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The chloroplast genome encodes proteins required for photosynthesis, gene expression, and other essential organellar functions. Derived from a cyanobacterial ancestor, the chloroplast combines prokaryotic and eukaryotic features of gene expression and is regulated by many nucleus-encoded proteins. This review covers four major chloroplast posttranscriptional processes: RNA processing, editing, splicing, and turnover. RNA processing includes the generation of transcript 5' and 3' termini, as well as the cleavage of polycistronic transcripts. Editing converts specific C residues to U and often changes the amino acid that is specified by the edited codon. Chloroplasts feature introns of groups I and II, which undergo protein-facilitated cis- or trans-splicing in vivo. Each of these RNA-based processes involves proteins of the pentatricopeptide motif-containing family, which does not occur in prokaryotes. Plant-specific RNA-binding proteins may underpin the adaptation of the chloroplast to the eukaryotic context.

A predominantly plant-based family of genes encoding RNA binding proteins is defined by the presence of a highly conserved RNA binding motif first described in the mei2 gene of the fission yeast Schizosaccharomyces pombe. In silico analyses reveal nine mei2 -like genes in Arabidopsis thaliana and six in Oryza sativa. These predicted genes group into four distinct clades, based on overall sequence similarity and subfamily-specific sequence elements. In situ analysis show that Arabidopsis genes from one of these clades, TEL1 and TEL2, are specifically expressed in central zone of the shoot apical meristem and the quiescent center of the root apical meristem, suggesting that they may somehow function to maintain indeterminacy in these tissues. By contrast, members of two sister clades, AML1 through AML5, are expressed more broadly, a trend that was confirmed by Q-PCR analysis. mei2 -like transcripts with similar sequences showed similar expression patterns, suggesting functional redundancy within the four clades. Phenotypic analyses of lines that contain T-DNA insertions to individual mei2 -like genes reveal no obvious phenotypes, further suggesting redundant activities for these gene products.

A modification of a previously described tissue print technique has been developed in which soft tissues are frozen and sectioned in a cryostat prior to direct collection on nitrocellulose or nylon membranes. The inexpensive embedding technique described here allows accurate orientation of specimens prior to mounting, and mounted material may be stored easily after initial sectioning for future reexamination. Standard hand tissue prints of soft specimens exhibit tissue distortion and uneven delivery of material to the membrane, which limits resolution and makes interpretation difficult. Cryostat sections retain tissue fragments in their original arrangement relative to each other during printing and deliver a consistent and quantitative amount of material from all parts of the specimen. The cryostat tissue print technique is applied here to immature floral buds, demonstrating the tissue-level histochemical localization of beta-glucuronidase in transgenic plants and immunolocalization of a novel protein present only in mutant plants. This modified technique is applicable for examining both plant and animal tissues.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Transcripts encoding ATP synthase subunit 6 (ATP6) in petunia mitochondria were shown to be edited at 15 sites, leading to 14 amino acid changes. Certain sites are partially edited, including a site that introduces a new translation termination codon that is 13 codons upstream of the genomically encoded stop codon. Transcripts lacking the new stop codon are present in an approximately 2.5:1 ratio to transcripts carrying the stop codon created by RNA editing. To investigate whether partially edited transcripts are represented as proteins, we generated an antibody against a 12-residue peptide that is specific for translation products of unedited transcripts. This antibody did not recognize any ATP6 protein in either total mitochondrial protein preparations or ATP6 samples purified by organic solvent extraction and reverse phase HPLC procedures. According to analysis by mass spectrometry, only one form of ATP6 protein accumulates in mitochondria despite the presence of abundant partially edited transcripts. Partially edited atp6 transcripts were associated with ribosomes, suggesting that a screening mechanism(s) acts cotranslationally or post-translationally to exclude the expression of incompletely edited transcripts.

The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. Pcf transcripts are modified by editing at 11 sites. Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region. Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989). N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa. Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence. Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots. Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes. Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.

Repeated sequences known as recombination repeats are present in the majority of plant mitochondrial genomes. Two recombination repeat sequences from Petunia have been analyzed. The two repeats are virtually identical over 1.42 kb. One of the repeats is truncated and is likely to have arisen from a rare recombination event in the full-length repeat. Two sequence-blocks within the Petunia repeat are highly similar to sequences in the 5' flank of several plant mitochondrial genes. No sequence motifs are shared by the Petunia repeat and other sequenced plant mitochondrial recombination repeats, suggesting that the recombination occurs by an homologous, rather than a site-specific, mechanism.

