Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1901-1905, April 1987 Botany

Chlorophyll catabolism in senescing tissues: In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves (chlorophyliide/pheophorbide//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 " (11, 12) were not con- used to separate 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. a and another polar, dephytylated of investigation. derivative accumulated in large amounts in senescing Citrus (i) The chlorophyllase 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 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 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 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 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. 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 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; in 7.2 min). The chromatogram was developed with 80:20 (vol/vol) solvent phytylated derivatives are eluted much later ( 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). next 12 min. At 22 min the A/B solvent ratio was changed to Removal of the Mg causes some delay; thus, pheophorbide a 50:50 (vol/vol), and at 60 min, to 20:80 (vol/vol), which was used until the compounds of interest were eluted. The total a development time was 75 min. 0 Ten-microliter samples were injected each time. The flow rate was 0.4 ml/min, the pressure did not exceed 2500 pounds a. 5 Dz~~~~~~~Cr a per square inch (1 psi = 6.89 kPa), and the system was operated at room temperature (ca. 25°C). Principal absorb- ance-detector wavelengths were 405 nm, which suits chlo- rophyll a and its derivatives, and 436 nm, which suits chlorophyll b and its derivatives. Peaks were tentatively U IR 448 identified by their retention time (RT) and by the absorbance spectra produced during the HPLC run, in comparison with 0 markers. Chlorophyllase Preparation and Assay. The outer, colored 4% layer of Citrus fruit peel was removed and homogenized in 0 ice-cold 50 mM Tris-HCl, pH 8.0/400 mM sucrose. The floating chloroplast pellets (16, 36) were subsequently ho- 0 20 40 6i0 mogenized at - 15°C in acetone for the preparation ofacetone powders. Acetone powders (50 mg) were extracted by stirring FI. a. a.oaormfaeoe xrcsfo eecn with 10 ml of 5 mM phosphate buffer, pH 7.0/50 mM KCl/0.24% Triton X-100 for 60 min at 30°C. The extract was filtered through glass wool and centrifuged at 12,000 x g for 10 min; the supernatant was used for enzyme assays. The protein content of the enzyme extract that contained Triton X-100 was determined by the biuret method (37). The enzymatic assay reaction mixture (5, 16) contained 5 ml of 100 mM phosphate buffer (pH 7.0), 0.24% Triton X-100, RETENTION TIME (min) 1.0 ml of enzyme, and 0.2 ml of diethyl ether containing 0.7 Fio. 1. Chromatograms of ace-tone extrarct from seniescing pmol of chlorophyll a, which was dissolved in the aqueous Citrus fruit peel exposed for 48 hr to a stream of air (Upper) or solution by vigorous shaking. Incubation was in a shaking ethylene (Lower), scanned spectrophotometrically at 405 nm. Names bath at 35TC. Aliquots (1 ml) were transferred into centrifuge of markers are indicated at their respective RT along the chromato- tubes containing 11 ml of acetone/hexane/10 mM KOH, gram. The arrow points to the "RT 4.8" peak. Downloaded by guest on October 5, 2021 Botany: Amir-Shapira et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1903

CHLORO - PHYLLIDE a PHEOPHORBIDE a CAROTENOIDS CHLOROPHYLL a PHEOPHYTIN a RT 13. T 7.2 RT 25.7 | T 47.0 RT 61.4

300 500 nm I 1 300 500 nm 300 500 nm I

300 500 nm 300 500 nm FIG. 2. Absorbance spectra (260-600 nm) of markers and of the chlorophyll a derivatives with RT of 4.8 and RT of 62.5.

