Breakdown of Chlorophylls by Soluble Proteins Extracted from Leaves of Chenopodium album

J Plant Physiol. Vol.

145. pp. 416-421 (1995)

Breakdown of Chlorophylls by Soluble Proteins Extracted from Leaves of Chenopodium album Yuzo SHIOI! \

TATSURU MASUDA!, KENICHIRO TAKAMIYA!,

and KEISHI SHIMOKAWA2

1

Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226, Japan

2

Faculty of Agriculture, Miyazaki University, Miyazaki 889-16, Japan

Received April 18, 1994 . Accepted September 14, 1994

Summary

Chlorophylls were degraded by soluble proteins extracted from leaves of Chenopodium album. The bleaching (oxidative cleavage) of chlorophylls and accumulation of pheophorbides were observed. The accumulation of pheophorbides was not inhibited by anoxygenic conditions or by ascorbate, but the bleaching of chlorophylls was inhibited. It appears that there are two distinct degradation pathways: chlorophyll bleaching and pheophorbide accumulation in the process of chlorophyll breakdown. The bleaching reaction was not affected by hydrogen peroxide or hydrogen peroxide and 2,6-dichloroindophenol, but was slightly inhibited by linolenic acid. Catalase had no effect, but superoxide dismutase inhibited weakly. This bleaching was strongly inhibited by tiron and ascorbate. These results suggest that active oxygen, probably the superoxide radical, may be involved, but the enzymes catalase, peroxidase, and lipoxidase are not responsible for the bleaching reaction. The accumulation of pheophorbide derivatives is discussed in relation to the inhibition of bleaching activity.

Key words: Chenopodium album, Chlorophylls, Chlorophyll bleaching, Enzymatic chlorophyll degradation, Pheophorbide accumulation.

Abbreviations: Chi

=

chlorophyll; HPTLC

Introduction

The loss of chlorophyll (Chi) from leaves and fruits occurs during the senescence process and also during the normal turnover of the pigment-protein complex. The degradation process can be divided into two reaction types as proposed by Brown et al. (1991). Type I includes the loss of phytol, magnesium, and the modification of the side chains of an isocyclic ring of the Chi structure by the action of several enzymes. Type II accompanies the oxidative cleavage (bleaching) of the tetrapyrrole macrocycle in a rapid process that appears to involve molecular oxygen. The enzyme that acts on the ring opening is still unclear. The accumulation of the pheophorbide species as part of type I reaction has been reported in the breakdown of Chis

*

All correspondence to this author.

© 1995 by Gustav Fischer Verlag, Stuttgart

=

high-performance thin-layer chromatography. in various materials such as algae, higher plants, and bacterial cells; for instance, tobacco cells (Schoch and Vielwerth, 1983), rape cotyledons (Langmeier et aI., 1993), Euglena cells (Schoch et al., 1981), Skeltonema extracts (Owens and Falkowski, 1982), Chlorella cells (Ziegler et aI., 1988), and Rhodobacter sphaeroides cells (Haidl et al., 1985). Besides these reports, we showed that in addition to the Chi bleaching, the accumulation of pheophorbide derivatives occurred during the breakdown of ChIs in crude extracts of Chenopodium album (Shioi et al., 1991). We determined the end product of ChI degradation as pyropheophorbide and also the sequence of their formation by using high-performance thin-layer chromatography (HPTLC) analyses with a CIS bonded silica-gellayer and 14C-Chla as the substrate (Shimokawa et aI., 1990). The processes involved in the degradation of pigments are poorly understood in contrast to those for the biosyhnthesis

