Livia Merendino

Phage-Type RNA Polymerase RPOTmp Transcribes the rrn Operon from the PC Promoter at Early Developmental Stages in Arabidopsis

The plastid genome of higher plants is transcribed by two different types of RNA polymerases named nucleus encoded RNA polymerase (NEP) and plastid encoded RNA polymerase. Plastid encoded RNA polymerase is a multimeric enzyme comparable to eubacterial RNA polymerases. NEP enzymes represent a small family of monomeric phage-type RNA polymerases. Dicotyledonous plants harbor three different phage-type enzymes, named RPOTm, RPOTp, and RPOTmp. RPOTm is exclusively targeted to mitochondria, RPOTp is exclusively targeted to plastids, and RPOTmp is targeted to plastids as well as to mitochondria. In this article, we have made use of RPOTp and RPOTmp T-DNA insertion mutants to answer the question of whether both plastid-located phage-type RNA polymerases have overlapping or specific functions in plastid transcription. To this aim, we have analyzed accD and rpoB messenger RNAs (mRNA; transcribed from type I NEP promoters), clpP mRNA (transcribed from the −59 type II NEP promoter), and the 16S rRNA (transcribed from the exceptional PC NEP promoter) by primer extension. Results suggest that RPOTp represents the principal RNA polymerase for transcribing NEP-controlled mRNA genes during early plant development, while RPOTmp transcribes specifically the rrn operon from the PC promoter during seed imbibition.

Plastid RNA polymerases: orchestration of enzymes with different evolutionary origins controls chloroplast biogenesis during the plant life cycle

Chloroplasts are the sunlight-collecting organelles of photosynthetic eukaryotes that energetically drive the biosphere of our planet. They are the base for all major food webs by providing essential photosynthates to all heterotrophic organisms including humans. Recent research has focused largely on an understanding of the function of these organelles, but knowledge about the biogenesis of chloroplasts is rather limited. It is known that chloroplasts develop from undifferentiated precursor plastids, the proplastids, in meristematic cells. This review focuses on the activation and action of plastid RNA polymerases, which play a key role in the development of new chloroplasts from proplastids. Evolutionarily, plastids emerged from the endosymbiosis of a cyanobacterium-like ancestor into a heterotrophic eukaryote. As an evolutionary remnant of this process, they possess their own genome, which is expressed by two types of plastid RNA polymerase, phage-type and prokaryotic-type RNA polymerase. The protein subunits of these polymerases are encoded in both the nuclear and plastid genomes. Their activation and action therefore require a highly sophisticated regulation that controls and coordinates the expression of the components encoded in the plastid and nucleus. Stoichiometric expression and correct assembly of RNA polymerase complexes is achieved by a combination of developmental and environmentally induced programmes. This review highlights the current knowledge about the functional coordination between the different types of plastid RNA polymerases and provides working models of their sequential expression and function for future investigations.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Transcriptional organization of the large and the small ATP synthase operons, atpI/H/F/A and atpB/E, in Arabidopsis thaliana chloroplasts

The ATP synthase is a ubiquitous enzyme which is found in bacteria and eukaryotic organelles. It is essential in the photosynthetic and respiratory processes, by transforming the electrochemical proton gradient into ATP energy via proton transport across the membranes. In Escherichia coli, the atp genes coding for the subunits of the ATP synthase enzyme are grouped in the same transcriptional unit, while in higher plants the plastid atp genes are organized into a large (atpI/H/F/A) and a small (atpB/E) atp operon. By using the model plant Arabidopsis thaliana, we have investigated the strategy evolved in chloroplasts to overcome the physical separation of the atp gene clusters and to coordinate their transcription. We show that all the identified promoters in the two atp operons are PEP dependent and require sigma factors for specific recognition. Our results indicate that transcription of the two atp operons is initiated by at least one common factor, the essential SIG2 factor. Our data show that SIG3 and SIG6 also participate in transcription initiation of the large and the small atp operon, respectively. We propose that SIG2 might be the factor responsible for coordinating the basal transcription of the plastid atp genes and that SIG3 and SIG6 might serve to modulate plastid atp expression with respect to physiological and environmental conditions. However, we observe that in the sigma mutants (sig2, sig3 and sig6) the deficiency in the recognition of specific atp promoters is largely balanced by mRNA stabilization and/or by activation of otherwise silent promoters, indicating that the rate-limiting step for expression of the atp operons is mostly post-transcriptional.

