Changes in Amount of Polyphenols and Activity of Related Enzymes during Growth of Tobacco Flower and Capsule

Developmental stages of tobacco (Nicotiana tabacum L. cv. Burley 21) flower and capsule were correlated with tissue contents of polyphenols and activities of phenylalanine ammonialyase, polyphenoloxidase, and peroxidase. Chlorogenic acid, scopolin, and scopoletin were present in most tissues, whereas rutin and two dihydroxyphenolic glycosides concentrated primarily in the corolla and placenta, respectively. Ovules contained only chlorogenic acid. As development progressed, polyphenols accounted for nearly 15% of the dry weight in the green capsule of field-grown plants. Fertilization triggered a rapid increase of chlorogenic acid in the ovary. When l-phenylalanine-U-(14)C was fed to the detached green capsules and capsule parts, an incorporation of radioactivity into chlorogenic acid and dihydroxyphenolic glycosides occurred which suggested in situ synthesis of these compounds. This was subtantiated by a positive correlation between phenylalanine ammonia-lyase activity and polyphenol accumulation. High polyphenoloxidase activity was associated mainly with the ovary, whereas peroxidase activity was maximal during senescence of all tissues. Polyacrylamide gel slab electrophoresis revealed five cathodic bands and one diffuse zone with poly-phenoloxidase activity in flower extracts. Two anodic poly-phenoloxidase isozymes appeared only in the fertilized ovary. Among 17 peroxidase isozymes, six cathodic forms were present throughout floral development, and the anodic ones increased in number and activity at the later stages of capsule growth.     

Ontogeny and Hormonal Control of Polyphenoloxidase Isozymes in Tobacco Pith

Isozymes of tobacco pith polyphenoloxidases (o-diphenol oxidase, EC 1.10.3.1) were separated electrophoretically from fresh pith of intact plants and from cultured pith sections. Extracts of fresh pith contained a poorly resolved complex of two to three anodic bands after starch gel electrophoresis at alkaline pH. This anodic complex was more active with chlorogenic acid than with 3,4-dihydroxyphenylalanine and was found in greater activity per gram fresh weight of tissue in younger internodes than in older ones. The longitudinal gradient of activity was thus the opposite of that found for the constitutive isozymes of peroxidase.A well defined cathodic band of polyphenoloxidase activity appeared after culture of pith in modified White's medium with shaking. This band, which was more active with 3,4-dihydroxyphenylalanine than with chlorogenic acid, could be detected after 1 to 2 days of incubation. Its appearance was enhanced by the addition of 10 mum indoleacetic acid; kinetin (1 mum tended to prevent this indoleacetic acid effect). Such hormonal control is opposite to that previously reported for the rapidly appearing new isozymes of peroxidase.The pattern of the major isozymes associated with polyphenoloxidase activities differs from that of peroxidase.     

Solanum berthaultii trichomes contain unique polyphenoloxidases and a peroxidase

Glandular trichomes of Solanum berthaultii leaves contain two unique forms of polyphenoloxidase (α- and β-PPO) and a separate peroxidase. The β-PPO appeared to be a polymer of α-PPO and exhibited a similar K_m but an apparently higher specific activity with chlorogenic acid and caffeic acid. These data are directly related to studies on the insect defensive properties of the trichomes.     

Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots

Peroxidase (EC 1.11.1.7) activity is associated with suberization during endodermal development and metacutization in roots of white spruce (Picea glauca [Moench] Voss) seedlings. Histochemical analysis indicates a relationship between suberization and peroxidase activity, but peroxidase is ubiquitous. Increased peroxidase activity results from the induction of four anodic peroxidase isozymes in addition to quantitative increases in two anodic peroxidase isozymes. Four of these polymerized eugenol. Cold temperatures induce formation of two anodic isozymes and result in suberization. The increased peroxidase activity associated with suberization is correlated to residual respiration. In an attempt to elucidate this relationship, the effect of respiratory inhibitors on respiration and peroxidase activity are compared.     

Peroxidase isozymes of first internodes of sorghum: tissue and intracellular localization and multiple peaks of activity isolated by gel filtration chromatography

Electrophoretic analyses using Sepraphore III strips indicate the presence of a minimum of five bands of peroxidase activity detectable with o-dianisidine and H₂O₂ in extracts from first internodes of Sorghum vulgare var. Wheatland milo. Three of these isozymes were anodic and two were cathodic forms at pH 8.3. The relative amounts of these forms are compared in zero time and incubated excised internodes, stelar and cortical tissues of internodes, and in other parts of the plant. Localization of these isozymes with respect to walls and cytoplasm was characterized by differential centrifugation after grinding of the internodes and by an in situ extraction of walls by centrifugation after vacuum infiltration. Using the latter in situ method, 32% of the total activity of the fast moving cathodic form was exchanged from the wall after infiltration with 50 mm CaCl₂. Only trace amounts of the other isozymes were localized in the walls of the cortex. The isozymes were eluted as two peaks from columns of Sephadex G-100 and three peaks from Agarose A-15m. Although such groupings may be due to asymmetric molecules and ionic interactions as well as to molecular weight differences, they may indicate associations with complexes or membranes of different cytoplasmic constituents.     

