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First published online April 25, 2008; 10.1105/tpc.107.055202 The Plant Cell 20:856-874 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein[W],[OA]Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany 1 Address correspondence to rbock{at}mpimp-golm.mpg.de.
Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts.
Tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) is one of the world's most important vegetable crops and has long been a classical model species for plant genetics. It belongs to the Solanaceae (nightshade) family and has its center of genetic diversity in South America. Recently, a tomato genome project was initiated by the international Solanaceae Genomics Network (SOL Genomics Network; http://www.sgn.cornell.edu/). The tomato nuclear genome has a haploid set of 12 chromosomes and comprises  950 Mb.
Among the three genomes of higher plants, the plastid genome (plastome) is the most gene-dense, with >100 genes in a genome of only 120 to 210 kb (for review, see Sugiura, 1992
The genome organization and mechanisms of gene expression in plastids very much resemble those of their cyanobacterial ancestors. Clusters of genes are arranged in operons and cotranscribed as polycistronic mRNAs, translation occurs on 70S ribosomes, and most components of the translational apparatus have homologs in eubacteria (Zerges, 2000
In spite of the small size of the plastome, the transcriptional apparatus of plastids is much more complex than that of prokaryotes and has a dual evolutionary origin. A plastid-encoded RNA polymerase (PEP) is homologous with eubacterial RNA polymerases (Igloi and Kössel, 1992
Plastid gene expression is also extensively regulated at the posttranscriptional level, most importantly at the level of protein biosynthesis (Gillham et al., 1994
Plastid gene expression and its regulation have been intensely studied in chloroplasts, the plastid type present in photosynthetically active tissues. By contrast, knowledge about gene expression in nongreen plastid types has remained scarce. Since a large fraction of plastid protein-coding genes is involved in photosynthesis, it is generally believed that plastid gene expression is drastically downregulated in nonphotosynthetic tissues (Piechulla et al., 1986 The objective of this study was to systematically analyze the expression of the tomato plastid genome and to identify regulatory patterns of gene expression in different tissues and plastid types. To separate tissue-specific regulation from regulation triggered by plastid differentiation (during the chloroplast-to-chromoplast conversion), we have (1) compared gene expression in fruits with that in leaves and (2) analyzed a developmental series during tomato fruit ripening. In addition to a plastome-wide analysis of the transcriptome and the translatome, we have also determined developmental changes in two key RNA-processing steps: intron splicing and RNA editing. Our data reveal the contributions of transcriptional, posttranscriptional, and translational processes to the regulation of plastid gene expression. They also uncover a single plastid gene, accD, as probably the only protein-coding gene in the plastome for which plastid gene expression is maintained in chromoplasts. In addition, our data sets identify expression elements suitable for triggering high-level transgene expression from the plastid genome in fruit chromoplasts.
Design of an Oligonucleotide Microarray to Analyze Plastid Gene Expression in Solanaceae Solanaceous plants have become important model species for studying plastid biology, not the least because their plastid genomes are amenable to genetic manipulation by chloroplast transformation (Svab and Maliga, 1993  ; Sidorov et al., 1999; Ruf et al., 2001). Knowledge about the regulation of plastid gene expression in response to developmental programs, environmental stimuli, and genetic perturbations is central to our understanding of photosynthesis and other plastid-localized metabolic pathways, plastid–nucleus communication by anterograde and retrograde signaling, and the application of plastid transformation in functional genomics and biotechnology. Therefore, we were interested in developing a plastome microarray for Solanaceae. As our primary interest was to provide a microarray that was applicable to the three most important solanaceous model species, tobacco (Nicotiana tabacum), tomato, and potato (Solanum tuberosum), we used the sequenced plastomes from those species to design an oligonucleotide microarray. We preferred an oligonucleotide array over a cDNA-based microarray for several reasons. First, an oligonucleotide microarray allows the strand-specific detection of transcripts. This is a significant advantage, because transcription from divergent promoters resulting in overlapping complementary transcripts is frequently observed in plastids (Kohchi et al., 1988; Haley and Bogorad, 1990; Meng et al., 1991; Vera et al., 1992). Second, an oligonucleotide microarray is much less prone to contamination and artifacts caused by nonspecific background amplification.