Chlorophyll breakdown by Chenopodium extract

of ChI in the greening process (see reviews: Hendry et al., 1987; Matile et al., 1989; Rudiger and Schoch, 1989). In the past few years, however, some progress has been made by MatiIe's group (Duggelin et al., 1988; Bortlik et al., 1990; Krautler et al., 1991, 1992). They found requirements for ATP and reduced ferredoxin for the generation of a putative catabolite of ChIs, FCC-4 in chloroplasts of senescent barley leaves (Schellenberg et al., 1990, 1993). They also reported several putative catabolites of ChI that were identified from senescent leaves of meadow fescue and barley as secoporphonoid derivative of pheophorbide a. Although the existence of two reactions, ChI bleaching and accumulation of pheophorbide derivatives, has been suggested (Brown et al., 1991) as described above, relatively few studies have characterized the enzymes involved in the breakdown of ChIs, and the properties of these activities are largely unknown. To characterize the degradation reaction, we have used soluble proteins that degraded ChIs extracted from leaves of C. album. With the soluble proteins, we have studied the effect of several chemicals and anoxia on ChI bleaching and the accumulation of pheophorbides. The accumulation of pheophorbide is discussed in relation to the inhibition of bleaching activity.

Materials and Methods

Plant materials Matured and fully expanded leaves of Chenopodium album were collected on the campus of Miyazaki University, Miyazaki, and from the uncultivated fields of Kamakura, Kanagawa.

Chlorophylls Chis a and b were extracted from spinach leaves with absolute acetone and were partially purified by precipitation with dioxane (Iriyama et al., 1974). The dioxane-precipitated Chis dissolved in absolute acetone were used for enzyme assays. Partially purified Chlsa and b were separated and further purified by sugar-column chromatography (Perkins and Roberts, 1962), when necessary.

(pH 7.0), and the solution, clarified by centrifugation at 12,000 x g for 10 min, was used directly or after dialysis against the same buffer as the partially purified enzyme.

Assay of chlorophyll degradation The degradation of Chis was assayed in soluble proteins (3.2 mg) and Chis dissolved in acetone in a total volume of 1.5 mL in the standard assays. In some experiments, purified Chi a was used instead of dioxane precipitated Chis. In this case, a polar lipid fraction isolated from a spinach extract was supplemented to activate chlorophyllase (Terpstra and Lambers, 1983). The Chi concentration was adjusted to 3.75 absorbance at 663 nm, and the final acetone concentration was 20 % (v/v). The reaction mixture was incubated at 30°C in complete darkness for 15 to 30 min in the standard assays. An aliquot (0.4 mL) was removed, and pigments were completely extracted by adding 1.1 mL of acetone to give 80 % (v/v) acetone (final concentration), and the absorbance difference before and after incubation was measured with a Shimadzu UV-2200 spectrophotometer. Pheophorbide formation was determined according to the method of Lavar-Martin (1985) by measuring the absorbance at 665, 663, and 655 nm before and after pheophytinization with 5 mL of 3 M HCl. Chi bleaching was also determined spectroscopically by the absorbance difference at 663 nm before and after incubation. Note that the activity determined by this method includes partly the absorbance decrease caused by pheophorbide formation.

Results

In the breakdowns of ChIs, the bleaching of ChIs (type II reaction) and formation of pheophorbide (type I reaction) were observed to coincide with the previous results of leaf extracts (Shioi et al., 1991) when ChIs and soluble proteins extracted from leaves of C. album were incubated (Figs. 1 and 2). As shown in Figure 1, bleaching of Chla occurred rapidly up to 30 min, after which proceeded slowly, whereas 10~----------------------~

Preparation of soluble proteins An acetone powder was prepared by the treatment of leaves with acetone. Washed leaves (ca. 100 g) were homogenized in 300 mL of cold (- 20 0C) acetone in a blender. The homogenate was filtered through a Buchner funnel, and the acetone was removed. The residue was mixed with 300 mL of cold acetone, homogenized and filtered a second time. Additional washing of the residue with 200 to 400 mL resulted in a powder which was dried at room temperature. About 15 g of dry powder was obtained 100 g of fresh leaves. We used the freshly prepared acetone powder because of a lability of the bleaching activity. The soluble proteins were extracted from the acetone powder by suspending them in 15 volumes of 20 mM Na-K phosphate buffer (pH 7.0). The suspension was stirred for 30 min in an ice-bath and then filtered through three layers of Miracloth. The filtrate was centrifuged at 20,000 x g for 30 min, and the resulting supernatant was used as the source of soluble proteins either with or without further purification by acetone fractionation as follows. The enzyme extracts were fractionated with 60 % (v/v) acetone. The precipitate formed was dissolved in a small volume of 20 mM phosphate buffer

