Biosynthesis and Accumulation of Microgram Quantities of Chlorophyll by Developing Chloroplasts in Vitro

Abstract

Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, 6-aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro. Rebeiz et al. (12,13,18) have shown that cell-free homogenates and etioplasts isolated from cucumber cotyledons and incubated with '4C-ALA' and cofactors, were capable of synthesizing '4C-Mg protoporphyrin monoester, "'C-Protochlorophyll and "C-chlorophyll a and b. These findings led to the conclusion that etioplasts and developing chloroplasts contained the biosynthetic machinery required for chlorophyll biosynthesis from ALA (12, 13). The foregoing results were subsequently

Kinetics and regulation of synthesis of the major polypeptides of thylakoid membranes in Chlamydomonas reinhardtii y-1 at elevated temperatures

“…This suggestion is supported by the observation that wild type cells, which synthesize chlorophyll in the dark (24,49), also are capable of making b and c in the dark. However, since the site of synthesis of chlorophyll is within chloroplasts (42,43), a mechanism by which synthesis ofb and c on cytoplasmic ribosomes can be controlled by chlorophyll or its precursors is not obvious. Moreover, it is not known whether b and c are primary products of translation or whether they are derived from a larger precursor.…”

“…THE JOURNAL OF CELL BIOLOGY" VOLUME 70, 1976" pages 326-337 other membrane lipids are synthesized within the chloroplast (8,11,42,43), but the membrane proteins are synthesized both inside and outside the organelle (12,17,30). Indeed, as work on the effects in vivo of inhibitors of protein synthesis (10,13,17,21,30) and with isolated chloroplasts (4, 9) has shown, the major integral polypeptides of this membrane are synthesized outside the chloroplast on cytoplasmic ribosomes.…”

Protochlorophyll and Chlorophyll Biosynthesis in Cell-Free Systems from Higher Plants

“…This is indicated by the re cent findings of Henningsen & Kahn (66), who reported that protochloro phyll(ide) subunits of 60,000 to 100,000 molecular weight and presumably con taining one protochlorophyll chromophore per subunit were photoconverted. (128,130). 14(;-chlorophyll a formed from 14(;-ALA was purified to constant specific radioactivity and degraded to 14(; maleirnides (125).…”

“…The enzyme was assayed in the presence of 0.1 % Triton X-lOO. Protoporphyrin IX was methylated at one tenth the rate of Mg protoporphyrin.In conclusion, all the evidence available so far derived fr om R. sp heroides chromatophores(43, 16 5), the solubilized Euglena enzyme (25), chloroplast preparations ( 119 ), and crude homogenates (3 1) fr om higher plants, indicate that Mg protoporphyrin is the best substrate fo r the methylation reaction.FORMATION OF PROTO CHLOROPHYLLAll the enzymes necessary for the formation of protochlorophyll fr om added ALA are obviously present in isolated etioplasts (Rebeiz & Caste1franco 124) and developing chloroplasts(Rebeiz et al 128,130). It was shown that crude cotyledonary homogenates as well as isolated etioplasts were capable of convert ing HC-ALA into HC-protochlorophyllide (IXb) and HC-protochlorophyllideester (IXc) (124).…”

Chlorophyll Metabolism in Etiolated Gherkin Seedlings I. Photoinhibition of Chlorophyll Accumulation

“…The total amount of Chl was calculated after Bruinsma (1963) and is given in mg per 30 cotyledons. We were unable to extract either Pchl or Chl from plants irradiated for periods up to 8 h. Rebeiz et al (1973) could not extract Chl completely with organic solvents from isolated Cucumis chloroplasts after in oitro incubation, whereas Hardy et al (1970) extracted Chl with acetone from Cucumis cotyledons. In those cases where pigments could not be extracted, the relative pigment content was obtained by measuring the absorbance of the cotyledons in uivo.…”