is eluted after chlorophyllide a, and pheophytin a, after -15°C acetone extracts were analyzed by HPLC immediate- chlorophyll a. The spectral characteristics ofthe peaks eluted ly after extraction instead of drying the extracts in N2, as in 25-30 min (Fig. 2) suggested that they were carotenoids. described in Materials and Methods. The Citrus System. Analyses of acetone extracts from The accumulation of large amounts of nonphytylated senescing Citrus fruit peel (Fig. 1) revealed the presence of chlorophyll derivatives in senescing Citrus peel was verified large amounts ofchlorophyllide a, tentatively identified by its also by detailed kinetics, using the phase separation between RT (3.3 min; compare Fig. 3) and by its absorbance spectrum, acetone and hexane (Fig. 5). At harvest the fruit had only a which was identical with that of the chlorophyllide a marker small portion of its pigment absorbance in the (Fig. 2). Pheophorbide a (RT of7.2 min) did not appear in our acetone chromatograms. However, another dephytylated derivative fraction. Ethylene-treated fruit, which showed an immediate with a RT of 4.8 min (Fig. 1 arrow) accumulated in consid- drop in chlorophyll, revealed a concurrent increase in the erable amounts. Its absorbance spectrum indicates lack of absorbance ofthe acetone fraction, reflecting the appearance Mg2+ and is identical with that ofpheophorbide a (Fig. 2). The of polar dephytylated derivatives. Fruit held in air did not large, initial off-scale peak (RT of 1.0-2.5 min) appeared also show a loss of chlorophyll for 5 days, and throughout this in acetone extracts of nonsenescent fruit. Detailed spectral period there was no increase in the absorbance ofthe acetone analysis showed that it contained mainly UV-absorbing fraction. When the decline in chlorophyll began, there was materials with peaks at 280 and 320 nm and no peaks in the also an increase in the absorbance of the acetone phase. visible spectral range. Ethylene-treated fruit (Fig. 1 Lower) The appearance of large amounts of chlorophyllide a in lost most of their chlorophyll and accumulated more senescing Citrus peel in vivo has prompted us to reassess the chlorophyllide a and "RT 4.8" derivative than fruit held in air chlorophyllase activity of the same tissue in vitro as affected (Fig. 1 Upper). Chlorophyll b (RT of 38.4 min), which is not by treatment with ethylene (Table 1). Chlorophyllase activ- prominent in the 405-nm scan, also decreased in ethylene- ity, which was already high at harvest, increased 5.8-fold on treated fruit. An unidentified peak at a RT of 45 min became an acetone-powder basis or 4.2-fold on a protein basis upon more prominent in ethylene-treated fruit. Pheophytin a (RT treatment with ethylene. of 61.4 min) appeared in minimal amounts in both air- and The Melia System. Mature green leaves already contained ethylene-treated fruit. Scanning of the HPLC run at 280 and a considerable amount of chlorophyllide a (Fig. 6). The 350 nm did not reveal the presence of additional UV- decline in chlorophyll a during autumnal senescence ap- absorbing materials. Comparison of integrated peak areas peared to be associated with changes in the nonphytylated showed the differences between ethylene- and air-treated end ofthe chromatogram; mainly an accumulation ofa broad fruit even more clearly (Fig. 4). Whereas "in air" the peak corresponding to pheophorbide a (RT of 6.1 min in this nonphytylated derivatives (chlorophyllide a and the 4.8-min peak) accounted for about 20% of the absorbance, "in ethylene" the nonphytylated derivatives accounted for 70% of the absorbance. Identical results were obtained also when

w z 4

0o E

co0

w-J I I I L. . - V Z 4 6p 8 RETENTION TIME (min) FIG. 4. Peak areas of chlorophyll a and major chlorophyll a derivatives in chromatograms of acetone extracts obtained from air- FIG. 3. Chromatogram of a chlorophyllide a marker, scanned and ethylene-treated Citrus fruit. Data are means from three repli- spectrophotometrically at 405 nm. cates; bars on the top of columns indicate the SEM. Downloaded by guest on October 5, 2021 1904 Botany: Amir-Shapira et al. Proc. Natl. Acad. Sci. USA 84 (1987)

3D 0.4 o , MATURE LEAVES . z 4 -Ja-a .4-- > cr 0 a.>.wiLz m w ~0 0'0 0oa.a: 01 ix0 WU~~ - W 1fi0.2 J _j-r u -J tI -J ~~~Li Li a.d.. Li

- 0 Ix E 00 c $ 0 0

o' 40 z z 0 cnw 0 o SENESCING LEAVES D len Wa cr -W U -J -J IL 20 W _Joi >. W >- 0 W z m~~~~a a- 0 ama- a. Wi 0 0a. cr W IO ~° ~ ~~~I0 Li Z LI C

0 I I . - I I DAYS

FIG. 5. Changes in total chlorophyll, as determined spectropho- tometrically in 80% acetone extracts (Upper), and the percentage of _j WI chlorophyll absorbance remaining in the acetone fraction (Lower) in the peel of Citrus fruit senescing in air or ethylene.

