During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run‐on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5‐methylcytosine was most abundant. The presence of 5‐methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run‐on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts.

Transcription of amyloplast DNA in a heterotrophic line of cultured cells of sycamore (Acer pseudoplatanus L.) appeared to be greatly suppressed. A mutant cell line obtained from the heterotrophic line is green and autotrophic.Heavy modification of amyloplast DNA with a variety of methylated bases was demonstrated by analysis of the acid hydrolysate of DNA by high-performance liquid chromatography, but little modification of chloroplast DNA from the green line was detected. When plastid DNAs from the original and green cell lines were digested with methyl-sensitive restriction enzymes, DNA methylation was detected in regions containing the genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), subunits of chloroplast coupling factor 1 (atpA, -B, and -E), the apoprotein of P700 (psaA), and ribosomal protein S4 (ips4) but not the genes for 16S rRNA and the 32-kDa QB protein (psbA) in the original line, whereas no methylation was observed in the green line. The genes for which methylation was not detectable were found to be active as templates for in vitro transcription by Escherichia coli RNA polymerase, but the methylated genes were apparently inactive. Methylation of DNA is a likely mechanism for the regulation of expression of amyloplast DNA in sycamore cells.

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon escukntum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

Green mutant cells of sycamore (Acer pseudoplatus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type ceUls, mutant cells exhibit photosynthetic Or-evolution activity dwuing their growth coincident with inceses of (a) chlorophyll, (b) protein, and (c) In view of the fact that amyloplasts are the sites of starch synthesis in storage organs such as seeds and roots (21), it is imperative to examine the structure and function of the amyloplast genome and to elucidate the regulatory mechanism(s) which control its expression (10). It is frequently postulated that amyloplasts and chloroplasts are ontogenically related (9), although neither the functional nor the structural nature of the former organelle has been substantively characterized in comparison with that of the latter which has

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.The amyloplast is a uniquely differentiated plastid-type which synthesizes and accumulates starch in the stromal matrix. It has long been hypothesized that this organelle is ontogenically and I