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© The Author(s) 2018. Published by Oxford University Press. The future of agricultural research depends on data. The sheer volume of agricultural biological data being produced today makes excellent data management essential. Governmental agencies, publishers and science funders require data management plans for publicly funded research. Furthermore, the value of data increases exponentially when they are properly stored, described, integrated and shared, so that they can be easily utilized in future analyses. AgBioData (https://www.agbiodata.org) is a consortium of people working at agricultural biological databases, data archives and knowledgbases who strive to identify common issues in database development, curation and management, with the goal of creating database products that are more Findable, Accessible, Interoperable and Reusable. We strive to promote authentic, detailed, accurate and explicit communication between all parties involved in scientific data. As a step toward this goal, we present the current state of biocuration, ontologies, metadata and persistence, database platforms, programmatic (machine) access to data, communication and sustainability with regard to data curation. Each section describes challenges and opportunities for these topics, along with recommendations and best practices.

International audience; MADS-box transcription factors play crucial roles in organ and cell differentiation in organisms ranging from yeast to humans. Most of the work on plant MADS-box proteins focused on their roles in floral development whereas less information is available on their function in fruit maturation. We cloned three distinct tomato cDNAs using a RT-PCR approach, encoding LeMADS1, LeMADS5 and LeMADS6 factors and whose mRNAs mostly accumulate in tomato flowers and fruits. Phylogeny analysis indicates that LeMADS1, 5 and 6 belong to the MEF2-like family. When transiently expressed in tobacco leaves or in human cells, LeMADS1, 5 and 6 are targeted to the cell nucleus. As the endogenous target genes of these putative transcription factors are unknown, the transcriptional activity of these proteins was characterized in a heterologous system and we showed that, when fused to a Gal4-DNA-binding domain, they repress the transcription of heterologous reporter genes. Since histone deacetylases control MEF2 transcriptional activity and since a putative histone deacetylase binding site was present in LeMADS1, 5 and 6, we tested the potential interaction between these factors and HDAC5 deacetylase. Surprisingly, in this heterologous system, LeMADS1, 5 and 6 interacted with HDAC5 N-terminal region. Our data suggest that, like mammalian MEF2A, plant MADS-box transcriptional activity might be regulated by enzymes controlling chromatin acetylation.

International audience; Degradation induced by thermal (50-110 °C) and radio-oxidation of low T g epoxy-amine networks has been studied. It has been found that oxidation leads mainly to amide groups formation at the vicinity of tertiary amines whatever ageing conditions (thermal or radio-oxidation at 200 Gy h -1 ). In addition, some species as acids, peracids or formates have been revealed indicating a chain scission process. Physical modifications as T g decrease and soluble fraction increase due to chain scission process, have been correlated with chemical modifications.

International audience; Summary The role of a subfamily of lipid globule-associated proteins, referred to as plant fibrillins (FIB1a, -1b, -2), was determined using a RNA interference (RNAi) strategy. We show that Arabidopsis plants with reduced levels of these plastid structural proteins are impaired in long-term acclimation to environmental constraint, namely photooxidative stress imposed by high light combined with cold. As a result, their photosynthetic apparatus is inefficiently protected. This leads to the prevalence of an abnormal granal and stromal membrane arrangement, as well as higher photosystem II photoinhibition under stress. The visible phenotype of FIB1-2 RNAi lines also includes retarded shoot growth and a deficit in anthocyanin accumulation under stress. All examined phenotypic effects of lower FIB levels are abolished by jasmonate (JA) treatment. An atypical expression pattern of several JA-induced genes was observed in RNAi plants. A JA-deficient mutant was found to share similar stress phenotypic characteristics with FIB RNAi plants. We conclude a new physiological role for JA, namely acclimation of chloroplasts, and that light/cold stress-related JA biosynthesis is conditioned by the accumulation of plastoglobule-associated FIB1-2 proteins. Consistent correlative data suggest that this FIB effect is mediated by plastoglobule (and triacylglycerol) accumulation as the potential site for initiating the chloroplast stress-related JA biosynthesis.

International audience; FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.

