Basic Isoperoxidases Research Articles

EFFECTS OF HEAT STRESS ON SOME BIOCHEMICAL TRAITS OF SMALL REDDISH BEAN

The effects of heat stress on malondialdehid (MDA) content, ascorbic acid (AsA), isoperoxidases, total soluble protein (TSP) and SDS-PAGE protein profiles of small reddish bean (Phaseolus vulgaris L. cv. Keklik) leaves were investigated. For this purpose, the collected leaf samples (preferably the third from the apex) were placed into pyrex tubes with caps closed and incubated in a water bath: after a 30-minutes habituation at 30ºC, to apply heat stress the leaves were subjected to 35, 40, 45, 50, 55 and 60°C with gradual increments every 30-minutes. According to the results, the MDA content increased with escalating temperatures, starting with 40°C. Although there was fluctuation, the AsA content generally decreased with high temperatures. Besides, TSP content decreased after 50°C. Moreover, three asidic and four basic isoperoxidase bands were identified. There was a marked difference at acidic isoperoxidase band intensities under 40 and 45°C heat treatment. Protein bands with molecu1ar weight of approximately 14.5-195 kDa were determined. In conclusion, especially 40°C is critical for small reddish bean plants, besides 40°C and above temperatures causes oxidative stress in small reddish bean plant. Additionally, isoperoxidases and proteins may be an effective mean on heat stress tolerance.

THE ROLE OF ANTIOXIDATIVE ENZYMES IN TOMATO LEAVES UNDER HEAT STRESS AT TWO DEVELOPMENTAL PHASES

The role of antioxidative enzymes in response to heat stress tolerance (HST) of three tomato cultivars was investigated in this research. The leaves were obtained from tomato plants at the first bloom and yield stages and then were subjected to controlled high temperature treatments in water bath. The temperature in the water bath was increased 5 °C systematically for every half an hour from 35 to 60°C to generate a heat-stressed condition. The plants in yield stage exhibited an increase in HST when compared with the plants in first bloom stage and heat stress increased hydrogen peroxide (H2O2) level in leaves. Among the performed enzyme analysis, only catalase (CAT) involved in HST of tomato cultivars, however it may not be correlated with the degree of HST. In addition, two acidic and one basic isoperoxidases appeared in relatively heat tolerant cultivars in the yield stage, therefore isoperoxidases may be associated to HST in tomato plant.

HEAT STRESS-INDUCED ALTERATIONS IN ANTIOXIDATIVE ENZYMES OF SOME PLANTS OF CUCURBITACEA FAMILY

The effects of high temperatures on melon cvs. Miranda and Poli, watermelon cv. Crimson Tide and zucchini cv. Asma leaves. The leaves obtained from plants were subjected to 35, 40, 45, 50, 55 and 60°C temperatures with gradual increments every 30-minutes. Samples, obtained at each treatment, were analyzed for ascorbic acid content, NADP(H) oxidase, catalase, gluthatione reductase, peroxidases activities and isoperoxidase patterns. The ascorbic acid content slightly increased parallel to temperature increment in zucchini but did not change in watermelon and in both melon cultivars. Melon cv. Poli exhibited comparatively less oxidative damage than cv. Miranda with a lower NAD(P)H oxidase activity. Heat stress induced NAD(P)H activity in watermelon and zucchini comparing to control plants. Results revealed that antioxidative enzyme activities were increased generally up to 50°C then decreased gradually in melon cultivars. Besides cv. Poli generally had higher enzyme activities than cv. Miranda. The activity of catalaes become prominent in watermelon while the activity of ascorbate peroxidase become prominent in zucchini. Acidic isoperoxidase bands with different relative mobility values were found in all species. Besides, basic isoperoxidase band could not be determined in both melon cultivars and watermelon while a basic isoperoxidase band was found in zucchini.

