Regulated Chlorophyll Degradation in Spinach Leaves during Storage

Degradation of chlorophyll in spinach (Spinacia olearacea L. cv. Hybrid 612) appeared to be regulated through the peroxidase-hydrogen peroxide pathway, which opens the porphyrin ring, thus resulting in a colorless compound. This conclusion was arrived at from the analysis of chlorophylls (Chls) and their metabolizes by HPLC and of enzyme activities catalyzing the degradative reactions. Chls decreased at 25C but not at 1C. The chlorophyll oxidase pathway was not active, as noted by the lack of accumulation of a reaction product named Chl a-1. Lipid peroxidation increased with storage, but the products of the reaction. did not degrade chlorophyll, as noted by the lack of increase in Chl a-1. Chlorophyllase activity increased, but chlorophyllide, the expected product of the reaction, changed minimally during senescence. Ethylene at 10 ppm did not alter the pathway that degraded chlorophyll in spinach. One of the symptoms of senescence in harvested leafy vegetables is loss of greenness with the degradation of chlorophyll (Chl). Quantitative changes in Chl and degradation products of Chl and/or the hydrolyzing enzymes have been monitored in vegetables, but the mechanism or pathways of degradation are not clear and appear to differ among commodities. Chlorophyllase activity, which catalyzes the release of the phytol chain from Chl to form chlorophyllide (Chd), has been reported to increase with yellowing in barley and oat leaves (Rodriguez et al., 1987; Sabater and Rodriguez, 1978), but in tobacco and radish leaves the activity decreases with senescence (Phillips et al., 1969; Shimizu and Tamaki, 1963). In ethylenetreated citrus fruit, chlorophyllase activity increased with concomitant decrease in Chl content (Amir-Shapira et al., 1987; Shimokawa et al., 1978). However, in regreening of Valencia oranges, chlorophyllase activity increased with the enhancement of-Chl, and the authors reported that the enzyme was involved in the biosynthesis of Chl (Aljuburi et al., 1979). Thus, chlorophyllase is involved in both biosynthetic and degradative reactions of Chl, but not necessarily in all commodities. Chlorophyll oxidase, which requires linoleic acid to catalyze the reaction, is located in the thylakoid membranes of the chloroplast. It is operative in the oxidation of Chl to a metabolize identified as Chl a-1, which is suspected to be an intermediate in chlorophyll degradation rather than a final product (Luthy et al., 1984; Schoch et al., 1984). Chloroplast lipids, such as monogalactosyldiglyceride, digalactosyldiglyceride, and phosphatidylglycerol, decreased markedly in senescing spinach leaves (Yamauchi et al., 1986, 1987), and the decrease was probably caused by lipolytic acyl hydrolase in the chloroplast, since its activity increased with senescence. The free fatty acids released by the hydrolase could be for publication 4 Jan. 1990.  W of the series “Mechanism ophyll Degradation in Harvested Leafy Vegetables”. We gratefully dge Willard Douglas for his kind and technical assistance. Use of a or product name by the USDA dots not imply approval or recomn of the product to the exclusion of others that may also be suitcost of publishing this paper was defrayed in part by the payment of rges. Under postal regulations, this paper therefore must be hereby dvertisement solely to indicate this fact. ddress: Himeji College of Hyogo, Shinzaike-honcho, Himeji, Hyogo n. reprint requests should be addressed. oxidized by lipoxygenase and form hydroperoxides. Free radicals, as one of the end products of unstable hydroperoxides, have the potential of degrading Chl. In parsley, Chl is degraded by a peroxidase-hydrogen peroxide system containing apigenin, a major flavone required to catalyze this reaction (Yamauchi and Minamide, 1985). We report here research on analyses of Chls and their degradation products and activities of enzymes known to catalyze the reactions that degrade Chl in spinach. We also determined if ethylene altered the degradation pathway.  