Florence Courtois

Regulation of plastid rDNA transcription by interaction of CDF2 with two different RNA polymerases

The plastid genome is known to be transcribed by a plastid‐encoded prokaryotic‐type RNA polymerase (PEP) and by a nucleus‐encoded phage‐type RNA polymerase (NEP). The spinach plastid rrn operon promoter region harbours three different, overlapping promoters. Two of them are of the prokaryotic type. The third promoter is a non‐consensus‐type NEP promoter. We separated three different transcriptional activities from spinach chloroplasts: PEP, the phage‐type RNA polymerase NEP‐1, and a third, hitherto undescribed transcriptional activity (NEP‐2). NEP‐2 specifically transcribes the rrn operon in the presence of the transcription factor CDF2. CDF2 was previously shown to recruit PEP to the rrn promoter to repress transcription. Together, our results suggest the existence of a third RNA polymerase in plastids and a mechanism of rDNA transcriptional regulation that is based on the interaction of the transcription factor CDF2 with two different transcriptional systems.

Building Up of the Plastid Transcriptional Machinery during Germination and Early Plant Development

The plastid genome is transcribed by three different RNA polymerases, one is called plastid-encoded RNA polymerase (PEP) and two are called nucleus-encoded RNA polymerases (NEPs). PEP transcribes preferentially photosynthesis-related genes in mature chloroplasts while NEP transcribes preferentially housekeeping genes during early phases of plant development, and it was generally thought that during plastid differentiation the building up of the NEP transcription system precedes the building up of the PEP transcription system. We have now analyzed in detail the establishment of the two different transcription systems, NEP and PEP, during germination and early seedling development on the mRNA and protein level. Experiments have been performed with two different plant species, Arabidopsis (Arabidopsis thaliana) and spinach (Spinacia oleracea). Results show that the building up of the two different transcription systems is different in the two species. However, in both species NEP as well as PEP are already present in seeds, and results using Tagetin as a specific inhibitor of PEP activity demonstrate that PEP is important for efficient germination, i.e. PEP is already active in not yet photosynthetically active seed plastids.

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.

Intraplastidial trafficking of a phage-type RNA polymerase is mediated by a thylakoid RING-H2 protein

The plastid genome of dicotyledonous plants is transcribed by three different RNA polymerases; an eubacterial-type enzyme, PEP; and two phage-type enzymes, RPOTp and RPOTmp. RPOTp plays an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation. RPOTmp fulfills a specific function in the transcription of the rrn operon in proplasts/amyloplasts during seed imbibition/germination and a more general function in chloroplasts during later developmental stages. In chloroplasts, RPOTmp is tightly associated with thylakoid membranes indicating that functional switching of RPOTmp is connected to thylakoid association. By using the yeast two-hybrid system, we have identified two proteins that interact with RPOTmp. The two proteins are very similar, both characterized by three N-terminal transmembrane domains and a C-terminal RING domain. We show that at least one of these proteins is an intrinsic thylakoid membrane protein that fixes RPOTmp on the stromal side of the thylakoid membrane, probably via the RING domain. A model is presented in which light by triggering the synthesis of the RING protein determines membrane association and functional switching of RPOTmp.

Transcription factor IIB (TFIIB)-related protein (pBrp), a plant-specific member of the TFIIB-related protein family.

ABSTRACT Although it is now well documented that metazoans have evolved general transcription factor (GTF) variants to regulate their complex patterns of gene expression, there is so far no information regarding the existence of specific GTFs in plants. Here we report the characterization of a ubiquitously expressed gene that encodes a bona fide novel transcription factor IIB (TFIIB)-related protein in Arabidopsis thaliana. We have shown that this protein is the founding member of a plant-specific TFIIB-related protein family named pBrp (for plant-specific TFIIB-related protein). Surprisingly, in contrast to common GTFs that are localized in the nucleus, the bulk of pBrp proteins are bound to the cytoplasmic face of the plastid envelope, suggesting an organelle-specific function for this novel class of TFIIB-related protein. We show that pBrp proteins harbor conditional proteolytic signals that can target these proteins for rapid turnover by the proteasome-mediated protein degradation pathway. Interestingly, under conditions of proteasome inhibition, pBrp proteins accumulate in the nucleus. Together, our results suggest a possible involvement of these proteins in an intracellular signaling pathway between plastids and the nucleus. Our data provide the first evidence for an organelle-related evolution of the eukaryotic general transcription machinery.