International audience; The protein termed fibrillin is involved in the formation of lipoprotein structures, such as plastoglobules and fibrils in certain chromoplast types, which have been implicated in the over-production of pigments due to a sink effect. In order to examine its effect in differentiating chromoplasts of a non-fibrillar type, the pepper fibrillin gene was expressed in tomato fruit. Both the transcript and protein were found to accumulate during tomato fruit ripening from an early mature green stage. However, formation of carotenoid deposition structures in tomato chromoplasts, such as fibrils, was not observed. Nevertheless, a two-fold increase in carotenoid content and associated carotenoid derived flavour volatiles (6-methyl-5-hepten-2-one, geranylacetone, beta-ionone and beta-cyclocitral) was observed. An unexpected phenotypic observation in the transgenic fruit was the delayed loss of thylakoids in differentiating chromoplasts, leading to the transient formation of plastids exhibiting a typical chromoplastic zone adjacent to a protected chloroplastic zone with preserved thylakoids. An in vitro assay has been developed to monitor fibrillin activity on thylakoids: data were obtained suggesting a membrane protection role for fibrillin, more specifically against moderate uncoupling effects.

The aim of this paper is to present a complete description of the structure of subsets S of an orderable group G satisfying | S 2 | = 3 | S | − 2 and 〈 S 〉 is non-abelian.

We used quantitative phase tomography with synchrotron radiation to elucidate the 3D structure of Arabidopsis seeds in their native state. The cells are clearly distinguished, and their internal structure is revealed through local variations in electron density. We visualized a 3D network of intercellular air space that might allow immediate gas exchange for energy supply during germination and/or serve for rapid water uptake and distribution during imbibition.

Plastid transformation technologies have developed rapidly over the last few years, reflecting their value in the study of the principal mechanisms of plastid gene expression and commercial interest in using plastids as bioreactors. Application of this technology is still limited by the difficulty of obtaining regulated, selective expression of plastid transgenes. The plastid genome is transcribed by two different types of RNA polymerase. One of them is of the eubacterial type of polymerase, and its subunits are encoded in the plastid genome [plastid-encoded RNA polymerase (PEP)]. The other one is of the phage type and nucleus-encoded [nucleus-encoded RNA polymerase (NEP)]. To obtain selective transgene expression, we have made use of the similarities and differences between the eubacterial and the plastid eubacterial type transcription systems. We created a hybrid transcription system in which the transgene is placed under the control of a eubacterial promoter which does not exist in the plastid genome and which is not recognized by the plastid endogenous transcriptional machinery. Selective transcription of the transgene is achieved by the supply of a chimeric transcription factor that interacts with PEP and directs it specifically to the foreign eubacterial-type transgene promoter. This hybrid transcription system could be used for biotechnological and fundamental research applications as well as in the characterization of the evolutionary differences between the eubacterial and the plastid eubacterial-type transcription systems.

Plant plastids contain a circular genome of approximately 150 kb organized into approximately 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.

An ultrasonic reflectometry method has been used successfully to detect elasticity changes following the curing of 100 $$\upmu $$ m thick epoxy films (DGEBA-PAA) coated onto 1.5 mm thick steel plates. The method employs a goniometric apparatus to measure the reflection coefficient amplitude around 5 MHz followed by a standard FFT analysis of the reflected signal. A specific ultrasonic wave mode was identified which was dependent on, and correlated with, the presence of the coating layer. The goniometer angle associated with this mode was different from that associated with Lamb modes of the plate, enabling the new mode to be detected reliably. The sensitivity of the new mode to variations in the paint mass density, longitudinal and transverse velocities and thickness has been quantified numerically by using Brekhovskikh’s model with due account taken of the finite width of the ultrasonic fields of the transducers. The method was tested for the detection of the evolution of the coating elasticity during curing at 80 $$^{\circ }$$ C for 400 h. Compensation was applied to correct for the effect of the natural swelling of the paint layer on the angular position of the tracked mode, and this was validated experimentally. The evolution of the angular position was found to offer a reliable means to detect elasticity changes during the cure of the coating. The mass density variation in the coating during cure only weakly affected the angular position. This method will provide a promising tool for the non destructive evaluation of paint coatings, particularly in service for the detection of ageing effects in the longer term.