An oligonucleotide microarray containing all genes and conserved open reading frames present in solanaceous plastid genomes was designed using the complete sequences of the tobacco, tomato, and potato plastid genomes (Yukawa et al., 2005
Two options exist for visualization of the data obtained with our microarrays. The TIGR (for The Institute for Genomic Research) MeV software (MultiExperiment Viewer; Saeed et al., 2003 ) can be used to display simple heat maps listing all plastome-encoded genes, providing a global overview of the expression changes (Figures 2
and 3
). To be able to evaluate changes in plastid gene expression in a more pathway-dependent manner, we also adapted the MapMan software (Thimm et al., 2004) with a plastid genome output mask (downloadable at http://gabi.rzpd.de/database/java-bin/MappingDownloader). This provides a simple tool for visualizing the specific changes in different gene classes that reflect processes involving plastid-encoded genes (Figure 1C).
Transcriptional Regulation To assess the relative contributions of transcriptional and translation regulation to the changes in plastid gene expression upon fruit development and chloroplast-to-chromoplast conversion, we first determined the changes in RNA accumulation for all plastid genome–encoded genes and open reading frames. In addition to green leaves, we analyzed a developmental series of tomato fruits (green, turning, light red, and ripe red tomatoes; Figure 2). All developmental stages were analyzed in triplicate, each representing an independent RNA isolation. We recorded the changes in plastid gene expression in two ways. First, we used the expression levels in leaves as a reference and, in this way, analyzed the changes in the expression of all genes in fruits compared with leaves (Figures 2A and 2B). This analysis determined the changes triggered by the fruit developmental program. In order to dissect the relative contributions of fruit development and plastid differentiation, we also used the expression levels in green, chloroplast-containing fruits as a reference (Figures 2C and 2D). This allowed us to identify those changes that are induced by chloroplast-to-chromoplast differentiation and thus are independent of the initiation of the fruit developmental program.
When gene expression in fruits was compared with that in leaves, it became apparent that most plastid genes are strongly downregulated in fruits. This is already the case in green fruits, suggesting that the downregulation is triggered by the developmental program rather than the chloroplast-to-chromoplast conversion. Interestingly, the photosynthesis-related genes were much more strongly downregulated than the genetic system genes, the latter including, for example, the ribosomal proteins and the subunits of the plastid-encoded E. coli–type RNA polymerase PEP (Figures 2A and 2B; the photosynthesis-related genes are at the top, ranging from psbA to ycf10, and the genetic system genes then follow from rpoA to clpP and may additionally include the conserved reading frames ycf1 and ycf2) (Drescher et al., 2000 Interestingly, the chloroplast-to-chromoplast conversion was not accompanied by drastic changes in transcript abundance (Figures 2C and 2D). For most plastid genes, only minor (statistically insignificant) changes were seen, suggesting that, at the level of RNA accumulation, plastid differentiation has a much lower impact on the regulation of plastid gene expression than the developmental program. Notable exceptions include the trnA gene (encoding the tRNA-Ala) and the rpoC2 gene (encoding an RNA polymerase subunit), which tended to be upregulated. Remarkably, the protein-coding gene that displayed the strongest change in expression was accD, which encodes a subunit of the acetyl-CoA carboxylase and is the only known plastid-encoded gene involved in fatty acid biosynthesis. Its transcript abundance increased during the chloroplast-to-chromoplast conversion (Figure 2D), correlating with the high demand for lipid biosynthesis in fruit ripening to provide the storage matrix for the accumulating carotenoids. To confirm that the microarray data faithfully represented the developmental changes occurring in mRNA accumulation, selected genes were analyzed by RNA gel blot experiments (using plant material generated independently from the material used in the microarray experiments; Figure 2E). We tested two photosynthesis genes (psbB and psbD), two genetic system genes (rpoB and matK), and accD, the gene displaying the strongest upregulation during fruit ripening. In all cases, the mRNA accumulation patterns seen in the RNA gel blots corresponded exactly to the data obtained by microarray analyses, confirming that our microarray reliably detected changes in plastid gene expression patterns.
