HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

Peroxidase and Senescence

Kinetin and a, á-dipyridyl prevented the rapid decreaseof chlorophyll content in detached oat leaves senescing in thedark. In the light, detachment caused a 27–40% rise in peroxidaseactivity and kinetin enhanced the enzyme in the segments byabout 80%. Darkness prevented any detachment-induced rise ofthe activity and decreased the stimulating action of kinetinand mechanical injury. The effect of dipyridyl on peroxidaseactivity in the dark was similar to that of kinetin. Kinetin enhanced the same distinctive isoperoxidases under lightand dark conditions. Neither horseradish peroxidase nor that extracted from oat leavesshowed any ability to hydroxylate free proline in vitro. A systemwhich supposedly led to peroxidase-catalysed proline hydroxylationyielded small amounts of hydroxyproline in the absence of theenzyme. Staining with Fast Blue BB salt in the presence of IAA as asubstrate after electrophoresis indicated that all detectedoat isoperoxidases had an IAA oxidase activity visually parallelingtheir peroxidase activity. Crude extracts contained IAA oxidaseinhibitors that could be partially or fully removed by dialysis. The possible significance of the rise in peroxidase activityduring senescence is discussed.     

Repression of isoperoxidase formation in excised tobacco pith by exogenous, auxin-controlled RNA

Previous work has shown that tobacco pith tissue contains two constitutive isoperoxidases migrating toward the anode at pH 9·0. Within 24 hr of aseptic culture on basal medium, such tissue develops five new isoperoxidases, three cathodic and two anodic. The appearance of the new isoperoxidases involves de novo protein formation; it is inhibited by anaerobic conditions, by such inhibitors as Actinomycin D, and by the plant hormone indole-3-acetic acid (IAA). We now find that phenol RNA extracted from parent pith and injected or vacuum infiltrated into cultured pith explants prevents the appearance of the new isozymes; RNA from cultured pith has no such effect. Hydrolysis with 0·3 N KOH, ribonuclease or proteolytic enzymes partially destroys this activity, while treatment with both ribonuclease and proteolytic enzymes completely destroys it. Fractionation of the RNA indicates that part of the repressor activity is associated with an mRNA-like fraction.     

Peroxidase and IAA oxidase activities and peroxidase isoenzymes in the pericarp of seeded and seedless “Redhaven” peach fruit

Parthenocarpic peach fruit (Prunus persica L. Batsch., cv. Redhaven) were induced with 1-(3-chlorophthalimide)-cyclohexane carboxamide (AC 94377). The activities of soluble, and ionically and covalently bound peroxidase and indole-3-acetic acid (IAA) oxidase in the pericarp of both seeded and parthenocarpic fruit were determined from 21–43 days after anthesis. Seedless fruit grew faster during early stage I and ceased growth earlier than seeded fruit. Total peroxidase and IAA oxidase activities increased with development on both types of fruit, but higher values were found in seedless fruit. The ionic fraction showed the greatest increase for both enzyme activities. Isoperoxidase profile showed new cationic isoenzymes and higher levels of the less anionic isoenzymes in the pericarp of seedless fruit, whereas the seeded fruit contained higher levels of the more acidic isoperoxidases.     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid     

Influence des régulateurs de croissance sur la prolifération,l'activité peroxydasique et les isoperoxydases de la suspension cellulaire deSilene alba

Growth ofSilene alba (MILLER) E. H. L. KRAUSE cells, as well as their peroxidase pattern and activity are studied. Cells were grown in the presence and absence either of IAA, or NAA or 2,4-D. The subculture is dependent upon the growth regulator used to sustain the growth of cells. For 14 days' passages, subculture is possible with 2,4-D (5 x 10^-7M) or NAA (10^-5M) but impossible with IAA or without any growth regulators. Cells grown using 2,4-D or NAA in the medium contain a smaller number of isoperoxidases and have lower activities than those grown using IAA or no growth regulator. The nature of growth substances does not affect the compartimontation of the peroxidase; in fact the bulk of the peroxidaso activity is always liable to the ionic wall bound fractions. Tho electrophorotic mobilities of peroxidase isoenzymes detected in the modium are not the same as those of tho eytoplasmic isoenzymes. Cell cultures grown with and without growth regulators show different patterns of modium peroxidase activities. Some forms are present both in cells and media and some other only in the media; this may indicate that there is some selection made in tho cells for retention of particulars forms; the others could be secreted as exoenzymes shortly after they are synthesized in the cells. The nature of the growth regulator used could act on the release of certain isoperoxidases. These results are discussed from the viewpoint of the correlation of isoperoxidase patterns with the possibility of subculture.     

