Developing shoots of rape seedlings (Brassica napus 1.) were excised and fed with 4-['4C]5-aminolevulinic acid to label the pyrroles in chlorophyll (Chl) synthesized during the final phase of expansion and greening of the cotyledons. About 80% of 14C taken up into the cotyledons was incorporated into Chl. The subsequent incubation of labeled shoots in permanent darkness caused the rapid loss of labeled Chl while increasing proportions of 14C appeared in the fradion of water-soluble compounds. Reversedphase high performance liquid chromatography resolved three nonfluorescent polar catabolites of Chl-porphyrin that were progressively accumulated as senescence advanced. At intermediate stages of senescence, the cotyledons contained a fluorescent radioadive derivative of Chl that was also detectable, together with traces of other putative fluorescent catabolites, in isolated senescent chloroplasts. l h e nonfluorescent catabolites, identified by means of radiolabeling, were also found to accumulate in attached cotyledons senescing under photoperiod; under these conditions, one of the compounds, NCC-1, was particularly abundant. The catabolites of rape exhibited the same ultraviolet spedra, characterized by a maximum at 320 nm, as a previously reported seco-

The chemical strudures of some colorless catabolites that accumulate in senescent leaves have been established recently (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile Such studies suggest that oxygenolytic cleavage of chlorophyllporphyrin may occur by the action of a dioxygenase. We have attempted to demonstrate such an enzyme activity and to explore the requirements of the cleavage reaction in a reconstituted system of chloroplast (Chlpl) components prepared from senescent rape (Brassica napus 1.) cotyledons. lntact senescent Chpls (also referred to as gerontoplasts) contain small amounts of two fluorescent chlorophyll catabolites, Bn-FCC-1 and Bn-FCC-2, probably representing primary cleavage produds. Upon the incubation of Cpls in the presence of glucose-6-phosphate (Clc6P) or ATP, these catabolites (predominantly FCC-1) were produced in organello. In a reconstituted system of thylakoids and stroma fraction the FCCs (predominantly FCC-2) were produced in the presence of ferredoxin (Fd) and cofactors (NADPH, Clc6P) helping to keep Fd in the reduced state. Reduced Fd could not be replaced by other eledron donors, suggesting that the putative dioxygenase requires Fd for the operation of its redox cycle. Produdion of FCC-2 did not occur in the absence of oxygen and it was inhibited by chelators of Fez+. The contributions to the production of FCCs from both parts of the reconstituted system, thylakoids and stroma, are heat labile. The enzymic process in the thylakoids yields pheophorbide a, the presumptive precursor of FCCs. However, native senescent thylakoids could not be replaced as a "substrate" by free pheophorbide a. The stromal enzyme appears to have an affinity for senescent thylakoids; thus, "loaded" thylakoids capable of FCC production in the presence of Fd and cofactors were obtained upon homogenization of senescent cotyledons in a medium containing sorbitol and ascorbate. Such thylakoids were inactive if prepared from mature green cotyledons. As senescence was induced, the capacity to generate FCCs appeared and peaked when about half of the chlorophyll had disappeared from the cotyledons. The effediveness of a relevant inhibitor showed that cytoplasmic protein synthesis was required for inducing the catabolic machinery in the loaded thylakoids. Thylakoids from mature Chlpls were ineffective as substrate of the stromal enzyme prepared from Cpls. However, senescent thylakoids yielded FCCs if challenged with stroma from either Chlpls or Cpls. Therefore, the stromal part of the system is likely to be a constitutive enzyme, and the pace-setting step of the This work was financially supported by the Swiss National Science Foundation.

