Cell Wall and Protoplast Isoperoxidases of Corn Leaves in Relation to Cut Injury and Infection with Helminthosporium maydis

Young leaves of two corn (Zea mays) inbreds with normal and Texas male-sterile cytoplasm, which differed in their susceptibility to Helminthosporium maydis Nisikado and Miyake race T, showed no significant qualitative or quantitative differences in their isoperoxidase patterns. Of six cathodic and four anodic isoenzymes present in the soluble fraction, four and two, respectively, comprised the fraction ionically bound to the cell wall. Peroxidase fractions ionically and covalently bound to the wall constituted about 20% of the total peroxidase activity. No new isoperoxidases were detected in either inbred line in response to cutting, infection, or detachment only and exposure to darkness for 40 hours. Three isoperoxidases, all cathodic, mainly reacted to cutting injury as well as fungal infection. One of the isoperoxidases appeared responsible for the increase in the peroxidase activity of the soluble fraction while the other two were responsible for the increase in that of the fraction ionically bound to the walls. The relative increase in the latter fraction was greater for infected leaves than for mechanically injured ones. No significant differences were found between the two inbreds in their peroxidase reactions to cutting injury or infection. Thus, the corn leaf isoperoxidases were distinctive in their distribution in the cell and in their reaction to injury. Changes in their activity induced by infection may result from a nonspecific response to injury.     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation

The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 μg/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Changes in isoperoxidases induced by hydroxyphenylacetic acids

The effects ofo-, m- andp-hydroxyphenylacetic acids on total peroxidase activity and isoperoxidase patterns of wheat coleoptile sections were studied.o-andm-HOPA reduced the total peroxidase activity whilep-HOPA increased it. Electrophoresis in polyacrylamide gel revealed thato- andm-HOPA diminish the intensity of some anodic and cathodic bands, but thatp-HOPA increases anodic and reduces cathodic isoperoxidases. These results may explain the apparently paradoxical positive effects ofp-HOPA on both growth and total peroxidase activity.     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

Time course and spatial distribution of phenylalanine ammonia-lyase and peroxidase activity in wounded potato tuber tissue

The time course and spatial distribution of wound-induced activities of phenylalanine ammonia-lyase and peroxidase were determined to establish correlations between molecular and cellular aspects of the wound-induced pattern of cell differentiation in potato (Solanum tuberosum L.) tissue. A high correlation between peroxidase activity and suberization was observed. Peroxidase activity increased for several days after wounding. Peroxidase content of suberizing cells was more than 10 times higher than that of the immediately adjacent dividing cells. Suberizing and dividing cells contained different isoperoxidases. Neither time course nor spatial distribution of the wound-induced activity of phenylalanine ammonia-lyase was directly correlated with the wound-induced pattern of cell differentiation.     

Changes in the activities of catalase, peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

PEROXIDASE LOCALIZATION,ACTIVITY, AND ISOZYME PATTERNS IN THE DEVELOPING SEEDLING OF VANDA (ORCHIDACEAE)

Peroxidase activity in the seedling of Vanda was investigated at various stages of development. Active sites were demonstrated histochemically and soluble proteins and peroxidase isozymes were resolved by disc electrophoresis at progressive stages of growth. Activity, which is highest in the early stages of development and lowest at the stage when maximum differentiation occurs, is confined to the epidermal layer and outer surface of the seedling in early stages of development, It is also present in the vascular tissues of the leaves, root, and parenchymatous region at later stages. Indoleacetic acid raises peroxidase activity when present in the growth medium in physiological concentrations. The number of isoperoxidases varies with developmental stage and is lowest in the stage at which leaves and roots are initiated. These observations are discussed in relation to the hypothesis that the peroxidases play a role in morphogenesis as a part of the indoleacetic acid-oxidase system.     