International audience; Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

International audience; Upon imbibition, dry seeds rapidly gain metabolic activity and the switching on of a germination-specific transcriptional programme in the nucleus goes ahead, with the induction of many nucleus-encoded transcripts coding for plastid-localized proteins. Dedifferentiated plastids present in dry seeds differentiate into chloroplasts in cotyledons and into amyloplasts in the root and in the hypocotyl, raising the question of whether the beginning of a new plant's life cycle is also characterized by specific changes in the plastid transcriptional programme. Here the plastid transcriptome is characterized during imbibition/stratification, germination, and early seedling outgrowth. It is shown that each of these three developmental steps is characterized by specific changes in the transcriptome profile, due to differential activities of the three plastid RNA polymerases and showing the integration of plastids into a germination-specific transcriptional programme. All three RNA polymerases are active during imbibition; that is, at 4 °C in darkness. However, activity of plastid-encoded RNA polymerase (PEP) is restricted to the rrn operon. After cold release, PEP changes specificity by also transcribing photosynthesis-related genes. The period of germination and radicle outgrowth is further characterized by remarkable antisense RNA production that diminishes during greening when photosynthesis-related mRNAs accumulate to their highest but to very different steady-state levels. During stratification and germination mRNA accumulation is not paralleled by protein accumulation, indicating that plastid transcription is more important for efficient germination than translation.

Class II WSCP proteins (Watersoluble Chlorophyll binding Proteins) are soluble proteins that interact with chlorophyll and its derivatives in Brassicaceae. These proteins belong to the protease inhibitor family and they are induced by abiotic stress under light conditions. To date, their functions in plants are not well documented. The aim of this study is to characterize the physiological function of a WSCP protein in model plants Arabidopsis thaliana. We demonstrated initially that theWSCP protein is not induced under stress conditions during photomorphogenesis. However, the protein is expressed in dark grown (etiolated) seedlings as well as in the flower transmitting tract, the path of pollen tubes growth. In the flower, we have demonstrated a WSCP regulation by HEC and NTT transcription factors involved in floral development. By a reverse geneticapproach, we have highlighted a regulatory WSCP function in transmitting tract cell death. Consequently, the pollen tubegrowth is impaired and the resulting nonspecific cell death influence seed development. In etiolated seedlings, WSCP transcript is expressed in the apical hook, a structure which protects seedlings during emergence from soil. Interestingly, HEC and NTT transcription factors are also expressed in etiolated seedlings and regulate WSCP gene expression differently. Furthermore, WSCP protein accumulates in the presence of the stress hormone ethylene in apical hook and WSCP gene promoter activity increases during mechanical wounding. Taken together, these experiments suggest a protective role of WSCP proteins during skotomorphogenesis., Les protéines WSCP (Water-soluble Chlorophyll binding Proteins) de classe II sont des protéines solubles capables de fixer la chlorophylle et ses dérivés chez les Brassicaceae. Ces protéines font partie de la famille des inhibiteurs de protéases et elles présentent la particularité d'être induites en conditions de stress abiotiques à la lumière. Leurs fonctions in planta sont à ce jour très peu documentées. Cette thèse présente l'étude de la fonction physiologique d'une WSCP chez la plante modèle Arabidopsis thaliana. Nous avons montré dans un premier temps que la protéine WSCP n'est pas induite en conditions de stress pendant la photomorphogénèse. En revanche, elle est exprimée dans les plantules à l'obscurité (étiolées) et dans le tissu de transmission des fleurs, lieu de passage des tubes polliniques. Dans la fleur, nous avons mis en évidence la régulation de WSCP par les facteurs de transcriptions HEC et NTT impliqués dans le développement floral. Par une approche de génique inverse, nous avons montré un rôle de WSCP dans le contrôle de la mort cellulaire du tissu de transmission. En conséquence, la progression des tubes polliniques est altérée et la mort cellulaire non spécifique influence le développement des graines. Chez les plantules étiolées, le transcrit WSCP est exprimé au niveau de la crosse apicale, structure dont la fonction est de protéger les plantules au cours de l'émergence hors du sol. De manière intéressante, les facteurs NTT et HEC sont également exprimés dans les plantules étiolées mais régulent l'expression du gène WSCP de façon différente. De plus, la protéine WSCP s'accumule en présence de l'hormone de stress éthylène dans la crosse apicale et l'activité du promoteur du gène WSCP augmente en conditions de stress mécaniques. Prises ensembles, ces expériences laissent présager d'un rôle protecteur de la protéine WSCP au cours de la skotomorphogénèse.

