Peroxidase Zymograms Research Articles

Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness

A recently published method to separate protein isoforms by means of a flat-bed agarose native gel was adapted for identifying simultaneously both acid and basic isoforms of plant peroxidases. These were evidenced by in situ activity staining using alternative substrates for which the isoforms showed specific preference. Such approach allowed the detection of a significantly higher number of horseradish peroxidases than the conventional methods based on sample separation by acrylamide gel and the single bands were clearer to observe. Samples recovered from different plant species and with variable level of purity were successfully analyzed for their peroxidase (POX) isoform pattern. We expect that the innovative electrophoretic methodology illustrated in this work will strongly improve the output of experiments that aim at relating the activity and expression variation of specific enzyme biomarkers with physiological and pathological conditions.

Classification of floating rice varieties by acid phosphatase and peroxidase zymograms.

Classification of floating rice varieties by acid phosphatase and peroxidase zymograms.

Overexpression of a basic peroxidase in transgenic tomato ( Lycopersicon esculentum Mill. cv. Pera) hairy roots increases phytoremediation of phenol

In recent years, genetic engineering and tissue culture techniques have produced significant biotechnological advances in phytoremediation. Numerous peroxidases (E.C. 1.11.1.7) have been implicated in the phytoremediation of phenol, a common and highly toxic contaminant present in industrial wastewaters. To explore the possibility of enhancing phenol removal and to find out if tpx1, a basic (p I 9.6) peroxidase from tomato, is involved in phenol removal, we established transgenic tomato hairy root clones which overexpress tpx1 gene, following an unusual procedure: successive transformation with Agrobacterium tumefaciens and Agrobacterium rhizogenes. The overexpression of tpx1 correlated with higher in vivo and in vitro peroxidase activity in crude extracts (CE) of transgenic hairy root clones than in those of the wild type culture. Furthermore, peroxidase zymograms of transgenic clones showed higher activity of a band with p I 9.6 in the fraction of ionically bound to cell wall proteins. The transgenic clones with the highest peroxidase activity (clones 8 and 9) were selected and tested for phenol removal from aqueous solutions. Removal efficiency of the transgenic clone 8 was significantly higher than the wild type hairy roots. The data presented here provide evidence of the utility of successive transformation for obtaining stable transgenic hairy roots and demonstrate that engineered hairy root cultures overexpressing tpx1 peroxidase enhance phenol removal and could be useful tools in phytoremediation process.

Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.

Root peroxidases and Rhizobium, Bradyrhizobium nodulation affinities of Phaseolus vulgaris L. and P. acutifolius A. Gray congruity backcross populations

Four parents [P. acutifolius var.acutifolius (A19), P. a.var. latifolius `Serowi' (A9),P. a. var. latifolius(A10), and P. vulgaris `Red Cloud' kidney (V1)] were used to create six interspecific recurrent and congruity backcross pedigrees (V1 × A9, A9 × V1, V1 × A10, A10 × V1, V1 × A19, A19 × V1) for evaluation of Rhizobium, Bradyrhizobium nodulation affinities and root peroxidase banding patterns. Most primary hybrids nodulated with all Rhizobium, Bradyrhizobium strains, while recurrent generations nodulated with strains of the recurrent parent, suggesting a number of independent loci and random assortment for strain-specific nodulation. A proximity matrix of nodulation phenotypes produced a cluster with V1, two recurrent and two congruity backcross hybrids with V1 as the cytoplasmic parent; a separate cluster with all primary interspecific and balanced, congruity backcross hybrids. A9 and A10were clustered the farthest away from V1. Root peroxidase zymograms of primary hybrids were the summation of the parents and controlled by independent loci. Advanced hybrids exhibited new root peroxidases, matching those found for otherPhaseolus spp., including ancestralP. coccineus. A proximity matrix of root peroxidase phenotypes produced a cluster with only A9 3V1F1, a cluster containing only A19 3V1 F2, and a large cluster with all P. acutifoliusparents and hybrids with P. acutifolius as the cytoplasmic parent in balanced hybrids or the majority parent in unbalanced hybrids. Estimation of genetic distances showed primary, interspecific hybrids to be more closely related with the cytoplasmic parent. Recurrent and congruity backcross generations varied in genetic distances between each respective parent. This is the first report of congruity backcrossing creating hybrids with biochemical traits of nonparental species.

