Peroxidase Isoenzyme Patterns Research Articles

Antioxidative and antifungal response of woody species to environmental conditions in the urban area.

The complexity of ecological conditions in urban areas imposes the plant species need for the development of various biochemical and physiological adaptive strategies. The aim of our research was to examine the antioxidative and antifungal metabolism of species Pinus nigra, Picea omorika, Tilia cordata and Betula pendula from the area of Banja Luka City (urban area) during two vegetation seasons (spring and autumn) and compared with the same species from forest habitats. Changes in the protein concentration, activity and isoenzyme profiles of peroxidases (POD, EC 1.11.1.7), content and antioxidative activity of total phenols and antifungal activity in leaves and needles of the plants from the urban area and forest habitats were monitored. The obtained results indicate that urban areas induce changes in antioxidative metabolism in all examined species, but that the response is species specific. The most sensitive parameter that indicates different adaptation strategy of Pinus nigra, Picea omorika, Tilia cordata and Betula pendula to environment conditions in the urban area were peroxidase isoenzyme patterns. Less specific parameter was phenol content even though there are some indications for role of their antioxidative capacity in the adjustment to specific habitat. In addition, each species had different metabolic strategy to cope with the changes caused by the urban environment.

Macromolecular Toxins Secreted by Botrytis cinerea Induce Programmed Cell Death in Arabidopsis Leaves

Botrytis cinerea causes grey mold disease in crops and horticultural plants. It is suspected to kill plant cells via secreted toxins and to derive nutrients from dead or dying cells. However, whether macromolecular phytotoxins (MPs) secreted by B. cinerea induce necrosis or also trigger a programmed cell death (PCD) remains to be determined. We have previously partially characterized MPs secreted by B. cinerea. Here we isolated MPs from B. cinerea culture and applied them to leaf cells, assessing PCD over the following 120 h. Cell death was assessed by propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. Catalase (CAT), peroxidase (POD) activity and the cytochrome c/a ratio were assessed by spectrophotometer. POD isomers were measured using the benzidine acetate method. In Arabidopsis thaliana (L.) Heynh. exposed to B. cinerea MPs, we observed chromatin condensation and marginalization, nuclear substance leakage and accumulation of autofluorescent materials in the cell wall. Furthermore, B. cinerea MPs induced release of cytochrome c from the mitochondria into the cytosol. Moreover, CAT and POD activity was upregulated and the POD isoenzyme pattern was altered. In conclusion, A. thaliana exposed to B. cinerea MPs exhibits multiple hallmarks of PCD, suggesting that B. cinerea induces PCD in host cells through secreted macromolecules.

Heat stress alleviation by exogenous ascorbic acid application: Changes in peroxidase isoenzyme and protein patterns of common bean plants

Heat stress alleviation by exogenous ascorbic acid application: Changes in peroxidase isoenzyme and protein patterns of common bean plants

Long-term exposure treatments revert the initial SA-induced alterations of phenolic metabolism in grapevine cell cultures

Vitis vinifera L. cv. Monastrell calli were used to evaluate the effects of long-term exposure to different salicylic acid (SA) concentrations (0.00, 0.01, 0.10 and 1.00 mM) on cell growth and phenolic metabolism. The impaired cell growth caused by high SA concentrations after the first sub-culture of cells was reverted to normal growth even in the presence of 1 mM SA after a few cycles of repeated culture in the same conditions. The initial alterations in the levels of total soluble phenolics, and in the peroxidase (PRX) activity, especially when resveratrol was used as substrate, were also counteracted upon long-term exposure to the phytohormone. Moreover, the recovering of PRX enzymatic activity paralleled the restoration of the PRX isoenzyme pattern. Since phenolic compounds and PRXs can be considered as markers of stress the restoration of their constitutive levels could be considered as indicators of cessation of stress allowing cells to reassume their normal growth and metabolic functions, even in the stressing initial conditions.

Microalgae respond differently to nitrogen availability during culturing.