ppm ethylene or at lC without ethylene. Air with or without ethylene was metered at a sufficient rate to keep the CO2 level below 0.5%. Leaves (≈100 g) were removed at scheduled intervals during the 6 days and blades without midribs were used for the analyses. The study was repeated with spinach obtained at a later harvest date. Pigments were extracted by grinding 2.5 g leaves in 20 ml cold acetone and 2.5 ml distilled water with a mortar and pestle, filtering the homogenate, rewashing the residue with 80% cold acetone until the residue was colorless, and bringing the final volume to 50 ml in low light. Aliquots of the combined extracts (which were kept in darkness) were used for photometric analysis or passed through a millipore filter (0.22-μm pores) for HPLC analysis. The apparatus for the HPLC consisted of Waters Model 6000 pumps (Waters, Milford, Mass.) with automated gradient controller and Model 712 WISP interfaced into a Hewlett-Packard 1040A rapid-scanning UV/visibIe photodiode array detector (Hewlett-Packard, Rockville, Md.) or Perkin-Elmer LC 95 spectrophotometer (Perkin-Elmer, Rockville, Md.). The absorption spectra of the pigments were recorded between 200 and 600 nm at the rate of 12 spectra/rein. Pigments were separated by a Beckman C18 ultrasphere column (Beckman, Columbia, Md.), 4.6 x 250 mm, using two solvents: A, 80 methanol : 20 water, and B, ethyl acetate in a gradient. Solvent B was added to A at a linear rate until a 50:50 mixture was attained at the end of 20 min. The 50:50 mixture then was used isocratically for an additional 25 rein, as described by Eskin and  Chromatogram of pigments extracted from spinach leaves and analyzed by HPLC with a photodiode array detector. Chd = chlorophyllide, Pb = pheophorbide, Chl = chlorophyll, Po = pheophytin. Harris (1981). Flow rate was 1 ml·min -1 and injection volume was 50 μl. Data from the photodiode array detector were stored and processed by means of a Hewlett-Packard 9000/series 300 computing system. Individual pigments were purified from the acetone extract for use as standards. Chls a and b were purified by adding 3 ml 1,4dioxane and 3 ml distilled water to 20 ml of the 80% acetone extract, allowing the mixture to stand until a precipitate formed, centrifuging the mixture at 12,000× g for 10 min, and dissolving the pellet in ?5 ml of acetone (Yoshiura and Iriyama, 1979). Part of this acetone mixture was used to prepare Chds a and b by treating it with chlorophyllase that was extracted from the acetone powder of spinach leaves, as described below. Pheophorbides a and b were prepared by adding one drop of 2 N HCl to the Chd solution. Another part of the acetone mixture was used to prepare pheophytins a and b by adding one drop of 2 N HCl. The individual pigments were separated by TLC using silica gel base with 60 nhexane :20 acetone :5 t-butyl alcohol solvent, scraping the base from the plate, and extracting it with 80% acetone before analysis. Carotenoids were extracted from spinach leaves with cold 80% acetone and also separated by the same TLC method as for Chl pigments. Each carotenoid was identified by its absorption spectrum in ethyl alcohol (Davies, 1976). For the crude enzyme extract, an acetone powder was prepared by macerating 5 g frozen spinach leaves ( – 80C) with 50 ml cold acetone ( – 18C) using a cold mortar and pestle, filtering the macerate with a Buchner funnel, washing the macerate with a small volume of cold acetone, remacerating the residue, refiltering, rewashing with a small volume of cold diethyl ether, and vacuum-drying the residue. Enzymes were extracted by suspending the acetone powder in 20 ml of 50 mM phosphate buffer (pH 7) containing 0.6% 3-[(3-Chlamido-propyl) dimothylammonio]-1-propanesulfonate (CHAPS) (a zwitterionic detergent) for 1 h at 4C, filtering the mixture through Miracloth, and centrifuging the filtrate at 24,000× g for 20 min. The crude extract was desalted by passing a 4-ml aliquot of the supenatant through  G-25 in a 1.6 × 18cm column. Peroxidase activity was determined as described by Yamauchi et al. (1985). Chlorophyllase activity was determined by a modification of the method by Shimokawa et al. (1978). The reaction mixture consisted of 1 ml crude enzyme extract, 1 ml acetone, and 100 kg chlorophyll in 10 mM phosphate buffer (pH 7) for a total volume of 2 ml. The reaction was carried out at 25C for 45 min and was stopped by adding 5 ml of a 3 acetone : 7 hexane mixture. Activity was based on a decrease in absorbance by chlorophyll at 663 nm. One unit of peroxidase or chlorophyllase activity was defined as a change of 0.01 absorbance unit per minute or hour, respectively. Total Chl content was determined by the method of Amen (1949), that of protein by the method of Lowry et al. (1951). Thiobarbeturic acid (TBA) assay, as described by Stewart and Bewley (1980), was used to determine the concentration of malondialdehyde (MDA), an indicator of the degree of lipid peroxidation.