The plastid genome is transcribed by two distinct RNA polymerases, the PEP encoded by the plastid genome and the NEP encoded in the nucleus. Initial models of plastid transcription held that the NEP is responsible for the transcription of housekeeping genes needed early in development, and that the PEP transcribes genes required for photosynthesis. Recently, this model was challenged by the discovery that all plastid genes are transcribed by NEP in PEP-deficient tobacco plastids, suggesting that mRNA turnover may have a strong role in previously observed transcription patterns. In this study, we provide evidence that the NEP enzyme level decreases as plastids mature. In contrast, production of mRNAs by NEP increases as plastids mature, yet their accumulations remain constant. These results suggest that as plastids mature NEP may become more active, and that mRNA turnover varies between transcripts synthesized by NEP and PEP.

BackgroundThe purpose of this study was to sequence and assemble the tobacco mitochondrial transcriptome and obtain a genomic-level view of steady-state RNA abundance. Plant mitochondrial genomes have a small number of protein coding genes with large and variably sized intergenic spaces. In the tobacco mitogenome these intergenic spaces contain numerous open reading frames (ORFs) with no clear function.ResultsThe assembled transcriptome revealed distinct monocistronic and polycistronic transcripts along with large intergenic spaces with little to no detectable RNA. Eighteen of the 117 ORFs were found to have steady-state RNA amounts above background in both deep-sequencing and qRT-PCR experiments and ten of those were found to be polysome associated. In addition, the assembled transcriptome enabled a full mitogenome screen of RNA C→U editing sites. Six hundred and thirty five potential edits were found with 557 occurring within protein-coding genes, five in tRNA genes, and 73 in non-coding regions. These sites were found in every protein-coding transcript in the tobacco mitogenome.ConclusionThese results suggest that a small number of the ORFs within the tobacco mitogenome may produce functional proteins and that RNA editing occurs in coding and non-coding regions of mitochondrial transcripts.

Light-independent protochlorophyllide reduction leading to chlorophyll formation in the dark requires both chloroplast and nuclear gene expression in Chlamydomonas reinhardtii . Mutations in any one of the plastid ( chlL , chlN , and chlB ) or nuclear ( y-1 to y-10) genes required for this process result in the phenotype of the yellow-in-the-dark or y mutants. Analysis of the chlL , chlN , and chlB transcript levels in both light-and dark-grown wild-type and y mutant cells showed that the y mutations have no effect on the transcription of these plastid genes. Protein gel blot analysis showed that the CHLN and CHLB proteins are present in similar amounts in light-and dark-grown wild-type cells, whereas CHLL is present only in wild-type cells grown in the dark or at light intensities р 15 mol m Ϫ 2 sec Ϫ 1 . Analysis of chlL transcript distribution on polysome profiles and rates of protein turnover in chloramphenicol-treated cells suggested that CHLL formation is most probably blocked at translation initiation or elongation. Furthermore, treatment of cells with metabolic inhibitors and uncouplers of photosynthetic electron transport showed that regulation of CHLL formation is linked to the physiologic status of the chloroplast. Similar to wild-type cells, y mutants contain nearly identical amounts of CHLN and CHLB when grown in either light or darkness. However, no CHLL is present in any of the y mutants except y-7 , which contains an immunoreactive CHLL smaller than the expected size. Our findings indicate that CHLL translation is negatively photoregulated by the energy state or redox potential within the chloroplast in wild-type cells and that nuclear y genes are required for synthesis or accumulation of the CHLL protein. INTRODUCTIONTwo distinct mechanisms have become established for the reduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide), a key step in the chlorophyll biosynthesis pathway. One mechanism, which is catalyzed by the enzyme NADPH:PChlide oxidoreductase (POR), depends completely on light for its activity (Reinbothe and Reinbothe, 1996;Timko, 1998). Light-dependent POR activity is present in cyanobacteria, green algae, and most nonvascular and vascular plants, and it is the only mechanism used for chlorophyll formation in angiosperms. The second mechanism, which is present in anoxygenic photosynthetic bacteria, cyanobacteria, nonvascular plants, ferns, and gymnosperms, can reduce PChlide to Chlide in a light-independent manner (Fujita, 1996;Armstrong, 1998). Organisms containing this PChlide reduction mechanism are all capable of chlorophyll formation in the dark. Although a large amount of information is now available on the regulation of POR biosynthesis and activity, little is known about the enzyme that mediates light-independent PChlide reduction (designated LIPOR), the factors that regulate its formation, and the enzyme's requirements for catalytic function.Previous studies have shown that the products of three chloroplast genes (designated chlL , chlN , and chlB ) and at lea...