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus minutus were cultured in the absence of N in the medium and subsequent resupply of N to the starved cells. Culture growth and in-gel changes in isoenzyme pattern and activity of glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were studied. The results demonstrated that the algae responded to the fully N-depleted and N-replete culture conditions by species-specific metabolic enzyme changes, suggesting differential regulation of both enzyme activity and cellular metabolism. Substantial differences in the activities of the antioxidant enzymes between N-depleted and N-replete cells of each species as well as between the species were also found. In the present work, besides the more general responses, such as adjustment of growth and pigmentation, we report on the involvement of specific metabolic and antioxidant enzymes and their isoforms in the mechanisms operating during N starvation and recovery in P. cruentum, T. minutus and S. incrassatulus.

Effect of cadmium stress on antioxidative enzymes during the germination of Serbian spruce ([Picea omorika (Panč.) Purkynĕe)

When considering the effect of heavy metals on trees generally and on forest ecosystems especially, importance is given to their influence on seed germination in metal polluted soil. There is insufficient data on this subject, especially when conifers are concerned. In this work, the influence of high cadmium concentrations on percentage germination, specific activities and isoenzyme patterns of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) during the germination of Serbian spruce [Picea omorika (Pan..) Purkynĕ] was studied. Cadmium chloride concentrations of up to 0.1 mM did not cause an inhibition of germination, while 1 mM concentration inhibited germination and the activities of catalase, superoxide dismutase and peroxidase. The isoenzyme profile of catalase and superoxide dismutase did not change at high cadmium concentrations, while peroxidase expression of basic peroxidase (B5) with pI value of 9.1 increased. This isoform of POD can play an important role in the early development of Serbian spruce and its defense mechanism against heavy metals. Key words : Seed germination, catalase, peroxidase, superoxide dismutase, heavy metals.

Lead-contaminated soil induced oxidative stress, defense response and its indicative biomarkers in roots of Vicia faba seedlings

Seeds of Vicia faba. L were grown in increasing concentrations of lead (Pb)-added soils (0-2,000 mg/kg). After germination of 25 days, roots were harvested to investigate oxidative stress, defense response and indicative biomarkers based upon chemical analyses and biological measurements. The results showed that higher concentrations of Pb-polluted soils led to seedling growth inhibition, indicative of phytotoxicity. O (2) (*-) and lipid peroxidation were increased with the increase of available Pb in soils and Pb contents in roots, displaying a "J"-shaped dose response curve, whereas H(2)O(2) showed a biphasic dose response curve (a consecutive "J"-shaped and inverted "U"-shaped curve). Superoxide dismutase (SOD), guaiacol peroxidase (POD) and ascorbate peroxidase (APX) enzymes were activated by soil Pb, displaying biphasic curves. The upregulated POD and APX enzymes might be major scavengers of excessive H(2)O(2) when CAT activities were drastically reduced with the increasing soil Pb. The enhanced glutathione (GSH) and APX activities suggested that GSH-ascorbate cycle also participated in eliminating H(2)O(2). Moreover, obvious changes were observed in SOD, CAT and POD isoenzyme patterns, but not in APX except increasing intensities of bands. HSP70 synthesis was significantly induced by extraneous Pb from 125 to 1,000 mg/kg and showed a biphasic curve in this experiment. Comparatively, HSP70 and lipid peroxidation might be more sensitive than other parameters in response to Pb stress, suggesting that these two parameters in the roots might be potential biomarkers for early bioassay of Pb-contaminated soils.

Morphological features and isoenzyme characterization of endosymbiotic algae from green hydra

Symbiotic associations are of a wide significance in evolution and biodiversity. Green hydra is a typical example of endosymbiosis. In its gastrodermal myoepithelial cells, it harbors individuals of unicellular green algae. Morphological characteristics of isolated algae determined by light and electron microscopy are presented. Cytological morphometric parameters (cell area, cell radius, chloroplast area) of isolated algae from green hydra (Cx), as well as from reference species Chlorella kessleri (Ck) and Chlorella vulgaris (Cv), revealed similarity between the isolated endosymbiont and C. kessleri. Isoenzyme patterns of esterase (EST), peroxidase (POX), and catalase (CAT) were used for the investigation of genetic variability in endosymbiotic algae isolated from green hydra. Out of 14 EST isoenzymes observed in Cx species, 9 were expressed in the Cx sample. Results of the EST isoenzyme analysis indicated a higher degree of similarity between Cx and Cv than between Cx and Ck. Due to much higher heterogeneity, EST isoenzymes seem to be more suitable genetic markers for identification of different Chlorella species than CAT and POX isoenzymes. Results obtained suggest that symbiogenesis in green hydra has probably not been terminated yet.

