Acidic Isoperoxidases Research Articles

Biochemical investigations on peroxidase contents of male and female inflorescences of date palm ( Phoenix dactylifera L.)

Peroxidases (EC 1.11.1.7) were extracted from male and female inflorescences of date palm ( Phoenix dactylifera L.). Enzyme extraction by a progressive solubility method gave rise to soluble peroxidases (S) and ionically wall-bound peroxidases (I). When expressed in units g −1 fresh weight, peroxidase was present in inflorescence rachillae essentially as soluble fraction, where it represents 94% and 89% of the total activity (S + I) in male and female rachillae, respectively. In flowers, a tendency to increase of peroxidases I was observed, where this fraction, represents 51% and 78% of the total activity in female and male flowers, respectively. Low peroxidase activity was found in male flowers. When subjected to polyacrylamide gel electrophoresis, both S and I enzyme fractions exhibited each one, two forms based on their electric charges that occurred in basic and acidic gel media. Acidic peroxidase forms, resolved on basic gels, are more represented than basic forms, well separated on acidic gels. Acidic soluble peroxidases showed two migration zones on 6–15% gradient polyacrylamide gels. Acidic bound peroxidases are characterized by fast isoforms. Basic peroxidases, separated on 11% polyacrylamide gels and represented in fractions I, exhibited two major isoforms within three to five bands. Differences between female and male inflorescences are more marked on the basis of acidic peroxidases. Female inflorescences showed two major acidic isoperoxidases in rachillae with R f 0.32 and 0.35 in enzyme fractions S and I, while male material showed one major band of R f 0.35. Evaluation of peroxidases as sex markers in young date palm not yet at their reproductive stage is discussed in this study.

Changes in peroxidase activity and isoperoxidase pattern during strawberry ( Fragaria × ananassa) callus development

Summary Changes in peroxidase (EC. 1.11.1.7) activity and isoperoxidase pattern were analysed during strawberry ( Fragaria × ananassa ) callus development. Peroxidase activity per g fresh weight using 4-methoxy-α-naphthol, 3,3′,5,5′-tetramethyl bencidine, coniferyl alcohol or ferulic acid as substrates was highest at 40 days of culture, which coincided with the exponential phase of growth. While the specific peroxidase activity with the first three substrates was highest during the exponential growth phase, the highest specific enzymatic activity with ferulic acid as substrate was reached after 80 days of culture when callus growth had ceased. Several isoperoxidases were constitutively expressed during the complete callus growth cycle, unlike a group of acidic isoperoxidases, which were only present when callus had ceased growing. The high reactivity of the acidic isoperoxidases with ferulic acid as substrate, as judged by the k 1 and k 3 values of the peroxidase catalytic cycle, strongly suggests that these acidic isoenzymes are mainly involved in the formation of diferuloyl bridges between non-cellulosic components of the cell wall, thus contributing to cell wall rigidification, which, in turn, is partly responsible for the cessation of growth.

Peroxidase involvement in the defense response of red raspberry to Didymella applanata (Niessl/Sacc.)

Peroxidase activity of red raspberry canes was dependent on the cultivar and influenced the subsequent lignification. After inoculation with Didymella applanata, responsible for the spur blight cane disease, the activity of soluble cytoplasmic enzyme increased in the moderately resistant ‘Latham’ and susceptible ‘Malling Promise’, similarly for syringaldazine and guaiacol as hydrogen donors. Systemic induction found in ‘Latham’ was recognized as a symptom of defence mechanism responsible for fungal restriction. Locally enhanced peroxidase activity in the ‘M.Promise’ tissues was related to the local lignification and/or may be associated with the loss of cell integrity caused by pathogen penetration. Pathogen-induced changes of cell wall peroxidases were similar in both cultivars mentioned above. No influence of the infection was found in the high susceptible Zeva cultivar. Using native-PAGE analysis and horizontal starch electrophoresis of soluble fraction five constitutive acidic isoperoxidases were detected in ‘Latham’ and three in ‘M. Promise’. The infection process was accompanied by the appearance of two new anodic isoforms.

