SUMMARYSolubilization of senescent thylakoids from rape cotyledons in the presence of Triton X-100 was employed to establish an in vitro system that allowed the assessment of enzymatic conversion of phaeophorbide a into an uncoloured fluorescent chlorophyll catabolite, Bn-FCC-2. The action of the putative dioxygenase responsible for the cleavage of the porphyrin macrocycle depends on reduced ferredoxin as reductant. Apart from this thylakoidal catalyst, stromal protein is also required for the production of FCC-2 in vitro. The cleavage reaction does not occur with phaeophorbide b as substrate. Saturation kinetics with phaeophorbide a as substrate yielded an apparent K^-value of c. 200 //M. The enzyme contains iron as suggested by inhibitory effects of appropriate chelators. Enzyme activity lost upon treatment with bipyridyl was partly restored in the presence of Fe-ions; other metal ions such as Cu, Zn and Co were ineffective. The enzyme is absent in the thylakoids of mature green cotyledons. It appears upon the induction of foliar senescence and reaches the highest levels towards the end of the yellowing process.

Abstract.Chlorophyllase catalyzes the first step in the catabolic pathway of chlorophyll. It is a constitutive enzyme located in chloroplast membranes. In isolated plastids the hydrolysis of the endogenous chlorophyll does not take place unless the membranes are solubilized in the presence of detergent. The structural latency of chlorophyllase activity appears to be due to the differential locations of substrate and enzyme within the plastids. Envelope membranes prepared from both chloroplasts and gerontoplasts contain chlorophyllase activity. The isolation of envelopes is associated with a marked increase in chlorophyllase activity per unit of protein.Yields of chlorophyllase and of specific envelope markers in the final preparations are similar, suggesting that the enzyme may be located in the envelope. It is hypothesized that the breakdown of chlorophyll during leaf senescence requires a mechanism that mediates the transfer of chlorophyll from the thylakoidal pigment-protein complexes to the sites of catabolic reactions in the envelope.

SUMMARYChlorophyll breakdown in senescent leaves proceeds in essentially three steps: dephytylation by the action of ehlorophyllase; conversion of chlorophyllide to phaeophorbide by Mg-dechelatase; and oxygenolytic cleavage of the chlorin-macrocycle by a newly discovered dioxygenase. The metabolic lesion responsible for high retention of chlorophyll during foliar senescence in a mutant genotype of meadow fescue {Festuca pratensis Huds.) was located in the third step of the breakdown pathway. Senescent leaves of both the normally yellowing reference genotype, cv Rossa, and the non-yellowing mutant Bf993 were shown to be competent with regard to ehlorophyllase and Mg-dechelatase, On the other hand, thylakoids isolated from senescent leaves of ev, Rossa were able to carry out oxygenolysis of phaeophorbide into a colourless fluorescent catabolite in vitro, whereas Bf993 thylakoids were deficient in this activity. It is concluded that the Sid locus, a mutant allele of which is responsible for the stay-green eharacter, encodes or regulates the gene for, phaeophorbide a dioxygenase.

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.