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

Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts

International audienceChloroplasts 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

Specific function of a plastid sigma factor for ndhF gene transcription

The complexity of the plastid transcriptional apparatus (two or three different RNA polymerases and numerous regulatory proteins) makes it very difficult to attribute specific function(s) to its individual components. We have characterized an Arabidopsis T-DNA insertion line disrupting the nuclear gene coding for one of the six plastid sigma factors (SIG4) that regulate the activity of the plastid-encoded RNA polymerase PEP. This mutant shows a specific diminution of transcription of the plastid ndhF gene, coding for a subunit of the plastid NDH [NAD(P)H dehydrogenase] complex. The absence of another NDH subunit, i.e. NDHH, and the absence of a chlorophyll fluorescence transient previously attributed to the activity of the plastid NDH complex indicate a strong down-regulation of NDH activity in the mutant plants. Results suggest that plastid NDH activity is regulated on the transcriptional level by an ndhF-specific plastid sigma factor, SIG4

Characterization of plastid psbT sense and antisense RNAs

The plastid psbB operon is composed of the psbB, psbT, psbH, petB and petD genes. The psbN gene is located in the intergenic region between psbT and psbH on the opposite DNA strand. Transcription of psbN is under control of sigma factor 3 (SIG3) and psbN read-through transcription produces antisense RNA to psbT mRNA. To investigate on the question of whether psbT gene expression might be regulated by antisense RNA, we have characterized psbT sense and antisense RNAs. Mapping of 5′ and 3′-ends by circular RT–PCR and /or 5′-RACE experiments reveal the existence of two different sense and antisense RNAs each, one limited to psbT RNA and a larger one that covers, in addition, part of the psbB coding region. Sense and antisense RNAs seem to form double-stranded RNA/RNA hybrids as indicated by nuclease digestion experiments followed by RT–PCR amplification to reveal nuclease resistant RNA. Western immunoblotting using antibodies made against PSBT protein and primer extension analysis of different plastid mRNA species and psbT antisense RNA suggest that sequestering of psbT mRNA by hybrid formation results in translational inactivation of the psbT mRNA and provides protection against nucleolytic degradation of mRNA during photooxydative stress conditions

Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psbN gene in plastids

We have investigated the function of one of the six plastid sigma-like transcription factors, sigma 3 (SIG3), by analysing two different Arabidopsis T-DNA insertion lines having disrupted SIG3 genes. Hybridization of wild-type and sig3 plant RNA to a plastid specific microarray revealed a strong reduction of the plastid psbN mRNA. The microarray result has been confirmed by northern blot analysis. The SIG3-specific promoter region has been localized on the DNA by primer extension and mRNA capping experiments. Results suggest tight regulation of psbN gene expression by a SIG3-PEP holoenzyme. The psbN gene is localized on the opposite strand of the psbB operon, between the psbT and psbH genes, and the SIG3-dependent psbN transcription produces antisense RNA to the psbT–psbH intergenic region. We show that this antisense RNA is not limited to the intergenic region, i.e. it does not terminate at the end of the psbN gene but extends as antisense transcript to cover the whole psbT coding region. Thus, by specific transcription initiation at the psbN gene promoter, SIG3-PEP holoenzyme could also influence the expression of the psbB operon by producing psbT antisense RNA

Function of plastid sigma factors in higher plants: Regulation of gene expression or just preservation of constitutive transcription?