Ethylene in relation to protein, peroxidase and polyphenol oxidase activities during rooting in hazelnut cotyledons

During formation of adventitious roots, the effects of 2-chlorethylphosphonic acid (CEPA), 1-aminocyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG) added to a Cheng basal medium, supplemented with indole-3-butyricacid (IBA) and kinetin were determined on peroxidase (PO; EC 1.11.1.7) and poiyphenol oxidase (PPO; EC 1.10.3.1) activities in cotyledon explants of hazelnut ( Corylus avellana L. cv. Casina). CEPA stimulated PO and PPO activities while AVG inhibited. SDS-polyacrylamide gel electrophoresis of preparations from hazelnut cotyledons showed a correlation between proteins and rooting. Ethylene seems to modify total protein content and the activities of PO and PPO. As compared to the control extracts, AVG inhibited the anodic (53, 30.7 and 27 kDa) and cathodic (66.2 and 53.4 kDa) isoperoxidases and anodic (27.5 and 21 kDa) and cathodic (67.5 kDa) isopolyphenol oxidases, whereas CEPA promoted the anodic (30.7, 28.8 and 27 kDa) and cathodic (53.4 and 27.2 kDa) isozymes with PO activity. The increased PO activity during rooting in hazelnut cotyledons could enhance isozymes with lignin biosynthetic activity.     

Photocontrol of chlorogenic acid biosynthesis in potato tuber discs

The appearance of phenylalanine ammonia-lyase activity and the accumulation of chlorogenic acid in potato tuber discs are stimulated by illumination with white light, whereas the appearance of cinnamic acid 4-hydroxylase activity is unaffected by illumination. The photosensitive step in chlorogenic acid biosynthesis may be by-passed by treatment of discs with exogenous supplies of cinnamic acid, whereas treatment of discs with phenylalanine does not isolate the photosensitive step. Therefore, the site of photocontrol of chlorogenic acid biosynthesis in potato tuber discs is the reaction catalysed by phenylalanine ammonia-lyase. Cinnamic acid 4-hydroxylase activity in vitro is unaffected by p-coumaric acid, caffeic acid or chlorogenic acid. Phenylalanine ammonia-lyase activity in vitro is sensitive to inhibition by cinnamic acid. The in vitro properties of the two enzymes are also consistent with the hypothesis that phenylalanine ammonia-lyase rather than cinnamic acid 4-hydroxylase is important in the regulation of chlorogenic acid biosynthesis in potato tuber discs.     

Changes in isoperoxidases induced by hydroxyphenylacetic acids

The effects ofo-, m- andp-hydroxyphenylacetic acids on total peroxidase activity and isoperoxidase patterns of wheat coleoptile sections were studied.o-andm-HOPA reduced the total peroxidase activity whilep-HOPA increased it. Electrophoresis in polyacrylamide gel revealed thato- andm-HOPA diminish the intensity of some anodic and cathodic bands, but thatp-HOPA increases anodic and reduces cathodic isoperoxidases. These results may explain the apparently paradoxical positive effects ofp-HOPA on both growth and total peroxidase activity.     

Studies on polyphenol content, activities and isozymes of polyphenol oxidase and peroxidase during air-curing in three tobacco types

The change in polyphenol content in the primed leaves of burley, flue-cured, and Turkish tobaccos during air-curing was related to the activities and isozymes of polyphenol oxidase and peroxidase. The quantity of chlorogenic acid was rapidly reduced during the first week of curing. The decrease in rutin content during curing was less significant, especially when the concentration of chlorogenic acid was high in leaf tissues. This result was further confirmed by in vitro assays with partially purified tobacco polyphenol oxidase.     

Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 ± 2°C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (−)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.     

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.     

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.     

Hormonal control of isoperoxidases in lentil embryonic axis

Detachment of the cotyledons from the lentil (Lens culinaris Med.) embryonic axis causes in the latter an increase in total peroxidase activity which is shown to be due to enhancement of specific cathodic isoperoxidases. Kinetin treatment of attached or detached axes promotes activity of essentially the same cathodic isoperoxidases. In addition kinetin enhances the activity of two anodic peroxidases and represses specifically that of a cathodic one. Abscisic acid inhibits the production of all isoenzymes in the presence or absence of kinetin. Cytokinin and abscisic acid actions are discussed in relation to the nature of a wounding hormone and the role of natural inhibitors in cotyledons during germination. Indoleacetic acid stimulates the activity of certain isoenzymes which are also stimulated by kinetin, whereas in others the effects of the two hormones are different. Specific inverse effects of indoleacetic acid and kinetin are demonstrated on the two most cathodic isoperoxidases. Indoleacetic acid-kinetin interactions on the cathodic isoperoxidases have been found in the literature and are discussed as a possible mechanism for explaining interactions of the two regulators on growth and other physiological processes.     

The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.     

The isozymic similarity of indoleacetic Acid oxidase to peroxidase in birch and horseradish

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.     

Absorption of polyphenols by polyvinylpyrrolidone and polystyrene resins

Absorption of polyphenols by insoluble polyvinylpyrrolidone (PVP) and by polystyrene resins has been examined. Dowex-1 was the most efficient absorbent of polyphenols extracted from leaves of spinach, bean or tobacco. Dowex-50 and Amberlite XAD-2 were more effective than PVP for the removal of leaf polyphenols from solution. With purified polyphenols, Dowex-1 efficiently absorbed chlorogenic acid, flavonol glycosides and catechins but did not absorb condensed proanthocyanidins. PVP absorbed all classes of polyphenols examined but showed a low affinity for chlorogenic acid.     

Characterization of isoperoxidase activity in Arachis hypogaea cultivars

Isoperoxidases (both anodic and cathodic) from individual seeds of several peanut cultivars (Arachis hypogaea), after ammonium sulfate precipitation of the major storage protein, arachin, from the extracts were characterized by polyacrylamide gel electrophoresis with respect to seed development, maturity and germination and the geographic areas where grown. All cultivars had a major cathodic isozyme near the origin of the gels. Much anodic intra- and inter-varietal isozyme polymorphism was observed in mature seeds collected from different geographic areas. These polymorphic isozymes were consistently present during the earlier stages of seed development (the activities decreased quantitatively and became variable during the later stages of maturation), and most were observed in each peanut upon germination, the latter showing quantitative increases in activity.     

Biosynthesis of cyanidin in buckwheat hypocotyls

Aminooxyacetate (AOA), an inhibitor of phenylalanine transamination and deamination in vitro, inhibits the light-induced formation of chlorogenic acid, leucoanthocyanin, rutin and anthocyanin (cyanidin glycosides) in buckwheat hypocotyls. Anthocyanin production is inhibited 87 ± 4%, when excised hypocotyls are incubated in 0.5 mM AOA in Petri dishes. AOA is also effective when taken up through the roots or sprayed onto seedlings. In the presence of biosynthetic precursors of cyanidin (l-phenylalanine, trans-cinnamic acid, p-coumaric acid, naringenin, eriodictyol, dihydrokaempferol. and dihydroquercetin) the inhibition of anthocyanin formation caused by AOA is completely or partially reversed. The general applicability of a complementation technique involving AOA or a similar inhibitor of phenylpropane synthesis is proposed to investigate the biosynthesis of natural products derived from cinnamic acid.     

Ueber den abbau von flavonolen durch pflanzliche peroxidasen

Peroxidases have been shown to catalyse the degradation of flavonols via 2,3-dihydroxyflavanones to benzoic acids. Incubation of (U-¹⁴C)-kaempferol with pure horseradish peroxidase leads to the same reaction products (2,3,4,5,7,4′-pentahydroxyflavanone, p-hydroxybenzoic acid, ¹⁴CO₂, several polar, water soluble catabolites as given by enzyme preparations from various plant species. Further reactions of flavonols and their glycosides with peroxidases are discussed. All peroxidase isoenzymes of Sinapis alba and Cicer arietinum, obtained by isoelectric focusing, have been shown to degrade flavonols at the same rate. The peroxidase catalysed degradation of polyphenols is discussed in relation to IAA oxidase.     

Root characteristics of grafted peppers and their resistance to <Emphasis Type="Italic">Fusarium solani</Emphasis>

Root rot caused by Fusarium solani, is one of the most severe diseases in pepper (Capsicum annuum L.). Grafting has been attempted as an effective means to control the disease, but little is known about the disease resistance mechanism in grafted pepper. Therefore, we investigated the changes of biomass, cell structure, and the secondary metabolism in roots of control (non-grafted pepper) and grafted peppers using cvs. Weishi and Buyeding as rootstocks and the cv. Xinfeng 2 as a scion. After a manual inoculation, less F. solani invaded grafted pepper roots and consequently less serious injury to the root cell ultra-structure compared with the control was found. The roots of grafted pepper infected with F. solani exhibited greater biomass production and root activity than the roots of infected controls. Grafting led to an increased content of salicylic acid, benzoic acid, vanillin, lignin, and polyamines, as well as activities of phenylalanine ammonia lyase, polyphenoloxidase, and peroxidase. These results suggest that grafting improved the resistance of peppers to root rot.