Translational Regulation When the polysome association of plastid mRNAs in fruits was compared with that in leaves, a strong downregulation was seen for most plastid genes (Figures 3A and 3B). However, there were two notable exceptions. Three of the four plastid-encoded subunits of the E. coli–like RNA polymerase (PEP) were only weakly downregulated in fruits (rpoB, rpoC1, and rpoC2; Figures 3A and 3B). The only gene whose polysome association remained essentially unchanged during fruit development was accD, the acetyl-CoA carboxylase subunit gene that we had found to be the most strongly upregulated gene at the RNA level during the chloroplast-to-chromoplast conversion (Figure 2D). While our analysis of plastid RNA accumulation revealed no significant overall changes during chromoplast development (Figure 2D), there was a pronounced general trend in polysome association (Figures 3C and 3D). Polysome association successively declined during fruit ripening, suggesting that, while the RNA levels remain largely constant, plastid translation is gradually downregulated during chloroplast-to-chromoplast differentiation. This trend was particularly pronounced in the photosynthesis gene group (genes psbA to ycf10; Figures 3C and 3D), correlating with the gradual loss of photosynthesis during the chloroplast-to-chromoplast conversion. A single exception was seen: accD, whose polysome association did not only stay high in fruits (Figures 3A and 3B) but even increased at the onset of ripening (Figures 3C and 3D). In order to confirm that accD is actively translated in chromoplasts, we analyzed fractionated polysome gradients by RNA gel blotting (Figure 3E). As a control for an mRNA that is translationally downregulated during fruit ripening, we also determined ribosome association for psbD (Figure 3E). These experiments revealed that, while accD was still strongly loaded with ribosomes even in ripe red tomatoes, the ribosome association of the psbD mRNA declined gradually during ripening and was almost undetectable in red fruits. The translational upregulation of accD during the onset of the chloroplast-to-chromoplast conversion (transition from the green to the turning stage) was seen equally clearly in both the microarray analysis and the RNA gel blot experiments (cf. Figures 3C and 3D with Figure 3E). It must be borne in mind that ribosome association does not necessarily correlate directly with translational regulation. This is because any change in mRNA accumulation can potentially translate into a corresponding change in polysome association. For example, in the extreme case that solely the amount of transcript limits gene expression, a transcriptional upregulation would be seen proportionally as an increase in polysome association, simply because any available mRNA molecule would become translated. Thus, the polysome association data reflect the net gene expression levels in that they directly correlate with the total amount of protein synthesized. In this sense, the data set presented in Figure 3 provides an overview of the changes in plastid gene expression during fruit development. Three main conclusions can be drawn. First, the expression of all photosynthesis-related genes is already drastically downregulated in green, chloroplast-containing fruits and declines further during fruit ripening and chloroplast-to-chromoplast differentiation. Second, the genetic system genes (i.e., genes involved in gene expression) show a similar trend, although the downregulation is somewhat less intense than for the photosynthesis genes (Figures 3A and 3B). Third, the only protein-coding gene that is expressed at constitutively high levels throughout fruit development is the fatty acid biosynthesis gene accD (Figures 3A and 3B). This raises the possibility that the main reason for maintaining some protein biosynthetic capacity in fruit plastids is to sustain fatty acid biosynthesis. To confirm the significance of the sustained accD expression in fruit ripening at the protein level, protein gel blot analyses with antibodies against the AccD protein were conducted. These analyses revealed that the AccD protein accumulated in all fruit ripening stages and even increased slightly during the onset of chloroplast-to-chromoplast conversion (transition from the green to the turning stage; Figure 4 ). By contrast, PsbD, a photosystem II protein, was only detectable in green, chloroplast-containing tomato fruits (Figure 4). The acetyl-CoA carboxylase enzyme consists of four subunits, three of which are encoded in the nuclear genome (AccA, AccB, and AccC) and one of which is encoded in the plastid genome (AccD). To test whether the nucleus-encoded subunits also accumulate in chromoplasts, protein gel blot analysis with an anti-AccC antibody was performed. As expected, AccC protein accumulation was seen in all fruit ripening stages (Figure 4), suggesting that both the plastid-encoded and the nucleus-encoded components of the enzyme are present in chromoplasts. The accumulation of AccC did not strictly parallel the accumulation of AccD in all ripening stages (cf. green and turning fruits in Figure 4), indicating that some subunits can stably accumulate even if not immediately incorporated into the complex.