Cell wall and protoplast isoperoxidases in tobacco plants in relation to mechanical injury and infection with tobacco mosaic virus

Leaves and pith of Turkish, Wisconsin 38, and Samsun NN tobacco (Nicotiana tabacum) varieties, which differ in their sensitivity to tobacco mosaic virus, showed the same qualitative isoperoxidase patterns and a similar distribution of distinctive isoperoxidases between the cell protoplast and wall-free, ionically, and covalently bound fractions. No changes in the qualitative isoenzyme spectrum were found in relation to age, mechanical injury, or leaf infection with tobacco mosaic virus. The distinctive isoperoxidases which reacted to infection were the same as those responsive to mechanical injury, confirming that the enzyme reaction to infection results from a nonspecific response to injury. The increase in peroxidase activity in response to infection or mechanical injury, or both, was greater in young tissue than in the older ones. The great increase in Samsun NN leaves and no increase in those of the two other varieties in response to infection may be due to differences in the degree to which the pathogen affected processes controlling the nonspecific peroxidase reaction to injury. Peroxidase development in the infected Samsun NN leaves was due to isoenzymes which form the wall-bound fraction in very young tissues, and to those which increase in activity with aging in the protoplast and wall-free fractions. In mechanically injured tissue, only the first group of isoenzymes increased in activity. In Samsun NN plants, the increased peroxidase activity in upper intact leaves above the infected ones was only due to isoenzymes whose activity increases with both normal and virus-accelerated senescence. Peroxidase reaction to challenge inoculation in these leaves was the same whether the lower ones were intact, infected and/or mechanically injured. Thus, the induced systemic resistance to tobacco mosaic virus may be due to other than peroxidase factors.     

Soluble peroxidase from winter wheat seedlings with phenoloxidase-like activity

Peroxidase isozymes from winter wheat (Triticum aestivum L. cv Orso) seedlings extracts showed phenoloxidase-like activity, becoming visible on polyacrylamide gels also in the absence of hydrogen peroxide. The results obtained after a characterization of the two activities, based on their substrate specificity, on their selective inhibition, and on the possible occurrence of artifacts, suggested the existence of polyfunctional peroxidase isozymes. Different isozymes possessing only phenol oxidase activity were not found in the same plant material. This appears to be the first evidence of phenoloxidase-acting isoperoxidases in winter wheat.     

The Association of Peroxidase Activity and Resistance of Maize to Exserohilum turcicum

Peroxidase activity in leaves of maize (Zea mays L.) differing in susceptibility to Exserohilum turcicum has been investigated in relation to their resistance to Northern Leaf Blight (NLB) caused by the fungal pathogen E. turcicum. In non-inoculated plants, high peroxidase activity was detected in leaves of the resistant isolines B37HtN and B73HtN as compared with the susceptible isolines B37 and B73 and the sweet corn variety Jubilee. After inoculation with E. turcicum, peroxidase activity increased in both susceptible and resistant isolines B73 and B73HtN. However, marked enhancement of peroxidase activity was detected 6 days after inoculation and became remarkable in isoline B73HtN, although symptomes started to show up in both susceptible and resistant plants only 10 days after inoculation. Using polyacrylamide gel electrophoresis separations, different banding pattern of isoperoxidases was found in the susceptible plants as compared with the resistant ones. In non-inoculated plants, three differential bands which appeared in the resistant isoline B37HtN, were absent in the susceptible Jubilee plants, and were as traces in the isoline B37. These bands first appeared in Jubilee and as clear bands in B37, only after inoculation with E. turcicum. The association of these isoperoxidases and resistance of maize to E. turcicum is discussed.     

"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.     