A number of cytosines are altered to be recognized as uridines in transcripts of the NADH-dehydrogenase subunit 3 (nad3) gene in the mitochondria of the higher plant Petunia hybrida. Here we show that the extent of editing for three of the edit sites, all of which change the encoded amino acid, varies between different Petunia lines. Genetic analysis indicates that a single nuclear gene is responsible for this variation. Interestingly, according to RNA blot hybridization analysis, RNA editing extent and transcript abundance are correlated. This observation is consistent with the hypothesis that RNA editing is a post-transcriptional event.

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.

Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.

The mitochondrial gene encoding NADH dehydrogenase subunit 1 (nad1) in Petunia hybrida is split into five exons, a, b, c, d, and e. With the use of a complete restriction map of the 443-kb Petunia mitochondrial genome, we have cloned these exons and mapped their location. Exon a is located 130 kb away from and in the opposite orientation from exons b and c. Exon d maps 95 kb away and in the opposite orientation from exons b and c. Exons d and e are separated by 190 kb. By performing the polymerase chain reaction on Petunia cDNAs, we have shown that transcripts from these five exons are joined via a series of cis- and trans-splicing events to create a mature nad1 transcript. In addition, we have found 23 C----U RNA edit sites in Petunia nad1. RNA editing changes 19 of the amino acids predicted by the genomic sequence.

Transcripts of the Petunia mitochondrial cytoplasmic male sterility (CMS)-associated S-Pcf locus, which consists of three co-transcribed genes (pcf, NADH dehydrogenase subunit 3, and ribosomal protein S12), have been characterized in reproductive tissues of CMS and fertility restored (Rf) Petunia lines by nuclease protection experiments and by RNA blot hybridization. Three 5' transcript termini have been previously described. Two 3' transcript termini and an additional S-Pcf locus transcript have now been identified. The relative abundance of the three 5' transcript termini is influenced by the presence of the nuclear Rf gene. A decrease in the abundance of the -121 5' transcript terminus relative to the -266 and -522 termini is consistently seen in Petunia lines which are restored to fertility by the Rf gene, compared to CMS Petunia lines. An additional transcript with a 5' terminus within the urf-s region of pcf is much more abundant in immature bud and anther tissue than in leaf or suspension cells. The total abundance of pcf transcripts varies greatly between plants of different nuclear backgrounds which lack the nuclear Rf allele, indicating that other nuclear genes also influence expression of the S-Pcf locus.

The rps19 ribosomal protein gene, which has not been previously reported in any mitochondrial genome, was identified by sequence analysis in the mitochondrial DNA of the higher plant Petunia hybrida. According to the sequence of eight rps19 cDNAs, seven C to U conversions with respect to the genomic sequence are present in rps19 transcripts. Not all transcripts are fully edited at these seven sites. Six of the seven C to U conversions change the encoded amino acid sequence by altering four codons. The rps19 gene is located entirely within a repeat sequence which is present in three copies on the 443 kb genome. Due to intragenomic recombination across these repeats, Petunia rps19 is present in nine different genomic environments.

A comparative analysis of the plant intron-containing mitochondrial cytochrome oxidase subunit II (coxII) genes provides an indication that four conserved sequence motifs, present in exon 1 (intron-binding sequences; IBS), and complementary motifs (exon-binding sequences; EBS), present in domain I of the group II intron, may be involved in splicing of the intron. Two of these potential IBS motifs (IBS1 and IBS2) have been previously discussed. Two further potential IBS motifs (IBSa and IBSb), which occur twice within exon 1, could be involved in specification of the 5' splice site and of a 5' cryptic splice site. Nuclease-protection experiments and DNA sequence analysis of a spliced coxII cDNA have confirmed the predicted positions of the petunia coxII 5' and 3' splice sites. Evidence for the occurrence of splicing in vivo at the putative 5' cryptic splice site in petunia is provided by the detection of a nuclease-protected fragment corresponding to the size which is predicted if splicing at the proposed cryptic splice site occurs. The existence and location of a cryptic splice site, upstream of the normal coxII 5' splice site, is consistent with the proposed derivation of the cytoplasmic male sterility (CMS)-associated pcf gene from an abnormally spliced coxII transcript (Pruitt and Hanson 1989).

