The Substrate Preference and Histochemical Localization Argue against the Direct Role of Cucumber Stress-Related Anionic Peroxidase in Lignification

The substrate preference and the localization of cucumber (Cucumis sativus L.) stress-related anionic peroxidase (srPRX) were investigated in order to assess whether this activity correlates with the lignification. The results showed that none of the purified srPRX isoenzymes (PRX 1 –3) could oxidize the lignin monomer analog syringaldazine. The srPRX immunospecific signal was found to be highly abundant in both the extrafascicular and fascicular phloem regions in cucumber stem and leaf petiole. In Nicotiana, Petunia and Dahlia, the srPRX homologs were specifically deposited in both outer and inner phloem elements of stem and in both abaxial and adaxial phloem of leaf stems. The srPRX mRNA expression analysis showed similar pattern as for immunolocalization. The subcellular localization of immunospecific srPRX demonstrated that at least part of the peroxidase could be ionically-bound to phloem cell wall.     

Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies

The cellular location of three peroxidase isoenzymes (PRX) in mature leaf tissue of Petunia and their affinity for Concanavalin A-Sepharose were investigated. The isoenzymes PRXa, PRXb and PRXc were identified by their positions in starch-gel zymograms. The fast-moving anodic and cathodic peroxidase bands, the isoenzymes PRXa and PRXc respectively, were the most active peroxidases in extracellular extracts. The molecular forms of PRXa showed a tissue-specific distribution between midrib and remaining leaf tissue. An intermediate-moving anodic peroxidase band, the isoenzyme PRXb, was the most active peroxidase released after extraction of isolated mesophyll protoplasts. Small amounts of the peroxidase isoenzymes were present in cell-wall-bound fractions. Incubation of a crude protein fraction with Concanavalin A-Sepharose showed that the isoenzyme PRXb bound more firmly to Concanavalin A-Sepharose than the isoenzymes PRXa and PRXc, of which only one molecular form bound partly. The results are discussed with respect to a possible function of one of the peroxidase isoenzymes, and a possible role of oligosaccharide chains in determining the cellular location of plant peroxidases is suggested.Abbreviations Con A Concanavalin A - PRX peroxidase (isoenzyme)     

Light-induced Changes in Expression of Pathogenesis-related Anionic Peroxidase in Cucumber Seedlings

The effect of irradiance on the expression of the Cucumis sativus pathogenesis-related (srPRX) anionic peroxidase was studied in germinating seeds at the period when seedlings start to be photosynthetically active. The diversity in the expression patterns of srPRX was observed in both dark- and light-grown seedlings using activity staining and immunoblotting: beside the three srPRX isoenzymes also other three, serologically unrelated, peroxidase isoforms were accumulated in dark-grown seedlings and one in light-grown seedlings. Furthermore, in light-grown seedlings, it was observed a marked difference in the expression of particular srPRX isoenzymes at higher irradiance (up to 260 W m-2, 400 - 700 nm) in comparison to low irradiance. By tissue printing on nitrocellulose paper it was demonstrated that irradiance during germination induced changes in the temporal and spatial distribution of the srPRX.     

Serologically-Related Anionic Peroxidases from Petunia and Cucumber can Substitute Flavonoid Antioxidants

In this study we have investigated whether naturally occurring flavonoid-deficient mutant Red Star of Petunia hybrida is capable of metabolizing H₂O₂ by invoking other antioxidant enzyme system. We demonstrated that reduced flower pigmentation due to a reduction in the chalcone synthase mRNA expression results in strong H₂O₂ accumulation accompanied by the induction of a specific set of anionic peroxidase (PRX), serologically-related to main cucumber srPRX. We found correlation between rate of H₂O₂ accumulation and qualitative, as well as quantitative changes in the srPRX expression which seems to be determined by flower phenotype. In detached flower buds cultured in vitro both abscisic acid and anther extirpation prevented anthocyanin pigmentation, and thus flavonoid biosynthesis, resulting in a marked accumulation of immunoprecipitable srPRX. In contrast, pigmented flowers cultivated under the same conditions did not accumulate corresponding srPRX. The results suggest that a specific set of anionic PRX can substitute for the absence of flavonoid antioxidants.     

