Isoperoxidase Patterns Research Articles

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (Cn) appeared in the medium within 2 days after the cells were placed in the dark. Cn was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). Cn increased and new anodic isoperoxidases Aa, Ab, Ad and Ae appeared in the media as the cells approached death in darkness.

Occurrence of Some Enzymes in Starchy Endosperm and Hormonal Regulation of Isoperoxidase in Aleurone of Wheat

Peroxidase, indoleacetic acid-oxidase, alkaline, and acid phosphatases were detected in dry starchy endosperm (minus aleurone) of wheat grain. The isoperoxidase pattern differed in different parts of the dry grain. Several new isoperoxidases were found in embryos after soaking. The intensity of isoperoxidases in aleurones was enhanced in the presence of embryo or 2 muM GA(3) after 24 hours of soaking, but decreased after 72 hours. Indoleacetic acid and kinetin had no effect on isoperoxidase of aleurone. Actinomycin D and cycloheximide had no effect on isoperoxidases of aleurones from embryonectomized or naturally occurring embryoless grains. However, these two inhibitors increased the intensity of isoperoxidases in aleurones of intact embryonated grains after soaking.

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.

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.In infected tissues, peroxidase was detected in the endoplasmic reticulum, Golgi apparatus, vacuole, cell wall, and intercellular spaces. The Golgi vesicles were often localized near the tonoplast and plasmalemma, fusing with membranes and secreting their contents. The possible "rejuvenating" effects of injury on synthesis and transport of distinctive isoperoxidases are discussed.

Cell Wall and Protoplast Isoperoxidases in Relation to Injury, Indoleacetic Acid, and Ethylene Effects

In tobacco (Nicotiana Tabacum) pith, sweet potato (Ipomoea batatas), and carrot (Daucus carota) storage roots, differences were found between cell wall and protoplast peroxidases in their isoenzyme patterns, activity, and reaction to tissue injury.In the pith of elongating tobacco internodes, 90% of total activity was associated with the walls, 80% of which was due to the ionically and covalently bound fractions. With senescence, the increase in activity occurred mainly in the protoplast and the free fraction in the walls. The major protoplast isoperoxidases formed the free wall fraction, and the minor ones formed the wall bound fractions. The minor isoperoxidases were the only ones whose activities were affected by cut injury. Neither senescence nor injury caused qualitative changes in the cell isoperoxidase pattern. In the potato root cells, the isoperoxidases were also distinctive in their distribution and in their reactions to injury as well as to ethylene.In potato and carrot roots, the wall fractions contributed 30 and 95%, respectively, to total peroxidase activity. The injury-induced increase in peroxidase activity occurred mainly in the protoplast of tobacco pith and potato roots and almost entirely in the ionically bound fraction of the walls in carrot roots. Ethylene stimulated peroxidase development in potato roots, especially in the protoplast, but had no effect in either tobacco pith or carrot roots. Ethylene did not affect the inhibiting action of indoleacetic acid on peroxidase activity in tobacco pith, nor did indoleacetic acid significantly affect the stimulating action of ethylene in potato roots.Actinomycin D inhibited the injury-dependent development of peroxidase in tobacco pith, stimulated the injury- and ethylene-dependent peroxidase development in potato root, and had no effect on peroxidase development in carrot root. Cycloheximide prevented the increase in peroxidase activity in tobacco pith and potato root, but not in the carrot root. Thus, the enhancement of peroxidase activity resulted from either enzyme activation exclusively or from enzyme synthesis. RNA synthesis seems to be only indirectly related to isoperoxidase synthesis caused by injury or ethylene.