Changes in activity of antioxidative enzymes in wheat (Triticum aestivum) seedlings under cold acclimation

Activated oxygen species such as superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radicals can be produced in plants exposed to low, non‐freezing, non‐injurious temperatures. To prevent or alleviate oxidative injury, plants have evolved several mechanisms which include scavenging by natural antioxidants and enzymatic antioxidant systems such as superoxide dismutases, catalase and peroxidases.Although overproduction of hydrogen peroxide and increased tolerance to oxidative stress can be induced in wheat by low‐temperature treatments, data concerning changes in the enzymatic antioxidant systems are almost absent.With the aim to provide this information, antioxidant enzyme (superoxide dismutases, catalase and peroxidases) activities were analysed in leaves and roots of Triticum aestivum cvs Brasilia (frost resistant in field) and Eridano (less frost resistant in field) seedlings grown at day/night temperatures of 24/22°C (control treatment) and 12/5°C (low‐temperature treatment).Our data showed that superoxide dismutase activities were unaffected by low‐temperature treatment both in leaves and roots. Catalase activity in leaves and roots was decreased in 12/5°C‐grown seedlings, but Brasilia maintained higher catalase activity than Eridano. Differences were also observed in guaiacol peroxidase activities between control and acclimated seedlings: Higher guaiacol peroxidase activities were found in the leaves of 12/5°C‐grown seedlings while in roots these activities were lower. Moreover, Brasilia guaiacol peroxidase activities were higher than Eridano. Superoxide dismutase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by low‐temperature treatment.Changes in the activities of antioxidant enzymes induced by cold acclimation support the hypothesis that a frost‐resistant wheat cultivar, in comparison with a less frost‐resistant one, maintains a better defence against activated oxygen species during low‐temperature treatment.

Peroxidase zymograms at constant and gradient pH electrophoresis as an analytical test in the identification of potato varieties

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPeroxidase zymograms at constant and gradient pH electrophoresis as an analytical test in the identification of potato varietiesAngel R. Nieto, Ana C. Sancho, Miguel V. Barros, and Julio L. GorgeCite this: J. Agric. Food Chem. 1990, 38, 12, 2148–2153Publication Date (Print):December 1, 1990Publication History Published online1 May 2002Published inissue 1 December 1990https://doi.org/10.1021/jf00102a010RIGHTS & PERMISSIONSArticle Views145Altmetric-Citations6LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Esterase and peroxidase zymograms from barley ( Hordeum vulgare L.) callus as a biochemical marker system of embryogenesis and organogenesis

Based on visual appearance three different tissue types could be distinguished in callus of Hordeum vulgare L.: soft pale callus, compact pale callus and compact green callus. Soft pale callus contains highly vacuolated and enlarged cells. Compact callus, pale as well as green, consists of hard nodules with small thin walled cells. Esterase (EC 3.1.1.2) and peroxidase (EC 1.11.1.7) isoenzyme patterns were analyzed in the different types of barley callus, before any sign of differentiation was visible. The esterase system is very sensible for detection of embryogenesis before somatic embryos are formed. Peroxidase indicates both leaf and root morphogenesis before primordia can be seen. Transfer of callus on regeneration medium with lower auxin content confirmed the date obtained after enzymatic analysis, although lower percentages of plant regeneration were obtained, than those predicted after enzymatic analysis due to necrosis and suboptimal culture conditions. The results of the isozyme analyses further confirm that no difference should be made between hard green and hard pale callus. Based on the enzymatic data, it was concluded that soft callus and both types of hard callus display a low capacity of organogenesis (2–3%). The embryogenic capacity is much higher in pale and green hard callus (43–48%) compared to soft pale callus (5%). To optimize the in vitro culture of barley, it is useful to make a selection for the hard parts of the callus while eliminating the soft callus tissue.

パーオキシダーゼ•アイソザイムによるサザンカ品種とツバキ属の種との類縁関係について

Peroxidase isozyme activities of the genus Camellia were examined to study the interrelationship between cultivars of Camellia sasanqua and species of the genus Camellia, and to identify the process of differentiation of cultivars in C. sasanqua. For the analysis, 13 species, 1 subspecies, 2 varieties, 4 intersecific hybrids, and 97 cultivars of C. sasanqua and C. japonica were used.Peroxidase zymogram patterns of Camellia species showed clear differences between sections in many cases, although there were a few exceptions which showed no significant differences. Especially, there were marked differences in the zymogram patterns between section Paracamellia including C. sasanqua and section Camellia including C. japonica. Peroxidase zymograms of interspecific hybrids generally showed the bands common to their parents.Peroxidase zymogram patterns of cultivars of C. sasanqua showed certain tendencies depending on four groups. Cultivars of“Sasanqua group”showed zymogram patterns similar to wild C. sasanqua. Cultivars of“Vernalis group”shared common bands with C. sasanqua and C. japonica. In“Hiemalis group”, some cultivars showed the same zymogram patterns as in“Sasanqua group”and others showed one of the patterns of“Vernalis group”. These results would indicate that the variation of cultivars in C. sasanqua has developed from interspecific hybrids between C. sasanqua and C. japonica.′Tagoto-no-tsuki′, a cultivar of“Obscura group”, showed the same zymogram pattern as C. oleifera, but no significant differences could be observed from other species of section Paracamellia.

