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

Pure preparations of intact amyloplasts and chloroplasts, free from mitochondrial contamination, were isolated from cultured cells of the white-wild and green-mutant lines of sycamore (Acer pseudoplatanus L.), respectively. A specific rabbit antiserum against yeast mitochondrial cytochrome c1 only cross-reacted with mitochondrial membranes from the white-wild sycamore cells. The outer and inner envelope-membranes of the two plastid-types were isolated and subsequently analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to characterize polypeptide pattems in each fraction. Analysis by immunoblotting clearly showed that antiserum against the 29-kilodalton inorganic orthophosphate translocator isolated from pea chloroplasts cross-reacted with a 31-kilodalton polypeptide residing in the inner-envelope membranes from both sycamore chloroplasts and amyloplasts. In contrast, antiserum against the ADP/ATPtranslocator isolated from mitochondria of Neurospora crassa yielded a positive signal with a 32-kilodalton polypeptide in the inner-membranes isolated from amyloplasts, but not green-mutant chloroplasts. We propose that this 32-kilodalton polypeptide in the amyloplast envelope is a putative ATP/ADP-translocator and its possible functional significance is discussed.assimilates (triose-P) to the cytosol via the Pi-translocator, carbon-partitioning in amyloplasts will be directed to the uptake ofcarbon-compounds from the cytosol into the stroma and their eventual conversion to starch. In both cases, the Pitranslocator in the inner-envelope membranes is thought to play a crucial role (16), although it remains unclear whether its function in the amyloplasts is truly identical to that operating in the chloroplast envelope. Another key component which possibly resides in the amyloplast inner-envelope is a putative ATP/ADP-translocator (16); since this plastid type lacks the energy-producing machinery associated with the thylakoid membranes in chloroplasts, the ATP which is required for the progress of gluconeogenesis (i.e. ADP-glucose formation) must be imported from the cytosol.To address the question of whether or not an ATP/ADPtranslocator is specifically present in the inner membrane of the amyloplast envelope, we have exploited immunoblot analysis using antiserum against the ADP/ATP-translocator isolated from the mitochondrial membranes of Neurospora crassa (22). Throughout this investigation related to the biochemistry of the amyloplasts, we have used functional chloroplasts isolated from the green-mutant cell-line of sycamore as a critical control system (16,17).The non-green amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates starch in the stromal matrix, possesses a unidirectional gluconeogenic pathway in contrast to the vast functional diversity of the chloroplast, including energy (ATP) formation, photosynthetic C02-fixation and gluconeogenesis among several others. Based on these reasons, we view the chloroplast and amyloplast each as a "'source," "sink," a...

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