case). The small amount of pheophytin a present in mature leaves (RT of 58 could not be found in senescing ones. min) 0 20 40 60 The Parsley System. In contrast with senescing Citrus fruit RETENTION TIME (min) and Melia leaves, senescing parsley leaves did not show any increase in chlorophyllide a or other nonphytylated chloro- FIG. 6. Chromatograms of acetone extracts from mature-(dark phyll derivatives (Fig. 7). The rapid loss of chlorophyll a was green) and senescing (yellow-green) leaflets of Melia azedarach accompanied by some increase in pheophytin a and mainly in trees, scanned spectrophotometrically at 405 nm. Names of markers an adjacent peak, having an absorbance spectrum identical are indicated at their respective RT along the chromatograms. with that of pheophytin a (Fig. 2). Several additional peaks appeared at 2 days and more clearly at 4 days in the vicinity tissues. The demonstration of the in vivo accumulation of of chlorophyll a and pheophytin a. Chlorophyll b practically large amounts of chlorophyllide a in senescing Citrus peel disappeared at 4 days. 1-Carotene (RT of 63 min) and other (Figs. 1 and 4) is therefore ofmajor significance. The function carotenoids (RT of 25-30 min) also decreased gradually. By of the "chlorophyllase pathway" in Citrus peel is not 6 days chlorophyll a and most other peaks had disappeared, unexpected, however, in view of the previously obtained while pheophytin a and its adjacent peak accounted for most correlations between the ethylene-induced enhancement of of the remaining absorbance. senescence and the upsurge of chlorophyllase activity in this tissue (18-20). The ethylene-induced activation of chloro- DISCUSSION phyllase has been reconfirmed in the present study (Table 1). The picture revealed by autumnal Melia leaves is basically Lack of evidence for the in vivo accumulation of breakdown similar to that obtained with Citrus. Senescing Melia leaves intermediates has been a major obstacle in the elucidation of contain, besides traces of chlorophylls, only polar dephytyl- the pathway of chlorophyll degradation in senescing plant ated derivatives (Fig. 6). On the other hand, the chromatograms from dark-senesc- Table 1. Chlorophyllase activity of ethylene-treated Citrus fruit ing parsley leaves indicate the accumulation of pheophytin a peel, as compared with air-treated and harvest-day controls and other phytylated derivatives (Fig. 7). Thus, the evidence Chlorophyllide a, Iumol for parsley suggests a different degradative pathway involv- ing in its initial steps phytylated ring-modified derivatives per per g of acetone mg of that are similar, perhaps, to the 132-hydroxychlorophyll Treatment powder protein identified recently in several systems (29). Harvest day 6044 + 320 (100) 130.0 (100) Similar differences in in viva breakdown intermediates 72 hr in air 4787 ± 72 (79) 88.7 (68) were detected also during heat stress-induced chlorophyll 72 hr in ethylene 35093 ± 317 (581) 544.1 (419) degradation; whereas Citrus accumulated chlorophyllide- Acetone powders prepared from chloroplast fragments were and pheophorbide-like materials, parsley revealed numerous extracted and assayed for protein and chlorophyllase activity as phytylated derivatives (unpublished data). Autolysis of chlo- described. Data are averages from three replicates + SEM. Numer- rophyll in chloroplast fragments in vitro also suggested als in parentheses indicate activity as a percentage ofthe harvest-day differences between the Citrus and parsley systems (40). We control. do not consider it unlikely, therefore, that Citrus and parsley Downloaded by guest on October 5, 2021 Botany: Amir-Shapira et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1905 detergents and other membrane-disrupting treatments (40). The enhancement of chlorophyll destruction in Citrus fruit by ethylene is correlated with elevated chlorophyllase activity (refs. 18-20 and the present study). It is presently not clear, ,0 w _ however, whether the increase in chlorophyllase arises by de novo synthesis of the enzyme protein or by some other form of X activation. Thus, the field ofchlorophyll catabolism is still wide open and invites further study. We thank Prof. K. V. Thimann for careful review of the manu- script and constructive criticism. The expert technical assistance of 0~~~~~~~0I Mr. David Galili is gratefully acknowledged. This research was supported by a grant from the United States-Israel Binational Science Foundation, Jerusalem, Israel. 