PEROXIDASE ZYMOGRAMS OF 16 APPLE ROOTSTOCKS

Shoot bark and root extracts of 16 different apple (Malus domestica Borkh.) root-stocks were analyzed for multiple electrophoretic forms of peroxidase (EC 1.11.1.7) using polyacrylamide gel electrophoresis (PAGE). Thirteen bands were seen in the peroxidase zymograms of shoot bark extracts and eight in the peroxidase zymograms of root extracts. The root peroxidase bands corresponded to ones in the shoot bark peroxidase zymograms. Two of the bands were common to all 16 rootstock patterns. The other 11 bands formed six patterns which were characterized by the presence of specific peroxidase bands. The 16 rootstocks were grouped according to banding characteristics.Key words: Apple, Malus domestica Borkh., apple rootstocks, PAGE-peroxidase zymograms

The production of fertile, triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr(-)) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.

Hybrid embryo formation in an intersubgeneric cross of soybean (Glycin max MERILL) with a wild relative (G. tomentella HAYATA).

Percentage of successful pod setting in the intersubgeneric hybridization between G.max(2n=40, maternal) and G.tomentella(2n=80, paternal) was estimated to be 2.5 to 7.6% Germinability of the G.tomentella pollens of G.max stigmas, elongation of the pollen tube in the style and arrival of the pollen-tube tips at the ovules were detected by SEM and fluorescence microscopy. Aneuploidy (2n=64) of the hybrid embryo cells and peroxidase zymogram pattern specific to the hybrid-derived calli were observed by light microscopy (smear method) and polyacrylamide gel electrophoresis, respectivly. Moreover, histlogecal observation of the hybrid embryo was carried out to investigate the extant of the hybrid embryo growth. It was concluded that morphologically normal growth may continue at least until heart or a more advanced developmental stage, but markedly retarded growth accompanies it and eventually it ceases growing and death of the hybrid embryo results. Though, we couldn't get intact hybrid plant, all of the facts revealed proved formation of the genuine hybyid embryos between G.max and G.tomentella.

Variations in peroxidase isozyme of Japanese lawn grass (Zoysia japonica STEUD.) populations in Japan.

Peroxidase isozymes in 24 populations of the Japanese lawn grass (Zoysia japonica Steud.), collected from all over Japan, were analyzed by thin layer gel electrofocusing. A peroxidase zymogram pattern of the leaf extracts revealed about 20 bands. Intra-population variability was estimated by the mean of the correlation coefficients based on peroxidase zymograms of ten plants in each population. Of the 24 populations analyzed, those from pastures generally showed a very low intra-population variability. The inter-population relationships among the 24 populations were assessed by cluster analysis using the correlation coefficients obtained from the average isozyme activity. No clear trend was noted in the relation between the dendrogram and the geographical location. However, three populations in Hokkaido were significantly distant from all other populations, suggesting a difference at the gene level. Two populations in Miyazaki Prefecture were also differ from all other. The populations in Honshu and Shikoku were close to one another and the similarity among the Oozasa, Sotoyama and Morioka populations was particularly striking.

The use of peroxidase polymorphism in the identification of apple scion cultivars

Polyacrylamide gel electrophoretic separations of peroxidase isoenzymes from shoot bark tissues of apple show patterns that are consistent for cultivar and independent of season, age or eco-physiological condition of the plant. This permits cultivar identification both throughout the year and during the juvenile phase of growth. Based on peroxidase zymograms, 108 apple cultivars from collections at the National Fruit Trials, Brogdale, and East Malling Research Station were reproducibly classified into 26 phenotypic groups.

Peroxidase isozyme differences in tall fescue cultivars and allopolyploid accessions

The use of isoelectric focusing for the isozymic identification of cultivars and wild forms in tall fescue (Festuca arundinacea Schreb.) and determination of ploidy effects was investigated. Peroxidase zymograms from allopolyploid accessions (4x, 6x, 8x, and 10x, x=7) and randomly sampled plants of the cultivars ‘Kenhy’, ‘Kentucky 31’, and ‘Missouri 96’, were compared for band number, position, and staining intensity. There was little isozymic variation among plants in the tetraploid and hexaploid, but considerably more in the octoploid and decaploid members of the series. Yet no significant effects of ploidy on isozyme complexity or band number were observed. There were no consistent differences in band number, position, or frequencies among the three cultivars in spite of their diverse parentage. Isoclectric focusing can be used successfully in tall fescue, but the limited variability of the peroxidases encourages investigation of other isozyme systems in the future.