International audience; Powering future generations of implanted medical devices will require cumbersome transcutaneous energy transfer or harvesting energy from the human body. No functional solution that harvests power from the body is currently available, despite attempts to use the Seebeck thermoelectric effect, vibrations or body movements. Glucose fuel cells appear more promising, since they produce electrical energy from glucose and dioxygen, two substrates present in physiological fluids. The most powerful ones, Glucose BioFuel Cells (GBFCs), are based on enzymes electrically wired by redox mediators. However, GBFCs cannot be implanted in animals, mainly because the enzymes they rely on either require low pH or are inhibited by chloride or urate anions, present in the Extra Cellular Fluid (ECF). Here we present the first functional implantable GBFC, working in the retroperitoneal space of freely moving rats. The breakthrough relies on the design of a new family of GBFCs, characterized by an innovative and simple mechanical confinement of various enzymes and redox mediators: enzymes are no longer covalently bound to the surface of the electron collectors, which enables use of a wide variety of enzymes and redox mediators, augments the quantity of active enzymes, and simplifies GBFC construction. Our most efficient GBFC was based on composite graphite discs containing glucose oxidase and ubiquinone at the anode, polyphenol oxidase (PPO) and quinone at the cathode. PPO reduces dioxygen into water, at pH 7 and in the presence of chloride ions and urates at physiological concentrations. This GBFC, with electrodes of 0.133 mL, produced a peak specific power of 24.4 microW mL(-1), which is better than pacemakers' requirements and paves the way for the development of a new generation of implantable artificial organs, covering a wide range of medical applications.

In angiosperms, singlet oxygen is a prominent form of reactive oxygen species that is produced during photosynthesis. Its excess causes photooxidative damage leading to cell death, demonstrated in mutants impaired in the chlorophyll biosynthetic pathway. In the present work, we used mutants of Arabidopsis thaliana that exhibit a conditional seedling lethal phenotype caused by the absence of the outer plastid envelope protein, OEP16-1. This protein is involved in the transport of amines, amino acids and is also implicated in the import of the key enzyme of chlorophyll synthesis, NADPH: Protochlorophyllide oxydoreductase A (PORA), into the plastids. Using a reverse genetic approach, four independent Atoep16-1 mutants were isolated and characterized, with different combinations of cell death properties and presence/absence of PORA. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark and died after illumination. Pchlide operated here as a photosensitizer triggering singlet oxygen formation. The other two mutants avoided excess Pchlide accumulation and greened normally. Using the mutant of barley, tigrina d12 as reference, we show that cell death induced in the photobleaching Atoep16-1 mutants occurs in a flu-independent pathway. Translation initiation at 80S ribosomes was identified to be a major target of singlet oxygen in the early hours of greening. At a delayed stage, singlet oxygen caused ribosome dissociation. We provided evidence that both effects on translation are genetically linked and they can be further studied using the Atoep16-1 mutant that we isolated and the previously described flu mutant., Chez les angiospermes, l'oxygène singulet est la forme majoritaire des espèces réactives de l'oxygène, étant produite lors de la photosynthèse. Son excès provoque le dommage photooxidatif conduisant à la mort cellulaire, observée chez les mutants affectés dans la voie de la biosynthèse de la chlorophylle. Dans ce travail, nous avons utilisé des mutants d'Arabidopsis thaliana qui manifestent le phénotype conditionnel de la mort cellulaire, causée par l'absence d'une protéine de l'enveloppe externe des plastes, OEP16-1. Cette protéine est impliquée dans le transport des amines, acides aminés et également dans l'import d'une enzyme clé de la synthèse de la chlorophylle, NADPH: Protochlorophyllide oxydoreductase A (PORA), dans les plastes. Une approche génétique inverse a permis d'isoler et de caractériser quatre mutants indépendants Atoep16-1, ayant différentes combinaisons des propriétés de la mort cellulaire et de la présence/absence de la PORA. Deux des mutants accumulent en excès de la protochlorophyllide libre (Pchlide) à l'obscurité et meurent après illumination. Dans ce cas, la Pchlide agit comme un photosensibilisateur déclenchant la production de l'oxygène singulet. Les deux autres mutants évitent la surproduction de la Pchide et verdissent normalement. En utilisant le mutant de l'orge, tigrina d12 comme référence, nous avons montré que la mort cellulaire induite lors du photoblanchiment chez les mutants Atoep16-1, intervient dans la voie flu-indépendante. L'initiation de la traduction sur des ribosomes 80S, a été identifiée comme étant une cible majeure de l'oxygène singulet, au cours des premières heures du verdissement. Dans un stade plus tardif, l'oxygène singulet a provoqué la dissociation des ribosomes. Nous avons ainsi fourni des preuves que les deux effets sur la traduction sont génétiquement liés et qu'ils peuvent être ensuite étudiés à l'aide des mutants Atoep16-1 que nous avons isolé et du mutant flu, préalablement identifié.