Peroxidase Isoenzymes in Strawberry Roots Infected with Binucleate Rhizoctonia spp. and their Implication in Disease Resistance

Infection of strawberry plants with binucleate Rhizoctonia spp. results in an increase in peroxidase activity and the appearance of new isoforms of the enzyme. In healthy and diseased roots of two different strawberry genotypes seven peroxidase isoenzymes were found. In healthy strawberry cv. Senga Sengana, which was moderately resistant to infection, four isoenzymes (1, 2, 5, and 6) were found. Moreover the activity of these isoenzymes was increased and three new isoenzymes (3, 4, and 7) were found in infected roots. In the strawberry hybrid 3/2/86/88/R, which is very susceptible to infection, only isoenzyme 2 was present in the roots of healthy plants. Following infection, the activity of isoenzyme 2 was increased and five new isoenzymes (1, 4, 5, 6, and 7) were detectable. The results obtained indicate that strawberry resistance to binucleate Rhizoctonia may be correlated with peroxidase isoenzyme profile with particular reference to isoform 3, which is only present in infected roots of the moderately resistant cv., Senga Sengana.     

Immunohistochemical localization of the stress-related anionic peroxidase in germinating cucumber seeds

The distribution of the stress-related anionic peroxidase in the course of cucumber (Cucumis sativus L.) seed germination was determined by tissue printing and immunoblotting. Of the three molecular forms of cucumber stress-related anionic peroxidase, the form PRX 1 was temporally accumulated in developing seedlings. Up to 6 d of germination PRX 1 was localized mainly in roots. As germination progressed, the immunoreactive PRX 1 signal was found in the transition zone between roots and stem, as well as in the lower epidermis of expanding cotyledons at the midrib. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Expression of cucumber stress-related anionic peroxidases during flower development or a cryptic infective process

The striking diversity in the expression pattern of the stress-related anionic peroxidase was observed during development of female cucumber flower. While the isoenzyme Prx3 was accumulated constitutively in the course of flower development, the expression patterns of other two isoenzymes (Prx1 and Prx2) were restricted to the period after flower opening. The virus infection was simulated by careful opening of the intact female flower buds 3 d before anthesis followed by exposition to the glasshouse environment for 3 d. The results obtained in this experiment revealed a marked accumulation of the isoenzyme Prx1 and Prx2 at anthesis. Under normal flower development, the pistils did not accumulate these isoenzymes at this stage. In contrast, the pattern of expression of Prx3 as well as of the pistil-specific peroxidase isoenzyme remained unchanged, confirming a constitutive type of expression. Beside the pistil, a 3-d exposition of the stripped flowers resulted in a marked accumulation of Prx1 and Prx2 isoenzymes also in both adjacent flower organs - the ovary and the pedicel. At the same time of the normal development of female flower these organs did not accumulate these isoenzymes.     

Localization and General Properties of Developing Peach Seed Coat and Endosperm Peroxidase Isoenzymes

Changes of soluble and ionically bound peroxidase and indoleacetic acid (IAA) oxidase activities were followed during peach seed development. Soluble peroxidase activity was located mainly in the embryo plus endosperm tissue, whereas wall ionically bound activities were found predominantly in the integument tissue. The different peroxidase isoenzymes present in the extracts were characterized by polyacrylamide gel electrophoresis and isoelectric focusing; the main soluble isoenzyme of embryo plus endosperm tissue was an anionic isoperoxidase of R _F 0.07. Basic ionically bound isoenzymes were located only in the integument tissue, but two soluble anionic isoenzymes of R _F 0.23 and 0.51 were also present in this tissue. In parallel, peroxidase protein content was estimated specifically using polyclonal antibodies. The kinetic data and the changes of seed IAA oxidase activity during fruit development suggested that basic peroxidase isoenzymes from ionically bound extracts of integument might be involved in IAA degradation. Received September 11, 1997; accepted October 21, 1997     

Peroxidase and IAA-oxidase in crude and partially purified enzyme extracts of growing and differentiating root cells

Crude enzyme extracts from the zones of division, elongation and differentiation of cells of primary maize (Zea mays) root show peroxidase activity but lack IAA-oxidase activity. After partial purification of the extracts by gel filtration on Sephadex G-25, the specific peroxidase activity increases almost twice and a high IAA-oxidase activity appears. The partial purification of the enzyme extracts does not change the electrophoretic pattern of the peroxidase isoenzymes, but significantly improves the separation and the visualization of isoenzymes with IAA-oxidase activity. The data obtained were interpreted in connection with the different modifying effect of the low molecular compounds (mainly phenolics) on the activity and the isoenzyme patterns of the peroxidase and the IAA-oxidase.     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

Do S allele-specific peroxidase isoenzymes exist in self-incompatible Nicotiana alata?

Summary In order to test Pandey's hypothesis that peroxidase isoenzymes determine S-gene specificity in Nicotiana alata, peroxidase isoenzymes in styles and pollen from various plants of an inbred- and a cross progeny were compared by means of starch gel electrophoresis and electrofocusing.No relation between the S-genotype and the peroxidase isoenzyme patterns of pollen or of styles could be established. The differences between the isoenzyme patterns of different S-genotypes were ascribed to differences in the genetic background of various plants that had the same S-genotype.     