RNA Polymerase Activity and Promoter Usage Having seen complex patterns of changes in RNA accumulation in fruits versus leaves (Figure 2), we were interested in determining whether these changes could be attributed to developmental changes in the relative activities of the two distinct transcription systems existing in plastids: the plastid-encoded E. coli–like RNA polymerase (PEP) and the nucleus-encoded bacteriophage-type RNA polymerase (NEP). To this end, we analyzed the transcription initiation sites of four plastid genes: rbcL, a gene predominantly transcribed by PEP; atpI, a gene transcribed by both PEP and NEP; clpP, a gene predominantly transcribed by NEP; and rrn16, the gene for the 16S rRNA, which is normally predominantly transcribed by PEP but is transcribed by NEP in the absence of PEP (as demonstrated by the analysis of PEP knockout plants) (Allison et al., 1996 ; Hajdukiewicz et al., 1997). RNA 5' ends were mapped by primer extension analysis in leaves, green fruits, and ripe red fruits. No significant changes in the patterns of RNA 5' ends were seen for rbcL, rrn16, and clpP (Figure 5
). Analysis of atpI transcriptional start sites provides the most informative data on the relative activities of the two polymerases, because atpI possesses both a PEP and a NEP promoter that are about equally strong (Miyagi et al., 1998). Primer extension analysis of atpI transcripts revealed that both promoters are used in leaves and fruits, suggesting that both plastid RNA polymerases are active during fruit development. We observed, however, a change in the relative polymerase activities in that, in leaves, the PEP promoter was more intensively used than the NEP promoter, whereas in red fruits, transcription from the NEP promoter prevailed (Figure 5). This may suggest that the plastid-encoded RNA polymerase PEP is more strongly downregulated in fruit development than the nucleus-encoded RNA polymerase NEP. Together with the earlier finding that PEP transcribes mainly photosynthesis-related genes whereas NEP transcribes mainly genetic system genes (Hajdukiewicz et al., 1997; Maliga, 1998), this may provide a mechanistic explanation for our observation that photosynthesis genes are generally more strongly downregulated in fruits than genetic system genes (Figure 2).
Intron Splicing The expression of a number of genes in plastids is dependent upon the removal of intervening sequences (introns) that have to be removed by splicing to obtain a functional gene product. With a single exception, all introns in higher plant plastid genomes belong to the group II introns (Michel et al., 1989 ; Sugita and Sugiura, 1996; Bonen and Vogel, 2001). Group II introns are the likely evolutionary ancestors of the spliceosomal introns present in the nuclear genomes of eukaryotes (Copertino and Hallick, 1993; Hetzer et al., 1997). Compelling evidence has accumulated that most, if not all, introns in the plastomes of higher plants do not self-splice but instead require the assistance of proteinaceous splicing factors to be excised in vivo. While most splicing factors for plastid group II introns are encoded by the nuclear genome (Jenkins et al., 1997; Perron et al., 1999, 2004; Vogel et al., 1999; Jenkins and Barkan, 2001; Rivier et al., 2001; Till et al., 2001; Ostheimer et al., 2003), at least one appears to be encoded in the plastome. The matK reading frame present in the group II intron that interrupts the plastid trnK gene displays homology with intron maturases in fungal mitochondrial genomes (Neuhaus and Link, 1987; Mohr et al., 1993; Hess et al., 1994; Liere and Link, 1995).
