Polyphenol Oxidation by Vicia faba Chloroplast Membranes

Abstract

The mechanism whereby light effects polyphenol oxidation was examined with Viia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3',4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6 idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H20 as electron donor for the photoreduction of NADP, (b) NADP replaced 02 as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chioroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncycic electron transport chain on the oxidizing side of photosystem II and to react with 02 at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks. The physiological mechanism for the activation of latent polyphenol oxidase remains an unanswered question. Present results suggest that activation could occur through either acidification or the release of free fatty acids. It is well-established that chloroplasts isolated from certain plants contain a latent membrane-bound PPO3 that can be solu-bilized and activated by detergents such as SDS (1 1, 12, 18, 22). Chloroplast membranes from such plants can also oxidize poly-phenols photochemically in the absence of SDS and the function of light has been ascribed to an activation of the latent PPO ' 3Abbreviations: PPO, polyphenol oxidase; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DOPA, L-dihydroxyphenylalanine; DPC, diphenyl carbamate. enzyme (24). No mechanism has been proposed to account for this type of activation. To satisfy our interest in the role of light in enzyme regulation, we investigated the mechanism whereby light effects polyphenol oxidation by chloroplast membranes of this type. We now report evidence that the light-dependent oxidation of polyphenols by such chloroplast membranes (isolated in this case from Viciafaba) is achieved independently of the latent membrane-bound PPO. The evidence indicates that the electrons released in the photo-chemical oxidation of polyphenols enter the oxidizing side of PSII in a manner analogous to known PSII donors and are transferred via noncyclic electron transport to 02 at a site on the system I side of the DCMU and DBMIB blocks. Photochemical polyphenol oxidation by chloroplast membranes isolated from plants with an active PPO (for example, spinach) seems to occur via this same route. The mode of activation of the latent PPO enzyme in vivo in plants such as V faba thus seemingly is independent of light. MATERIALS AND METHODS Plant Material. V faba (Broad Windsor horsebean, Northrup King and Co., Fresno CA) was greenhouse-grown in University of California mix under natural environmental conditions (2). Reagents. Biochemicals were purchased from Sigma Chemical Co. All other reagents were purchased from commercial sources and were of the highest quality available. Ferredoxin and ferre-doxin-NADP reductase (a gift from R. Chain) were isolated as described by Buchanan and Arnon (4) and Shin et al. (21), respectively. Preparation of Chloroplast Fragments and Chloroplast Extract. Chloroplasts were isolated from freshly harvested chilled leaves in a preparative solution containing 0.35 M sucrose and 25 mm Tris-HCI buffer (pH 7.9). Fifty g of leaflets were blended and the slurry was filtered and centrifuged as described by Kalberer et al. (10). The sedimented intact chloroplasts were osmotically broken by suspension in a solution containing 50 mm Tris-HCl buffer (pH 7.9) and 1 mM MgCl2 (henceforth called buffer B) and the chlo-roplast membrane fragments were collected by centrifugation for 5 min at 27,000g. For obtaining the soluble enzyme fraction, the Chl concentration of the resuspended intact chloroplasts was adjusted to 1.0 mg/ml (1) before centrifugation and the resultant supernatant fraction (chloroplast extract) was saved. For obtaining chloroplast membrane fragments, the pellet from the centrifuga-tion step was washed by suspension in 40 ml buffer B and collected by centrifugation as above. The pelleted membranes were resus-pended in buffer B to a Chl concentration of I mg/ml. For aging, the washed chloroplast membranes were stored overnight at 6 C. Assay Methods. PPO was assayed either spectrophotometrically or polarographically with DOPA as substrate. In the spectropho-1150