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases.

Incubation of oat coleoptile segments with 40 µm indoleacetic acid (IAA) induced a decrease of 35-60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0-5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V(max)/s(0.5) value of 2.4 x 10(-2) min(-1). g fw(-1), 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 x 10(-2) min(-1). g fw(-1)). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H(2)O(2) (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking.Key Words. IAA-Auxin-Wall peroxidases-Oat-Avena sativa-Diferulic acid-Ligninhttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p25.html

Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance

The effect of NaCl on total peroxidase activity, induction of isoperoxidases and lipid peroxidation in 5-day-old seedlings of two contrasting genotypes of Setaria italica L . (Prasad, a salt tolerant cultivar and Lepakshi, a salt susceptible cultivar), was studied. Total peroxidase activity increased under NaCl salinity and the degree of elevation in the activity was salt concentration dependent. Nevertheless, a greater activity was recorded in the tolerant cultivar (cv Prasad) compared to the susceptible (cv Lepakshi) one in all days of sampling. Further, the pattern of isoperoxidases was modified during stress conditions as evident from the electrophoregrams. Although, five acidic isoforms were detected in both cultivars, differences were found between the cultivars. Furthermore, it was observed that acidic isoperoxidases were strongly expressed and an acidic isoperoxidase, A 3p (27 kDa) is specifically found in the tolerant cultivar (cv Prasad) under NaCl stress. This isoform was partially purified and found to be thermostable with p I 5.5 and the optimum pH 7.4. A close correlation exists between the rate of lipid peroxidation in terms of malonaldehyde (MDA) content and total peroxidase activity per gram fresh weight with salt tolerance of the two cultivars. The tolerant cultivar (cv Prasad) had low MDA content and high total peroxidase activity than the susceptible variety (cv Lepakshi) during salinity stress.

An extensin peroxidase is associated with white-light inhibition of lupin (Lupinus albus) hypocotyl growth

The spatial distribution of the major basic (B2; pI 8.8) peroxidase of the intercellular fluid has an inverse relation with extension rate in etiolated hypocotyls of Lupinus albus L., suggesting its possible role in the control of cell expansion. White-light irradiation of etiolated hypocotyls resulted in growth inhibition and the induction of B2 and acidic (A2, pI 4.7–5.2) isoperoxidases (EC 1.1.11.7) to higher physiological activities. However, only the activities of the B2 isoperoxidases underwent quantitative changes in both space and time which suggested their role in growth-retardation. We have purified the B2 and A2 (pI 5.2) peroxidases to apparent electrophoretic homogeneity. To corroborate evidence obtained elsewhere that growth cessation coincides with cell wall structural changes and cell wall rigidification, we have shown that the B2 peroxidase, and not A2 peroxidase, cross-links tomato extensin in vitro. The B2 peroxidase may therefore catalyse the developmentally and light regulated formation of a covalently cross-linked cell wall extensin matrix in lupin hypocotyls. The cell wall would be more rigid or more recalcitrant to wall-loosening and subsequently contribute to the control of cell expansion.

Gibberellic acid stimulation of isoperoxidase from pedicel of grape

Soluble peroxidase activity from pedicels of cv. Thompson seedless grape was highly stimulated by post-bloom gibberellic acid (GA 3) applications to vines. This stimulation was dependent on the dose employed and increased with berry ripening. In control vines, i.e. non GA 3 treated, the level of peroxidase activity remained constant during berry ripening. Isoelectrofocusing (IEF) analysis of soluble peroxidases from pedicel extracts performed in a 3–10 pH range, showed the presence of a main band that corresponded to an acidic isoperoxidase of (pI 3.5) and the presence of a minor band of (pI 3.9). It is a well-known fact that GA 3 applications to cv. Thompson seedless grape beside producing berry enlargement cause berry-drop which has been associated with pedicel thickening. Here we found a positive correlation between pedicel thickness and peroxidase activity within the rates of GA 3 used. The involvement of the acidic isoperoxidases in pedicel thickening and its relation to berry-drop are discussed.

Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco

When subjected to micropropagation by tissue culture, the two reputed cultivars of date palm (Phoenix dactylifera L.); Bou-Sthammi noire, resistant to Bayoud disease and Bou-Feggous, of high fruit quality, give rise to three types of calli, called white and root-forming callus, hyperhydric and degenerating callus and friable and embryogenic callus. All explant sources, calli and germinated embryos were analysed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for acid soluble protein composition. Phenol-oxidizing enzymes; peroxidase and polyphenoloxidase, were also, evaluated and the isoforms separated by polyacrylamide gel electrophoresis. When compared with the explant and germinated embryos, embryogenic calli of the two date palm cultivars could be identified by a concentrated polypeptide of molecular weight 27 500 and polypeptides of molecular weights 70 000 and 11 500. Hyperhydric and degenerating callus contained the polypeptide exhibiting the molecular weight 32 000. Embryogenic calli showed high levels of soluble, ionically and covalently bound peroxidases. The soluble acidic isoperoxidase of R f 0.60, revealed in these calli and germinated embryos could be a marker of the two tissues. White and root-forming calli of Bou-Feggous cultivar were typified by soluble acidic isoperoxidases with high mobility (R f 0.75) and anodic ionically wall-bound polyphenoloxidases similar to those of the explant sources. Polyphemoloxidase activities detected in calli and embryos were very low when compared with those of explants. Used as an early test to screen embryogenic calli of date palm, acid soluble proteins, peroxidase and polyphenoloxidase data could lead to introduce lightening and economy in the tissue culture technique.

The Cell Wall Localization of two Strongly Basic Isoperoxidases in Etiolated Lupinus albus Hypocotyls and its Significance in Coniferyl Alcohol Oxidation and Indole-3-Acetic Acid Catabolism

Summary Two strongly basic peroxidase (EC 1.11.1.7) isoenzymes, B3 and B4, of isoelectric points 9.5 and 9.7, are localized in cell walls of young Lupinus albus hypocotyls. The binding of B3 and B4 isoenzymes to cell walls is specific and, unlike that observed for acidic isoperoxidases, is maximal at neutral pH values. By comparison with A1 and A2 acidic isoperoxidases, cell wall-bound basic B3 and B4 isoperoxidases are little involved in coniferyl alcohol oxidation and indole-3-acetic acid catabolism. However, due to the localization of these isoenzymes in vacuoles also, they are probably involved in some type of H2O2-dependent oxidation reaction common to both lytic compartments. These results are discussed on the basis of a possible participation of these isoenzymes, as indicated by other authors, in the oxidative transformation of tetracyclic bisquinolizidine-type alkaloids in lupin.

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.

A Biochemical and Cytochemical Study of the Cuticle-associated Peroxidases in Lupinus

A biochemical and cytochemical study of peroxidases (EC 1.11.1.7) associated with the outermost cell layers of Lupinus albus hypocotyls has been performed. Cytochemical investigations showed that in the epidermis of lupin hypocotyls, peroxidatic activities could be detected mainly at the level of the endoplasmic reticulum, Golgi apparatus, ground substance of the cytoplasm, intercellular spaces between adjacent epidermal cells, and in the cuticle layer. No peroxidatic reactions were seen at the level of either the radial or the tangential walls. The cuticle peroxidatic reaction was due to two acidic isoperoxidases, also found in cell walls of vascular tissues, and was further characterized through a study of its catalytic activities. These studies showed that cuticle-associated peroxidases were able to oxidize genistein, but unable to oxidize ascorbic acid. These results suggest that extracellular peroxidases associated with the outermost cell layers of lupin hypocotyls are involved more in the metabolism of isoflavones of the cuticle layer than in the growth responses of the whole organ.

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 (1)H-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.

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

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.