Changes in peroxidase activities and soluble proteins in strawberry varieties under salt-stress

Effects of salt stress on the activity of peroxidase (PRX) isozyme and leaf proteins were studied in the three strawberry varieties, Camarosa, Tioga and Chandler. Plants were watered with modified 1/3 Hoagland nutrient solution containing 0 (control), 8.5, 17.0 and 34.0 mM NaCl for 30 days fallowing 20 days acclimation. PRX activity was assayed in leaf extracts and PRX profiles of control and salt treated plants were performed by native polyacrylamide gel electrophoresis (PAGE). During the salt stress Tioga and Camarosa tolerated the cellular damage with less electrolyte leakage while Chandler exhibited a severe cellular damage with the highest (87.5 % in 34.0 mM NaCl treatment) electrolyte leakage. Total soluble protein content was decreased by salinity in Tioga (from 11.35 to 5.86 mg g−1 DW) and Chandler (from 9.35 to 3.90 mg g−1 DW) while it was almost unchanged in Camarosa. Salt stress increased total and specific PRX activity as compared with the control in all the varieties. In native PAGE, one basic isoperoxidase band (Rf=0.27) was observed commonly with different band intensity in all the treatments. Therefore, it might be associated with lignification and recovery of cell membrane damage in strawberry plants under salt stress.

Metabolism of avenanthramide phytoalexins in oats.

Oat leaves produce phytoalexins, avenanthramides, in response to infection by pathogens or treatment with elicitors. The metabolism of avenanthramides was investigated using low molecular weight, partially deacetylated chitin as an elicitor. When oat leaf segments are floated on the elicitor solution, avenanthramides accumulate in the solution. The transfer of elicited oat leaves to solutions containing stable-isotope-labeled avenanthramides resulted in a rapid decrease in the labeled avenanthramides, suggesting the metabolism of avenanthramides. The rate of decrease was enhanced by elicitor treatment, and was dependent on the species of avenanthramides, with avenanthramide B decreasing most rapidly. The rates of biosynthesis and metabolism of avenanthramides A and B were measured using a model of isotope-labeling dynamics. Avenanthramide B was found to be more actively biosynthesized and metabolized than avenanthramide A. Radiolabeled avenanthramide B was incorporated into the cell wall fraction and 99% of incorporated activity was released by alkaline treatment. Gel filtration indicated that high-molecular-weight compounds derived from avenanthramide B were released by alkaline treatment. The decrease in stable-isotope-labeled avenanthramides was suppressed by catalase, salicylhydroxamic acid, and sodium ascorbate, suggesting the involvement of peroxidase in the metabolism. Consistent with this, peroxidase activity that accepts avenanthramide B as a substrate was induced in apoplastic fractions by elicitor treatment. The appearance of multiple basic isoperoxidases was observed by activity staining with 3-amino-9-ethylcarbazole coupled with isoelectric focusing of proteins from elicitor-treated leaves. These findings suggest that accumulated avenanthramides are further metabolized in apoplasts in oat leaves by inducible isoperoxidases.

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.

Diversity of leaf peroxidases in date palm ( Phoenix dactylifera L.) as revealed in an example of marginal (seedling derived) palm groves

Peroxidases (EC 1.11.1.7) were extracted from fresh leaves of date palm ( Phoenix dactylifera L.) harvested from 90 non-fruit-producing male trees and 75 adult female plants growing all in the Marrakech (South Morocco) marginal palm grove of unknown seedling origin. Two enzyme fractions were obtained by a progressive solubility method leading to soluble peroxidases (S) and ionically wall-bound peroxidases (I). When subjected to electrophoresis on polyacrylamide gels, S and I peroxidase fractions exhibited each one, two enzyme forms based on their electric charges occurred in basic and acidic gel media. Acidic peroxidase forms, well separated on basic gels, showed two migration zones with a maximum of nine isoperoxidases. Basic forms, more represented in fractions I and resolved on acidic gels, were typified by two major isoforms. When combining patterns of acidic and basic isoperoxidases, enzyme fractions S and I of individual trees, showed, respectively, 10 and nine isoenzyme phenotypes. No sex-specific isoforms were found at the electrophoresis-based qualitative study. However, some phenotypes were found to be highly represented in male trees. Evaluation of peroxidases as markers in date palm is discussed in this study.

Plant regeneration from callus of Cuscuta reflexa – an angiospermic parasite – and modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid

Callus induction and plant regeneration system for an obligate parasite, Cuscuta reflexa (family Cuscutaceae), has been established. Callusing was induced from explants of basal portion of seedlings as well as from shoots (vines) in a modified Murashige and Skoog (MMS) media supplemented with 1 mg·L –1 benzyl adenine (BA) and 3 mg·L –1 naphthalene acetic acid (NAA) (MMS-1). The MMS media containing 1 mg·L –1 BA (MMS-2) was found to be better than the MMS-1 media for callus proliferation and long term maintenance. Use of glucose in place of sucrose enhanced callus induction and growth. The browning of callus was substantially reduced by supplementing 0.1 mg·L –1 gibberellin to the MMS-1 media. Shoot regeneration from the green callus was achieved in the MMS media containing 3 mg·L –1 NAA and 1 mg·L –1 kinetin. Cuscuta callus was characterised by having several folds higher peroxidase activity than those of culture-raised and field-grown vines. Catalase activity also revealed a pattern similar to that of peroxidase except for the fact that it could not be detected in field-grown vines. Developmental profiles of peroxidase and catalase, during the growth of the callus in the absence and presence of NAA, revealed inhibition of both the enzymes by NAA, though inhibition of peroxidase was stronger as compared to that of catalase. The inhibitory effect of NAA was also evident when in-gel activity staining of the two enzymes were performed. In callus, three acidic and four basic isoperoxidases were present, while in field-grown vines, two acidic and one basic isoperoxidases were present. Investigation of the effect of salicylic acid on catalase and peroxidase revealed inhibition of both enzymes in a concentration dependent manner during seedling growth. The inhibitory effect of salicylic acid was also evident when in-gel enzyme activity staining of the two enzymes was done. The results are discussed in light of the proposed mode of action of salicylic acid.

Purification and stability of a basic peroxidase from strawberry callus culture

A basic isoperoxidase from strawberry ( Fragaria × ananassa cv. Chandler) callus culture was purified by sequential CM-Cellulose, Concanavalin-A Sepharose 4B and Sephacryl S-200 chromatographies. Isoelectrofocusing of the purified peroxidase activity revealed the presence of one unique basic isoperoxidase (Faprx1, pI 9.2). Native cathodic electrophoresis and SDS-PAGE analysis of the basic isoenzyme yielded a single protein band. The molecular mass of the basic isoenzyme estimated by SDS-PAGE and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was 35 and 32.6 kDa, respectively. The low molecular mass of the isoenzyme may be due to the low degree of glycosylation of its polypeptide chain. Faprx1 showed unusual instability even at room temperature. The effects of bovine serum albumin (BSA), Ca 2+, hematin and pH on the stability of the basic isoperoxidase were assayed. The presence of the three stabilising agents showed a remarkable protective effect on Faprx1 activity in a wide pH range.

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

Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a nonrooting mutant of tobacco

The rooting recalcitrant rac Nicotiana tabacum cv Xanthi mutant has been multiplied in vitro under the form of shoots in parallel to wild-type. rac Shoots grew at a lower rate and did not root whatever the treatments when compared to those of wild-type shoots. They were characterized by a higher lignin level, a higher total specific peroxidase activity with higher activity of both acidic and basic isoperoxidases (although missing and supernumerary isoenzymes were observed), and higher ethylene production. These observations might be causally related to growth inhibitions as similar incidences have been noted in different stress-induced growth limitation, through cell wall rigidification and auxin catabolism. The relationship between these aspects and rooting recalcitrance remains to be explored.

Phytohormone regulation of isoperoxidases in catharanthus roseus suspension cultures

Peroxidase (POD) activity was investigated in Catharanthus roseus cell suspensions cultured under different hormonal conditions. Depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) from the culture medium enhanced POD activity in cells and spent medium. Addition of phytohormones, in particular the auxin 2,4-D, reduced POD activity in medium and cellular compartments and enhanced ionically cell-wall bound POD. The differential modulation of POD is due to hormone effects on synthesis and⧹or accumulation of POD, rather than on the secretion process. Qualitative analysis showed that 2,4-D, but not cytokinins, regulated the synthesis of a basic isoform. The cytokinin treatment seemed to affect acidic rather than basic isoforms. The presence of basic POD is correlated with the capacity of cells to produce indole alkaloids. The major extracellular basic isoperoxidase was purified to homogeneity from culture medium of Catharanthus roseus cell suspensions. The isolated peroxidase is a haem protein with a M r of 33,000 and a pI close to 9. The effect of pH on peroxidase activity was studied using guaiacol as substrate and the optimum pH determined at 25° was 6.0. This enzyme acted on guaiacol, 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine ( o-PD) and pyrogallol, but had no effect on syringaldazine or coniferyl alcohol substrates.