In this paper, we describe the complete chloroplast genome of Lolium arundinaceum. This sequence is the culmination of a long-term project completed by >400 undergraduates who took general genetics at Middle Tennessee State University from 2004-2007. It was undertaken in an attempt to introduce these students to an open-ended experiential/exploratory lesson to produce and analyze novel data. The data they produced should provide the necessary information for both phylogenetic comparisons and plastome engineering of tall fescue. The fescue plastome (GenBank FJ466687) is 136048 bp with a typical quadripartite structure and a gene order similar to other grasses; 56% of the plastome is coding region comprised of 75 protein-coding genes, 29 tRNAs, four rRNAs, and one hypothetical coding region (ycf). Comparisons of Poaceae plastomes reveal size differences between the PACC (subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae) and BOP (subfamilies Bambusoideae, Oryzoideae, and Pooideae) clades. Alignment analysis suggests that several potentially conserved large deletions in previously identified intergenic length polymorphic regions are responsible for the majority of the size discrepancy. Phylogenetic analysis using whole plastome data suggests that fescue closely aligns with Lolium perenne. Some unique features as well as phylogenetic branch length calculations, however, suggest that a number of changes have occurred since these species diverged.

Dinoflagellates are a group of unicellular protists with immense ecological and evolutionary significance and cell biological diversity. Of the photosynthetic dinoflagellates, the majority possess a plastid containing the pigment peridinin, whereas some lineages have replaced this plastid by serial endosymbiosis with plastids of distinct evolutionary affiliations, including a fucoxanthin pigment-containing plastid of haptophyte origin. Previous studies have described the presence of widespread substitutional RNA editing in peridinin and fucoxanthin plastid genes. Because reports of this process have been limited to manual assessment of individual lineages, global trends concerning this RNA editing and its effect on the biological function of the plastid are largely unknown. Using novel bioinformatic methods, we examine the dynamics and evolution of RNA editing over a large multispecies data set of dinoflagellates, including novel sequence data from the peridinin dinoflagellate Pyrocystis lunula and the fucoxanthin dinoflagellate Karenia mikimotoi. We demonstrate that while most individual RNA editing events in dinoflagellate plastids are restricted to single species, global patterns, and functional consequences of editing are broadly conserved. We find that editing is biased toward specific codon positions and regions of genes, and generally corrects otherwise deleterious changes in the genome prior to translation, though this effect is more prevalent in peridinin than fucoxanthin lineages. Our results support a model for promiscuous editing application subsequently shaped by purifying selection, and suggest the presence of an underlying editing mechanism transferred from the peridinin-containing ancestor into fucoxanthin plastids postendosymbiosis, with remarkably conserved functional consequences in the new lineage.

In maize, the chloroplast chromosome encodes 104 genes whose roles are primarily in photosynthesis and gene expression. The 2,000-3,000 nuclear gene products that localize to plastids are required both to encode and regulate plastid gene expression as well as to underpin each aspect of plastid physiology and development. We used a new "three-genome" maize biogenesis cDNA microarray to track abundance changes in nuclear, chloroplast and mitochondrial transcripts in stage 2 semi-emerged leaf blades of one month-old maize plants. We report the detection and quantification of 433 nuclear, 62 chloroplast, and 27 mitochondrial transcripts, with the majority of the nuclear transcripts predicted or known to encode plastid proteins. The data were analyzed as ratios of expression of individual transcripts in the green tip (mature chloroplasts) versus the yellow base of the leaf (etioplasts). According to the microarray data at least 51 plastid genes and 121 nuclear genes are expressed at least two-fold higher in the tip of the leaf. Almost all (25) mitochondrial and 177 nuclear transcripts were expressed at least 2-fold higher in the leaf base. Independent quantification of a subset of each transcript population by RNA gel blot analysis and/or quantitative real time RT-PCR concurred with the transcript ratios determined by the array. Ontological distribution of the transcripts suggests that photosynthesis-related RNAs were most highly abundant in the leaf tip and that energy use genes were most highly expressed in the base. Transcripts whose products are used in plastid translation constituted the largest single ontological group with relatively equal numbers of genes in the three expression categories, defined as higher in tip, higher in base, or equally expressed in tip and base.

The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival. In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes. We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants. We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression.

Nearly all land plants post-transcriptionally modify specific nucleotides within RNAs, a process known as RNA editing. This adaptation allows the correction of deleterious mutations within the asexually reproducing and presumably non-recombinant chloroplast and mitochondrial genomes. There are no reports of RNA editing in any of the green algae so this phenomenon is presumed to have originated in embryophytes either after the invasion of land or in the now extinct algal ancestor of all land plants. This was challenged when a recent in silico screen for RNA edit sites based on genomic sequence homology predicted edit sites in the green alga Chara vulgaris, a multicellular alga found within the Streptophyta clade and one of the closest extant algal relatives of land plants. In this study, the organelle transcriptomes of C. vulgaris and Chlamydomonas reinhardtii were deep sequenced for a comprehensive assessment of RNA editing. Initial analyses based solely on sequence comparisons suggested potential edit sites in both species, but subsequent high-resolution melt analysis, RNase H-dependent PCR (rhPCR), and Sanger sequencing of DNA and complementary DNAs (cDNAs) from each of the putative edit sites revealed them to be either single-nucleotide polymorphisms (SNPs) or spurious deep sequencing results. The lack of RNA editing in these two lineages is consistent with the current hypothesis that RNA editing evolved after embryophytes split from its ancestral algal lineage.