As intron removal is an essential step in gene expression and a prerequisite for obtaining a functional gene product, it seems conceivable that splicing could be employed to regulate gene expression. The recent finding that some splicing factors act intron-specifically in that they promote the splicing of only a single intron in the plastid genome (Till et al., 2001 Splicing efficiency was compared in leaves and four different stages of fruit ripening by separating RNA samples on denaturing gels and hybridizing them to specific probes for intron-containing plastid genes. Five transcripts containing eight introns in total were investigated: petB/D (harboring one intron in petB and one in petD), rps12 (containing one intron spliced in cis and one spliced in trans), clpP (containing two introns), trnI-GAU, and ndhB. For most of these genes, there were no significant changes in the relative ratios of intron-containing precursor transcripts versus mature spliced RNAs (Figure 6 ). This was also the case for the only plastid mRNA that is processed by trans-splicing (rps12; Figure 6). By contrast, the intron in the ndhB transcript, encoding a subunit of the plastid NAD(P)H dehydrogenase, showed a strong decline in splicing during fruit development. In ripe red tomatoes, almost all ndhB transcripts remained unspliced, indicating that fruit development is accompanied by a gradual loss in ndhB intron splicing activity.
In order to investigate this splicing deficiency in more detail, RNA gel blot experiments with an ndhB intron probe were conducted. These experiments revealed that the first intermediate of the group II intron splicing reaction, the lariat structure resulting from the first transesterification step, accumulated throughout fruit development and was still present in essentially unchanged amounts in the red fruit (Figure 6). Together with the nearly complete absence of the free intron and the ligated exons, this finding suggests that a splicing factor specifically mediating the second transesterification reaction of ndhB intron excision is absent from chromoplasts. In addition to intron splicing, our RNA gel blot analyses also provided information about the processing patterns of plastid transcripts during fruit development. Even for transcripts displaying very complex RNA accumulation patterns in leaves (such as the transcripts from the rps12 and the pentacistroinc psbB/petBD operons), no significant changes were observed during fruit ripening, suggesting that the cleavage of polycistronic transcripts into monocistronic or oligocistronic mRNAs is probably not under developmental regulation.
mRNA Editing
We analyzed 23 of the 36 RNA editing sites encoded in the plastid genome of tomato (Kahlau et al., 2006
However, editing in three transcripts was found to be altered in fruits compared with leaves. Interestingly, all three of these mRNAs encode subunits of the plastid NAD(P)H dehydrogenase, an enigmatic multiprotein complex of unclear physiological function. The complex is present in the thylakoid membranes of angiosperm plants (but not in gymnosperms and green algae) and has been implicated in cyclic electron flow and some abiotic stress responses (Burrows et al., 1998 ; Sazanov et al., 1998; Shikanai et al., 1998; Horváth et al., 2000; for a recent review, see Rumeau et al., 2007). The ndhB transcript contains nine RNA editing sites in tomato, tobacco, and deadly nightshade. While seven of these sites were constitutively fully edited in all analyzed tissues and developmental stages, two of the sites (sites 196 and 277) displayed strongly reduced editing efficiency in at least some fruit stages (Figure 7). Site 196 was nearly unedited in small green fruits. As fruit development proceeded, editing at site 196 first increased (in full-sized green tomatoes) and then declined during ripening, with 50% of the mRNA molecules remaining unedited in ripe tomatoes. By contrast, site 277 was still fully edited in small green fruits, but editing efficiency then gradually declined, reaching only 50% in ripe red tomatoes. RNA editing at the single editing site present in the ndhF transcript was already affected in small green fruits (with about half of the RNA molecules remaining unedited), and no pronounced further decline in editing efficiency was observed (Figure 7). The most striking changes were seen in the ndhD transcript. Here, editing at two of six sites was affected. Editing at site 1 creates a canonical AUG translation initiation codon from a genomically encoded ACG codon, whereas editing at site 200 changes a genomically encoded TCA Ser codon to a UUA Leu codon at the RNA level. Editing at site 1 declined gradually during fruit development and was undetectable in ripe red fruits. An even more dramatic effect was seen at site 200. Editing was already completely lost in small green fruits, and no editing was seen at all later stages of fruit development (Figure 7). The early loss of editing at this site indicates that it is not an effect of chloroplast-to-chromoplast differentiation but rather the result of switched-off editing at the onset of fruit development. To further substantiate this conclusion, we also analyzed different tissues from small green fruits (fruit peel and pericarp). Site 200 remained unedited in the ndhD transcripts independent of the tissue type, indicating that the developmental switching off of editing is an organ-wide process affecting all fruit tissues (see Supplemental Table 2 online). The immediate consequence of this developmental regulation in RNA editing is that the NdhB, NdhF, and NdhD proteins in fruits differ from the proteins made in leaves. The NdhB and NdhF protein populations are probably heterogeneous in fruits due to partial editing in single amino acid positions. In the case of the NdhD protein, all protein molecules made in fruits are different from those in leaves. Whereas the NdhD protein in leaves carries a Leu at position 200, the protein in fruits has a Ser at that position. No novel RNA editing sites were detected in fruits, possibly suggesting that the editing pattern seen in leaves represents the maximum RNA editing capacity of the plastid. However, as we have not sequenced fruit cDNAs for all plastid genes, there remains a slight possibility that some fruit-specific or chromoplast-specific RNA editing events could exist.