International audiencePlastid gene expression is rather complex. Transcription is performed by three different RNA polymerases, two of them are nucleus-encoded, monomeric, of the phage-type (named RPOTp and RPOTmp) and one of them is plastid-encoded, multimeric, of the eubacterialtype (named PEP). The activity of the eubacterial-type RNA polymerase is regulated by up to six nucleus-encoded transcription initiation factors of the sigma-type. This complexity of the plastid transcriptional apparatus is not yet well understood and raises the question of whether it is subject to any regulation or just ensures constitutive transcription of the plastid genome. On the other hand, considerable advances have been made during the last years elucidating the role of sigma factors for specific promoter recognition and selected transcription of some plastid genes. Sigma-interacting proteins have been identified and phosphorylation-dependent functional changes of sigma factors have been revealed. The present review aims to summarize these recent advances and to convince the reader that plastid gene expression is regulated on the transcriptional level by sigma factor action

A new concept to engineer specific transcription of transplastomic genes

Plants are increasingly becoming attractive bioreactors for production of commercially important proteins. Plastid transformation, which has been recently developed for this purpose, offers several advantages. (1) Each molecular unit of the chloroplast genome has a relatively small size, ca. 150 kbp, which facilitates homologous recombination and hence permits introduction of DNA modifications such as deletions or insertions at precise locations into the chloroplast chromosome. This is in contrast with the random insertion of DNA fragments into the nuclear genome obtained using the well-known method of gene transfer using Agrobacterium tumefaciens. (2) The overall organization of the chloroplast genome is of prokaryotic type, and therefore complex phenomenon such as position effects and epigenetic controls encountered in nuclear gene regulation do not exist. (3) The chloroplast chromosome has a large number – up to one hundred – of copies per mature chloroplast. For example, 10 000 chromosome copies per cell are present in mesophyll, which have approximately 100 chloroplasts/cell. Consequently, the amount of heterologous protein corresponding to a given transgene can reach a relative high level, e.g., in some cases up to 45 % of total soluble proteins.1 (4) Finally, most plant species of agronomic interest do not transmit the chloroplast genome by pollen, as their chloroplasts are maternally inherited. Maternal inheritance avoids gene dispersion and transfer to other, non-transformed, related plant species. Application of plastid transformation technology is limited by the difficulty of obtaining regulated, selective expression of the transgenes. Except for the blue light-inducible psbD promoter, there are no specific inducible endogenous promoters available; hence transgenes are constitutively expressed throughout plant development. As a consequence, plant growth is often heavily disturbed by th

Mise en place d'un système spécifique de l'expression d'un transgène plastidial

Mon travail a visé à mettre au point un système permettant l'expression dans les chloroplastes d'une protéine à haute valeur ajoutée, de manière spécifique et contrôlée. Pour cela, la réalisation d'une double transformation (nucléaire et plastidiale) a été envisagée. Une première avancée dans ce domaine a été réalisée en 1994 par la mise en place d'un système impliquant l'ARN polymérase T7 (McBride et al., 1994). L'originalité de mon travail réside en l'utilisation du système transcriptionnel propre au chloroplaste, en le re-programmant en quelque sorte, pour le forcer à transcrire, lorsque l'ordre lui en serait donné, un gène d'intérêt. La cible de cette re-programmation est le système transcriptionnel PEP et le moyen du re-programmation est un facteur sigma chimérique (hybride) ayant une spécificité de reconnaissance pour un promoteur absent du génome plastidial normal. Des plantes de tabac transplastomiques contenant la construction E- YFP reconnue par le facteur hybride après transformation plastidiale ont été régénérées. Elles ont été ensuite agro-infiltrées par la construction contenant le facteur sigma hybride. Des résultats préliminaires indiquent une faible expression du gène rapporteur dans des structures associées à des chloroplastes.D'autre part, afin de pouvoir tester in vitro les interactions du facteur sigma hybride avec la PEP core, un système de transcription utilisant l' ARN polymérase PEP a été mis au point.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Ontologie de l'appareil transcriptionnel plastidial au cours de la germination et du développement de la jeune plantule

