CHLOROPHYLL BREAKDOWN IN SENESCENT CHLOROPLASTS - CLEAVAGE OF PHEOPHORBIDE A IN 2 ENZYMATIC STEPS

The cleavage of pheophorbide (Pheide) a into primary fluorescent chlor ophyll (Chi) catabolites (pFCCs) in senescent chloroplasts was investi gated. Chloroplast preparations isolated from senescent canola (Brassi ca napus) cotyledons exhibited light-dependent production of pFCC when assay mixtures were supplemented with ferredoxin (Fd). pFCC productio n in detergent-solubilized membranes was dependent on the presence of an Fd-reducing system. Pheide a cleavage required the action of two pr oteins, Pheide a oxygenase and a stroma protein. In the absence of str oma protein, Pheide a oxygenase converted Pheide a into a red Chi cata bolite (RCC), the presumptive intermediary product of Pheide a cleavag e. Incubation of the stroma protein (RCC reductase) together with chem ically synthesized RCC resulted in the production of three different F CCs. Two of these catabolites were identical to the pFCCs from canola or barley (Hordeum vulgare) (pFCC-1) and sweet pepper (Capsicum annuum ) (pFCC-2), respectively. Thus, the conversion of Pheide a to pFCC cou ld be demonstrated to proceed in two consecutive steps, and both react ions depended on reduced Fd as the source of electrons. The function o f Fd in Chi breakdown in vivo is corroborated by the marked retention of this protein until the late stages of senescence, as demonstrated b y immunoblotting.