In the course of this work, we developed an oligonucleotide microarray suitable for obtaining complete transcriptomics and translatomics data sets for the plastid genome. We then used microarray-based analysis of RNA accumulation and polysome association to study the expression of the plastid genome during tomato fruit development and showed how transcriptomics and translatomics data sets can be integrated to assess the relative contributions of RNA metabolism and translation to the regulation of plastid gene expression. Our results demonstrate that both the regulation of RNA accumulation and translational regulation contribute to the regulation of gene expression in plastids. Interestingly, the global changes in gene expression in fruits compared with leaves are mainly brought about by changes in RNA accumulation, with translational regulation playing only a relatively modest role (cf. Figures 2A and 2B with 3A and 3B). By contrast, the global changes in gene expression during chloroplast-to-chromoplast conversion in fruit ripening are not accompanied by major changes in RNA abundance and, instead, are predominantly the result of translational regulation (cf. Figures 2C and 2D with 3C and 3D). It should be borne in mind that the expression changes seen in transcriptomics-type microarray experiments reflect the sum of transcriptional regulation and the regulation of RNA stability and degradation. Thus, any change in RNA accumulation seen on the array could be due to either altered transcription or changes in RNA turnover (or both). Whether, for example, the strong global decline in RNA accumulation in fruit plastids (Figures 2A and 2B) is predominantly the result of downregulated plastid transcription or is, at least in part, also caused by an increase in RNA turnover remains to be established. From our data, however, it is clear that if the global downregulation of plastid RNA levels in fruits is transcriptional in nature, it cannot be specifically attributed to one of the two RNA-polymerizing activities in plastids, PEP and NEP. Although their relative activities change in fruit development, both are still active in ripe red tomatoes (Figure 5).
It is also important to note that most plastid genes are part of operons and transcribed as polycistronic mRNAs. Although most polycistronic precursor transcripts are posttranscriptionally processed into monocistronic or oligocistronic units (presumably by specific endonucleolytic cleavage) (Westhoff and Herrmann, 1988
Finally, it must be kept in mind that the translation rate is not the only determinant of protein accumulation. Posttranslational processes, including the differential stability of proteins, may additionally contribute to regulating the final protein accumulation levels. Protein degradation may become particularly relevant to all of those proteins that are components of multiprotein complexes and whose stability depends on the accumulation of the other subunits in stoichiometric amounts. It is well established, for example, that many photosystem subunits are condemned to rapid degradation if they are not properly incorporated into the complex (Goldschmidt-Clermont, 1998
One of the most remarkable findings from our transcriptomics and translatomics analyses is that only a single protein-coding gene circumvented the global decline in gene expression during tomato fruit development: accD, the only gene on the plastome that is involved in fatty acid biosynthesis (Figures 3 and 4). This makes biological sense in that high fatty acid biosynthetic capacity must be sustained in fruit development to provide the membrane lipids that accommodate the storage carotenoids that are massively synthesized during fruit ripening. It is noteworthy in this respect that the accD gene is essential and cannot be deleted from the plastome (Kode et al., 2005 The requirement for accD expression in fruit plastids makes it necessary that all plastid genes involved in gene expression (genetic system genes) also continue to be expressed. This is consistent with our observation that these genes are, on average, less strongly downregulated in fruits than are the photosynthesis genes (Figures 3A and 3B). Since, in green leaves, the photosynthesis genes are the most highly expressed plastid genes, it seems reasonable to assume that if no significant expression of the photosynthesis genes occurs, a low translational capacity is sufficient to (1) translate the genetic system gene mRNAs at a low level and (2) sustain the high expression of the accD gene in fruit plastids.
