Role of Apoplastic and Cell-Wall Peroxidases on the Stimulation of Root Elongation by Ascorbate

Elongation of onion (Allium cepa L.) roots was highly stimulated by ascorbate (ASC) and its natural precursor I-galactone-[gamma]-lactone (GL). When incubation media were supplemented with lycorine (Lyc), an inhibitor of the ASC biosynthesis, root growth was negligible even in the presence of ASC or GL. ASC completely inhibited in vitro guaiacol peroxidase activities that were isolated from both the apoplast and the cell wall. However, ferulic-acid-dependent peroxidase from the cell wall was partially inhibited by ASC, whereas ferulic acid peroxidase activity from the apoplastic fluid was completely inhibited by ASC as long as ASC was present in the assay medium. ASC content in cells was increased by preincubations with ASC or GL, whereas Lyc reduced it. On the other hand, ASC or GL treatments decreased both apoplast and cell-wall-bound peroxidase activities, whereas Lyc had a slight stimulating effect. These results are discussed on the basis of a possible control of root elongation by ASC via its action on peroxidases that are involved in the regulation of cell-wall extensibility.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

Localization and Characterization of Peroxidases in the Mitochondria of Chilling-Acclimated Maize Seedlings

We present evidence of two peroxidases in maize (Zea mays L.) mitochondria. One of these uses guaiacol and the other uses cytochrome c as the electron donor. Treatments of fresh mitochondria with protease(s) indicate that ascorbate and glutathione peroxidases are likely bound to the mitochondria as cytosolic contaminants, whereas guaiacol and cytochrome peroxidases are localized within the mitochondria. These two mitochondrial peroxidases are distinct from contaminant peroxidases and mitochondrial electron transport enzymes. Cytochrome peroxidase is present within the mitochondrial membranes, whereas guaiacol peroxidase is loosely bound to the mitochondrial envelope. Unlike other cellular guaiacol peroxidases, mitochondrial guaiacol peroxidase is not glycosylated. Digestion of lysed mitochondria with trypsin activated mitochondrial guaiacol peroxidase but inhibited cytochrome peroxidase. Isoelectric focusing gel analysis indicated guaiacol peroxidase as a major isozyme (isoelectric point 6.8) that is also activated by trypsin. No change in the mobility of guaiacol peroxidase due to trypsin treatment on native polyacrylamide gel electrophoresis was observed. Although both peroxidases are induced by chilling acclimation treatments (14[deg]C), only cytochrome peroxidase is also induced by chilling (4[deg]C). Because chilling induces oxidative stress in the maize seedlings and the mitochondria are a target for oxidative stress injury, we suggest that mitochondrial peroxidases play a role similar to catalase in protecting mitochondria from oxidative damage.     

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.     

Ascorbic acid improves conversion of white spruce somatic embryos

Summary The effects of exogenous applications of ascorbic acid on white spruce somatic embryogenesis were examined. Increasing concentrations of ascorbate (1 μM to 100 μM) in the germination medium enhanced somatic embryo conversion in a linear fashion. At the optimal ascorbate level (100 μM) the number of embryos able to undergo normal conversion, i.e., emergence of both root and shoot, increased from 34% (control) to 58%. The effect of ascorbate had a more pronounced effect on shoot growth than on root emergence; and at 100 μM ascorbate, the percentage of embryos able to produce new leaf primordia increased from 47% (control) to 79%. Root emergence increased slightly from 64% in the control embryos to 74% in the presence of ascorbic acid. The ascorbate-treated embryos were characterized by an enlarged apical region, presumably due to a larger number of leaf primordia produced, and by dark green leaves. When allowed to grow further, these embryos were able to develop into normal plantlets.     

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.     

Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce

Somatic embryo quality is still a problem for many researchers. To improve the efficiency of germination, special procedures are used, such as partial drying of somatic embryos at high relative humidity or desiccation in the presence of supersaturated solutions of salt. In this work, cotyledonary somatic embryos of Norway spruce (Picea abies) and Serbian spruce (P. omorika) were placed on culture media (ME or BM-5) to germinate. We found that after 4 weeks of incubation on these media, hypocotyl and radicle growth of control (non-dried) somatic embryos of both species was not adequate to yield seedlings able to acclimatize to greenhouse conditions. Therefore, somatic embryos were partly dried at relative humidity of 97 % or desiccated at relative humidity of 79 %, for 2 or 3 weeks, and then placed on the Margara (ME) medium. Partial drying of somatic embryos at the higher relative humidity (97 %) enabled an improvement of radicle growth of germinating somatic embryos in both species. The highest conversion rate (45 %) was obtained for embryos of Norway spruce maintained for 2 weeks at relative humidity of 97 %. This treatment contributed to the improvement of germination and conversion efficiency of somatic embryos of Norway spruce, regardless of the drying period. Improved radicle growth facilitated development of better quality seedlings of this spruce species. In Serbian spruce, we did not obtain seedlings of sufficient quality, due to poor hypocotyl growth. Desiccation at humidity of 79 % for 3 weeks proved to be lethal to somatic embryos of both species.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Changes in Peroxidase Activity Associated with Cell Walls During Pine Hypocotyl Growth

Peroxidase active against 2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulphonicacid] (ABTS) and guaiacol were found in the apoplastic fluid,as well as ionically and covalently associated with pine cellwalls. The highest activity was found covalently bound to cellwalls, while the lowest activity was in the apoplastic fluid.Both ABTS and guaiacol peroxidases increased with the hypocotylage in the three fractions, apoplastic, ionically and covalentlybound. Furthermore, the changes in both peroxidases along thehypocotyl were also studied. Both apoplastic ABTS- and guaiacol-peroxidasesincreased from the apical towards the basal region of the hypocotylsof 10-d-old seedlings. A relation between peroxidase activityin the apoplastic fluid and the cell wall stiffening in pinehypocotyls is proposed.Copyright 1995, 1999 Academic Press Cell wall, growth, hypocotyl, peroxidase, pine, Pinus pinaster Aiton     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heart-shaped embryos. The transient increase in GSH contents also correlated with the changes in measured γ-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii

The effect of salinity on the antioxidative system of root mitochondria and peroxisomes of a cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) was studied. Salt stress induced oxidative stress in Lem mitochondria, as indicated by the increased levels of lipid peroxidation and H(2)O(2). These changes were associated with decreased activities of superoxide dismutase (SOD) and guaiacol peroxidases (POD) and contents of ascorbate (ASC) and glutathione (GSH). By contrast, in mitochondria of salt-treated Lpa plants both H(2)O(2) and lipid peroxidation levels decreased while the levels of ASC and GSH and activities of SOD, several isoforms of ascorbate peroxidase (APX), and POD increased. Similarly to mitochondria, peroxisomes isolated from roots of salt-treated Lpa plants exhibited also decreased levels of lipid peroxidation and H(2)O(2) and increased SOD, ascorbate peroxidase (APX), and catalase (CAT) activities. In spite of the fact that salt stress decreased activities of antioxidant enzymes in Lem peroxisome, oxidative stress was not evident in these organelles.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

Purification, characterization and stability of barley grain peroxidase BP 1, a new type of plant peroxidase

The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and Jaboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 m M C_aCl₂, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (m M mg⁻¹ min⁻¹): coniferyl alcohol (930±48), caffeic acid (795±53), ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-(6)-sulfonic acid]) (840±47), ferulic acid (415±20), p -coumaric acid (325±12), and guaiacol (58±3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399–402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75°C at pH 6.6, and 67°C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.     

Germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes

A selenium-containing peroxidase from the germinating barley grown on a selenium-containing artificial medium was isolated and purified by means of cold acetone precipitation, Sephadex-G150 filtration, followed by DEAE-Sepharose chromatography and sodium dodecyl sulfate — polyacrylamid gel electrophoresis. The form of selenium existing in the peptide assayed with paper chromatography was selenomethionine. The amino acid composition of this enzyme was similar to those peroxidases from other sources except amino acids Glu, Val Phe, Lys, and Arg. Electron-spin resonance (ESR) spectra recorded at −136°C showed that both the selenium-containing peroxidase from germinating barley and horseradish peroxidase had same the ESR signals as iron protoporphyine. Those results suggested that the germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes.     

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Hydrogen peroxide (H₂O₂) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K_m = 40 millimolar) and H₂O₂ (apparent K_m = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.