A Novel Type of Chlorophyll Catabolite in Senescent Barley Leaves

J. PlantPhysiol. Vol. 136. pp. 161-165 (1990)

A Novel Type of Chlorophyll Catabolite in Senescent Barley Leaves KARLHEINZ BORTLIK, CHRISTIAN PEISKER,

and PHILIPPE MATILE

Department of Plant Biology, University of Zurich, Zollikerstr. 107, CH-8008 Zurich, Switzerland Received December 4, 1989 . Accepted January 3, 1990

Summary A group of novel non-green catabolites of chlorophyll is presented. The native compounds are colourless but on thin layer silicagel plates they are readily oxidized to rust-coloured pigments (RPs). In senescent primary leaves of barley the predominant RP, RP 14, is progressively accumulated. It is located in the vacuoles of senescent mesophyll cells. The origin of RP 14 from chlorophyll has been demonstrated by means of radio labelling of porphyrins during the greening of etiolated barley seedlings and tracing of the label during subsequent induction of senescence. RPs appear to represent secondary or even final products of chlorophyll breakdown.

Key words: Hordeum vulgare, foliar senescence, chlorophyll, catabolite, vacuole. Abbreviations: Chi = chlorophyll; FC = lipofuscin-like fluorescent compound; RP = rust-coloured pigment; HPLC = high-performance liquid chromatography; TLC = thin layer chromatography.

Introduction Senescent leaves of meadow fescue (Duggelin et al. 1988 a), barley (Duggelin et al. 1988 b) as well as of a number of other plant species (unpublished results) contain lipofuscin-like fluorescent compounds (FC) which have tentatively been identified as products of chlorophyll breakdown. This identification was originally deduced from the absence of FCs in senescent leaves of a genotype of Festuca pratensis which is defective in its ability to degrade chloroplastic porphyrins (Thomas 1987). The abundance of FCs in senescent leaves was found to be positively correlated with the rate of chlorophyll breakdown. A group of pink-coloured compounds which has been identified earlier with catabolites of chlorophyll (Matile et al. 1987), turned out to represent derivatives of FCs formed upon the extraction of leaves with acidic solvents (Matile et al. 1989). Hence, a number of arguments favouring the origin of pink pigments from chlorophyll (Matile et al. 1987, 1988) are also valid with regard to FCs. Both pink pigments and FCs have been localised in the vacuoles of barley mesophyll cells (Matile et al. 1988, Duggelin © 1990 by Gustav FIscher Verlag, Stuttgart

et al. 1988 b). Yet, of the two principal FCs present in senescent barley leaves, FC 1 and FC 2, a small but significant pool of FC 2 is located in the chloroplasts (Duggelin et al. 1988 b). The chloroplastic FC 2 appears to represent a transient pool of primary product of Chi breakdown. This is suggested by the recent discovery of an ATP-dependent reaction in isolated senescent chloroplasts yielding FC 2 at the expense of chlorophyll ide a (Schellenberg et al. 1990). Since the bulk of FC 2 is located in the vacuoles it must be assumed that the primary product(s) of chloroplastic porphyrin breakdown are exported from the chloroplasts and transported across the tonoplast. Levels of FCs in barley primary leaves decline as rates of Chi breakdown slow down at advanced stages of foliar senescence (Duggelin et al. 1988), suggesting that reactions taking place in the cell sap result in the conversion of FCs into secondary Chi catabolites which hitherto remained undiscovered. We now present data about a novel group of compounds occurring in senescent leaves and having features of secondary, perhaps even final products of porphyrin breakdown in plants.

162

KARLHEINZ BORTLIK, CHRISTIAN PEISKER, and PHIUPPE MATILE

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Materials and Methods Plant material, induction of senescence The cultivation of barley, Hordeum vulgare cv. Gerbel, as well as the experimental conditions of dark-induced senescence in the excised primary leaves have been described elsewhere (Matile et al. 1987).

