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Chlorophyll Catabolism in Senescing Plant Tissues

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Chlorophyll Catabolism in Senescing Plant Tissues Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1901-1905, April 1987 Botany Chlorophyll catabolism in senescing plant tissues: In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves (chlorophyliide/pheophorbide/pheophytin/ethylene/chlorophyliase) DEKEL AMIR-SHAPIRA, ELIEZER E. GOLDSCHMIDT*, AND ARIE ALTMAN Department of Horticulture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel Communicated by Kenneth V. Thimann, November 3, 1986 ABSTRACT High-pressure liquid chromatography was Reports on "changed chlorophylls" (11, 12) were not con- used to separate chlorophyll derivatives in acetone extracts firmed by more recent studies. from senescing Citrus fruit peel, autumnal Melia azedarach L. Renewed interest in the area of chlorophyll catabolism in leaves, and dark-held detached parsley (Petroselinum sativum recent years has produced developments along several lines L.) leaves. Chlorophyllide a and another polar, dephytylated of investigation. derivative accumulated in large amounts in senescing Citrus (i) The chlorophyllase enzyme has been thoroughly puri- peel, particularly in fruit treated with ethylene. Ethylene also fied and characterized (13, 14), and its localization in induced a 4-fold increase in the specific activity of Citrus chloroplasts seems well established (15, 16). Correlative chlorophyllase (chlorophyll chlorophyllidohydrolase, EC evidence in senescing leaves (17) and the upsurge of 3.1.1.14). Detailed kinetics based on a hexane/acetone solvent chlorophyllase in ethylene-treated Citrus fruit (16, 18-20) partition system showed that the in vivo increase in dephytyl- further support its role in senescent chlorophyll catabolism. ated derivatives coincided with the decrease in total chloro- (ii) Several oxidative and peroxidative enzyme systems phyll. Polar, dephytylated derivatives accumulated also in capable of degrading chlorophyll have been described (21, senescingMelia leaves. Senescing parsley leaves revealed a very 22), including a model system in which thylakoids fortified different picture. The gradual disappearance of chlorophyll a with linolenic acid rapidly degraded chlorophyll (23). The was accompanied by an increase in pheophytin a and by the presence of an enzyme system responsible for removal ofthe transient appearance of several phytylated derivatives. Only Mg2+ from the tetrapyrrole ring has also been hinted (24, t). pheophytin a and an adjacent peak were left when all the (iii) In vivo spectroscopy of senescing fruit has been chlorophyll a had disappeared. The pathways for breakdown used of chlorophyll in the Citrus and parsley senescence systems are for detection of biochemical/biophysical pigment changes discussed. associated with ripening and senescence (25). (iv) Characterization and identification of small amounts of breakdown products have been achieved in a cases Chlorophyll plays a central role in the life processes ofplants. few Little is known, however, about its degradative metabolism. (26-30). Of particular interest is the recent identification of The chlorophyllase system (chlorophyll chlorophyll- 132-hydroxychlorophyll a as a breakdown intermediate in a idohydrolase, EC 3.1.1.14) was discovered more than 70 model system (29) and probably also in senescing bean and years ago by Willstatter and Stoll (1), who suggested that barley leaves (30). removal of the phytyl group could be the first step in The progress in detection of breakdown intermediates has chlorophyll catabolism. Difficulties in the study of chloro- been made possible mainly through the introduction ofHPLC phyllase, mainly because of the insolubility of both enzyme systems (31-33). In the present study, HPLC was used to and substrate in aqueous media, raised questions as to its screen for breakdown intermediates in senescing Citrus fruit biochemical significance (3). The progress in the study of peel and in Melia and parsley leaf systems. In the Citrus and membranal enzymes through the use of detergents opened Melia systems, HPLC and additional corroborating evidence the way for modem assay and characterization of this point to the in vivo function of chlorophyllase, whereas in enzyme system (4, 5). Doubts still prevailed, however, parsley the HPLC data suggest a different path of degrada- regarding the physiological significance of chlorophyllase, tion. and its role in the natural breakdown of chlorophyll during MATERIALS AND METHODS senescence has not been unequivocally demonstrated (3, 6, Plant Material. Mature green tangerine fruit (Citrus 7). reticulata hybrids var. Murcott and Topaz) were harvested The problem of chlorophyll catabolism in plant tissues has from the orchard and allowed to senesce in glass cylinders been around for many years without any significant progress. under a humid stream of air containing 20 A.l of ethylene per The major difficulty seems to lie in the fact, already noted by liter at 250C in the