Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Δrpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.

Bacterial group II introns encode maturase proteins required for splicing. In organelles of photosynthetic land plants, most of the group II introns have lost the reading frames for maturases. Here, we show that the plastidial maturase MatK not only interacts with its encoding intron within trnK -UUU, but also with six additional group II introns, all belonging to intron subclass IIA. Mapping analyses of RNA binding sites revealed MatK to recognize multiple regions within the trnK intron. Organellar group II introns are considered to be the ancestors of nuclear spliceosomal introns. That MatK associates with multiple intron ligands makes it an attractive model for an early trans -acting nuclear splicing activity.

Plant mitochondrial genomes are split into sub-genomes, i.e. genes are distributed across various sub-genomic molecules. To investigate whether copy numbers vary between individual mitochondrial genes, we used quantitative real-time PCR in combination with flow cytometric determination of nuclear DNA quantities to determine absolute per-cell-copy numbers of four mitochondrial genes in various Arabidopsis organs and the leaves of tobacco (Nicotiana tabacum) and barley (Hordeum vulgare). The copy numbers of the investigated mitochondrial genes (atp1, rps4, nad6 and cox1) not only differed from each other, but also varied between organs and changed during the development of cotyledons and leaves in Arabidopsis. We found no correlation between altered gene copy numbers, transcript levels and O₂ consumption. However, per cell, both the number of mitochondria and the number of gene copies increased with growing cell size. Gene copy numbers varied from approximately 40 (cox1 in young leaves) to approximately 280 (atp1 in mature leaves), and the mean number of mitochondria was approximately 300 in young leaves and 450 in mature leaves. Thus, cells are polyploid with respect to their mitochondrial genomes, but individual mitochondria may contain only part of the genome or even no DNA at all. Our data supports structural models of the mitochondrial genome in non-dividing cells of angiosperms that predict localization of the genes on sub-genomic molecules rather than master chromosomes. The data indicate control of the number of individual genes according to the genotype and developmental program(s) via amplification and/or degradation of sub-genomic molecules.