Langmeier, M., Ginsburg, S. and Matile, Ph. 1993. Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity. -Physiol. Plant. 89: 347-353.The action of Mg-dechelatase was brought to light by incubating senescent rape cotyledons or chloroplasts under conditions which prevented the oxidative cleavage of chlorophyll-porphyrin. The accumulation of chlorophyllide and pheophorbide taking place under such conditions was considered as a measure of apparent activities of chlorophyllase and dechelatase, respectively. In excised cotyledons metal chelators such as 2,2'-dipyridyl and o-phenanthroline caused a marked accumulation of pheophorbide a, without affecting the apparent activity of chlorophyllase. Treatment of cotyledons with an inhibitor of cytoplasmic protein synthesis D-2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide (D-MDMP) caused a reduced accumulation of pheophorbide a in the presence of dipyridyl, suggesting that the appearance and maintenance of Mg-dechelatase activity in senescent cotyledons requires continuous cytoplasmic protein synthesis. In isolated senescent chloroplasts (gerontoplasts) the cleavage of chlorophyll-porphyrin requires the supplementation with glucose-6-phosphate (Glc6P). Upon the incubation of gerontoplasts in the absence of Glc6P, a conspicuous accumulation of pheophorbide a occurred. Much smaller pools of pheophorbide a were produced when porphyrin cleavage was allowed in the presence of Glc6P. These phenomena were not observed in pre-senescent chloroplasts. In contrast to the apparent Mg-dechelatase activity, chlorophyllase activity did not change in a senescent-specific fashion. The lysis of gerontoplasts by freezing and thawing caused an enhancement of apparent chlorophyllase activity whereas the activity of Mgdechelata.se was lower than in the intact organelles. In the pre-sene.scent chloroplasts, lysis evoked a small apparent Mg-dechelatase activity, suggesting that in a latent form this enzyme may be present even before the onset of foliar senescence.

Dedicated to Prof. Duilio Arzgoni on the occasion of his 65th birthday (30.1X.93) The experiments leading to the isolation and to the elucidation of the constitution of Bn-NCC-I, a colourless non-fluorescent chlorophyll catabolite from senescent cotyledons of rape (Brussicn nupus L,), are described. A series of fast-atom-bombardment (FAB) mass and 'H-and "C-NMR spectral experiments are used to determine the constitution of the catabolite Bn-NCC-I. The structural information available indicates Bn-NCC-I to be a l-formyl-19-oxobilane, structurally related to 'RP 14, isolated earlier from artificially aged primary leaves of barley. The major differences between the constitution of the metal-free chlorophyll pheophorbide a and that of Bn-NCC-I concern oxygenolytic opening of the porphinoid macrocycle at C(4)-C(5), saturation at the other meso positions, hydrolysis of the methyl-ester function, and functionalization by a malonic-acid unit of the side chain at C(8). This work provides for the first time the structural data of a chlorophyll-degradation product from senescent plant leaves formed under normal growth conditions. Introduction. -Little is known to date of the natural fate of the chlorophylls [l] [2]. The high visibility of the phenomenon of the colouring of the autumn leaves every year again provides motivation to unravel this enigma. The massive amounts of material involved in chlorophyll breakdown on this earth (estimated to be ca. lo9 t annually) 111 also demand knowledge on this subject. However, it has proven difficult to address this problem as the breakdown of chlorophyll appears to occur without leaving behind a trace [2]. A breakthrough was lately achieved in this field: in the primary leaves of barley (Hordeurn oulgare) that were aged artificially by storage in the dark, several colourless chlorophyll catabolites could be localized oia "C-labelling [3]; the constitution of one of these, of the nonfluorescent chlorophyll catabolite Ho-NCC-1 ('RP-14') could be deduced by spectroscopic means by a collaborative effort of our laboratories 141. In related recent studies, the red pigment from the green algae Chlorella protothecoides [5a] was identified as a linear tetrapyrrole, apparently derived from chlorophylls by cleavage of the chlorin macrocycle at the a-meso -position (C(5)) also [5b, c]. On the other hand, the light-emitting substance from krill (Euphausia pacfica) [6a] and the dinoflagellate luciferin [6b] were discovered to be related to the chlorophylls by oxidative cleavage of the chlorin macrocycle at the 6-meso-position (C(20)) [6c, d].We report here for the first time on the structure of a chlorophyll catabolite from senescent plant leaves that degreened under natural growth conditions: in the cotyledons of the dicot rape (Bvassica napus) which aged naturally under the photoperiod with loss of their chlorophylls, a correlated accumulation of several colourless pyrrolic compounds