417

Heat-treated, ChIs (il) and pheophorbides (£>-)

-10

_20L------L----~------~~

o

20

40

60

Time, min Fig. 1: Time course of Chla bleaching and pheophorbidea formation by soluble proteins extracted from leaves of C. album. The soluble proteins were incubated with Chi in air at 30°C for the indicated periods of time. The pigment concentration was measured spectroscopically under the conditions described in the text. Data are averages of duplicate samples (two to three experiments). In heat-treated samples, soluble proteins was heated at 100°C for 5 min.

418

Yuzo SHIOI, TATSURU MASUDA, KENICHIRO TAKAMIYA, and KEISHI SHIMOKAWA

1, Control (0 time) 2, Ascorbate addition 3, No addition

0. 5

600

500

Wavelength, nm

the formation of pheophorbide was linear and continued after the bleaching reaction almost ceased. These reactions did not require any organic solvent or detergent supplements although these chemicals accelerated the activity as demonstrated in the leaf extracts (Shioi et al., 1991). Triton X-100 could substitute for acetone, but we did not use Triton because of the formation of a chlorophyllide-Triton ester in the chlorophyllase reaction (Michalski et al., 1987). The degradative activities were completely eliminated by the heat treatment of soluble proteins. pH dependence of the bleaching reaction and pheophorbide formation both showed an almost symmetrical profile having a pH optimum at 6.5 to 7 in 60 mM MES-HEPES-Tricine buffer (data not shown). Incubation under N 2 instead of air reduced ChI bleaching to 59 %, whereas the accumulation of degradation products, pheophorbides, decreased by only 8 % of that of the oxygenic conditions (Table 1). When ascorbate (1 mM, pH 7.0) was added to the reaction mixture, the ChI-bleaching activity was inhibited almost completely; but the accumulation of the pheophorbide derivative still occurred. These results indicate that at least the bleaching of ChIs but not pheophorbide formation is an oxygen-dependent reaction.

Table 1: Effects of anoxia and ascorbate on Chi bleaching and pheophorbide accumulation. Addition

Chi bleaching Pheophorbide formed (n mollh· mg protein)

Complete (in air) Complete, + 1 mM ascorbate Complete, in N2

32.4 1.67

7.61 8.10

19.20

6.99

700

Fig. 2: Absorption spectra showing the effect of ascorbate on Chi bleaching and pheophorbide formation by the acetonefractionated protein fraction. Acetone-fractionated proteins were incubated Chis in the presence or absence of 1 mM ascorbate (PH 7.0) for 30 min in air at 30°C in the dark.

To examine the effect of ascorbate on the bleaching reaction in more detail, we measured the absorption spectra by using an acetone-fractionated protein fraction in the presence or absence of ascorbate (Fig. 2). Partially purified protein fractions from acetone fractionation also had the same degradation activities. In the absence of ascorbate, the spectrum showed a decrease in the ChI absorption at 433, 459, and 664 nm and an increase in peaks at 413 and 541 nm due to the formation of pheophorbide. On the other hand, sample with ascorbate (1 mM, pH 7.0) showed a slight decrease in the ChI peaks at 664 and 433 nm, indicating that the bleaching activity of ChIs was suppressed. In addition, it showed a slight increase in peaks at 413, 510, and 541 nm, due to the formation of pheophorbide derivative. A red shift of the 664 nm peak also indicates the formation of pheophorbide. The decrease of ChIs and the formation of pheophorbide was stoichiometric in the presence of ascorbate, but this was not the case in the absence of ascorbate (see Fig. 1). Thus, our idea of an oxygen-independent formation of pheophorbide was confirmed by the fact that only the Chlbleaching reaction was completely inhibited by ascorbate. Furthermore, a small amount of pheophorbide accumulation was observed under bleaching conditions in the absence of ascorbate, indicating that some part of pheophorbide was also degraded. As can be seen from the spectral data of Figure 2, the accumulation of pheophorbide derivative is obvious. To test whether the product is pheophytin or pheophorbide derivative, we used C I8 bonded HPTLC according to the previous methods (Shioi et al., 1991). It showed that the main degradation product had an Rf = 004. The major pigment isolated had absorption peaks at 408 and 666 nm in ether (Fig. 3).

