Studies on Polyphenol Content, Activities and Isozymes of Polyphenol Oxidase and Peroxidase During Air-Curing in Three Tobacco Types

Abstract

Abstract. 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.The polyphenol oxidase activity did not differ at any stage of curing in the 3 tobaccos. When the activity was measured by the oxidation of 3,4-dihydroxyphenylalanine it rose rapidly during the first day of curing and then decreased sharply so that in the fully cured leaf only 15 %/c activity remained. The increase in activity was not observed when chlorogenic acid was used as the substrate. A similar level of peroxidase activity was found in the 3 tobaccos before curing. Peroxidase activities increased rapidly during the first 24 hr of curing. declined thereafter, and remained highest in the flue-cured tobacco, less in the Turkish line, and least in the burley at the end of curing process.By polyacrylamide gel block electrophoresis, 10 peroxidase isozyme bands, 2 cationic and 8 anionic, appeared identical in all 3 tobaccos. When catechol replaced benzidine-2 HCI as the electron donor, 1 cationic and 2 anionic peroxidase isozvmes did not form. Of interest is that the same 10 peroxidase isozyme bands also exhibited polyphenol oxidese activities when treated with 3,4-dihydroxyphenylalanine or chlorogenic acid.

Ontogeny and Hormonal Control of Polyphenoloxidase Isozymes in Tobacco Pith

“…This contrasts with the data of Sheen (14,15), who reported that the zymograms were identical with either CA or DOPA as substrate and that the PPO patterns in a variety of tobacco tissues, including pith, were similar to those of peroxidase. His pith tissues also differed in containing as constitutive enzymes major activities of cathodic as well as anodic forms.…”

“…Both activities may involve isozymes and both are controlled, although in opposite ways, by IAA (1, 4, 10, 11). Re-cently, the pattern of isozymes of PPO' in tobacco tissues has been reported to be identical with or similar to that of peroxidase (14,15). In the present work, the possibility of a complex of enzymes catalyzing the oxidation of phenolic compounds and the hormonal control of the newly appearing isozymes has been explored in cultures of tobacco pith.…”

“…There appears to be no simple correlation in tobacco between phenolic content and the PPO activity (2,15). Chlorogenic acid is found in pith tissue (14,19) and could be considered a physiological substrate, but neither DOPA nor catechol has been identified in tobacco tissues, and tyrosine is apparently not hydroxylated to DOPA (3).…”

Alterations in biochemical components in mesta plants infected with yellow vein mosaic disease

“…In the enzyme assays, H 2 O 2 was used as the substrate for CAT (assay pH 7.0), indophenyl acetate for EST (assay pH 5.5), p-nitrophenyl phosphate for ACP (assay pH 5.2), orthodianisidine for POD (assay pH 6.0), o-catechol for PPO (assay pH 6.8), and diethylenetriamine pentaacetic acid, nitroblue tetrazolium and xanthine for SOD (pH 7.8). The isoenzyme profiles of CAT (Woodbury et al, 1971), EST (Brewbaker et al, 1968), ACP (Murray and Collier, 1977), POD (Sheen and Calvert, 1969), PPO (De Ascensao and Dubery, 2000) and SOD (Chen and Pan, 1996) were examined by native PAGE. Each experiment was replicated five times.…”

Detergent Extraction of Enzymes from Tobacco Leaves Varying in Maturity

“…However, Weston (37) reported that peroxidase and polyphenoloxidase activities fluctuate similarly during growth and senescence of tobacco leaves. The discrepancy resulting from similar banding patterns for peroxidase and polyphenoloxidase isozymes after electrophoresis of tobacco leaf extracts (28) has since been resolved by Van Loon (34) using improved staining techniques. Changes in activity and isozyme composition of peroxidase (4) and of polyphenol oxidase (31) are subject to hormonal control.…”