Correlation between Chlorophyll and Chlorogenic Acid Content in Tobacco Leaves

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-¹⁴C 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.     

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.     

Altered levels of antioxidant enzymes associated with two mutations in tomato

Activity of peroxidase, superoxide dismutase and catalase were examined in leaves, stems and roots of olivacea ( oli ) and monstrosa ( mon ) mutants of Lycopersicon esculentum Mill. The extent of the difference between the pattern of oxidative enzyme activities of the wild type (wt) and the mutants was determined. The high peroxidase activity during the developmental stages of the leaves and stems of oli and mon phenotypes is associated with high levels of 4 anodic peroxidases in leaves and of two isozymes in the stem. Leaves of oli exhibit higher activity of the cathodic peroxidase C2, while both mutations have a marked increase of peroxidase C1 in stems. A positive relation between high peroxidase activity and oxidative stress damage was found: in chilling experiments at 5°C, peroxidase level in mutants and wt leaves was negatively correlated with electrolyte leakage. Superoxide dismutase (SOD) activity rises in oli stems around flowering time due to the high activity of the chloroplast forms SOD-1 and SOD-2. Catalases (CAT) were detectable only in early stages of plant development; CAT-2 was nearly absent in wild type tissues but well represented in mon and oli. The oli and mon mutations may affect critical steps of a regulatory pathway controlling various classes of oxidative enzymes in tomato.     

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.     

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.     

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.     

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.     

山银花不同发育阶段花结构与绿原酸含量变化关系研究

利用HPLC测定了山银花不同发育阶段花中绿原酸的含量变化,同时采用荧光显微镜和石蜡制片法观察了花器官中绿原酸的分布部位及其在花发育过程中的结构变化,以探讨绿原酸含量变化与花结构间的相互关系。结果显示:绿原酸主要分布于萼筒和花冠筒的内、外表皮细胞及外表皮下的几层细胞中;在花发育过程中,当花蕾外观上呈绿色时含绿原酸细胞处于分化之中,而呈白色时则分化成熟,至花开放呈白色和黄色时则趋于衰亡;花中绿原酸含量在细胞分化早期较高,在细胞分化接近成熟时则开始急剧减少,至细胞趋于衰老时,绿原酸含量最低。这些结果说明,花中绿原酸含量的变化与花的发育过程密切相关;推测可能是花发育过程中的叶绿体变化直接影响了淀粉合成,进而对绿原酸含量积累产生了影响;本研靠结果为制定金银花药材的合理采收期标准提供了理论依据。     

Characterization of Muskmelon Fruit Peroxidases at Different Developmental Stages

An increase in exocarp peroxidase activity was observed in fruit at 5 to 30 days post pollination (DPP), and decreased at 40 and 50 DPP. Total peroxidase activity of the mesocarp was significantly lower than the exocarp in all developmental stages. Mesocarp peroxidase activity decreased consecutively from outer, to middle and, to inner tissue at every developmental stage. Total activity in the mesocarp peaked at 20 DPP. Native-PAGE of exocarp tissue showed at least two cathodic (basic) peroxidases and two anionic (acidic) peroxidases. The number of isozymes was greatest and bands most intense at 30 DPP. IEF-PAGE of the 5 to 50 DPP fruit exocarp showed at least 8 peroxidase isozymes (pI 4.6 to 9.6). Anion exchange chromatography showed only one peak of anionic peroxidase activity that was not evident until 15 DPP. This peak was greatest at 30 DPP and declined at 40 and 50 DPP. Cationic peroxidase isozymes appeared to be the predominant and most intense isoforms throughout fruit development. The changes in peroxidase activity corresponded to fruit formation and may be associated with susceptibility to fruit rot.     

Phenylalanine Ammonia-Lyase in Sliced Sweet Potato Roots

A marked rise in the phenylalanine ammonia-lyase activity and the polyphenol synthesis was observed in sliced roots of a sweet potato. The enzyme activity was found to be localized in the root tissue adjacent to the sliced surface. In this region, the synthesis of polyphenols was much higher compared to the inner tissues. When the specific inhibitors for the protein and nucleic acid biosynthesis such as an actinomycin D and blasticidin S were added to the tissues by vacuum infiltration technique, both the development of phenylalanine ammonia-lyase activity and the synthesis of polyphenols were severely prevented. These results suggest the important role of phenylalanine ammonia-lyase in the polyphenol synthesis and de novo synthesis of the enzyme protein molecule in the sliced tissues.     

Ethylene Production,Peroxidase Activity and the Change of Peroxidase Isozyme during the Ripening of Tomato Fruits

Climacteric rise, ethylene production, peroxidase activity and its isozyme and their interrelationships during the ripening of tomato fruits have been studied. It was found' that there was parallelism between ethylene production and climacteric rise. The climacteric rise of tomato fruits was hastened by ethylene applied at the mature green stage. The ethylene production was inhibited by low oxygen and high carbon dioxide partial pressure. The peroxidase activity in the tomato fruits appeared to be different at three stages, higher in the half red fruits and lower in both green mature and fully red fruits. This activity was increased by ethylene and decreased by lower partial pres- sure of oxygen. The peroxidase isozymes sppeared also different at different stages of ripening. There were 4 bands in young fruits, 3 in green mature fruits, 5 in half red fruits and 3 in fully red fruits. After the application of ethylene to the tomato fruits, there appear one new band of peroxidase isozyme.     

Effects of competitive inhibitors of phenylalanine ammonia-lyase on the levels of phenylpropanoid enzymes in Solatium tuberosum

Abstract The accumulation of chlorogenic acid in illuminated discs of Solanum tuberosum tuber tissue is accompanied by rapid but transient increases in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase and hydroxycinnamoyl-CoA : quinate hydroxycinna-moyl transferase. Exogenous D-phenylalanine and L-α-aminooxy-β-phenylpropionic acid, competitive inhibitors of phenylalanine ammonia-lyase, inhibit the accumulation of chlorogenic acid and presumably reduce the endogenous pools of pathway intermediates such as cinnamic acid. These treatments prolong the phase of increase in phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activities and indicate that product feedback modulation is important in maintaining the interrelationship between the levels of these two enzymes during the later stages of induction. In contrast,L-α-aminooxy-β-phenylpropionic acid inhibits the development of hydroxycinnamoyl transferase in illuminated discs supporting the idea that the light-stimulated increase in phenylalanine ammonia-lyase activity causes an increase in cinnamic acid production which mediates the light-stimulated increase in hydroxycinnamoyl transferase activity.     

Pyruvate kinase isozymes in man

Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.     

Function of phenolic substances in the degradation system of indole-3-acetic acid in strawberries

The homogenate of different strawberry organs inhibits the degradation of IAA in the presence of horse radish peroxidase, while intact strawberry tissues are able to degrade IAA. The chemical nature of peroxidase inhibitors present, in strawberry tissues was in vestigated. Using paper chromatography the following polyphenolic substances inhibiting peroxidase activity were identified: chlorogenic, caffeic, ellagic, gentisic, gallic, and vanillic acids, quercetin and pelarginidin. Monophenolic compounds, also present in strawberry, such as p-hydroxy-phenyloacetic acid and p-hydroxybenzoic acid, are strong stimulators of IAA oxidase. Abscisic acid in very high concentration (1×10^?4M) enhances degradation of IAA by peroxidase. When both poly-and monophenolic compounds at equimolar concentrations are present in the system, only the inhibition of IAA degradation occurs. Tissue explants from the strawberry leaves and petiole degrade less IAA if they are previously forced to synthetize more polyphenols under illumination. Although the difference in IAA-decarboxylation activity between the illumination and dark treated explants was relatively small, nevertheless it was consistent and appears to be very important from a physiological point of view suggesting that there exists a regulatory relationin vivo between IAA degradation and the presence of phenolsin plant tissue. Electron microscope data revealed that phenolic substances are specially isolated from cytoplasm of the receptacle cells.     

Isoperoxidase Activity and Induction in Cultured Tissues of Wild Carrot: a Comparison of Proembryos and Embryos

Several anodic isoperoxidases were found in embryonic tissues of cultured wild carrot, Daucus carota L., which were not present in the proembryo masses from which they originate. This difference is further reflected in the higher specific activity of peroxidase in embryo extracts as compared to proembryonic tissues. The absence of anodic isoperoxidases and depressed peroxidase activity in carrot tissue cultures in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) suggests a regulatory role for this plant growth regulator in controlling peroxidase activity.     

Effect of gamma irradiation on peroxidase isoenzymes during suberization of wounded potato tubers

A comparison of peroxidase isoenzymes in skin, cortex and pith tissues of the potato tuber by thin-layer isoelectric focusing in Sephadex revealed major differences in the isoenzyme patterns. Wounding induced several-fold increases in the peroxidase activity which were correlated with the increased amounts of specific isoenzymes. The anodic and cathodic forms with high activity, normally present in large amounts in skin, were found to be preferentially synthesized in suberizing tissues, suggesting a functional role for peroxidase in the suberization process. Cycloheximide treatment prevented the rapid increase in the content and activity of these specific isoenzymes, which indicated that the increase in peroxidase is due to a de novo synthesis of the enzyme. Suberization is not inhibited by gamma irradiation at sprout-inhibiting dose levels.     

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.