In angiosperms, singlet oxygen is a prominent form of reactive oxygen species that is produced during photosynthesis. Its excess causes photooxidative damage leading to cell death, demonstrated in mutants impaired in the chlorophyll biosynthetic pathway. In the present work, we used mutants of Arabidopsis thaliana that exhibit a conditional seedling lethal phenotype caused by the absence of the outer plastid envelope protein, OEP16-1. This protein is involved in the transport of amines, amino acids and is also implicated in the import of the key enzyme of chlorophyll synthesis, NADPH: Protochlorophyllide oxydoreductase A (PORA), into the plastids. Using a reverse genetic approach, four independent Atoep16-1 mutants were isolated and characterized, with different combinations of cell death properties and presence/absence of PORA. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark and died after illumination. Pchlide operated here as a photosensitizer triggering singlet oxygen formation. The other two mutants avoided excess Pchlide accumulation and greened normally. Using the mutant of barley, tigrina d12 as reference, we show that cell death induced in the photobleaching Atoep16-1 mutants occurs in a flu-independent pathway. Translation initiation at 80S ribosomes was identified to be a major target of singlet oxygen in the early hours of greening. At a delayed stage, singlet oxygen caused ribosome dissociation. We provided evidence that both effects on translation are genetically linked and they can be further studied using the Atoep16-1 mutant that we isolated and the previously described flu mutant.; Chez les angiospermes, l'oxygène singulet est la forme majoritaire des espèces réactives de l'oxygène, étant produite lors de la photosynthèse. Son excès provoque le dommage photooxidatif conduisant à la mort cellulaire, observée chez les mutants affectés dans la voie de la biosynthèse de la chlorophylle. Dans ce travail, nous avons utilisé des mutants d'Arabidopsis thaliana qui manifestent le phénotype conditionnel de la mort cellulaire, causée par l'absence d'une protéine de l'enveloppe externe des plastes, OEP16-1. Cette protéine est impliquée dans le transport des amines, acides aminés et également dans l'import d'une enzyme clé de la synthèse de la chlorophylle, NADPH: Protochlorophyllide oxydoreductase A (PORA), dans les plastes. Une approche génétique inverse a permis d'isoler et de caractériser quatre mutants indépendants Atoep16-1, ayant différentes combinaisons des propriétés de la mort cellulaire et de la présence/absence de la PORA. Deux des mutants accumulent en excès de la protochlorophyllide libre (Pchlide) à l'obscurité et meurent après illumination. Dans ce cas, la Pchlide agit comme un photosensibilisateur déclenchant la production de l'oxygène singulet. Les deux autres mutants évitent la surproduction de la Pchide et verdissent normalement. En utilisant le mutant de l'orge, tigrina d12 comme référence, nous avons montré que la mort cellulaire induite lors du photoblanchiment chez les mutants Atoep16-1, intervient dans la voie flu-indépendante. L'initiation de la traduction sur des ribosomes 80S, a été identifiée comme étant une cible majeure de l'oxygène singulet, au cours des premières heures du verdissement. Dans un stade plus tardif, l'oxygène singulet a provoqué la dissociation des ribosomes. Nous avons ainsi fourni des preuves que les deux effets sur la traduction sont génétiquement liés et qu'ils peuvent être ensuite étudiés à l'aide des mutants Atoep16-1 que nous avons isolé et du mutant flu, préalablement identifié.

Thèse préparée dans le laboratoire Plastes et Différenciation Cellulaire UJF-CNRS FRE 3017; Chloroplast division in plant cells uses proteins from both the prokaryotic and eukaryotic ancestors. The prokaryotic derived proteins include FtsZ, the progenitor of tubilin. When bacteria only use one FtsZ protein to divide, chloroplasts in higher plants use two distinct FtsZ proteins: FtsZ1 and FtsZ2. The characterization of FtsZ Arabidopsis thaliana mutants shows that plant FtsZ proteins in addition to be involved in plastid division have gained new functions in plant development during evolution. The observation that association of plastid FtsZ with thylakoid membranes is developmentally regulated suggests a function of FtsZ proteins during leaf development in chloroplast biogenesis. The number and size of starch granules in mutants together with FtsZ expression during the proplastid-amyloplast transition suggest a function in starch granules metabolism.; La division des chloroplastes des cellules végétales fait intervener des protéines d'origine procaryotique et eucaryotique. Parmi les protéines d'origine procaryotique on trouve la protéine FtsZ, l'ancêtre de la tubiline. Alors que les bactéries n'utilisent qu'une protein FtsZ pour se diviser, les chloroplastes de plantes supérieures possedent deux familles de protéines FtsZ: FtsZ1 et FtsZ2. La caractérisation des mutants FtsZ d'Arabidopsis thaliana montre que les protéines FtsZ en plus d'être impliquées dans la division des plastes ont acquis de nouvelles fonctions au cours de l'évolution. La localization des protéines FtsZ avec les thylacoïdes au cours du développement suggère un rôle des protéines FtsZ dans la biogenèse des chloroplastes au cours du développement des feuilles. Le nombre et la taille des grains d'amidon dans les mutants d'Arabidopsis, ainsi que l'expression des protéines FtsZ au cours de la transition proplastes-amyloplastes dans les cellules BY2 de tabac, suggèrent une fonction de ces protéines dans le metabolisme du grain d'amidon.

