Chloroplast biogenesis: Biosynthesis and accumulation of protochlorophyll by isolated etioplasts and developing chloroplasts

Abstract

Etioplasts and developing chloroplasts were isolated from etiolated Cucumis cotyledons that were irradiated with white fluorescent light for various periods of time. The endogenous porphyrins and phorbins of the isolated plastids were partitioned between hexane, hexane-extracted aqueous acetone and a lipoprotein precipitate. Spectrofluorometric determinations were performed on these fractions without further fractionation. For quantitative determinations, the fluorescence amplitudes of the various fluorescent components were corrected for fluorescence emission overlap by sets of simultaneous equations. Developing chloroplasts contained endogenous amounts of the following metabolites: Protochlorophyllide, protochlorophyllide ester, Mg-protoporphyrin monoester + longer-wavelength metalloporphyrins and protoporphyrin. The protochlorophyll pool consisted mainly of protochlorophyllide. The latter was heterogeneous and consisted of at least two chemically related protochlorophyllides. In contrast to developing chloroplasts, irradiated etioplasts contained mostly protochlorophyllide ester and smaller amounts of protochlorophyllide. Upon incubation of developing chloroplasts and irradiated etioplasts with δ-aminolevulinic acid and cofactors (coenzyme A, glutathione, adenosine triphosphate, nicotinamide adenine dinucleotide, methyl alcohol, magnesium, potassium and phosphate), a net synthesis and accumulation of protochlorophyllide, Mg-protoporphyrin monoester + longer-wavelength metalloporphyrins, protoporphyrin, coproporphyrin and uroporphyrin were observed. Small amounts of zinc-coproporphyrin and zinc-uroporphyrin were also formed. In some experiments a net synthesis of protochlorophyllide ester was also observed. This report represents the first account of the unambiguous net synthesis of protochlorophyll in vitro.