Analysis of the cDNA of the atp9-1 gene transcript from petunia mitochondria has revealed that ten C residues of the gene sequence are edited into U in the mRNA. Seven of these edits result in amino acid changes and one introduces a stop codon before the end of the open reading frame predicted from the gene sequence. The resulting protein is better conserved when compared to the same protein in other organisms. Comparison of the edited petunia sequence with other plant mitochondrial atp9 gene sequences idicates variation in the number and positions of edits required to obtain the same amino acids in ATP9 polypeptides of higher plants.

A characteristic of CMS mutations in plants, in contrast to the single base changes in human mitochondrial mutants (49, 140), is the presence of chimeric genes or chimeric loci; different open reading frames are joined together, or placed in proximal locations and cotranscribed with standard mitochondrial genes. Despite much progress, and the identification of several mitochondrial loci that specify CMS, the molecular basis of this defect is not understood in any plant species. Observations of altered electron transport in Petunia and toxin-mediated membrane disruption in maize plants, bacteria, and yeast expressing the maize urf13 gene product, provide clues to possible mechanisms for disruption of pollen development. Whether disruption in a particular mitochondrial function is at the root of CMS in all species, or whether defects in numerous mitochondrial activities can produce sterility, will only be revealed by further probing of physiological and biochemical defects present in CMS genotypes.

The mitochondrial genome of Petunia hybrida contains two transcribed cytochrome oxidase subunit II (coxII) genes. The coding region of both genes is split by a 1.3 kb group II intron. Unlike coxII-1, which is similar to other sequenced plant coxII genes, the coxII-2 coding region is extended by 48 codons. The cytoplasmic male sterile (CMS) Petunia contains one coxII gene similar in structure and transcript pattern to the coxII-1 gene found in the fertile genome. Comparison of the sequenced coxII genes from the fertile mitochondrial genome with the coxII sequences present in the CMS-associated pcf gene from the CMS genome (Young and Hanson 1987) suggests that pcf is a processed pseudogene. A model for the generation of pcf is presented.

DNA sequence analysis 3' to the Petunia S-pcf coding region has resulted in the identification of an open reading frame similar to mammalian mitochondrial genes for subunit 3 of the NADH dehydrogenase complex (nad3). Both the abnormal fused gene S-pcf and S-nad3 fall within the mitochondrial DNA region previously shown to be associated with cytoplasmic male sterility (CMS). The S-nad3 sequence, co-transcribed with S-pcf, is present in only one copy within the Petunia CMS genome. A homologous transcribed sequence from the mitochondrial genome of a fertile Petunia line has been identified. The coding region of the two genes are identical and they share homology for at least 800 bp downstream. The genes diverge 117 bp upstream of the nad3 start codon. Transcripts of the S-pcf/S-nad3 transcripts are similar in tissues of a fertility-restored line and a CMS line.

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

Sequencing of an open reading frame associated with cytoplasmic male sterility (CMS) in Petunia has revealed a gene fusion (the Pcf gene) containing the 5'-flanking and amino-terminal transmembrane segment of the ATP synthase proteolipid gene (atp9), parts of the cytochrome oxidase subunit II (coxII) coding region, and the carboxyl terminus and 3'-flanking region of an unidentified reading frame (urfS). The coxII region has several small deletions and tandem repeats that remove all of the segments coding for the residues involved in copper binding, but may possibly maintain the cytochrome c binding site. Normal atp9 and coxII genes and their transcripts are also present in the sterile cytoplasm. S1 nuclease protection studies identify fused gene transcripts only in CMS lines, with an increase in transcript amount in anthers relative to leaves.

At 443 kb, the map of Petunia hybrida line 3704 mitochondrial DNA is the largest yet produced from a dicot plant. Regions of similarity to known plant mitochondrial genes and to the chloroplast genome have been placed on a master circle. One long repeated sequence, apparently active in recombination, is present in three copies. Two copies of 6.6 kb occur in a direct orientation and are separated by 199 kb. A third truncated copy of 3.5 kb is inverted relative to the other two and is separated from the others by 99 and 145 kb. The presence of the recombination repeats predicts a multipartite molecular organization, consisting of four master circles and three subgenomic circles. Two other repeated regions were found not to be substrates for, or products of recombination. The absence of recombination at certain reiterated regions indicates that there is specificity of recombination at the recombination repeats.