On the Complexity of Chloroplast RNA Metabolism: psaA Trans-splicing Can be Bypassed in Chlamydomonas

In the chloroplast, the posttranscriptional steps of gene expression are remarkably complex. RNA maturation and translation rely on a large cohort of nucleus-encoded proteins that act specifically on a single target transcript or a small set of targets. For example in the chloroplast of Chlamydomonas, trans-splicing of the two split introns of psaA requires at least 14 nucleus-encoded proteins. To investigate the functional significance of this complex trans-splicing pathway, we have introduced an intron-less copy of psaA in the chloroplast genomes of three mutants deficient in trans-splicing and of the wild type. We find that the intron-less psaA gene rescues the mutant phenotypes. The growth of strains with the intron-less psaA is indistinguishable from the wild type under the set of different experimental conditions that were investigated. Thus, the trans-splicing factors do not appear to have any other essential function and trans-splicing of psaA can be bypassed. We discuss how these observations support the hypothesis that complex RNA metabolism in the chloroplast may in part be the result of a nonadaptive evolutionary ratchet. Genetic drift may lead to the accumulation of chloroplast mutations and the recruitment of compensatory nuclear suppressors from large preexisting pools of genes encoding RNA-binding proteins.

Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5′ and 3′ end-definition. The current model for processing proposes that the 3′ end of the upstream cistron transcripts and the 5′ end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3′ end, thus representing degradation products. We observe, instead, that the 5′ ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3′ ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple strategies to produce mature transcripts suitable for translation.

Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1

The albino pap7-1 mutant of Arabidopsis reveals the relative impact of light and plastid developmental stage on the expression of nuclear genes involved in metabolism and photosynthesis. Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.

Characterization of the psbH precursor RNAs reveals a precise endoribonuclease cleavage site in the psbT/psbH intergenic region that is dependent on psbN gene expression

The plastid psbB operon harbours 5 genes, psbB, psbT, psbH, petB and petD. A sixth gene, the psbN gene, is located on the opposite DNA strand in the psbT/psbH intergenic region. Its transcription produces antisense RNA to a large part of the psbB pentacistronic mRNA. We have investigated whether transcription of the psbN gene, i.e. production of antisense RNA, influences psbT/psbH intergenic processing. Results reveal the existence of four different psbH precursor RNAs. Three of them result from processing and one is produced by transcription initiation. One of the processed RNAs is probably created by site-specific RNA cleavage. This RNA is absent in plants where the psbN gene is not transcribed suggesting that cleavage at this site is dependent on the formation of sense/antisense double-stranded RNA. In order to characterize the nuclease that might be responsible for double-stranded RNA cleavage, we analysed csp41a and csp41b knock-out mutants and the corresponding double mutant. Both CSP41 proteins are known to interact physically and CSP41a had been shown to cleave within 3′-untranslated region stem-loop structures, which contain double-stranded RNA, in vitro. We demonstrate that the psbH RNA, that is absent in plants where the psbN gene is not transcribed, is also strongly diminished in all csp41 plants. Altogether, results reveal a site-specific endoribonuclease cleavage event that seems to depend on antisense RNA and might implicate endoribonuclease activity of CSP41a.

Correction: Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

The name of the first author was incorrectly represented in the Citation. The correct Citation is: Malik Ghulam M, Courtois F, Lerbs-Mache S, Merendino L (2013) Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts. PLoS ONE 8(11): e78265. doi:10.1371/journal.pone.0078265