished by immunological methods but were closely related. Antisera specific for either horseradish acidic isoperoxidase gave a lesser degree of cross reaction with the basic isoenzyme from this plant. Acidic isoperoxidases from turnip and radish were more closely related to acidic horseradish peroxidases than the basic isoperoxidase from horseradish as assessed by immunological cross-reaction. Basic isoperoxidases from carrot ( Daucus carota L. cv. Danvers), radish or turnip did not react with antisera prepared against acidic horseradish peroxidases. Finally, acidic horseradish peroxidases were shown to be poor immnnogens in rabbits in contrast to the basic horseradish isoenzyme.     

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and ¹H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.Abbreviations DCP 2,4-dichlorophenol - IAA indole-3-acetic acid - IM indole-3-methanol     

The relationship between the resistance of Aegilops umbellulata Zhuk. seedlings to Septoria nodorum Berk. and peroxidase isozyme pattern

The resistance of Aegilops umbellulata Zhuk. to blotch pathogen Septoria nodorum Berk. has been studied. Cytoplasmic peroxidase activity in normal seedlings could not be used for biochemical prediction of Ae. umbellulata resistance to Septoria blotch. At the same time, isoperoxidases with pI ～7.5 and infection-induced activity of anionic isoforms with pI ～3.5 were markers of the resistant Aegilops plants. Aegilops resistance to the fungus depended on the activity of cell wall peroxidases.     

Simultaneous separation of acidic and basic isoperoxidases in wounded potato tissue by acrylamide gel electrophoresis

Preparation and use of a newly developed pH 4.3 horizontal thin layer acrylamide gel which permits the simultaneous separation of acidic and basic isoperoxidases in up to 30 samples is described. Use of cytochrome c, horseradish peroxidase, and a purified potato isoperoxidase as internal standards for a range in isoelectric points of peroxidases from pH 3 to 11 is introduced to facilitate comparison of results obtained with different materials and different methods. Distribution of tissue-specific isoperoxidases in different cell layers of wounded potato (Solanum tuberosum L.) tissue is shown and their purification described. Evidence for the in vitro degradation of basic potato isoperoxidases resulting in more acidic forms similar to isoperoxidases occurring in wounded potato tissue is presented. The significance of this observation for the postulated differential function of different isoperoxidases is discussed.     

Induction of Chitin-Binding Proteins during the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

Previous work has shown that attachment of Vibrio harveyi to chitin is specific and involves at least two chitin-binding peptides. However, the roles and regulation of these chitin-binding peptides in attachment are still unclear. Here we show that preincubation with the oligomeric sugars composing chitin stimulated chitinase activity, cellular attachment to chitin, and production of chitin-binding peptides. One of these peptides, a 53-kDa peptide, is produced constitutively and appears to mediate initial attachment to chitin. Synthesis of another peptide, a 150-kDa chitin-binding peptide, is induced by chitin and thus may be involved in time-dependent attachment. Coordinated regulation of attachment and degradation of chitin may give bacteria like V. harveyi a selective advantage over other bacteria in nutrient-poor aquatic environments.