1. Willstdtter, R. & Stoll, A. (1910) Justus Liebigs Ann. Chem. 0. 378, c 18-72. 2. Terpstra, W. (1977) Z. Pflanzenphysiol. 85, 139-146. 3. Simpson, K. L., Lee, T. C., Rodriguez, D. B. & Chichester, C. 0. (1976) in Chemistry and Biochemistry of Plant Pigments, ed. Goodwin, T. W. (Academic, New York), 2nd Ed., Vol. 2, pp. 780-842. 4. Klein, A. & Vishniac, W. (1961) J. Biol. Chem. 236, 2544-2547. 5. McFeeters, R. F., Chichester, C. 0. & Whitaker, J. R. (1971) Plant Physiol. 47, 609-618. 6. Holden, M. (1967) Rep. Rothamsted Exp. Stn. 1966, 310-319. VI. 7. Goldschmidt, E. E. (1980) in Senescence in , ed. Thimann, ~ ~ ~ ~ A K. V. (CRC, Boca Raton, FL), pp. 207-217. 8. Seybold, A. (1943) Bot. Arch. 44, 551-568. w 9. Whitfield, D. M. & Rowan, K. S. (1974) Phytochemistry 13, 77-83. 10. Durand, M. & Laval-Martin, D. (1974) J. Chromatogr. 97, 92-98. 11. Goodwin, T. W. (1958) Biochem. J. 68, 503-511. 12. Schanderl, S. H. & Lynn, D. Y. C. (1966) J. Food Sci. 31, 141-145. 13. Terpstra, W. (1981) FEBS Lett. 126, 231-235. 14. Shimokawa, K. (1982) Phytochemistry 21, 543-545. 15. Terpstra, W. & Goedheer, J. C. (1975) Z. Pflanzenphysiol. 75, 405-414. 16. Hirschfeld, K. R. & Goldschmidt, E. E. (1983) Plant Cell Rep. 2, 117-118. 17. Sabater, B. & Rodriguez, Ma. T. (1978) Physiol. Plant. 43, 274-276. 18. Barmore, C. R. (1975) HortScience 10, 595-596. 19. Shimokawa, K., Shimada, S. & Yaeo, K. (1978) Sci. Hortic. 8, 129-135. 20. Purvis, A. C. & Barmore, C. R. (1981) Plant Physiol. 68, 854-856. 21. Martinoia, E., Dalling, M. J. & Matile, Ph. (1982) Z. Pflanzenphysiol. 107, 269-279. 20 40 22. Huff, A. (1982) Phytochemistry 21, 261-265. RETENTION TIME (min) 23. Luthy, B., Martinoia, E., Matile, Ph. & Thomas, H. (1984) Z. Pflanzenphysiol. 113, 423-434. FIG. 7. Chromatograms of acetone extracts from detached pars- 24. Owens, T. G. & Falkowski, P. G. (1982) Phytochemistry 21, ley leaves senescing in the dark for 0, 2, 4, and 6 days, scanned 979-984. spectrophotometrically at 405 nm. Names of markers are indicated 25. Gross, J. & Ohad, I. (1983) Photochem. Photobiol. 37, 195-200. at their respective RT along the chromatogram. 26. Schoch, S., Scheer, H., Schiff, J. A., Rudiger, W. & Siegelman, H. W. (1981) Z. Naturforsch. C 36, 827-833. 27. Schoch, S. & Vielwerth, F. X. (1983) Z. Pflanzenphysiol. 110, different routes of 309-317. represent chlorophyll breakdown in higher 28. Haidl, H., Knodlmayr, K., Rudiger, W., Scheer, H. & Schoch, S. plants. (1985) Z. Naturforsch. C 40, 685-692. The present study paves the way for further investigation 29. Schoch, S., Rudiger, W., Lfithy, B. & Matile, Ph. (1984) J. Plant of chlorophyll catabolism and its control in higher plants. Physiol. 115, 85-89. the identification of the reactions 30. Maunders, M. J., Brown, S. B. & Woolhouse, H. W. (1983) Following intermediates, Phytochemistry 22, 2443-2446. involved may be elucidated. Chlorophyllase is the only 31. Eskins, K. & Leland, H. (1979) Photochem. Photobiol. 33, 131-133. well-defined enzyme system shown so far to be involved in 32. Schwartz, S. J., Woo, S. L. & von Elbe, J. H. (1981) J. Agric. chlorophyll catabolism. However, chlorophyllase itself ex- Food Chem. 29, 533-535. ists in several forms in different 33. Fuesler, T. P., Hanamoto, C. M. & Castelfranco, P. A. (1982) plant systems (2, 10, 14, 41). Plant Physiol. 69, 421-423. Other enzyme systems await characterization (21-23, t) or 34. Bazzaz, M. B. & Rebeiz, C. A. (1979) Photochem. Photobiol. 30, are yet to be discovered. 709-721. The regulatory aspects of chlorophyll degradation are also 35. Hynninen, P. H. & Elifolk, N. (1973) Acta Chem. Scand. 27, intriguing. Chlorophyllase is present in green plant tissues 1463-1477. 36. Goldschmidt, E. E. (1977) Phytochemistry 16, 1046-1047. before the onset of senescence, and it is not clear how the 37. Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis (Aca- destruction of chlorophyll is turned on during senescence. demic, New York), 2nd Ed., Vol. 1, pp. 174-176. Some initial disintegration of the thylakoid membranes may be 38. Arnon, D. I. (1949) Plant Physiol. 25, 1-15. needed to expose the chlorophyll to hydrolysis by chlorophyll- 39. Bruinsma, J. (1963) Photochem. Photobiol. 2, 241-249. 40. Amir-Shapira, D., Goldschmidt, E. E. & Altman, A. (1986) Plant ase (or other enzyme systems). Autolysis of chlorophyll in Sci. 43, 201-206. chloroplast fragments has indeed been set into motion by 41. Ogura, N. (1972) Plant Cell Physiol. 13, 971-979. Downloaded by guest on October 5, 2021