Adjustments of embryonic photosynthetic activity modulate seed fitness in Arabidopsis thaliana.

In this work, we dissect the physiological role of the transient photosynthetic stage observed in developing seeds of Arabidopsis thaliana. By combining biochemical and biophysical approaches, we demonstrate that despite similar features of the photosynthetic apparatus, light absorption, chloroplast morphology and electron transport are modified in green developing seeds, as a possible response to the peculiar light environment experienced by them as a result of sunlight filtration by the pericarp. In particular, enhanced exposure to far-red light, which mainly excites photosystem I, largely enhances cyclic electron flow around this complex at the expenses of oxygen evolution. Using pharmacological, genetic and metabolic analyses, we show that both linear and cyclic electron flows are important during seed formation for proper germination timing. Linear flow provides specific metabolites related to oxygen and water stress responses. Cyclic electron flow possibly adjusts the ATP to NADPH ratio to cope with the specific energy demand of developing seeds. By providing a comprehensive scenario of the characteristics, function and consequences of embryonic photosynthesis on seed vigour, our data provide a rationale for the transient building up of a photosynthetic machinery in seeds.

Sub-plastidial localization of two different phage-type RNA polymerases in spinach chloroplasts

Plant plastids contain a circular genome of ∼150 kb organized into ∼35 transcription units. The plastid genome is organized into nucleoids and attached to plastid membranes. This relatively small genome is transcribed by at least two different RNA polymerases, one being of the prokaryotic type and plastid-encoded (PEP), the other one being of the phage-type and nucleus-encoded (NEP). The presumed localization of a second phage-type RNA polymerase in plastids is still questionable. There is strong evidence for a sequential action of NEP and PEP enzymes during plant development attributing a prevailing role of NEP during early plant and plastid development, although NEP is present in mature chloroplasts. In the present paper, we have analysed two different NEP enzymes from spinach with respect to subcellular and intra-plastidial localization in mature chloroplasts with the help of specific antibodies. Results show the presence of the two different NEP enzymes in mature chloroplasts. Both enzymes are entirely membrane bound but, unlike previously thought, this membrane binding is not mediated via DNA. This finding indicates that NEP enzymes are not found as elongating transcription complexes on the template DNA in mature chloroplasts and raises the question of their function in mature chloroplasts.

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.

Plastid gene expression during chloroplast differentiation and dedifferentiation into non-photosynthetic plastids during seed formation

Arabidopsis seed formation is coupled with two plastid differentiation processes. Chloroplast formation starts during embryogenesis and ends with the maturation phase. It is followed by chloroplast dedifferentiation/degeneration that starts at the end of the maturation phase and leads to the presence of small non-photosynthetic plastids in dry seeds. We have analysed mRNA and protein levels of nucleus- and plastid-encoded (NEP and PEP) components of the plastid transcriptional machinery, mRNA and protein levels of some plastid RNA polymerase target genes, changes in plastid transcriptome profiles and mRNA and protein levels of some selected nucleus-encoded plastid-related genes in developing seeds during embryogenesis, maturation and desiccation. As expected, most of the mRNAs and proteins increase in abundance during maturation and decrease during desiccation, when plastids dedifferentiate/degenerate. In contrast, mRNAs and proteins of components of the plastid transcriptional apparatus do not decrease or even still increase during the period of plastid dedifferentiation. Results suggest that proteins of the plastid transcriptional machinery are specifically protected from degradation during the desiccation period and conserved in dry seeds to allow immediate regain of plastid transcriptional activity during stratification/germination. In addition, results reveal accumulation and storage of mRNAs coding for RNA polymerase components and sigma factors in dry seeds. They should provide immediately-to-use templates for translation on cytoplasmic ribosomes in order to enhance RNA polymerase protein levels and to provide regulatory proteins for stored PEP to guaranty efficient plastid genome transcription during germination.

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long-Term Response of Arabidopsis to Light Quality Shifts

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.