POLYACRYLAMIDE GEL ELECTROPHORESIS OF RAT BRAIN ACETYLCHOLINESTERASE: ISOENZYME CHANGES FOLLOWING PARATHION POISONING1

Brain acetylcholinesterase (EC 3. 1. 1. 7) isoenzymes of 15- and 30-day-old rats were found to be inhibited by 2.5 mg/kg and 1.25 mg/kg dosage levels of intraperitoneally administered parathion (E-605; O, O-diethyl-p-nitrophenyl phosphorothionate). With 2.5 mg/kg dose level, the response of isoenzymes in 15- and 30-day-old rats was similar. At both ages, there was no significant sex difference in the degree of depression of the isoenzymes. There were no significant regional differences in the degree of inhibition of acetylcholinesterase isoenzymes in the rat brain. At 1.25 mg/kg dosage level, a differential isoenzyme inhibition was evident, with the major isoenzyme (isoenzyme 3) exhibiting the greatest sensitivity to the inhibitor in all brain areas examined. The course of isoenzyme depression and recovery following the administration of parathion differed in brain, serum and skeletal muscle. Whereas brain isoenzymes exhibited most marked inhibition at 2 h after injection, inhibition of serum and skeletal muscle isoenzymes was more prolonged. At 4 h after injection, these isoenzymes were still inhibited while brain isoenzymes had recovered to a substantial degree. Twenty four h following the injection of parathion, when brain and serum acetylcholinesterase isoenzymes had returned to control activity levels, isoenzymes of skeletal muscle demonstrated only minimal recovery.     

The development of glutathione S-transferase and glutathione peroxidase activities in human lung

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight     

Isoenzymes of Japanese-radish Peroxidase

Japanese-radish root contained eighteen isoenzymes of peroxidase distinguishable on polyacrylamide gel electropherograms. The isoenzymes were found to be quite similar to those of horseradish peroxidase, although their quantities were different between two plants. The acidic components were the major isoenzyme in Japanese-radish peroxidase, while the neutral ones were the major one in horseradish. The chromatographic purification of the isoenzymes was performed on CM- and DEAE-Sephadex columns to characterize the components. The components in the preparations purified by the previously reported procedures of Morita et al. were also identified.     

The Subcellular Localization of Isoperoxidases in Capsicum annuum Leaves and their Different Expression in Vegetative and Flowered Plants

The subcellular localization of leaf peroxidases (EC 1.11.1.7)and their expression in vegetative and flowered plants has beenstudied in Capsicum annuum (var. annuum) in order to assesswhether the expression of new peroxidase isoenzymes can characterizethe floral state which determines the beginning of reproductivedevelopment. The results showed that floral development is accompaniedby a significant increase in the level of soluble (non-sedimentable)leaf peroxidase, independently of leaf position along the internodes,and therefore independently of the leaf age. An analysis ofthe leaf peroxidase isoenzyme patterns along the internodesfor vegetative and flowered plants shows that the increase inperoxidase activity is due to a general increase in the activityof all the pre-existing peroxidase isoenzymes, although isoenzymeB₂ and, especially, isoenzyme A₁ showed a distinctive and majorincrease in activity. These two isoenzymes are mainly ionically-boundto cell walls, probably in equilibrium with the same isoenzymesmoving freely in the cell-wall free spaces. The differs fromother peroxidase isoenzymes, such as isoperoxidase B₆, whichis mainly located in the covalently-bound cell-wall fractionand in mesophyll vacuoles. These results are discussed in thelight of a possible role of cell wall peroxidases as markersof the floral state in Capsicum annuum morphogenesis.Copyright1993, 1999 Academic Press Capsicum, floral state, leaf peroxidases, subcellular localization, vegetative state     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

Role of carbohydrate moieties in peanut (Arachis hypogaea) peroxidases.

The activities of a cationic (C.PRX) and an anionic peroxidase isolated from peanut (Arachis hypogaea)-cell suspension culture were drastically reduced when they were deglycosylated with glycopeptidase F or oxidized by 10 mM-periodate. In contrast with the controls, the deglycosylated or the oxidized peroxidases were much more susceptible to proteolytic degradation. In radiolabelling experiments with [35S]methionine, the non-glycosylated C.PRX was synthesized in the tunicamycin-treated cultures and secreted into the medium. Examination of the C.PRX polypeptides by SDS/polyacrylamide-gel electrophoresis followed by fluorography showed that the non-glycosylated form had an Mr of approx. 31,000, which is about 78% of that of the glycosylated form. Our results suggest that carbohydrates may not be essential for peroxidase secretion, but that stabilization of the peroxidase molecules and acquisition by these isoenzymes of a catalytically active conformation is linked directly or indirectly to glycosylation.