Changes in activities of antioxidant enzymes during Chenopodium murale seed germination

The activities and isoenzyme pattern of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) have been studied during germination of Chenopodium murale seeds. CAT and SOD activities were similar in dry seeds and during first 2 d of imbibition. CAT activity increased during radicle protrusion and early seedling development. The maximum SOD activity was found at final stages of germination and early seedling development. POD activity was not detected until the 6th day of germination, indicating POD involvement not until early seedling development. Gibberellic acid (GA3, 160 µM) delayed and synchronized C. murale germination.

Response to chitin in suspension-cultured Citrus aurantium cells

Morphological changes and enzyme activity in suspension-cultured cells of Citrus aurantium were followed after chitin elicitation. Chitin oligomers presented a rapid effect with a maximum activity after 3 h followed by a gradual decrease to the original levels at 8 h of incubation. Cell walls presented a lignification process and the cytoplasmatic protein became less reactive to anionic stains. In the treated material a reduction in starch grain size, an increase in the number of autophagic vacuoles, deposition of secretion in the pericellular space and a defibrillation of the cell-wall polymers were observed. Chitin oligomers increased peroxidase and L-phenylalanine ammonia-lyase activities. Extracellular peroxidase activity increased from 0.20 U mL-1 after 1 h to 0.45 U mL-1 at 3 h followed by a gradual decrease up to 8 h. The peroxidase isoenzyme pattern revealed a predominance of basic isoenzymes in these cells.

Variability of antioxidant enzyme activity and isoenzyme profile in needles of Serbian spruce ( Picea omorika (Panč.) Purkinye)

Variations were studied of the activity and isoenzyme patterns of soluble peroxidase, catalase, catechol oxidase and superoxide dismutase, in needles of the Balkan endemic conifer Serbian spruce, Picea omorika (Panč.) Purkinye. The samples were collected from the natural habitat of the species, Mt. Tara. Seasonal changes were found to affect enzymatic activities and isoenzyme profiles. Total protein content was significantly lower in the summer than in other seasons. Several isoforms of peroxidase, catechol oxidase and superoxide dismutase (SOD), as well as two catalase isoenzymes were detected. The number of peroxidase isoenzymes was greatest during the vegetative season. Catalase and catechol oxidase peaked in summer and spring, respectively. Total SOD and Mn-SOD activities were significantly higher in the winter samples than the summer ones.

Antioxidative enzymes during germination of two lines of Serbian spruce [Picea omorika (Panc.) Purkyně

Two lines of Picea omorika (Panc.) Purkyn? were compared with respect to germination percentage as well as specific activity and isoenzyme pattern of catalase, superoxide dismutase, and peroxidase (POD) during germination. Line A had a higher germination percentage and higher enzyme activities in dry seeds and seedlings compared to line C. Peroxidase activity was not detected in dry seeds, but measured up to 10 U/g and 28 U/g on the 7th day of germination in lines C and A, respectively. The most abundant POD basic isoform in seedlings of both lines (pI 8.2) was not found previously in needles of adult Serbian spruce trees of the same lines. .

Somatic hybrids between potato Solanum tuberosum and wild species Solanum verneï exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum vernei (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.

Biochemical changes in barley plants after excessive supply of copper and manganese