Although accD may be the only plastid-encoded protein needed in chromoplasts, at least one plastid-encoded RNA molecule should also be essential: the glutamyl-tRNA encoded by the trnE gene. In addition to its role in translation, the tRNA-Glu is also required in the first step of tetrapyrrole biosynthesis (Schön et al., 1986 It is conceivable that, in addition to the regulation of RNA accumulation and translation, RNA processing also contributes to the regulation of plastid gene expression. Our analysis of the transcript patterns of several plastid operons revealed no evidence of a regulation at the level of intercistronic RNA processing and only a single example of developmentally downregulated splicing (Figure 6). The significance of our finding that the ndhB transcripts remain largely unspliced in chromoplasts is unclear, but the lack of splicing may contribute to the ndhB mRNA being the most strongly downregulated transcript (at the level of polysome association; Figures 3A to 3D) of all 11 plastid ndh genes. Moreover, the accumulation of the lariate intermediate suggests the existence of at least one splicing factor that is specifically involved in the second transesterification and/or exon ligation reaction in ndhB intron splicing.
Our comprehensive analysis of RNA editing in plastid transcripts during fruit development indicates that only three mRNAs undergo developmental changes in their editing patterns. Interestingly, all three mRNAs encode subunits of the plastid NAD(P)H dehydrogenase, a membrane protein complex of unclear physiological function (reviewed in Rumeau et al., 2007
Finally, our plastome-wide analysis of the transcriptional and translational regulation of gene expression in fruit plastids also provides a valuable data set for the design of expression cassettes for plastid transformation experiments. Plastid transformation is uniquely attractive for plant biotechnology because it offers high-level foreign protein accumulation, the absence of epigenetic effects, convenient transgene stacking in operons, and increased gene containment due to maternal plastid inheritance, which largely excludes transgenes from transmission via pollen (reviewed in Maliga, 2004
Plant Material Seeds from Solanum lycopersicum cv IPA-6, a commercially grown South American tomato cultivar, were germinated and grown in a greenhouse with supplementary lighting of 200 µmol quanta·m–2·s–1. Fruits were harvested at the different stages of ripening defined by the California Tomato Commission (http://www.tomato.org/Member/Content.aspx?id=20).
RNA Isolation and RNA Gel Blot Analyses
Isolation of Polysomes
cDNA Synthesis
Design and Production of a Plastome Oligonucleotide Microarray for Solanaceae
Microarray Hybridization Experiments
Microarray Data Evaluation
The normalized microarray data were read into R (R Development Core Team, 2008
PCR and DNA Sequencing
Primer Extension Analyses
Protein Analysis
Accession Numbers
Supplemental Data
We thank the Max-Planck-Institut für Molekulare Pflanzenphysiologie Green Team for plant care and cultivation; Dirk Hincha, Susanne Freund, and Kerstin Petersen for help with microarray design and spotting; and Björn Usadel for help with statistical evaluation of the microarray data. We are grateful to Yukio Nagano and Yukiko Sasaki (Saga University and Nagoya University) for generously providing antibodies against the AccC and AccD proteins. This research was supported by a grant from the European Union (FP6 Plastomics Project LSHG-CT-2003-503238) and by the Max Planck Society.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Ralph Bock (rbock{at}mpimp-golm.mpg.de).
[W] Online version contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.107.055202 Received August 20, 2007; Revision received March 19, 2008. accepted April 9, 2008.
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ABSTRACT
INTRODUCTION
factors, which are required for promoter recognition, are encoded by genes residing in the nuclear genome (Tanaka et al., 1996
-32P]dATP and T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. A total of 100 fmol of radiolabeled rbcL or 16S rRNA primers and 30 fmol of labeled atpI or clpP primers were used for gene-specific cDNA synthesis with the SuperScript III RNase H–free reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The cDNAs were denatured for 5 min at 95°C and separated by electrophoresis on denaturing 8% polyacrylamide gels. To determine the size of the extension signals, a radiolabeled 
X174 Hinf I DNA marker was run alongside the samples. 
