Ca2+ and Mn2+ mediated binding of the glycoprotein peroxidase to membranes of Pharbitis cotyledons

Ca²⁺ and Mn²⁺ promote the binding of the basic isoperoxidase to a crude membrane preparation in extracts from Pharbitis cotyledons. The Ca²⁺- or Mn²⁺-induced binding is resistant to high ionic strength and can be saturated by increasing the divalent ion or the isoperoxidase concentrations. Treatments in vitro with glucosaminidase or in vivo with tunicamycin show that the carbohydrate part of the isoperoxidase is necessary for the binding. The amino sugar galactosamine inhibits the binding at rather high concentrations. Pharbitis basic isoperoxidase can be bound to zucchini squash microsomes in the presence of Ca²⁺ and conversely.     

Soluble and cell wall peroxidases in reed canarygrass in relation to disease resistance and localized lignin formation

The relationship of peroxidases to an inducible disease-resistance mechanism involving lignification of leaf epidermal cell walls was studied. Reed canarygrass (Phalaris arundinacea L.) leaf discs were inoculated with Helminthosporium avenae Eidam and floated on water. In inoculated discs, the activity of soluble, ionic wall-bound and covalent wall-bound peroxidases was about twice the level of activity in noninoculated discs. The increase was attributable to increases in activity of three cathodic isoperoxidases and to the appearance of a new cathodic isoperoxidase. Peroxidase activity in cryostat microtome sections of inoculated discs was histochemically localized in the wall near the site of attempted penetration. When inoculated discs were floated on solutions of cycloheximide (25 μg/ml), increases in peroxidase activity were inhibited, and the fungus penetrated the tissue. The inhibition of peroxidase activity was related to inhibition of cathodic isoperoxidase activity. Anodic isoperoxidase activity did not show changes in response to inoculation or cycloheximide treatment.     

Effect of darkness on isoperoxidases in tobacco tissue cultures

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (C_n) appeared in the medium within 2 days after the cells were placed in the dark. C_n was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). C_n increased and new anodic isoperoxidases A_a, A_b, A_d and A_e appeared in the media as the cells approached death in darkness.     

Purification and characterization of a basic peroxidase from the medium of cell suspension cultures of chicory

A 34-kDa cationic peroxidase (Cicpx) with a pI of 8.9 was purified to homogeneity (RZ 3.5) from the medium of cell suspension cultures of chicory (Cichorium intybus L.) by a combination of ammonium sulphate precipitation, ultrafiltration, ion exchange and gel filtration chromatography. The partial amino acid sequence presented a low homology with other plant peroxidases. Antibody against spinach peroxidase was shown to cross react with chicory isoperoxidase on immunoblots. Unlike anionic peroxidases, Cicpx displayed a high reactivity towards guaiacol and no reactivity towards syringaldazine, indicating that Cicpx was not involved in the lignification process. Thus, further investigations are necessary to assign a specific function to this particular isoperoxidase.     

Ferulic acid: a substrate for two isoperoxidases from Nicotiana tabacum tissue cultures

An anodic isoperoxidase (A₂) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C₄) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H₂O₂. When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A₂ and C₄ appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The Kms for guaiacol are 4 and 4·5 mM for isoperoxidases C₄ and A₂, respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.     

Changes in enzyme activity and isoperoxidases in haploid tobacco callus during organogenesis

Haploid tobacco tissues cultured on Murashige and Skoog (MS) medium supplemented with 0.3 mg/l indole-3-acetic acid (IAA) and 1% sucrose remained undifferentiated. Increasing sucrose level (3%) at the same IAA concentration induced shoot differentiation in 9 days. Further increase in sucrose level (6%) resulted in root differentiation on day 12. Total specific activity of peroxidase, IAA oxidase, malate dehydrogenase (MDH) and phenylalanine ammonia-lyase (PAL) exhibited definite development variations among the three programmes. The number of the anodic and cathodic isoperoxidase bands changed in each case with time. Shoot formation was characterized by the synthesis of anodic peroxidases: AS₁ (Rm=0.41), AS₂ (Rm=0.44) and AS₃ (Rm=0.46), all three being synthesized prior to visual manifestation of shoots. Likewise, root differentiation was heralded in advance by synthesis of one anodic isoperoxidase AR (Rm=0.30). Cathodic isoperoxidases did not show any subtle correlation with shoot or root formation.     

4-Coumarate:Coenzyme A Ligase and Isoperoxidase Expression in Zinnia Mesophyll Cells Induced to Differentiate into Tracheary Elements

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.     

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F₂ data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.     

Scopoletin: A substrate for an isoperoxidase from Nicotiana tabacum tissue culture W-38

Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H₂O₂ only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent K_ms for scopoletin and H₂O₂ were 0·6 and 0·9 rnM respectively.     

Differential extraction of isoperoxidases from Pisum shoots

When green or etiolated Alaska pea shoots are extracted with water or 50 mM phosphate buffer, pH 6·5, or macerated without addition of buffer, the elution profile of the resulting supernatant lacks several isoperoxidase components which are readily extracted from the residue with 10 mM EDTA at pH 6·5. Subsequent extraction with 270 mM NaCl does not solubilize any further components. These results cast doubt on the validity of earlier reports on isoperoxidase patterns in Pisum and other plants.     