Chlorophyll breakdown by Chenopodium extract

0.2

chemicals such as resorcinol (40 11M) or p-coumaric acid (40IlM) had no stimulative effect (data not shown). The reaction was almost completeley inhibited by a potent quencher of radicals, ascorbate and superoxide anion radical scavenger, tiron at a concentration of 1 mM. However, superoxide dismutase inhibited weakly 32 % at 300 U / mL. KCN, an inhibitor of heme containing enzyme, inhibited 71 % of the activity at 1 mM.

408

OJ

()

c:

-eo

419

<0

.2 «

0.1

Discussion

666

400

500

600

700

Wavelength , nm

Fig. 3: Absorption spectrum of the major degradation product, pyropheophorbidea in ether isolated from the assay mixture. Soluble proteins and Chis were incubated in air at 30°C in the dark for 1 h. Pigment extraction and chromatography were performed according to the method described previously (Shioi et al., 1991).

The Rf value and the absorption peaks coincided with those of pyropheophorbide a (Shioi et aI., 1991). These results show that the major product accumulated during ChI breakdown by soluble proteins is pyropheophorbide a as demonstrated in various other materials (Schoch et aI., 1981; Schoch and Vielwerth, 1983; Owens and Falkowski, 1982; Haidl et aI., 1985; Ziegler et aI., 1988; Shimokawa et aI., 1990). To characterize the properties of the bleaching enzyme, we tested the effect of chemicals and enzymes on the Chlbleaching activity (Table 2). The reaction was not stimulated but rather slightly inhibited by the addition of H 20 z, HzO z and 2,6-dichloroindophenol, and linoleic acid. Similarly, linolenic acid (300-600/-lM) as well as H 20 z with other Table 2: Effect of certain chemicals and enzymes on the Chi bleaching activity of soluble proteins. Incubation conditions

Chi decreased

%

(~A663nm/h)

Complete Complete, heat-treated proteins - Soluble proteins Complete, + 1 mM HzO z Complete, + 1 mM H 20 2 + DCIP Complete, + 600 J.1M linolenic acid Complete, + 1 mM ascorbate Complete, + 1 mM tiron Complete, + 1 mM KCN Complete, + 20U/mL catalase Complete, + 300U/mL superoxide dismutase

0.54 0 0 0.51 0.52 0.42 0.021 0.043 0.15 0.55

100 0 0 95

8 29 102

0.35

68

96

78

4

DCIP = 2,6-dichloroindophenol. The values are means of results from the duplicate samples (two to four experiments).