Comparative Studies on Tobacco Pith and Sweet Potato Root Isoperoxidases in Relation to Injury, Indoleacetic Acid, and Ethylene Effects

Sweet potato (Ipomoea batatas) root parenchyma and tobacco (Nicotiana tabacum) stem pith, both known to increase peroxidase activity after excision, differed from each other in their isoperoxidase patterns and in the isoperoxidase responses to injury and exogenous ethylene.In potato root sections, the injury-dependent peroxidase increase was due to an induction of two isoenzymes, as well as to a promotion of some constitutive ones. In tobacco pith, this increase was entirely due to seven isoperoxidases not detectable, or detectable only in traces, immediately after excision. Actinomycin D did not inhibit the development of any isoperoxidases in the potato root sections and strongly repressed the development of all injury-induced isoenzymes in tobacco pith. Cycloheximide totally inhibited the development of all isoperoxidases in both species, with the exception of two injury-enhanced isoenzymes in root parenchyma.In root sections, indoleacetic acid had a weak inhibitory effect on one injury-induced isoperoxidase only, whereas in tobacco pith it inhibited the development of the injury-induced, as well as the constitutive, isoperoxidases.Exogenous ethylene did not induce, enhance, or significantly suppress any of the tobacco pith isoenzymes, whereas in potato root sections, it suppressed slightly the development of the injury-induced, had no effect on some of the injury-enhanced, and greatly promoted some of the injury-unaffected or-enhanced isoperoxidases. Removal of ethylene stopped the ethylene-dependent peroxidase increase without affecting the injury-induced increase. When applied to intact potato roots, ethylene did not induce any new isoperoxidases and promoted the same constitutive isoenzymes as it did in root sections.Thus, the tissue peroxidase response to ethylene seems independent of its response to injury. Differences between tissue species in their response to ethylene may depend on the presence or absence of isoperoxidases sensitive to ethylene. The inhibition of injury-dependent peroxidase development by indoleacetic acid cannot be explained by an ethylene-induced inhibition.

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F 2 data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.

Differential extraction of isoperoxidases from Pisum shoots

When green or etiolated Alaska pea shoots are extracted with water or 50 mM phosphate buffer, pH 6·5, or macerated without addition of buffer, the elution profile of the resulting supernatant lacks several isoperoxidase components which are readily extracted from the residue with 10 mM EDTA at pH 6·5. Subsequent extraction with 270 mM NaCl does not solubilize any further components. These results cast doubt on the validity of earlier reports on isoperoxidase patterns in Pisum and other plants.

Isoperoxidase Patterns in Tobacco Pith and their Alteration Following Tissue Excision

Isoperoxidase Patterns in Tobacco Pith and their Alteration Following Tissue Excision

EFFECTS OF CADMIUM ON ISOPEROXIDASE PATTERN AND ANTIOXIDANT ENZYME ACTIVITIES IN GERMINATING WHEAT SEEDS

The objective of the study was to investigate the changes in growth parameters and in enzyme activities during wheat seed germination, in laboratory conditions, in presence of different amounts of cadmium excess. Two varieties of wheat were studied (Triticum aestivum L.cv. Boema and cv. Simnic 30) in order to select plants which are tolerant to cadmium stress. The activity of ascorbate peroxidase, soluble peroxidase, isoperoxidase and catalase, enzymes involved in the scavenging of reactive oxygen species, were measured. Obtained results show cadmium induced changes in investigated enzyme activities and illustrate that cadmium uptake was positively correlated with some enzyme activities and negatively correlated with other ones. It is reasonable to think that enzyme stimulation plays a role in the cellular protection against metal toxicity, but it cannot be excluded that in some cases the increased enzyme activity might represent a toxic effect. Measurement of oxidoreductase activity, especially peroxidase and specific change in the isoperoxidase pattern, might be used as biomarkers to asses the phytotoxicity for wheat grown on cadmium contaminated media.

ISOPEROXIDASES PATTERN OF ROBINIA PSEUDOACACIA VAR. OLTENICA IN VITRO CULTURE ON DIFFERENT MEDIA

In this work is studied the isoperoxidase pattern of the in vitro subculture of Robinia pseudoacacia var. oltenica, on various culture media, with various content of myo-inositol, humic acids and magnetic fluids. The electrophoretic separation of the isoperoxidases was performed from extracts obtained from 30 days’old vegetal material. The isoperoxidase fractions were characterized in terms of quantity and quality and a study of their thermal stability was performed. The obtained data show that the isoperoxidase activity is modulated in various ways by the presence of the studied factors into the culture medium.