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.

Our group is interested in understanding the role of plant hormones in the development process of the plant model Arabidopsis thaliana. In a search for regulators of the expression of a gene involved in the differentiation of epidermal cells whose expression is controlled by Gibberellins (GA) and Cytokinins (CK) hormones, a new family of proteins named GeBP (GL1 enhancer Binding Protein), GPL1 (GeBP Protein Like 1), GPL2 and GPL3 was identified by our team. The aim of this PhD work was to make a functional and molecular analysis of the 4 members of this new family.First, we showed that the four GeBP/GPL proteins are nuclear localized and we demonstrated the ability of these proteins to form dimers through a new non-canonical Leucine-Zipper motif. The role of the GeBP/GPL family in plant development was assessed by studying simple mutant as well as combination of multiple mutant lines. We obtained several convergent physiological and molecular data that demonstrate a redondant role of the GeBP/GPL genes in the CK responses. This functional analysis has been completed by the transcriptomic analysis of the quadruple mutant gebp/gpl and of a surexpressor line of GPL2. This study allowed us to etablish a link between the GeBP/GPL family and CPR5, a gene involved in several processes such as pathogen responses and cell cycle. This link has been reinforced by ploïdy measurements which showed a decrease in DNA level of leaf cells of the quadruple mutant and the surexpressor of GPL2 lines as already mentioned for CPR5 mutants.Futur works will aim at confirming the role of the GeBP/GPL in the CPR5 pathway., Notre équipe de recherche étudie l'action des hormones gibbérellines (GA) et cytokinines (CK) dans les processus de développement chez la plante modèle Arabidopsis thaliana. En recherchant des régulateurs de l'expression d'un gène impliqué dans la différenciation des cellules épidermiques et dont l'expression est contrôlée par ces deux hormones, une nouvelle famille de quatre protéines nommées GeBP (GL1 enhancer Binding Protein), GPL1 (GeBP Protein Like 1), GPL2 et GPL3 a été identifiée au laboratoire. Le but de ce travail de thèse était de caractériser moléculairement et fonctionnellement les membres de cette nouvelle famille multigénique. Nous avons tout d'abord vérifié l'adressage nucléaire des protéines GeBP/GPL et démontré leur capacité à former des dimères in planta via un motif Leucine-Zipper non canonique. L'analyse fonctionnelle a été abordée par l'étude de lignées simples et multiples mutantes gebp/gpl. Différentes données physiologiques et moléculaires convergentes ont démontré un rôle redondant des gènes GeBP/GPL dans le contrôle de la réponse aux CK. Enfin, une analyse globale des transcriptomes d'une lignée surexpresseur de GPL2 et du quadruple mutant gebp/gpl, nous a permis d'établir un lien entre les GeBP/GPL et CPR5, un gène impliqué dans la réponse aux pathogènes, et le contrôle du cycle cellulaire. Ce lien a été conforté expérimentalement par des mesures de ploïdies des noyaux des cellules foliaires montrant une dérégulation du cycle cellulaire chez la lignée quadruple mutant et la lignée surexpresseur de GPL2.Les futurs travaux auront pour but de poursuivre l'étude de la fonction des GeBP/GPL et de confirmer le lien avec CPR5.

International audience

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.