The results of the present study by using inhibitors and cofactors suggest that lipoxygenase, peroxidase, and catalase are unlikely candidates for the bleaching (type II reaction) enzyme in this system, although there are reports for ChI degradation using these enzymes (Holden, 1970; Huff, 1982; Matile, 1980; Kato and Shimizu, 1985). At present, there is no information concerning the possibility of the participation of these enzymes in the seasonal breakdown of chloroplast pigments in the plant species, except that an increase in the levels of peroxidase during ethylene-induced senescence in Cucumis sativus cotyledons is not involved in the degradation of ChIs (Abeles and Dunn, 1989). Two other ChI-bleaching reactions have been reported. Luthy et a1. (1984) demonstrated that a latent chlorophyllbleaching activity, the so called Chi oxidase, is present in barley thylakoids. This reaction is strictly oxygen dependent and is activated by the presence of polyunsaturated fatty acids, particularly linolenic acid. Schoch et a1. (1984) determined the first product of the reaction of the ChI oxidase as 131-hydroxy Chla. Chenopodium enzyme is, however, different from the ChI oxidase in certain points, since no activation, but rather a slight inhibition of the bleaching activity was observed after the addition of linolenic acid. We could not detect hydroxy derivatives of ChI a in this system. Recently, the breakdown of thylakoid pigments by soluble proteins of developing chloroplasts was reported by Whyte and Castelfranco (1993). This activity required oxygen and a detergent such as Triton X-I00. The enzyme fraction can degrade not only plastidic pigments but also exogenous Mg-protoporphyrin IX mono methyl ester (Mgproto). The Chenopodium enzyme can be, however, distinct from the developing chloroplast enzyme based on the following facts: The Chenopodium enzyme did not require detergent for the activity and did not use Mg-proto as the substrate. The Chenopodium bleaching enzyme thus appears to be different from all reported enzymes. In the bleaching of ChIs in our system, free radical may be involved in some of the reactions from the effect of several chemical compounds. Inhibition by tiron, a scavenger of superoxide anion radical, and superoxide dismutase indicates the participation of superoxide anion radical for bleaching activity. A weak inhibition by superoxide dismutase is probably caused by the inaccessibility of the superoxide dismutase to the anion radical that is produced at the enzymes active site (Younes and Weser, 1978). It has been reported that ascorbate retards leaf senescence including ChI degradation (Grag et aI., 1972). In this study, we showed that ascorbate was a potent inhibitor of the bleaching activity. This is prob-

420

Yuzo SHIOI, TATSURU MASUDA, KENICHIRO TAKAMlYA, and KEISHI SHIMOKAWA

ably due to the fact that a low concentration of ascorbate acts as an anti-oxidant and quenches the formed anion radicals, although ascorbate at high concentration in the presence of certain transition metals can act as a pro-oxidant. In the enzyme extracted from leaves of C. album, pheophorbides formation can be separated from the ChI-bleaching reaction under conditions that prevent the bleaching in the anoxygenic condition or by the addition of reductant, ascorbate, as demonstrated in the present investigation (Fig. 2 and Table 1). Previously, Matile's group reported that pheophorbide accumulated in anoxygenic conditions in leaves of parsley and in barley senescing under N 2 (Peisker et al., 1989). Besides these reports, the accumulation of pheophorbide derivatives has previously been reported for a variety of materials from the photosynthetic bacteria to higher plants (Schoch and Vielwerth, 1983; Langmeier et al., 1993; Schoch et al., 1981; Owens and Falkowski, 1982; Ziegler et al., 1988; Haidl et aI., 1985). The breakdown of ChIs into pheophorbide derivatives in such diverse species suggests a general step of the pathway by which some of the ChIs are degraded. Recent studies on the establishment of the identification of bleaching products suggest that pheophorbide derivatives are considered to be precursors of secoporphonoid and bilin derivatives that are putative catabolites of parts of degraded ChIs after the cleavage of a macrocyclic ring system (Krautler et aI., 1991, 1992; Engel et al., 1991). Purification and characterization of the enzymes involved in the breakdown of ChIs will be necessary to elucidate the sequence of steps in the degradation pathway. We have already purified and partially characterized chlorophyllase from C. album that occurs in the first step of the pyropheophorbide pathway (Kita et al., 1993). We are now trying to purify the ChI-bleaching enzyme. Acknowledgements

The authors thank Dr. Gamini C. Kannangara of Carlsberg Laboratory for a critical reading of the manuscript and his encouragement during this work. This study was supported in part by Grantin-Aid for Scientific Research to Y.S. from the Ministry of Education, Science and Culture, Japan (01540566, 06640836).