La graine au cours de la germination utilise ses réserves puis acquiert des capacités d'autotrophie. Cette transition de mode de nutrition dépend de la différenciation des proplastes, présents dans les graines matures, en chloroplastes. Ces derniers organites possèdent un génome d'environ 150 kpb chez les végétaux supérieurs. Malgré cette taille réduite, la transcription du génome plastidial fait intervenir au moins 2 types d'ARN polymérases : les NEP, ARN polymérase de type phagique codées dans le noyau, et la PEP, d'origine procaryote et dont les sous unités catalytiques sont codées dans le plastome. Nous nous sommes intéressés au fonctionnement de ces ARN polymérases lors de la germination et des premières phases de développement de la jeune plantule, afm de savoir si, d'une part, ces polymérases sont exprimées et actives lors des stades précédant la mise en place de la photosynthèse et, d'autre part, d'évaluer l'importance de leurs activités dans le processus germinatif. Après avoir démontré que ces deux types d' ARN polymérases sont présentes dans les graines de deux plantes, chez Arabidopsis et l'épinard, nous avons cherché à caractériser le rôle de chacune d'entre elles. Nous avons utilisé pour cela plusieurs approches, combinant l'utilisation d'un inhibiteur spécifique de la PEP et de mutants d'expression des NEP, à des méthodes d'analyse à petite échelle (Northem blot, extension d'amorces) et à grande échelle (transcriptomique, protéomique).Nous montrons pour la première fois que les deux types d'ARN polymérases plastidiales sont actives simultanément et dès le début de la germination (imbibition) contredisant l'idée courante d'une activité séquentielle des polymérases.Nous montrons également pour la première fois l'expression antisens d'un groupe de gènes plastidiaux dont certains sont présents dans les graines.Nous montrons enfm que les toutes premières étapes de la transcription plastidiale concernent l'opéron ribosomique et que l'expression de cet opéron pendant la germination fait intervenir à la fois la PEP et une des NEP. L'absence d'activité de l'une ou l'autre provoque essentiellement une déficience de transcription des ARN ribosomiques plastidiaux et induit un retard de germination.En conclusion notre travail souligne l'importance et la nécessité de la transcription plastidiale au cours de la germination.During the germination process the seed uses all the reserves that it contains and only afterwards becomes autotrophic. This transition in the nutrition mode is dependent on the differentiation of proplastids (that are present in the mature seed) into chloroplasts.ln higher plants, chloroplasts contain a genome of around 150 kb. However, despite the small size, the genome is transcribed by two different types of RNA polymerases: the NEPs enzymes, which are phage-type polymerases and are encoded by the nucleus Œucleus-gncoded RNA rolymerase), and the PEP enzyme, which is a eubacterial-type polymerase and contains plastid-encoded subunits. Aim of this study is to investigate the role of the plastid RNA polymerases during germination and early plant development. First of all, we have analysed if all the RNA polymerases are expressed and active in the stages before the building up of the photosynthetic apparatus and if they are required by the germination process. We have indeed shown that both the NEP and the PEP polymerases are aIready present in the seeds oftwo plant species, Arabidopsis and Spinach. ln order to determine the role of each enzyme, we have analysed the transcriptome and the proteome of plants where the plastid polymerases were inactive. For this reason, we have either analysed plants that contain mutations in the genes coding for the NEP enzymes or that were treated with a specifie inhibitor of the PEP polymerases. Both large scale and small scale approaches were used.For the first time, we have shown that both types ofplastid RNA polymerases are active at the same time and since the beginning ofthe germination process (imbibition). Our data are in opposition to the CUITent opinion that suggests that NEP and PEP polymerases transcribe the plastid genome in a sequential fashion.For the first time, we have also shown the anti-sense expression of a group of plastid genes, in same cases even in the seeds.Finally, we have shown that during germination one of the fust gene-unit to be transcribed in the plastid is the ribosomal operon. Interestingly, the expression of the ribosomal operon needs both NEP and PEP polymerases. Therefore, the main consequence of the absence of either one of the two polymerases types is the reduction of the ribosomal RNAs synthesis that leads to a germination delay. ln conclusion, our study shows the importance and the requirement of the transcription of the plastid genome during the germination process.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

IDENTIFICATION ET CARACTERISATION DE FACTEURS D'INITIATION DE LA TRANSCRIPTION ASSOCIES AU PLASTE

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF