In a previous study by the authors, two isoenzymes of 6-phosphogluconate dehydrogenase were isolated from cultures of tobacco tissue Nicotiana tabacum W-38 and shown to be similar in their pH optima and MWs and in their affinities toward 6-phosphogluconate or NADP+. In an attempt to clarify the structural relationships between these two isoenzymes, peptide mapping of trypsin digests of the purified isoenzymes was performed. The maps were found to be similar, with at least 29 peptide groups from the trypsin digestion of each isoenzyme being alike. There were, however, definite minor differences in the peptide maps of the two isoenzymes.

Anodic polyacrylamide gel electrophoresis of extracts of cultures of tobacco tissue Nicotiana tabacum W-38 revealed the presence of two 6-phosphogluconate dehydrogenases (6PGD). The slow and the fast anodic migrating zones were designated I and II, respectively. After purification, enzymes from both zones exhibited no major differences in their affinity towards 6-phosphogluconate (6PG) or NADP+, and were found to have approximately the same pH optima and MWs (69 000–72 000). The coumarins scopoletin and esculetin showed some inhibitory effect on each isozyme at 0.4 mM. Below 0.3 mM, however, esculetin stimulated the activity of zone I when lower amounts of 6PG (S0.25) were used. The glucosylated compounds, scopolin and esculin, were much more inhibitory towards the 6PGDs than their respective aglycones. Ferulic, p-coumaric and caffeic acids seemed to have an inhibitory effect dependent on 6PG concentration. A larger inhibition was observed in each case at the lower 6PG levels used. Zone I activity appeared to be inhibited to a greater degree than zone II activity by 0.4 mM p-coumaric acid with low 6PG. Of the phenolic compounds tested, chlorogenic acid was most effective, completely inhibiting the enzyme activity at 0.4 mM. Of the non-phenolic compounds investigated, glucose 1,6-diphosphate inhibited both isoenzymes of 6PGD at lower 6PG concentrations. On the other hand, 2,3-diphosphoglycerate activated both isoenzymes up to 200% of their original activity.

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 m s for guaiacol are 4 and 13.3 mM for A f 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 A 3 from W-38 tobacco tissue culture.

Eight isoperoxidases from tobacco suspension culture WR-132 (termed Cn, C3, C4, Ac, and Af) and tobacco callus culture W-38 (termed A1, A2, and A3) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases Af and A3 are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.

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 (Cn) appeared in the medium within 2 days after the cells were placed in the dark. Cn 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). Cn increased and new anodic isoperoxidases Aa, Ab, Ad and Ae appeared in the media as the cells approached death in darkness.

AbstractIsoperoxidase A3 from tobacco tissue culture, Nicotiana tabacam W‐38, apparently does not have a subunit structure. Its molecular weight is approximately 54,000.The oxidation of scopoletin appears to occur in more than a single step since a blue intermediate is first formed. This is followed by the production of the final yellow product. A plausible explanation for this observation is the coupling of free radicals generated by the primary enzyme catalyzed reaction.

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.

The effect of indoleacetic acid, 2,4-dichlorophenoxyacetic acid, tryptophan, scopoletin, scopolin, and related compounds on the activity of a phenylalanine ammonia-lyase preparation from tobacco tissue WR-132 grown in suspension culture has been investigated. Under the experimental conditions used, IAA and its probable plant precursor trytophan, were found to inhibit the enzyme strongly. Scopoletin showed slightly lesser inhibition. The synthetic auxin, 2,4-D, produced little effect on the enzyme except at the higher concentration used. Scopolin, kinetin, gibberellic acid, and β-indole-3-propionic acid showed no effect. The concentration of β-mercaptoethanol used in each assay was found to affect the values obtained for the percentage inhibition of phenylalanine ammonia-lyase by IAA. The possible physiological significance of these inhibitions is discussed.

Time studies were made on the fate of scopoletin-4-14C taken up through the roots of whole tobacco seedlings. Of the total radioactivity recovered in one set of scopoletin-4-14C continuous feeding experiments, a range of 43–51% (av. 46%) was found to be in the 'soluble' portion of the roots and shoots. At one period (after 4 hr of continual feeding of scopoletin-4-14C), about 91% of the radioactivity in the 'soluble' fraction was found to be present as scopoletin-7-glucoside (scopolin); less than 3% as scopoletin; and approximately 5% as fabiatrin. The percentage of the total recovered radioactivity contributed by the 'soluble' compounds decreased with time, whereas the percentage of the radioactivity due to the 'insolubles' increased with time. The results also suggest that some of the radioactive scopolin (from scopoletin-4-14C not converted at an early stage to 'insoluble' material) may be converted with more time to other 'soluble' compounds and possibly also to some 'insoluble' material. In the roots, fabiatrin was formed slowly from scopolin. The half-life of scopolin was found to be 16 days. Fabiatrin had a slower turnover rate than did scopolin. No measurable amount of fabiatrin was found in the shoots. When a lower starting concentration of scopoletin-4-14C was fed to the seedling in continuous feeding experiments, a larger percentage of the radioactivity appeared in the 'insoluble' fraction of tobacco roots. A hypothesis based on compartmentation of metabolic and vacuolar pools is proposed.

The pH optimum for glucose-6-phosphate dehydrogenase from tobacco tissue culture was determined to be between 8 and 9. The plot of the reaction velocity vs. glucose-6-phosphate concentration showed a non-sigmoidal, hyperbolic saturation curve. A similar curve was obtained for the NADP + saturation function. Compounds that may be related to glucose-6-phosphate metabolism were tested for their effects on the enzyme. It was found that the phenolic compounds, scopoletin, scopolin, esculin and ferulic acid, inhibited the enzyme. Similar inhibitions by scopolin and scopoletin were observed for three other NADPH producing dehydrogenases from the same plant cells; namely, 6-phosphogluconate dehydrogenase, NADP + specific isocitrate dehydrogenase and NADP + specific malate dehydrogenase.

Through the use of one-dimensional paper chromatographic separation, followed by spectro-photometric analysis, chlorogenic acid, neochlorogenic acid and 4-O-caffeoylquinic acid concentrations have been determined in sections of field-grown Helianthus annuus L. Using similar procedures, relative concentrations of “isochlorogenic acid” were also determined. All these compounds decreased in concentration basipetally in stem and leaf sections except in leaves from the first six nodes where the monocaffeoylquinic acids increased in concentration down from the apex. These concentration changes are see to correlate with the postulated role of caffeoylquinic acids in lignification and in influencing internal regulatory mechanisms.

AbstractA significant depression of callus growth resulted from low concentrations of abscisic acid (ABA) added to the medium recommended by Linsmaier and Skoog. Low concentrations also decreased the chlorogenic acid and lignin content of the callus, and generally decreased amounts of scopolin and scopoletin in the tissue. Gibberellic acid (GA3) stimulated callus growth in a low concentration (0.1 mg/1) and inhibited growth at a high concentration (10.0 mg/1). Both levels of GA3 increased scopoletin accumulation in tobacco callus. A high concentration of GA3 increased the accumulation of scopolin and chlorogenic acids, whereas a low concentration decreased the amounts of these two phenolic compounds. In comparison with the control, lignin synthesis was stimulated by a low GA3 concentration, but a high GA3 concentration did not have a significant effect. Both low and high concentrations of GA3 overcame ABA inhibition of growth and lignin synthesis, and partially reversed ABA inhibition of scopoletin production. However, GA3 did not reverse the inhibitory effect of ABA on scopolin production. The low concentration of GA3 overcame the inhibition of chlorogenic acid production resulting from a 0.01 mg/1 concentration of ABA, but this was the only reversal of chlorogenic acid inhibition resulting from addition of GA3 to the medium.

AbstractThrough the use of paper chromatographic separation, and fluorometric and spectrophotometric analysis, three caffeoylquinic acid isomers and scopolin have been quantilaed in tobacco as a function of age and chilling temperature. Coldchilled plants exhibited nitrate deficiency symptoms and 4‐5 fold increases over control plants in chlorogenic acid (CGA) concentration in all plant sections except the roots. Varying lesser increases in neochlorogenic acid, “Band 510”, and scopolin concentration were also observed in the above ground sections of chilled plants. Roots of cold‐chilled plants contained lesser concentrations of CGA and scopolin than control plants after 15 days of treatment. Leaves from the same control plant were found to decrease in CGA, Band 510 and scopolin concentration with age, while neochlorogenic acid concentrations changed little. In stem sections CGA concentration also decreased basipetally while the concentrations of scopolin increased. Similar leaf age studies done with cold‐chilled plants revealed phenolic concentration changes comparable to those of control plants, even though their concentrations were appreciably higher.

Eighty-day-old tobacco plants were irradiated with 1000R, 2500R and 4400R of X-radiation, harvested 12, 21 and 29 days after treatment, and analyzed for chlorogenic acid, neochlorogenic acid, 4-0-caffeoylquinic acid (“band 510”) and scopolin. Dose dependent, post-irradiation accumulations of scopolin were observed in both young and old leaves. In roots and stems, scopolin concentrations first increased to 1·5–10 times that of comparable controls, and then declined in later harvests to levels below that of controls. Accumulations of caffeoylquinic acids were variable. Young leaves formed after treatment with 1000 R were twisted, malformed and contained lower caffeoylquinic acid concentrations on a fresh weight basis. Alternative speculations are discussed which could account for scopolin accumulations and the implications thereof.

Roots, stems, and leaves of tobacco plants grown on complete and on nitrogen-deficient nutrient solutions were each analyzed quantitatively for content of scopolin and of chlorogenic, neochlorogenic, and 4-O-caffeoylquinic acids. Increases in scopolin and chlorogenic acid concentrations were found in the nitrogen-deficient leaves, stems, and roots as compared to corresponding parts of control plants. These increases correlated approximately with the time of first observable deficiency symptoms.

Using a simple but reliable method involving one-dimensional paper chromatography for the quantitative determination of chlorogenic acid, neochlorogenic acid, 4-O-caffeoylquinic acid (“band 510”), and scopolin, the effect of varying u.v. light intensities on the concentration of these compounds in tobacco and sunflowers has been determined. With increased u.v. intensity, an increase in scopolin concentration was found in tobacco leaves and stems and in sunflower leaves. Chlorogenic acid concentration was higher in roots, stems, and leaves of tobacco plants treated with low, stimulatory u.v. radiation than in comparable plants grown under control or under higher u.v. conditions. Similar experiments with sunflowers indicated that the chlorogenic acid concentration of sunflower leaves was less under the low u.v. radiation than in comparable sunflower plants of control or of higher u.v. treatments. In stems and roots of sunflower, no consistent pattern of change in the chlorogenic acid concentration was found under the u.v. conditions tested. The concentration of chlorogenic acid increased in sunflower older leaves, but no consistent pattern of chlorogenic acid change with age was observed in sunflower stems and roots. Neochlorogenic acid and the third isomer (band 510) were found in both tobacco and sunflower, but in much higher concentrations in tobacco. Both of these isomers were found in all parts of the two test plants, except tobacco stems or roots. Postulates are presented which point out the possible significance of chlorogenic acid and/or scopolin in lignification and in mediating environmental conditions through effects on internal regulatory mechanisms. Internal concentrations of these compounds possibly are correlated with their release from the plant, and subsequently with allelopathic relations among plants.