References ABELES, F. B. and L. J. DUNN: Role of peroxidase during ethylene-induced chlorophyll breakdown in Cucumis sativus cotyledons. J. Plant Growth Regul. 8, 319-325 (1989). BORTLIK, K., C. PEISKER, and P. MATILE: A novel type of chlorophyll catabolite in senescent barley leaves. J. Plant Physiol. 136, 161165 (1990). BROWN, S. B., J. D. HOUGHTON, and G. A. F. HENDRY: Chlorophyll breakdown. In: SCHEER, H. (ed.): Chlorophylls, 465-489. CRC Press, Boca Raton, FL (1991). DUGGELIN, T., K. BORTLIK, H. GUT, P. MATILE, and H. THOMAS: Leaf senescence in Festuca pratensis: Accumulation of lipofuscin-like compounds. Physiol. Plant. 74, 131-136 (1988). ENGEL, N., T. A. JENNY, V. MOOSER, and A. GOSSAUER: Chlorophyll catabolism in Chlorella protothecoides. Isolation and structure elucidation of a red bilin derivatives. FEBS Lett. 293. 131-133 (1991).

GARG, O. P. and V. KApOOR: Retardation of leaf senescence byascorbic acid. J. Exp. Bot. 23, 699-703 (1972). HAIDL, H., K. KNODLMAYR, W. RUDIGER, H. SCHEER, S. SCHOCH, and J. ULLRICH: Degradation of bacteriochlorophyll a in Rhodopseu· domonas sphaeroides R26. Z. Naturforsch. 40 c, 685-692 (1985). HENDRY, G. A. F., J. D. HOUGHTON, and S. B. BROWN: The degradation of chlorophyll a biological enigma. New Phytol. 107, 255302 (1987). HOLDEN, M.: Chlorophyll bleaching system in leaves. Phytochemistry 9, 1771-1777 (1970). HUFF, A.: Peroxidase-catalyzed oxidation of chlorophyll by hydrogen peroxide. Phytochemistry 21, 261-265 (1982). IRIYAMA, K., N. OGURA, and A. TAKAMIYA: A simple method for extraction and partial purification of chlorophyll from plant material, using dioxane. J. Biochem. 76, 901-904 (1974). KATO, M. and S. SHIMIZU: Chlorophyll metabolism in higher plants VI. Involvement of peroxidase in chlorophyll degradation. Plant Cell Physiol. 26, 1291-1301 (1985). KITA, N., K. TAKAMlYA, and Y. SHIOI: Partial purification and characterization of a soluble chlorophyllase from Chenopodium album. Research in Photosynthesis 3, 115-118 (1993). KRXUUER, B., B. JAUN, K. BORUIK, M. SCHELLENBERG, and P. MATILE: On the enigma of chlorophyll degradation: The constitution of a secoporphinoid catabolite. Angew. Chern. IntI. Ed. 30, 1315-1318 (1991). KRXuUER, B., B. JAUN, W. AMREIN, K. BORTLIK, M. SCHELLENBERG, and P. MATILE: Breakdown of chlorophyll: Constitution of a secoporphinoid chlorophyll catabolite isolated from senescent barley leaves. Plant Physiol. Biochem. 30, 333 - 346 (1992). LANGMEIER, M., S. GINSBURG, and P. MATILE: Chlorophyll breakdown in senescent leaves: Demonstration of magnesium-dechelatase activity. Physiol. Plant. 89, 347 -353 (1993). LAVAL-MARTIN, D. L.: Spectrophotometric method of controlled pheophytinization for the determination of both chlorophylls and pheophytins in plant extracts. Anal. Biochem. 149, 121-129 (1985). LUTHY, B., E. MARTINOIA, P. MATILE, and H. THOMAS: Thylakoid-associated «chlorophyll oxidase»: Distinction from lipoxygenase. Z. Pflanzenphysiol. 113, 423-434 (1984). MATILE, P.: Catabolism of chlorophyll: Involvement of peroxidase? Z. Pflanzenphysiol. 99, 475-478 (1980). MATILE, P., T. DUGGELIN, M. SCHELLENBERG, D. RENTSCH, K. BORTLIK, C. PEISKER, and H. THOMAS: How and why is chlorophyll broken down in senescent leaves. Plant Physiol. Biochem. 27, 595-604 (1989). MICHALSKI, T. J., C. BRADSHAW, J. E. HUNT, J. R. NORRIS, and J. J. KATZ: Triton X-I00 reacts with chlorophyll in the presence of chlorophyllase. FEBS Lett. 226, 72-76 (1987). OWENS, T. G. and P. G. FALKOWSKI: Enzymatic degradation of chlorophyll a by marine phytoplankton in vitro. Phytochemistry 21,979-984 (1982). PEISKER, c., T. DUGGELIN, D. RENTSCH, and P. MATILE: Phytol and the breakdown of chlorophyll in senescent leaves. J. Plant Physiol. 135, 428-432 (1989). PERKINS, H. J. and D. W. A. ROBERTS: Purification of chlorophylls, pheophytins and pheophorbides for specific activity determinations. Biochim. Biophys. Acta 58,486-498 (1962). RUDIGER, W. and S. SCHOCH: Abbau von Chlorophyll. Naturwissenschaften 76, 453 - 457 (1989). SCHELLENBERG, M., P. MATILE, and H. THOMAS: Breakdown of chlorophyll in chloroplasts of senescent barley leaves depends on ATP. J. Plant Physiol. 136, 564-568 (1990).