The present study was undertaken to identify changes in some important proteins involved in CO 2 fixation (Rubisco, Rubisco activase (RA), Rubisco binding protein (RBP)), NH 4 + assimilation (glutamine synthetase (GS) and glutamate synthase (GOGAT)), using immunoblotting, and in the antioxidative defense as a result of Cu or Mn excess in barley leaves ( Hordeum vulgare L. cv. Obzor). Activities and isoenzyme patterns of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT), as well as the levels of ascorbate (ASC), non-protein sulfhydryl groups, hydrogen peroxide and oxidative damage to proteins were determined. Data were correlated to the accumulation of Cu or Mn in the leaves after 5 days supply of heavy metal (HM) excess in the nutrient solution. In the highest Cu excess (1500 μM), Rubisco LS and SS were reduced considerably whereas under the highest Mn concentrations (18,300 μM) only minor changes in Rubisco subunits were detected. The RBP was diminished under the highest concentrations of both Cu or Mn. The bands of RA changed differently comparing Cu and Mn toxicity. GS decreased and GOGAT was absent under the highest concentration of Cu. At Mn excess Fd-GOGAT diminished whereas GS was not apparently changed. The development of toxicity symptoms corresponded to an accumulation of Cu or Mn in the leaves and to a gradual increase in protein carbonylation, a lower SOD activity and elevated CAT and GPX activities. APX activity was diminished under Mn toxicity and was not changed under Cu excess. Generally, changes in the isoenzyme profiles were similar under both toxicities. An accumulation of H 2O 2 was observed only at Mn excess. Contrasting changes in the low-molecular antioxidants were detected when comparing both toxicities. Cu excess affected mainly the non-protein SH groups, while Mn influenced the ASC content. Oxidative stress under Cu or Mn toxicity was most probably the consequence of depletion in low-molecular antioxidants as a result of their involvement in detoxification processes and disbalance in antioxidative enzymes. The link between heavy metal accumulation in leaves, leading to different display of oxidative stress, and changes in individual chloroplast proteins is discussed in the article.

A Quantitative and Qualitative Analysis of Antioxidant Enzymes in Relation to Susceptibility of Apples to Superficial Scald

The activities and isoenzyme patterns of guaiacol-dependent peroxidase (POX), ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) were studied in yellow- and red-fruited crab apple [Malus (L.) Mill.] selections from a `White Angel' × `Rome Beauty' cross that show differential susceptibility to the physiological storage disorder, superficial scald. There were no consistent relationships between total enzyme activities and scald incidence, high activities of the enzymes being detected in selections with both high and low susceptibilities to scald. However, additional individual isoforms of some antioxidant enzymes were detected in the scald-resistant selections when compared with scald-susceptible selections. In a native gel system, four guaiacol-dependent POX isoenzymes were detected in both yellow and red scald-resistant selections compared with only two in scald-susceptible selections. Similarly, for anodic acidic POX assayed using benzidine, six isoenzymes were detected in both yellow and red scald-resistant selections compared with five in yellow and four in red susceptible selections. Ten SOD isozymes were detected in scald-resistant yellow-fruited selections compared with only five faint bands in scald-susceptible selections, but similar patterns were not detectable for red-fruited selections. Differences in the presence of various isoenzymes for CAT and APX were also detected among the selections, but associations with scald susceptibility were also affected by fruit color or were inconsistent. The presence or absence of individual isoenzymes may be a better indication of scald resistance or susceptibility than the total enzyme activities. Isoenzyme analyses, especially of POX, could be useful to breeders for the early detection of scald resistance/susceptibility in apples.

Tomato (Lycopersicon esculentum cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

Hairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.

Toxicity of nickel and cadmium in spruce seedlings: Effect of separated and combined treatments on peroxidase and superoxide-dismutase activity

We studied the soluble peroxidase and superoxid-dismutase activity peroxidase isoenzyme pattern and metal content in the needles of 2-year-old spruce grown on soils supplemented with cadmium and nickel concentrations from 3 to 30 mg kg -1 and 50 to 500 mg kg -1 respectively. The two metals were applied both separately and simultaneously. The kinetics of metal assimilation and total accumulated quantity depended on the type of treatment. Following metal exposure, an increase in peroxidase activity in the seedlings treated with Cd and Ni/Cd, and in superoxide-dismutase activity during Ni/Cd treatment was observed. A parallel change of peroxidase isoenzyme pattern occurred. There was a good correlation between accumulated metals in needles and enzyme activities. The effect of Cd on the measured parameters was shown to be stronger comparing to the effect of Ni. The results obtained show that peroxidase and superoxide-dismutase activity and peroxidase isoenzyme pattern could be used to evaluate the capacity of one part of the defense system in spruce seedlings to with-stand metal stress.