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.     

The vacuolar localization of basic isoperoxidases in grapevine suspension cell cultures and its significance in indole-3-acetic acid catabolism

The study of the subcellular localization of the basic isoperoxidases in grapevines was carried out by using cells cultured in suspension as a model system. Results from subcellular fractionation, isoenzyme analysis, enzyme binding and cytochemical probes suggest that basic isoperoxidases are localized mainly in the vacuolar sap of the suspension cultured cells, probably in equilibrium with a pool of the same basic isoperoxidases bound to the internal face of tonoplast membranes through a Ca²⁺-saline bridge. This vacuolar location of basic isoperoxidases raised the question of their function, since indole-3-acetic acid (IAA) oxidase activity of these isoperoxidases is almost totally inhibited by vacuolar anthocyanins in the in vivo concentration range of these compounds. Thus, a central role is proposed for these isoenzymes in the H₂O₂-dependent oxidative phenol metabolism which occurs in grapevines, discarding therefore a possible role of these isoperoxidases in the control of IAA levels during the later stages of development of anthocyanin-rich grapes.     

Soluble peroxidase gradients in lupin hypocotyls and the control of the level of polarly transported indole-3yl-acetic acid

The distribution of basic soluble isoperoxidases along the growth gradient of lupin hypocotyl was studied in order to establish the role of these isoenzymes in controlling polarly transported indole-3yl-acetic acid (IAA) levels. The observation that the levels of basic isoperoxidases, which diminish from the young (vascular differentiating) to the older (vascular differentiated) tissues, are related with previously reported IAA oxidation rates in decapitated plants, suggests that these isoenzymes can play a role in the oxidation of IAA during polar transport. The fact that the level of basic isoperoxidases is controlled by IAA in hypocotyl sections harvested from different growth zones is in accordance with the previously described adaptative activation of basic isoperoxidases to IAA content. This adaptative activation of basic isoperoxidases might constitute the basic characteristic of a system of subcellular oscillators, coupled at the cellular level, necessary to generate the supracellular auxinwave associated with auxin transport.     

Soluble peroxidase gradients in lupin hypocotyls and the control of the level of polarly transported indole-3yl-acetic acid

The distribution of basic soluble isoperoxidases along the growth gradient of lupin hypocotyl was studied in order to establish the role of these isoenzymes in controlling polarly transported indole-3yl-acetic acid (IAA) levels. The observation that the levels of basic isoperoxidases, which diminish from the young (vascular differentiating) to the older (vascular differentiated) tissues, are related with previously reported IAA oxidation rates in decapitated plants, suggests that these isoenzymes can play a role in the oxidation of IAA during polar transport. The fact that the level of basic isoperoxidases is controlled by IAA in hypocotyl sections harvested from different growth zones is in accordance with the previously described adaptative activation of basic isoperoxidases to IAA content. This adaptative activation of basic isoperoxidases might constitute the basic characteristic of a system of subcellular oscillators, coupled at the cellular level, necessary to generate the supracellular auxinwave associated with auxin transport.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Effect of gamma irradiation on peroxidase isoenzymes during suberization of wounded potato tubers

A comparison of peroxidase isoenzymes in skin, cortex and pith tissues of the potato tuber by thin-layer isoelectric focusing in Sephadex revealed major differences in the isoenzyme patterns. Wounding induced several-fold increases in the peroxidase activity which were correlated with the increased amounts of specific isoenzymes. The anodic and cathodic forms with high activity, normally present in large amounts in skin, were found to be preferentially synthesized in suberizing tissues, suggesting a functional role for peroxidase in the suberization process. Cycloheximide treatment prevented the rapid increase in the content and activity of these specific isoenzymes, which indicated that the increase in peroxidase is due to a de novo synthesis of the enzyme. Suberization is not inhibited by gamma irradiation at sprout-inhibiting dose levels.     