Application of Esterase and Peroxidase Zymograms to Breeding in Brassica with Reference to Nucleus Substitution

Application of Esterase and Peroxidase Zymograms to Breeding in Brassica with Reference to Nucleus Substitution

Isoperoxidases in Epidermal Layers of Tobacco and Changes during Organ Formation in vitro

AbstractThe content and pattern of soluble isoperoxidases were determined in epidermal explants taken from different internodes of tobacco plants in the vegetative and floral states. There were qualitative and quantitative differences in the isoperoxidases, with a decrease in content and fewer bands being observed acropetally, i.e., in going from the base of the stem towards the apex. Epidermal explants from floral branches were grown in in vitro culture, with various media moditications, to form de novo floral or vegetative buds, roots or callus. Changes in soluble isoperoxidases were followed electrophoretically in relation to these varying morphogenetic pathways. In each of them, the number of bands increased on both the anodic and cathodic sides with time in culture. Compared to each other these four morphogenetic programmes were different in their peroxidase zymograms, mainly through varying kinetics in the development of activity of the isoenzymes. The changes observed during root and vegetative bud formation agree with previously published data, and the changes during floral bud formation agree with those observed in vivo.

Visualization of peroxidase isozymes with eugenol, a noncarcinogenic substrate

Plant peroxidase isozymes utilize hydrogen peroxide to oxidize redox dyes. Benzidine, the substrate most commonly used for identification of peroxidase isozymes, is a potent carcinogen and has been banned from laboratory use. O-Dianisidine, the other common substrate for peroxidase isozymes, is structurally related to benzidine and is a possible carcinogen. A peroxidase zymogram stain has been developed which uses eugenol, a substrate which is safe to use in the laboratory. Peroxidase isozymes are recognized as bright blue fluorescent bands under ultraviolet light and develop within 1 min after staining. Eugenol may also be used in a quantitative fluorimetric assay of peroxidase activity.

Regulation of internodal lenght by peroxidase enzymes in grain sorghum

A relationship between height genes (dw locus) and perioxidase was demonstrated by extracting and determining peroxidase specific activity in internode tissue from different height isogenic lines of sorghum Sorghum bicolor (L.) Moench]. Tall plants (2 dwarf) had less peroxidase per gram tissue than their short counterparts (3 dwarf); their F1 offspring internodes were closer but had more peroxidase than the tall parent. Peroxidase in the F2 offspring was inversely related to their height and followed a simply-inherited pattern similar to that for height.Among different tissues analyzed, peroxidase concentration in roots was higher than in leaves and internodes, whole internode higher than in pith, and seed embryo higher than in endosperm. Peroxidase activity of nonviable seeds was negligible.Isoelectric focusing provided a more detailed peroxidase zymogram than did gel electrophoresis. Differences in peroxidase bands among tall and short parental plants, F1 and F2 segregating groups all appear to be reflected by intensity differences rather than by position or number of bands.Activities of nitrate reductase and acid phosphatase did not correlate with height. That finding provides a control and suggests that peroxidase activity is not associated with height by chance but may have a functional relationship.

Cytogenetical studies in the genus Persea (lauraceae). III. Electrophoretical studies on peroxidase isozymes.

Comparison of the peroxidase zymograms of the avocado, Persea americana between states from which a large number of strains were collected indicated that the strains from Michoacan, Mexico, Morelos, Puebla and Veracruz have 15, 13, 10, 12 and 12 active regions in the zymograms, respectively. The form having activities at more regions of zymograms would be regarded as more advanced ones than those possessing lower number of active regions. The percentage of strains in which statistically significant variation of zymograms within strains were found was high in Puebla (60.0%), Michoacan (43.1%) and Veracruz (37.4%). Also these states are the major places where avocado is cultivated more abundantly. Considering the higher frequencies of strains having significant differences of peroxidase zymograms within strains and lower number of active regions in the zymograms, the Puebla-Veracruz region was postulated as the possible center of the origin of the Mexican type of avocado. Archeological evidences found in the Valley of Tehuacan in Puebla, and the fact that P. schiedeana and P. floccosa, semicultivated and wild relatives of P. americana, respectively, grow in the Puebla-Veracruz area support this postulation.Differences of peroxidase zymograms between Mexican, Guatemalan and West Indian types were also found to be highly significant. From the frequency of strains which showed significant variations within strains (62.5%, 50.0% and 39.1% in Guatemalan, West Indian and Mexican types, respectively), it was presumed that Guatemalan type is the most ancient form.