Extraction and enrichment ofRPs For analytical purposes, the leaves were ground in the presence of 50% methanol (4mL per 500mg fw). The homogenate was transferred to a centrifuge tube, thoroughly mixed with chloroform (0.25 mL per mL extract) to remove the pigments and centrifuged (5min 2000 x g). The aqueous phase containing RPs and FCs was used for HPLC. For preparative purposes, frozen leaves sampled at day 5 of dark induced senescence were placed in a garlic squeezer and processed into a slightly turbid press sap. The sap was extracted 3 x with ethylacetate and extracts discarded. The sap was now adjusted to pH 0.5 -1 with HC] and RPs extracted 3 x with ethylacetate. RPs were subsequently partitioned into 200 mM phosphate buffer pH 7.0. After acidification of the aqueous phase with HCI to pH 0.5-1 RPs were loaded onto an RP-18 cartridge preequilibrated with 0.2 N KCI/HCI buffer pH 1. The cartridge was washed with dist. water acidified with HCI to pH 3, and finally the RPs eluted with methanol. TLC chromatography. Silicagel 60 plates (Merck) and a solvent system (chloroform - methanol - water 65: 25: 4 by vol.) described by Merzylak et al. (1983) were employed. Treatments with Ehrlich reagent (4-dimethylaminobenzaldehyde-HCI) were performed according to the procedure outlined in E. Merck, Darmstadt, Anfarbereagenzien (1984).

HPLC chromatography Aliquots of 20JLL were injected into the HPLC RP-12 column (Permacoat, 3 I'm, 125 x 4.6 mm, Stagroma, Wallisellen, Switzerland). Two mixtures of acetonitrile, tetrahydrofurane, formic acid, and water: A(10%, 8 %,1 %,81 %) and B (30%,8%,1 %, 61 %; v/v), were employed for elution. The column was equilibrated with solvent A prior to injection, which coincided with the start of a linear gradient from A to B within 10 min at a flow rate of 0.5 mL min -1.

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The subsequent isocratic run with solvent B took 20 min. RPs were monitored at 315 nm (Linear uvis 204) using a Shimadzu C-R 3 A integrator. Under the above conditions, the main component of RPs eluted after ca. 14 min.

Preparation ofprotoplasts, chloroplasts and vacuoles. It has been described elsewhere (Matile et al. 1988). Determination of chlorophyll. It followed the method of Lichtenthaler and WeJlburn (1983). Radiolabelling ofchlorophyll. Intact etiolated barley seedlings were exposed to light and fed during the greening with 4{14C}aminolaevulinic acid via the roots as described elsewhere (Peisker et al. 1990). For inducing senescence after completion of the greening process, the roots were excised and the shoots placed in permanent darkness. HPLC analysis and radiomonitoring of chlorophyll catabolites were performed as detailed in Peisker et al. (1990).

Results and Discussion As reported earlier (Matile et a1. 1987) TLC of extracts from senescent barley leaves resolves a number of compounds with properties distinct from those of FCs and pink pigments. Some of them are not coloured unless the developed silicagel plates are exposed to air and light. In the oxidised state they assume a colour reminding of rust and are, therefore, referred to as «rusty pigments» (RP). They are absent in the presenescent barley leaves as well as in the senescent leaves of the non-yellowing genotype Bf 993 of Festuca pratensis. These observations have suggested that the RPs represent products of ChI breakdown. As shown in Fig. 1 RPs react with acidic4-dimethylaminobenzaldehyde (Ehrlich reagent), areagent used for the detection of amines, pyrrols and derivatives thereof. One of the RPs is always particularly abundant in extracts from senescent leaves and was selected for a detailed study. It is referred to as RP 14 according to its retention time of ca. 14 min in the HPLC system used (see Fig.4). The kinetics of accumulation of RP 14 in the course of dark induced senescence is remarkably different from that of FCs. Whereas the levels of FCs peak when the rate of ChI de-