Lithium Inhibition of the Thigmomorphogenetic Response in Bryonia dioica

Pretreatment of young Bryonia dioica plants with lithium prevents the inhibition of elongation due to rubbing. Lithium treatment also suppresses the appearance of a specific cathodic isoperoxidase characteristic of rubbed plants.     

Effect of UV-C on peroxidase isoenzymes in axillary bud cultures ofVitis species differing in fungal resistance toPlasmopara viticola

The effect of shortwave (250 nm) UV radiation (UV-C) on the level of peroxidase activity and peroxidase isoenzyme patterns in leaves of resistant ([Vitis vinifera x Viris riparia] x Vitis rupestris andVitis rupestris) and susceptible (Vitis vinifera) grapevine species toPlasmopara viticola (downy mildew) was studied. The results show that although UV-C did not produce significant changes in peroxidase activity in susceptible species, and only minor changes in resistant species, treatment with UV-light induces an acidic isoperoxidase (isoperoxidase A₁), capable of oxidising 4-hydroxystilbenes in resistant species. It was named HSPrx 2. Since peroxidase is apparently the enzyme responsible for ε-viniferin synthesis from resveratrol in grapevines, a close relationship between this peroxidase isoenzyme and ε-viniferin synthesis which occurs in grapevine leaves after UV-C treatment must be expected.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Isoperoxidase and isopolyphenol oxidase spectra in male and female tissues ofActinidia deliciosa in vitro

The isoperoxidase and isopolyphenol oxidase patterns were studied in undifferentiated calli and regenerated shoots and roots of two cultivars ofActinidia deliciosa: one female cv. Hayward and one male cv. Matua. One characteristic, very stable isoperoxidase band (0.85<Rf<0.90) was found only in the male callus, shoots and roots cultured on media supplemented with zeatin (ZEA) or indole-3-butyric acid (IBA). In contrary, one isopolyphenol oxidase band (0.35<Rf<0.40) was typical for male callus and shoots cultured on the medium enriched with ZEA and another one (0.85<Rf<0.90) for male shoots and roots cultured on the medium with IBA. These specific bands had never been found in female cultivar.     

Simultaneous separation of acidic and basic isoperoxidases in wounded potato tissue by acrylamide gel electrophoresis

Preparation and use of a newly developed pH 4.3 horizontal thin layer acrylamide gel which permits the simultaneous separation of acidic and basic isoperoxidases in up to 30 samples is described. Use of cytochrome c, horseradish peroxidase, and a purified potato isoperoxidase as internal standards for a range in isoelectric points of peroxidases from pH 3 to 11 is introduced to facilitate comparison of results obtained with different materials and different methods. Distribution of tissue-specific isoperoxidases in different cell layers of wounded potato (Solanum tuberosum L.) tissue is shown and their purification described. Evidence for the in vitro degradation of basic potato isoperoxidases resulting in more acidic forms similar to isoperoxidases occurring in wounded potato tissue is presented. The significance of this observation for the postulated differential function of different isoperoxidases is discussed.     

RNA silencing of the anionic peroxidase gene impairs potato plant resistance to Phytophthora infestans (Mont.) de Bary

Potato Solanum tuberosum L. plants expressing an antisense M21334 fragment were obtained by agrobacterial transformation. A manifold decrease in activity of anionic isoperoxidase with pI ～ 3.5 in the transformed plants demonstrated that the enzyme is encoded by M21334. The transformed plants showed a decrease in lignin accumulation and a dramatically lower resistance to the late blight agent Phytophthora infestans, implicating the enzyme in the response to P. infestans infection.     

Changes in phenol content and peroxidase activity during in vitro organogenesis in Xanthosoma sagittifolium L.

Direct plant regeneration, multiple shoot formation and callogenesis were induced from cocoyam shoot tips cultured in vitro. At different stages of culture, phenol content, peroxidase activity and acidic soluble isoperoxidase patterns were analysed in plantlets. Results showed that phenol content of plantlets cultured on auxin-free media decreased with time, while it increased in those cultured on media supplemented with an auxin. Each form of morphogenesis induced with a growth regulator was preceded by an increase in total peroxidase activity. On hormone-free medium, organogenesis occurred (single shoot development and rhizogenesis), but there was no increase in total peroxidase activity. The appearance of isoperoxidase A2 was associated with root initiation, while the disappearance of isoperoxidase A5 and the appearance of isoperoxidase A6 preceded multiple shoot formation. These results indicate that total peroxidase activity was not a proper marker for organogenesis in cocoyam. Each form of morphogenetic differentiation is associated with an alteration of the acidic isoperoxidase pattern. These enzymes can be used as biochemical markers for rooting and multiple shoot initiation in cocoyam.     

Isolation and characterization of two isoperoxidases from tobacco tissue cultures

Two isoperoxidases (A_f and C_n) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C_n and A_f have MWs of ca 30 000 and 54 000, respectively. A_f has ca 5.1% carbohydrate, but none could be detected in C_n. Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K_ms for guaiacol are 4 and 13.3 mM for Af and C_n, respectively, while both isoperoxidases have a pH optimum at 6.5. C_n, is dissimilar to other isoperoxidases from tobacco tissue cultures, but A_f is very similar to isoperoxidase A₃ from W-38 tobacco tissue culture.