Involvement of ethylene in growth induction of stationary tobacco pith tissue in vitro

The effects of ethylene on growth initiation of tobacco pith tissue in vitro were investigated. Pith explants were incubated on a double inorganic modified White’s media containing 0.2 mg l⁻¹ kinetin with and without indole-3-acetic acid (IAA) and the ethylene synthesis inhibitor aminooxyacetic acid (AOA). The burst of wound ethylene had no effect on growth initiation, was not affected by the AOA, and decreased to its minimum level during the initial 24 h in culture. Tissue growth was initiated after 72 h and continued on IAA-containing media only. A marked increase in ethylene evolution occurred only in tissues subjected to an IAA-containing medium prior to growth initiation. AOA inhibited this ethylene synthesis and the following growth of the tissues. The initial water uptake by the pith explants occurring even in the absence of IAA was also inhibited by AOA. The metabolic indicators for growth initiation such as enhanced respiration, increased activity of nitrate reductase, and initiation of cathodic isoperoxidases were all inhibited by AOA. It was concluded that the primer function of IAA in growth initiation is via inducing the biosynthesis of a marked ethylene signal, which in the absence of which active growth will not occur. The inhibiting effect of AOA is continuous and a transfer of the pith explants to fresh IAA-containing media did not result in a new ethylene burst nor tissue growth induction. The morphological changes in the tissues and cells during the initial stages of their development on the different media are demonstrated.     

EFFECTS OF X IRRADIATION AND INDOLEACETIC ACID ON SPECIFIC PEROXIDASE ISOZYMES IN PITH TISSUE OF A NICOTIANA AMPHIPLOID

Two electrophoretically anodal peroxidase bands, designated A3 and A4, which normally develop in zymograms of incubated pith tissue of a Nicotiana amphiploid, were inhibited by the inclusion of 0.10 mg indoleacetic acid (IAA) in a liter of culture medium. The visibility of the bands was not inhibited, however, if the pith was irradiated or if unirradiated pith blocks were incubated in either irradiated IAA-containing media or in media supplemented with irradiated IAA. Irradiation inactivates IAA in media, and according to this isozyme test, damages the capability in irradiated tissue of using exogenously supplied IAA. Radiationinduced damage to IAA metabolism caused radiation-induced peroxidase stimulation, shown in the more rapid appearance of isozyme bands A3 and A4.     

Localization and General Properties of Developing Peach Seed Coat and Endosperm Peroxidase Isoenzymes

Changes of soluble and ionically bound peroxidase and indoleacetic acid (IAA) oxidase activities were followed during peach seed development. Soluble peroxidase activity was located mainly in the embryo plus endosperm tissue, whereas wall ionically bound activities were found predominantly in the integument tissue. The different peroxidase isoenzymes present in the extracts were characterized by polyacrylamide gel electrophoresis and isoelectric focusing; the main soluble isoenzyme of embryo plus endosperm tissue was an anionic isoperoxidase of R _F 0.07. Basic ionically bound isoenzymes were located only in the integument tissue, but two soluble anionic isoenzymes of R _F 0.23 and 0.51 were also present in this tissue. In parallel, peroxidase protein content was estimated specifically using polyclonal antibodies. The kinetic data and the changes of seed IAA oxidase activity during fruit development suggested that basic peroxidase isoenzymes from ionically bound extracts of integument might be involved in IAA degradation. Received September 11, 1997; accepted October 21, 1997     

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.

     

Ribosomal and mitochondrial peroxidase isoenzymes of the lentil (Lens culinaris) root

Summary Ribosome- and mitochondria-rich preparations were obtained from lentil roots and their peroxidase isoenzymes examined by starch gel electrophoresis. Seven isoenzymes were shown to be associated with both the ribosomal and mitochondrial fractions.The apparent similarity between ribosomal and mitochondrial isoenzyme patterns, together with the observation that a considerable amount of peroxidase activity dissociated from the ribosomes during sedimentation of the ribosomal preparation into a sucrose gradient, suggested that at least some of these isoperoxidases were attached to membranes.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

Isoperoxidases in jerusalem artichoke in relation to tuberization and dormancy

Peroxidase activity and isoenzyme pattern were investigated in buds and tubers of Jerusalem artichokes in relation to induction and breaking of dormancy. Peroxidase activity per unit soluble protein is the highest in the dormant stage. Conditions leading to growth,i.e. release of dormancy by the cold, stimulation of axial growth by gibberellic acid or stimulation of radial growth (tuberization) by kinetin, cause rapid loss of total peroxidase activity together with a decrease of intensity of the most cathodic isoperoxidases. Induction of dormancy by AMO-1618 increases peroxidase activity mainly through the same cathodic isoenzymes. The role of the cathodic isoperoxidases is discussed in relation to auxin catabolism and the genesis of oxygenation products inhibitory to plant growth.     

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.