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. We have found that transcription of atpH, was also strongly reduced in sig3 mutant plants. In addition, a group of other genes also showed a decrease in transcription levels in sig3 plants. Interestingly, all these genes belong to two distinct operons, atpA (containing the atpI/H/F/A genes) and atpB (containing the atpB/E genes), and code for subunits of the ATP synthase. We have characterized the 5' extremities of the transcripts coded by the two atp operons by using primer extension technique. Furthermore, in order to determine transcription starts, we have distinguished primary and secondary transcripts by performing 5'-RACE in combination with treatment of mRNAs with tobacco acid pyrophosphatase (TAP). We have also identified the transcriptional systems that are involved in synthesis of the atpA and atpB operon RNAs. By comparing transcripts in plants that were deficient in either the PEP (by treatment with spectinomycin, a inhibitor of chloroplast translation) we found that the PEP polymerase is the only responsible for the transcription of both atpA and atpB operons. Further investigations have shown that SIG3 is probably the main sigma factor responsible for the transcription of both operons, especially it recognizes the promoter sequences upstream to the atpH and atpB genes. We speculate that SIG3 may coordinate the synthesis of all the subunits of the ATP synthase in chloroplast., Trois ARN polymérases sont responsables de la transcription du génome plastidial. L'une d'entre elle, la PEP (Plastid-Encoded RNA Polymerase), est de type eubactérien et multimérique. Cette enzyme interagit avec des facteurs de transcription de type sigma au niveau des régions promotrices des gènes cibles. Chez Arabidopsis thaliana, six facteurs sigma sont codés par des gènes nucléaires et sont localisés dans les plastes. Nous avons étudié la fonction du facteur sigma 3 (SIG3), à l'aide de mutants d'insertion d'ADN-T d'Arabidopsis. L'analyse du profil d'expression des gènes plastidiaux (transcriptome) par puce à ADN nous a permis de montrer que le complexe PEP/SIG3 transcrit spécifiquement le gène psbN et régule l'expression des gènes atpH, atpA, atpE, atpF et atpB. Le gène psbN se trouve sur le brin opposé de l'opéron psbB, entre les gènes psbT et psbH. L'expression de psbN produit des ARNs antisens de psbT. Des expériences de protection à la RNase A/T1 nous permettent de suggérer que les transcrits sens et antisens de psbT forment in vivo un ARN double brin. Nous avons montré que chez le mutant sig3, le transcrit antisens de psbT est totalement absent alors que la protéine PsbT est plus abondante par rapport au sauvage. Ces résultats suggèrent que la formation d'un ARN double brin psbT sens/antisens diminue l'efficacité de la traduction de la protéine PsbT. Ainsi, le complexe SIG3-PEP, en reconnaissant spécifiquement le promoteur du gène psbN, permet la synthèse de transcrits complémentaires à psbT ce qui constitue un outil de régulation de l'expression de la protéine PsbT. Les gènes atpH et atpB font partie respectivement de l'opéron atpI/atpH/atpF/atpA et l'opéron atpB/atpE. En utilisant la spectinomycine, nous avons montré que l'expression des différents gènes codant les sous unités de l'ATP synthase plastidiale est exclusivement contrôlée par l'ARN polymérase plastidiale, PEP. Nous avons ensuite analysé l'expression de ces opérons dans le mutant sig3. Le gène atpH code la sous unité CF0-III du complexe de l'ATP synthase, 5-12 fois plus abondante que les autres sous unités. Le gène atpB code la sous unité β du complexe CF1 de l'ATP synthase. Nous avons observé par puce à ADN chez la plante sauvage que l'accumulation de l'ARNm atpH est 5 à 12 fois plus importante que les autres transcrits codant les sous unités de l'ATP synthase. Nous avons montré que l'ARNm atpH est produit soit par co-transcription des gènes atpI/atpH soit par transcription à partir d'un promoteur (PatpH-413) reconnu par le complexe SIG3-PEP. Nous suggérons ainsi que le complexe PEP/SIG3 pourrait participer par la régulation transcriptionnelle de l'expression du gène atpH à l'établissement de la stœchiométrie de l'ATP synthase plastidiale chez Arabidopsis thaliana. Par contre, l'opéron atpB/atpE est transcrit à partir de deux promoteurs: PatpB -520 et PatpB -467. Seul le promoteur PatpB -467 est sous le contrôle du facteur SIG3. Ces résultats suggérent donc que le facteur SIG3 régule de manière plus atténuée l'expression de l'opéron atpB/atpE.