Physiological significance of the binding of acidic isoperoxidases to cell walls of lupin

Intercellular acidic isoperoxidases (EC 1.11.1.7) isolated from exponentially growing lupin (Lupinus albus. L. cv. multolupa) hypocotyls are under the control of exogenously applied auxins. Application of auxins leads to a short‐term reduction in the level of free intercellular peroxidases, and this effect is associated with a binding of these free peroxidases to the cell walls, probably mediated by an acidification of the cell wall. The ratio of free intercellular peroxidases to the total intercellular peroxidase activity, varies along the axis of exponentially growing hypocotyls. It has a V‐shaped distribution with the minimum value in the elongation III‐zone, where high levels of auxins have previously been implied in differentiation. This minimum value coincides spatially with the first signs of cell wall thickening in the hypocotyl cells and, paradoxically, it is out of phase with respect to the maximal cell elongation. On the other hand, the ratio of free intercellular peroxidases reaches its maximal values in both the most undiffercntiated phloem cells and the differentiated xylem cells. High levels of free intercellular peroxidase activity in phloem cells are hard to explain, since phloem cell walls remain unlignified during almost all stages of differentiation. However, association of free intercellular peroxidase activity with xylem cells is clearly associated with the lignification of the xylem cell walls. The physiological significance of the binding vs release of intercellular peroxidase is discussed in relation to the catalytic properties and stability at acidic pH of both the bound and free forms of this enzyme.

Subcellular location of basic and acidic soluble isoperoxidases in Lupinus

The subcellular location of non-particulate (soluble) peroxidases (EC 1.11.1.7) was studied in lignifying lupin ( Lupinus albus L.) hypocotyls. Supernatants of cellular homogenates contain two acidic (A 1 and A 2) and two basic (B 1 and B 2) isoperoxidases. Studies of centrifugation on isopycnic sucrose gradients of cellular homogenates of plasmolyzed tissues, vacuum infiltration of hypocotyl sections, and binding probes to membranes and cell walls, suggest that while basic soluble isoperoxidases possess a cytoplasmic location, acidic isoperoxidases possess an extracellular location, as freely soluble forms in the intercellular spaces. These results are discussed on the basis of the subcellular location of the auxin catabolism, mediated mainly by the two basic isoperoxidases, which in lupin cells at least, seems to be restricted to the cytosolic compartment.

Lupin peroxidases. II. Binding of acidic isoperoxidases to cell walls

Extracellular acidic isoperoxidases (EC 1.11.1.7), isolated from both the cell walls and intercellular spaces of lupin (Lupinus albus L. cv. multolupa) hypocotyls, bound to water‐insoluble pectins of wall fragments also isolated from the hypocotyls. The binding was sáturable by increasing the isoenzyme concentration in the assay medium and it was dependent on the pH; neutral pH (6.0–7.0) favoured release, while acidic pH (4.0–5.0) favoured the attachment to the cell wall. Binding of acidic isoperoxidases to wall fractions was correlated with the in vitro acid‐induced growth of hypocotyl segments, and both were modulated in the same direction by the Ca2+/H+ ratio in the incubation media, although the two responses were clearly separated when the Ca2+/H+ ratio varied. Binding of acidic isoperoxidases of cell walls could operate as a fine control of the activity of these cell wall enzymes, although its physiological role in the cell wall stiffening remains unclear. Some aspects of Ca2+ on the control of peroxidase activity at this level are also discussed.

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.

Isoperoxidases of Japanese radish root infected by downy mildew fungus.

Lignin is formed in the cell wall of the downy mildew-infected Japanese radish root and peroxidase activity increases in the neibouring tissues. Attempts were made to study the functional role of this increased peroxidase activity in lignification in diseased tissues. The crude peroxidase prepared from the healthy tissue with ammonium sulfate precipitation and isoelectric point precipitation was fractionated into 11 fractions by cellulose column chromatography. The activity of the crude enzyme preparation increased gradually in response to the infection, reached a maximum 9 days after inoculation and then decreased. Elution profiles of peroxidase extracted from the root 9 days after inoculation showed that activities in acidic and neutral fractions apparently decreased while those of basic fractions increased. Polyacrylamide gel electrofocusing of the crude peroxidase preparation showed a decrease in activity of acidic isoperoxidases and an increase in activity of a part of basic and neutral isoperoxidases in the diseased tissue.