Changes in Isoperoxidases during Cold Treatment of Dormant Pear Embryo

The number of isoperoxidases and the intensity of certain isozymes increased with increasing periods of stratification of pear (Pyrus communis cv. Bartlett) embryos. The presence of GA₃ or kinetin during stratification enhanced the activity of certain isoperoxidases, and these enhancements were blocked in the presence of ABA which by itself had an inhibitory effect. Enhancement in isoperoxidases of pear embryos during stratification was inhibited by 6-methylpurine and cycloheximide; and in the presence of either of these two inhibitors, stratification failed to release the dormancy. Pear embryos germinated for 3 days showed changes in the pattern of isoperoxidases depending on the length of stratification.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Isoperoxidases in Epidermal Layers of Tobacco and Changes during Organ Formation in vitro

The content and pattern of soluble isoperoxidases were determined in epidermal explants taken from different internodes of tobacco plants in the vegetative and floral states. There were qualitative and quantitative differences in the isoperoxidases, with a decrease in content and fewer bands being observed acropetally, i.e., in going from the base of the stem towards the apex. Epidermal explants from floral branches were grown in in vitro culture, with various media moditications, to form de novo floral or vegetative buds, roots or callus. Changes in soluble isoperoxidases were followed electrophoretically in relation to these varying morphogenetic pathways. In each of them, the number of bands increased on both the anodic and cathodic sides with time in culture. Compared to each other these four morphogenetic programmes were different in their peroxidase zymograms, mainly through varying kinetics in the development of activity of the isoenzymes. The changes observed during root and vegetative bud formation agree with previously published data, and the changes during floral bud formation agree with those observed in vivo.     

Cell Wall Free Space of Cucumis Hypocotyls Contains NAD and a Blue Light-Regulated Peroxidase Activity

Solutions were obtained from the cell wall free space of red light-grown cucumber (Cucumis sativus L.) hypocotyl sections by a low-speed centrifugation technique. The centrifugate contained NAD and peroxidase but no detectable cytoplasmic contamination, as indicated by the absence of the activity of glucose-6-phosphate dehydrogenase from the cell wall solution. Peroxidase activity centrifuged from the cell wall of red light-grown cucumber hypocotyl section could be resolved into at least three cathodic isoforms and two anodic isoforms by isoelectric focusing. Treatment of red light-grown cucumber seedlings with a 10-minute pulse of high-intensity blue light increased the level of cell wall peroxidase by about 60% and caused a qualitative change in the anodic isoforms of this enzyme. The increase in peroxidase activity was detectable within 25 minutes after the start of the blue light pulse, was maximal at 35 minutes, and declined to control levels by 45 minutes of irradiation. The inhibitory effect of blue light on hypocotyl elongation was more rapid than the effect of blue light on total wall peroxidase activity, leading to the conclusion that growth and peroxidase activity are not causally related.     

Isoperoxidase Activity and Induction in Cultured Tissues of Wild Carrot: a Comparison of Proembryos and Embryos

Several anodic isoperoxidases were found in embryonic tissues of cultured wild carrot, Daucus carota L., which were not present in the proembryo masses from which they originate. This difference is further reflected in the higher specific activity of peroxidase in embryo extracts as compared to proembryonic tissues. The absence of anodic isoperoxidases and depressed peroxidase activity in carrot tissue cultures in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) suggests a regulatory role for this plant growth regulator in controlling peroxidase activity.     

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.     

Structural physiological, and biochemical gradients in tobacco pith tissue

Explants of tobacco pith taken at various distances from the apex of a mature stem show a sharp gradient in growth potential in vitro; growth is highest in the extreme apical and basal explants, and is minimal in explants removed ca. 75 cm from the apex. Calluses produced by the vigorously growing basal explants are harder and more compact than those produced from more apical explants. The gradient in growth potential is directly correlated with gradients in RNA, protein of cell sap and soluble N per unit fresh weight, but is inversely correlated with peroxidase activity. Cell size increases from apex to base of plants.

The peroxidase activity of pith explants is electrophoretically resolvable into 2 isoperoxidases, moving anodically at pH 9.0. During in vitro culture, this activity rises, due to the formation of several new isozymes moving toward the cathode. The appearance of these isozymes occurs most rapidly in apical and extreme basal explants.