Chloroplast culture: the chlorophyll repair potential of mature chloroplasts incubated in a simple medium.

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCl/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protocholorphyllide from exogenous delta-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between delta-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from delta-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of delta-amino[4-(14)C]levulinic acid into [14C]protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E430-F631) was shown to be neither protochlorophyllide nor zinc-prototochlorophyllide but and a fluorescence emission maximum at 631 nm (F631) in methanol/acetone (4 : 1, v/v). Cp(E430-F631) WAS SHOWN TO BE NEITHER PROTOCHLOROPHYLLIDE NOR ZINC-PROTOTOCHLOROPHYLLIDE BUT an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.