Hydrogen Peroxide-Dependent Oxidation of Flavonols by Intact Spinach Chloroplasts

Abstract

ABSTRACIExternally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H202. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H202, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN3. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H202. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.Flavonols are widely distributed pigments in vascular plants. Their subcellular localization has been investigated and they were found in chloroplasts (12,(14)(15)(16) MATERIALS AND METHODSIntact chloroplasts were isolated from spinach leaves obtained from a local market. Depetiolated spinach leaves were homogenized by a Waring Blendor in a medium which contained 0.5 mM KH2PO4, 1 mM MnCl2, 1 mM MgC12, 2 mm EDTA, 10 mM NaCl, 330 mM sorbitol, and 25 mM Mes-NaOH (pH 6.3). The homogenate was filtered through eight layers of gauze. The filtrate was centrifuged at 3,000 rpm for 1 min. The sediment was suspended into the above buffer solution, centrifuged at 3,000 rpm for 45 s, and suspended again into the same buffer solution. To separate intact chloroplasts from broken chloroplasts, the obtained chloroplast fraction was centrifuged in a Percoll density gradient. Percoll and the above buffer solution were mixed to obtain Percoll density gradient. At the bottom of the centrifugation tube, 2 ml of 40% Percoll (v/v) was added. Two ml of 30% and 20% Percoll were layered successively onto the 40% Percoll. Chloroplast suspension was layered onto 20% Percoll. After centrifugation at 4,000 rpm for 10 min, chloroplasts were separated into two bands. Chloroplasts between 20% and 30% Percoll were broken chloroplasts and chloroplasts between 30% and 40% Percoll were intact chloroplasts. The lower band was collected by a syringe and used for experiments. The intactness of the chloroplasts at the lower band was more than 80% by a ferricyanide test (6). Thylakoids were obtained from intact chloroplasts; hypotonically ruptured intact chloroplasts in the buffer solution without sorbitol were collected by centrifugation at 10,000g for 10 min, and suspended into the above buffer solution to use in experiments.Rates of flavonol oxidation were followed at 380 nm with a Hitachi 557 spectrophotometer at 25C. The basic reaction mixture (1 ml) contained chloroplasts equivalent to 5 to 9 pg of Chl, 0.5 mM KH2PO4, 1 mM MgC92, 10 mm NaCl, and 50 mM HepesNa...

Damage and protection of the photosynthetic apparatus from UV-B radiation. II. Effect of quercetin at different pH

“…On the other hand, several reports have suggested that the surface localization of flavonoids improves their antioxidant potential by blocking the interaction of free radicals with the membrane lipids, efficiently preventing peroxidation (Moliveanu et al, 2000;Erlejman et al, 2004;Oteiza et al, 2005). In addition, the investigations with intact chloroplasts and thylakoids showed that the exogenously added quercetin delays lipid peroxidation (Takahama, 1983(Takahama, , 1984Chanhan et al, 1992), suppresses carotenoid and chlorophyll photobleaching and prevents D1 protein degradation under abiotic stress (Singh and Singhal, 1999). As can be seen in Fig.…”

Reduction of Phenoxyl Radicals Mediated by Monodehydroascorbate Reductase

“…The reaction was initiated by the addition of glucose oxidase (1 g ml Ϫ1 ) which allowed the generation of H 2 O 2 at 0.37 M s Ϫ1 . The oxidation of phenols was followed spectrophotometrically using a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan) by monitoring a decrease in absorbances at 380, 310 and 260 nm for quercetin, ferulic acid (FA) and coniferyl alcohol (CA), respectively, using absorption coefficients of 8.7 (quercetin), 11.3 (FA) and 7.5 (CA) mM Ϫ1 cm Ϫ1 (36,37). At the same time, the oxidation rate of NADH was determined by following a decrease in the absorbance at 340 nm using an absorption coefficient of 6.2 mM Ϫ1 cm Ϫ1 (5).…”

Photosynthesis and photoprotection in mangroves under field conditions

“…This transfers the problem of scavenging from 02^ to H2O2, implying that none of the mechanisms is sufficient, and creating a need for alternative or additional peroxidase capacities. While no such models have been proposed using any species, peroxide-dependent oxidation of phenolics has been studied in spinach, both in chloroplasts and in the apoplast of leaves (Takahama 1984;Takahama & Oniki 1992), and R. stylosa leaves contain up to 20% by dry weight of such compounds, particularly under stressed conditions (Robertson 1988;Lovelock, Clough & Woodrow 1992).…”