163

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Fig.2: Changes of total chlorophyll and of the novel chlorophyll catabolite, RP 14, during dark-induced senescence in primary leaves of barley. For comparison the changes of a preciously described catabolite, FC 2 (Diiggelin et al. 1988 b), are marked with a dotted line. Table 1: Changes of Chi, FC 2 and RP 14 after exposure of senescent primary leaves to the natural daylight (L) of the laboratory for 10 h. Prior to the light treatment the leaves had been incubated in permanent darkness (DD) for 48 h. Contents of RP 14 and FC 2: integrated peak areas (315 nm, fluorescence) are given as percentage of the values measured after 48 h of incubation. 48h DD Chlorophyll FC 2 RP 14

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gradation is maximal and then decline, the accumulation of RP 14 is progressive and ceases towards the end of the senescence period when the ChI is largely broken down (Fig. 2). This observation suggests that RP 14 could represent a secondary or even final product of ChI breakdown. Light has a strong effect in retarding foliar senescence. When detached primary leaves were first induced to senesce in permanent darkness for 2 days and then transferred to light, the degradation of ChI was stopped or even reversed. During the light period the FCs disappeared whilst the accumulation of RP 14 continued (Table 1). This finding can be interpreted to indicate that FCs represent a pool of primary catabolites which in the light is no longer fed but only drained to yield the secondary catabolites, RPs. Although the purification of RP 14 in its native colourless form for studies of its chemical structure appears to be difficult because the compound is so readily oxidised, an effort was made to develop a convenient protocol for rapid isolation on a large scale. Chromatography on reversed phase

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columns requires an acidic solvent suggesting that RP 14 is acidic in nature. Indeed, it binds readily to anion exchange resins. Employing the procedure outlined in «Materials and Methods». RP fractions from HPLC runs (Fig. 3) were collected and used for the reading of spectra of the freshly prepared as well as of the oxidised compound (Fig. 4). The native form of RP 14 is characterised by its absorption maximum at the 315 nm. In the oxidized state the broad and flat minor maximum at ca 435 nm is responsible for the rustcoloured appearance of the compound on TLC plates. The lability of native RP 14 appears also from the observation that repeated rechromatography of the isolated compound yields similar chromatograms as depicted in Fig. 3. It seems that the series of minor peaks at the polar and apolar side of RP 14 are derivatives which in vitro are continuously produced from RP 14. Table 2: Localization of RP 14 in vacuoles prepared from mesophyll protoplasts. For comparison, the distribution of FC 2 in vacuoles and chloroplasts is also given. Protoplasts were prepared from excised leaves induced to senesce for 3 days in permanent darkness. The analysis of compartmentation was performed as detailed in Diiggelin et al. (1988 b). (*), peak detectable but not integrated. Distribution (%) chloroplasts vacuoles Chlorophyll a-Mannosidase RP 14 FC2

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164

KARLHEINZ BORTLIK, CHRISTIAN PEISKER, and PHILIPPE MATILE complexes (Thomas et ai. 1989). There follows an oxidative step and an ATP-dependent reaction resulting in the accumulation of FC 2 in the senescent chloroplasts (Schellenberg et ai. 1990). This step is probably decisive for both, the release of the porphyrin from the apoprotein and for the cleavage of the macrocycle. FC 2 is subsequently transported to the vacuole. Further reactions taking place in the cell sap may finally yield the secondary catabolites, RPs, described in the present paper. Whether the ATP-requirement of the production of FC 2 is connected with a modification of the chlorophyll-binding proteins resulting in the dissociation of the complexes, or else with a phosphorylation of the porphyrin cannot be decided on the basis of available data. It is now urgent to purify both FC 2 and RP 14 in order to analyse their structure and eventually deduce the nature of biochemical reactions involved. Work is currently being done to prepare native RP 14 sufficient in quantity and stability to eventually establish its chemical structure.