Scopoletin production and degradation in relation to resistance of Hevea brasiliensis to Corynespora cassiicola

Inoculation of leaves of resistant and susceptible Hevea brasiliensis clones with Corynespora cassiicola induced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Hevea phytoalexin. Foliar symptoms appeared, as soon as 24 h after infection, and precede Scp accumulation in inoculum droplets (peak: 48 h after inoculation). Scp concentration was 5-fold higher in the susceptible than in the resistant clone 48 h after infection. Nevertheless, a fungitoxic effect of Scp on spore germination and on mycelium growth was shown in bioassays, but the efficient concentrations were greater than those found with another foliar pathogenic fungus of rubber tree such as Microcyclus ulei and Colletotrichum gloeosporioides. Bioassay showed that the low Scp fungitoxicity to C. cassiicola could also be related to the ability of fungus to detoxify Scp. Isoelectric focusing analysis of peroxidase activity in Hevea infected leaflets has shown an increase in acidic and basic isoperoxidases that are able to use Scp as substrate. The rapid increase of Scpoxidase activity (as soon as 16 h after inoculation) was higher in the resistant than in the susceptible clone. In vitro testing of Scp peroxidation products has shown a toxic effect on C. cassiicola (conidia germination and mycelium growth), but not significantly higher than Scp. It appears that the level of Scp accumulation was a balance between its synthesis and its degradation by the pathogen or/and by foliar Scp-peroxidases. In conclusion, these results lead to the fact that Scp cannot be considered as a major defense mechanism of Hevea towards C. cassiicola disease. (Resume d'auteur)

Isoperoxidase Involvement in the Resistance of Capsicum annuum to Infection by Cucumber Mosaic Virus

Summary Cucumber mosaic virus (CMV) infection of Capsicum annuum leaves provokes a strong increase in soluble peroxidase activity. Isoelectrofocusing of the peroxidase activity in polyacrylamide gel patterns, after staining the soluble protein fractions from both healthy and infected leaves with benzidine/H2O2, revealed the presence of two acidic and one basic isoperoxidases, the increase in peroxidase activity being mainly due to the former. Staining of peroxidase activity in the gels with coniferyl alcohol/H2O2 showed that acidic isoenzymes were more capable of catalyzing coniferyl alcohol oxidation than basic isoenzymes. Native polyacrylamide gel electrophoresis of the soluble fraction showed that the increase in peroxidase isoenzymes. CMV elicitation of the acidic isoperoxidase through its involvement in the lignification process might be considered as a part of the defense mechanism of Capsicum annuum against viral infection, lignin deposition on plasmodesmata could act as a physical barrier against the cell-to-cell advance of viral particles.

Peroxidases during the course of callusing and organ differentiation from root explants ofCichorium intybus

Growth ofCichorium intybus root explants was accompanied by an important increase of fresh mass during the course of callusing and rooting. The absence of glucose in callus forming medium was compensated for by hydrolysis of storage carbohydrates of the tissues, inducing a decrease in dry mass. Protein content showed similar slight variations in explants during the course of budding and callusing, whereas an important increase of protein content was found during the first 48 h in explants cultured on root forming medium.Specific increase of soluble peroxidase activity during bud and root neoformation was found. A peak of peroxidase activity, proceeding the organ emergence, was always observed. This peak occurred earlier in bud forming than in root forming explants. Conversely, during callusing, the bulk peroxidase activity showed only weak variations, and no peak was detectable. Moreover one basic isoperoxidase was missing in these explants after a 6-d culture, whereas in both root and bud forming explants the isoperoxidase patterns were very similar. When explants growing on differentiation media were in darkness a supplementary basic isoperoxidase with the highest electrophoretic mobility was revealed.The data obtained show that isoperoxidase patterns cannot be correlated to a particular organ differentiation process, but that peroxidase activity and especially the moment of peak emergence can be a reliable marker of the differentiation process. Moreover, light seems to inhibit the expression of a basic peroxidase in explants growing in media promoting organ differentiation.

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.

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.

The Tonoplast Localization of Two Basic Isoperoxidases of High pI in Lupinus

AbstractThe ultrastructural localization of peroxidase (EC 1.11.1.7) activity in cambial initial and xylem parenchyma cells of etiolated Lupinus albus hypocotyls (cv. Multolupa) revealed that, unlike phloem tissues, most of the enzymatic activity is located as dark electron‐dense deposits on the tonoplast. Subcellular fractionation studies of plasmolyzed hypocotyls revealed that this enzymatic activity is apparently due to two basic peroxidase isoenzymes of isoelectric points 9.5 and 9.7 for B3 and B4, respectively. These isoenzymes are found mainly in young (5‐day old) seedlings, and are probably involved, as indicated by other authors, in the metabolism of quinolizidine alkaloids in Lupinus species.

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.

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.