No abstract

Red chlorophyll (Chl) catabolite (RCC) reductase, which catalyzes the reaction of an intermediary Chl catabolite (RCC) in the two-step cleavage reaction of pheophorbide (Pheide) a into primary fluorescent catabolites (pFCCs) during Chl breakdown, was characterized and partially purified. RCC reductase activity was present at all stages of barley leaf development and even in roots. The highest specific activity was found in senescent leaves, which were used to purify RCC reductase 1000-fold. Among the remaining three proteins, RCC reductase activity was most likely associated with a 55-kD protein. RCC reductase exhibited saturation kinetics for RCC, with an apparent Michaelis constant of 0.6 mM. The reaction depended on reduced ferredoxin and was sensitive to oxygen. Assays of purified RCC reductase with chemically synthesized RCC as a substrate yielded three different FCCs, two of which could be identified as the stereoisomeric pFCCs from canola (Brassica napus) (pFCC-1) and sweet pepper (Capsicum annuum) (pFCC-2), respectively. In the coupled reaction with Pheide a oxidase and RCC reductase, either pFCC-1 or pFCC-2 was produced, depending on the plant species employed as a source of RCC reductase. Data from 18 species suggest that the stereospecific action of RCC reductase is uniform within a plant family. ~~~~~~~~~~~~~~Of the enzymes that constitute the pathway of Chl breakdown in senescent leaves (for review, see , PaO deserves special attention for severa1 reasons. It is responsible for the oxygenolytic opening of the porphyrin macrocycle and therefore could be considered to be the center piece of the catabolic system. Indeed, the retention of Chl in senescent leaves of mutant genotypes of Festuca pratensis and Pisum sativum has been demonstrated to be due to deficient PaO activities. Its key role in Chl breakdown is also emphasized by the strictly senescence-specific regulation of its activity . In contrast, other catabolic enzymes appear to be present in the (potentially) active form at a11 stages of leaf development.PaO is located in the envelope of senescent chloroplasts and participates in a reaction in two steps by which Pheide a is converted to a linear tetrapyrrolic product (Fig. 1). As demonstrated in a preceding report (Rodoni et al., 1997), in the first step PaO '

In angiosperms the key process of chlorophyll breakdown in senescing leaves is catalyzed by pheophorbide a oxygenase and RCC reductase which, in a metabolically channeled reaction, cleave the porphyrin macrocycle and produce a colourless primary catabolite, pFCC. RCC reductase is responsible for the reduction of the C20/C1 double bond of the intermediary catabolite, RCC. Depending on plant species, RCC reductase produces one of the two C1 stereoisomers, pFCC‐1 or pFCC‐2. Screening of a large number of taxa for the type of RCCR revealed that the isomer produced is uniform within families. It also revealed that type RCCR‐2 is predominant; RCCR‐1 seems to represent a recent derivation which in unrelated lineages has evolved independently from RCCR‐2. A third type of pFCC was produced by RCCR from basal pteridophytes and some gymnosperms; its structure is unknown. Collectively, the data suggest that the pathway of chlorophyll breakdown is very conserved in vascular plants. RCCR appears to represent a decisive addition to the catabolic pathway: it allows terrestrial plants to metabolize the porphyrin part of the chlorophyll molecule to photodynamically inactive final products that are stored in the vacuoles of senescing mesophyll cells.

Key words: Carotenoids, chlorophyll, ripening, senescence, tomato. The gf tomato mutant, which retains chlorophyll during ripening, has been found to be affected in leaf senes-

During the yellowing of leaves the porphyrin moiety of chlorophyll is cleaved into colorless linear tetrapyrrolic catabolites, which eventually are deposited in the central vacuoles of mesophyll cells. In senescent cotyledons of rape, Brassica napus, three nonfluorescent chlorophyll catabolites (NCCs), accounting for practically all the chlorophyll broken down, were found to be located in the vacuoles (vacuoplasts) prepared from protoplasts. Transport of catabolites across the tonoplast was studied with vacuoles isolated from barley mesophyll protoplasts in conjunction with a radiolabeled NCC, Bn-NCC-1, prepared from senescent rape cotyledons. The uptake of Bn-NCC-1 into vacuoles was against a concentration gradient and strictly dependent on MgATP and it followed saturation kinetics with a K m of approximately 100 M. Although the hydrolysis of ATP was required, transport was apparently independent of the vacuolar proton pumps: accumulation of the NCC occurred both in the presence of the H ؉ -ATPase inhibitor bafilomycin and after destroying the ⌬pH between the vacuolar sap and the medium. ATP could be replaced by GTP or UTP, and the transport was inhibited in the presence of vanadate. Chlorophyll catabolites isolated from senescent barley leaves competed with the rape-specific substrate for uptake into the vacuoles. Compounds such as the glutathione conjugate of N-ethylmaleimide and taurocholate, which are known to be transported across the tonoplast in a primary active mode, did not significantly inhibit uptake of Bn-NCC-1. Although the heme catabolites biliverdin and bilirubin inhibited the uptake of the NCC, this effect is caused by unspecific binding to the vacuolar membrane rather than to the specific inhibition of carrier-mediated transport. Taken together, the results demonstrate that barley mesophyll vacuoles are constitutively equipped with a directly energized carrier that transports tetrapyrrolic catabolites of chlorophyll into the vacuole.