Chlorophyll breakdown by Chenopodium extract SCHELLENBERG, M., P. MATILE, and H. THOMAS: Production of a presumptive chlorophyll catabolite in vitro: requirement for reduced ferredoxin. Planta 191, 417 -420 (1993). SCHOCH, S., H. SCHEER, J. A. SCHIFF, W. RUDIGER, and H. W. SIEGELMAN: Pyropheophytin a accompanies pheophytin a in darkened light grown cells of Euglena. Z. N aturforsch. 36 c, 827 - 833 (1981).

SCHOCH, S. and F. X. VIELWERTH: Chlorophyll degradation in senescent tobacco cell culture (Nicotiana tabacum var. «Samsun»). Z. Pflanzenphysiol. 110, 309-317 (1983). SCHOCH, S., W. RUDIGER, B. LUTHY, and P. MATILE: 13 2-Hydroxychlorophyll a, the first product of the reaction of chlorophylloxidase. J. Plant Physiol. 115, 85-89 (1984). SHIMOKAWA, K., A. HASHIZUME, and Y. SHIOJ: Pyropheophorbidea, a catabolite of ethylene-induced chlorophyll a degradation. Phytochemistry 29,2105-2106 (1990).

421

SHIOJ, Y., Y. TATSUMI, and K. SHIMOKAWA: Enzymatic degradation of chlorophyll in Chenopodium album. Plant Cell Physiol. 32, 87 -93 (1991).

TERPSTKA, W. and J. W. J. LAMBERS: Interactions between chlorophyllase, chlorophyll a, plant lipids and Mt+. Biochim. Biophys. Acta 746, 23-31 (1983). WHYTE, B. J. and P. A. CASTELFRANCO: Breakdown of thylakoid pigments by soluble proteins of developing chloroplasts. Biochem. J. 290, 361-367 (1993). YOUNES, M. and U. WESER: Involvement of superoxide in the catalytic cycle of diamine oxidase. Biochim. Biophys. Acta 526, 644-647 (1978).

ZIEGLER, R., A. BLAHETA, N. GUHA, and B. SCHONEGGE: Enzymatic formation of pheophorbide and pyropheophorbide during chlorophyll degradation in a mutant of Chlorella fusca SHIHIRA et KRAUS. J. Plant Physiol. 132, 327 - 332 (1988).