dark. Controls were gassed with a humid Seybold many years ago (8), that chlorophyll seems to stream of air. disappear from plant tissues without leaving any visibly Leaves of Melia azedarach L., China tree, were collected detectable clues. Searches for breakdown intermediates in from individual trees growing on campus during late summer. senescing leaves (6, 9) and fruit (10) have revealed diminish- Parsley leaves (Petroselinum sativum L.), purchased daily ing amounts of chlorophylls a and b and traces of chloro- in the market, were allowed to senesce in the dark at 250C in phyllides and pheophytins (6), the latter often suspected to be Petri dishes containing moist filter paper covered with artefacts formed during extraction and chromatography. a Abbreviation: RT, retention time. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tZiegler, R., Guha, N. & Schnell, B. Meeting: Deutsche Botanische in accordance with 18 U.S.C. §1734 solely to indicate this fact. Gesellschaft, Sept. 12-18, 1982, Freiburg, F.R.G., Abstr. 473. Downloaded by guest on October 5, 2021 1901 1902 Botany: Amir-Shapira et al. Proc. Natl. Acad. Sci. USA 84 (1987) Saran net, on which the leaves were laid to avoid direct 4:6:1 (vol/vol), the mixture was shaken vigorously and contact with water. centrifuged at 12,000 x g for 10 min to separate the phases. Pigment Standards for HPLC. Chlorophylls a and b were Chlorophyllide a was determined in the acetone phase prepared from fresh parsley leaves as described by Bazzaz spectrophotometrically by using an extinction coefficient of and Rebeiz (34), stored dry at -15'C, and redissolved in 7.49 x 10-2 M-l cm-1 at 667 nm (5). Although the enzymatic diethyl ether prior to use. Chlorophyllides a and b were reaction progressed linearly for 30 min, the activity was prepared by treating their respective chlorophylls with calculated according to the 10-min readings. chlorophyllase obtained from Citrus fruit peel chloroplasts Estimation of Total Chlorophyll and Dephytylated Deriva- (see below). The chlorophyllides were transferred into ace- tives. For estimation of total chlorophyll contents, the tissue tone, dried under N2, and stored dry at -15'C. Pheophytins was extracted in cold 80% acetone, and the absorbance was a and b were obtained by adding 13% HCl to diethyl ether measured at 645 and 663 nm (38, 39). solutions of their respective chlorophylls (35). Pheophor- For estimation ofdephytylated chlorophyll derivatives, the bides a and b were prepared by the same method (35) from tissue was extracted in 20 vol of cold acetone/0.1 M NH40H, their respective chlorophyllides. 9:1 (vol/vol), in the dark (34). The extract was filtered Pigment Extracts for HPLC. The outer, colored layer of through Whatman no. 1 filter paper. Ten milliliters of hexane Citrus fruit peel and Melia and parsley leaflets were homog- was added to 10 ml of the extract. After vigorous shaking and enized in the dark at -15'C in acetone (0.5 g/5 ml; Ultra centrifugation, both the acetone and the hexane phases were Turrax homogenizer), dried under N2, and stored at -150C. ready for spectrophotometry. The spectral peak of the Samples were redissolved at -150C in acetone and filtered acetone phase, which contained the dephytylated deriva- through Schleicher & Schuell Dassel Millipore 0.45-gm tives, was at 662.4-663.3 nm, and the peak of the hexane pore-size filters prior to analysis by HPLC. fraction was at 659.3 nm. Relative amounts of pigments in the Reagents for HPLC. Absolute methanol, hexane, ethyl acetone and hexane fractions were calculated from these acetate, and acetone were purchased from Merck. PIC A (33) absorbance peaks, taking into account the volume ratios was purchased from Waters Associates and was diluted obtained (acetone/hexane, 5.2:14.8). according to the manufacturer's directions to give a 5 mM tetrabutylammonium phosphate solution of pH 7.0. Water RESULTS was distilled in glass. The HPLC Pigment Separation System. The system devel- HPLC Equipment and Operation. An HP 1090 liquid oped in the present study is based on previously published chromatography solvent delivery system equipped with an HPLC pigment separation programs, mainly HP 85B computer, an HP 3390 integrator, an HP 7470 plotter, those of and an HP 9121 diskette drive was used. A 125 x 4 mm (i.d.) Schwartz et al. (32) and Fuesler et al. (33). Our system Lichrosphere 100 CH-18 column by Merck was used. Mix- enables separation of both phytylated and nonphytylated tures of solvents were used in a stepwise elution program. chlorophyll derivatives on a single chromatogram (Fig. 1). Solvent A consisted of 7:3 (vol/vol) methanol/PIC A (Wa- The polar nonphytylated derivatives are eluted rather early ters). Solvent B consisted of ethyl acetate. First the (chlorophyllide a in 3.3 min; pheophorbide a in 7.2 min). The chromatogram was developed with 80:20 (vol/vol) solvent phytylated derivatives are eluted much later (chlorophyll b in A/solvent B for 10 min, followed by solvent A alone for the 38.4 min; chlorophyll a in 47 min; pheophytin a in 61.4 min).
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