Developmental regulation of the H 2O 2-producing system and of a basic peroxidase isoenzyme in the Zinnia elegans lignifying xylem

The lignin content, H 2O 2 production and peroxidase (EC 1.11.1.7) isoenzyme patterns of the lignifying xylem of the first internode from Zinnia elegans L. stems were analyzed at three different developmental stages. The results showed that both H 2O 2 production and the catalytic activity of a basic peroxidase isoenzyme were strongly regulated developmental processes, which coincided temporally with the onset of lignification. H 2O 2 production by the lignifying xylem was stimulated by the sulfhydryl blockers, N-ethyl-maleimide (NEM) and N-pyrenyl-maleimide (NPM), and this sulfhydryl blocker-stimulated H 2O 2 production was inhibited by imidazole, suggesting that it was performed by an NADPH oxidase-like enzyme. These distinctive features of H 2O 2 production by the lignifying Z. elegans xylem open up the possibility of unambiguously identifying this enzymatic system responsible for the delivery of H 2O 2 to xylem peroxidases from the vast array of plasma membrane oxidoreductases present in plant cells.

Efeito do extrato aquoso de leucena sobre o desenvolvimento, índice mitótico e atividade da peroxidase em plântulas de milho

A leucena (Leucaena leucocephala (Lam.) de Wit), quando usada como cobertura no solo, apresenta a propriedades de controlar plantas daninhas, sendo esse efeito resultante da presença de aleloquímicos, principalmente mimosina, encontrados na parte aérea da planta. A maioria dos estudos em alelopatia refere-se apenas ao efeito do aleloquímico sobre a germinação e o crescimento da planta-teste, sem considerar os eventos celulares relacionados às mudanças fisiológicas. Com este trabalho objetivou-se avaliar o efeito do extrato aquoso da leucena sobre o desenvolvimento, índice mitótico radicular e atividade da peroxidase e suas isoformas na parte aérea e raízes de plântulas de milho. Os bioensaios foram conduzidos em casa-de-vegetação utilizando-se sementes de milho híbrido desenvolvidas nas concentrações 0; 0,4; 0,8; 1,6; 3,2 e 6,4 % do extrato aquoso de leucena. Verificaram-se inibição do crescimento de raízes e redução do índice mitótico proporcional ao incremento na dose do extrato, não sendo observada divisão celular a partir da concentração de 1,6 %. A atividade da peroxidase, tanto em raízes como em folhas, cresceu com o aumento da concentração do extrato, sem, contudo, apresentar diferença nos zimogramas de suas isoformas. A maior atividade dessa enzima, nas raízes, foi correlacionada com a presença das isoformas aniônicas, pI 4,99 e 4,86, o que possivelmente esteja contribuindo para maior espessamento das raízes, o que foi verificado nas doses elevadas do extrato. As análises por HPLC revelaram teor de mimosina crescente com aumentos na concentração do extrato, sugerindo o envolvimento desse aleloquímico no desenvolvimento das plântulas.

Enhanced indole alkaloid production in suspension compact callus clusters of Catharanthus roseus: impacts of plant growth regulators and sucrose

A special culture system, compact callus clusters, was developed from Catharanthus roseus stem explants in a modified Murashige and Skoog (MS) liquid medium containing 5.37 µM α-naphthaleneacetic acid and 4.65 µM kinetin. Morphological and anatomical studies showed that the globular compact callus cluster cultures consisted of many cohesive callus aggregates displaying some level of cellular/tissue differentiation, which was also in agreement with the results from peroxidase and esterase isoenzyme pattern analysis. The compact callus cluster cultures could synthesise about 2-fold more indole alkaloids than the dispersed cell cultures, and this was postulated to be associated with their differential status. Plant growth regulators and sucrose concentration, as well as shaking speed significantly affected properties of the compact callus clusters. In detail, 2,4-dichlorophenoxyacetic acid destroyed the compact structure and reduced alkaloid production of the compact callus cluster cultures; but a high concentration of cytokinins was necessary to maintain the compact structure and high alkaloid production of the special cultures. The optimum sucrose (5–6%) gave the greatest alkaloid and biomass production, as well as the greatest degree of compaction of the compact callus clusters.