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Fig.4: Absorption spectra of RP 14. A, native form immediately after purification with HPLC. B, oxidised form obtained upon the storage of dry purified RP 14 in air for 2 weeks. The oxidised pigment was dissolved in methanol. The intracellular localisation of RP 14 in protoplasts prepared from the mesophyll of senescent leaves has yielded evidence for the absence in the chloroplasts. The data presented in Table 2 strongly suggest that RP 14 is located exclusively in the vacuoles. Of all the putative catabolites of chlorophyll described so far, only the lipofuscin-like fluorescent compound FC 2 is partially located in the chloroplasts (Table2, Diiggelin et al. 1988 b). An unambiguous identification of products of ChI breakdown in senescent barley leaves has been achieved through radiolabelling of ChI during greening of etiolated barley seedlings (Peisker et ai. 1990). Feeding 4-[l4C}aminolaevulinic acid providing label in the pyrrol rings only, we succeded to recover over 70 % of the 14C incorporated in the ChI. In the course of senescence radioactivity in the apolar ChIs gradually decreased concomitant with increasing proportions of label appearing in polar compounds. HPLC analysis of the aqueous phase of extracts from senescent leaves has yielded unambiguous evidence that RP 14 is one of the major compounds which carries radioactivity when ChI is broken down (Fig. 5). The identification of RP 14 as a vacuolar catabolite of ChI taken together with previous observations on ChI breakdown in chloroplasts may be converted into the following hypothetical pathway ChI catabolism. Breakdown is initiated by dephytylation to yield chlorophyllide which remains associated with apoproteins of the pigment-protein

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Fig. 5: Demonstration of radiolabelling of RP 14 in senescent primary barley leaves. A, Radiochromatogram; B, uv-absorption at 320 nm of the same chromatogram. The specific labelling of chlorophyll during greening of etiolated seedlings is detailed in Peisker et al. (1990). Five excised leaves senesced in the dark for 7 days (180 mg fw) were extracted in 500 ILL 50 % methanol. The extract was centrifuged and 20 ILL injected into the HPLC system. In 6 runs the eluates between 13.5 and 14.5 min were collected. The addition of NaCl to the combined eluates yielded a phase separation with an apolar phase of tetrahydrofurane containing RP 14. This phase was concentrated with a stream of N2 and injected to yield the chromatograms shown. The peaks of uv absorption appearing between 3 and 6 min are due to senescence-irrelevant secondary compounds which are abundant in barley leaves; they were only incompletely removed upon the initial HPLC runs with concentrated leaf extract.

Catabolite of chlorophyll

Acknowledgements We are indepted to Dr. Felix Keller and Dr. B. Kriiutler for technical advice, to E. Disler for help with the manuscript, and to Dr. W. Egger for technical assistance.

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- - - - Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells. Proc. Natl. Acad. Sci. U.S.A. 85, 9529-9532 (1988). MATlLE, PH., TH. DUGGELlN, M. SCHELLENBERG, D. RENTSCH, K. BORTLlK, CH. PEISKER, and H. THOMAS: How and why is chlorophyll broken down in senescent leaves? Plant Physiol. Biochem. 27,595-604 (1989). MERZLYAK, M. N., V. B. RUMYANTSEVA, V. V. SHEVYRYOVA, and M. V. GUSEV: Further investigations of liposoluble fluorescent compounds in senescing plant cells. J. Exp. Bot. 34,604-609 (1983). PEISKER, c., H. THOMAS, and PH. MATlLE: Radiolabelling of chlorophyll for studies of catabolism. J. Plant Physiol. (in press) 1990. SCHELLENBERG, M., PH. MATlLE, and H. THOMAS: Breakdown of chlorophyll in chloroplasts of senescent barley leaves depends on ATP. J. Plant Physiol. (in press) 1990. THOMAS, H.: Sid: a Mendelian locus controlling thylakoid membrane disassembly in senescing leaves of Festuca pratensis. Theor. Appl. Genet. 73, 551-555 (1987). THOMAS, H., K. BORTLlK, D. RENTSCH, M. SCHELLENBERG, and PH. MATlLE: Catabolism of chlorophyll In vivo: significance of polar chlorophyll catabolites in a non-yellowing senescence mutant of Festuca pratensts Huds. New Physologist, New Phytol. 111, 3 - 8 (1989).