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Gymnosperms, mosses and green algae are able to turn green even in the dark. In contrast, chlorophyll biosynthesis is blocked in angiosperms at its penultimate step, the reduction of protochlrophyllide into chlorophyllide. This reaction is catalyzed by the NADPH: protochlorophyllide oxidoreductase (POR), whose function is entirely light-dependent. Three POR isoforms (PORA, PORB and PORC) have been identified in Arabidopsis thaliana and their expression have been mainly studied at the transcript level. We have therefore decided to analyze both POR transcript and protein accumulation during plant development. We have confirmed that POR gene expression is tightly regulated by light: much like their transcripts, the PORA and PORB proteins are mainly accumulated in the dark while PORB and PORC are mainly observed in the photosynthetic tissues. However, weak levels of both PORA transcript and protein were detected in the light, raising the intriguing question of the possible role of PORA beyond skotomorphogenesis.Up to now, the studies concerning the role of the PORA enzyme are indirect. We have therefore decided to investigate the function of the protein more in details using independent porA mutants. Among them a knock out porA line presents a complex phenotype, in which defects in the photosynthetic process have been identified. We have also obtained a double mutant that is KO for both the PORA and PORB alleles. The comparison of the porA porB double mutant with the porA mutant has show that the PORA and PORB isoforms are functionally redundant in the establishment of the prolamellar bodies in cotyledons of etiolated plants. Our results also suggest that PORA may act as photoprotector.; Si les gymnospermes, les mousses, ou encore les algues vertes sont capables de verdir à l'obscurité, ce n'est pas le cas des angiospermes. Chez ces derniers, la voie de biosynthèse de la chlorophylle est bloquée au niveau de la pénultième étape qui correspond à la réduction de la protochlorophyllide en chlorophyllide. Cette réaction est catalysée par la NADPH : protochlorophyllide oxydoréductase (POR) dont l'activité est strictement dépendante de la lumière. Chez Arabidopsis thaliana, trois isoformes, PORA, PORB et PORC, ont été identifiées. Nous avons décidé d'étudier de façon systématique l'expression de ces gènes au cours du développement d'Arabidopsis thaliana. Nos résultats confirment que l'expression des gènes POR est soumise à un contrôle par la lumière. Alors que les protéines PORA et PORB sont principalement exprimées à l'obscurité, PORB et PORC sont les formes majoritaires dans les tissus photosynthétiques à la lumière. Cependant un taux faible mais non nul de transcrit PORA ainsi que de la protéine correspondante a été détecté à la lumière au cours du développement de la plante. Cette donnée pose donc la question du rôle éventuel de la protéine PORA au delà de la skotomorphogenèse.Nous avons entrepris la caractérisation de mutants porA, le rôle de cette protéine n'ayant été déduit qu'à partir d'études indirectes jusqu'à ce jour. Un allèle nul a été identifié qui présente un phénotype complexe. Nous avons cherché à caractériser les anomalies photosynthétiques dont il est l'objet. Par ailleurs, nous avons construit un double mutant porA porB. Son étude parallèlement à celle du simple mutant porA, nous a permis de confirmer la redondance fonctionnelle des protéines PORA et PORB dans l'établissement des corps prolamellaires des cotylédons de la plante étiolée. Ces études suggèrent également un rôle photoprotecteur pour la protéine PORA.

The plastid terminal oxidase (PTOX) is a plastoquinol oxidase whose absence in tomato (Solanum lycopersicum) results in the ghost (gh) phenotype characterized by variegated leaves (with green and bleached sectors) and by carotenoid-deficient ripe fruit. We show that PTOX deficiency leads to photobleaching in cotyledons exposed to high light primarily as a consequence of reduced ability to synthesize carotenoids in the gh mutant, which is consistent with the known role of PTOX as a phytoene desaturase cofactor. In contrast, when entirely green adult leaves from gh were produced and submitted to photobleaching high light conditions, no evidence for a deficiency in carotenoid biosynthesis was obtained. Rather, consistent evidence indicates that the absence of PTOX renders the tomato leaf photosynthetic apparatus more sensitive to light via a disturbance of the plastoquinone redox status. Although gh fruit are normally bleached (most likely as a consequence of a deficiency in carotenoid biosynthesis at an early developmental stage), green adult fruit could be obtained and submitted to photobleaching high light conditions. Again, our data suggest a role of PTOX in the regulation of photosynthetic electron transport in adult green fruit, rather than a role principally devoted to carotenoid biosynthesis. In contrast, ripening fruit are primarily dependent on PTOX and on plastid integrity for carotenoid desaturation. In summary, our data show a dual role for PTOX. Its activity is necessary for efficient carotenoid desaturation in some organs at some developmental stages, but not all, suggesting the existence of a PTOX-independent pathway for plastoquinol reoxidation in association with phytoene desaturase. As a second role, PTOX is implicated in a chlororespiratory mechanism in green tissues.

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.

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The plastid envelope of higher plant chloroplasts is a focal point of plant metabolism. It is involved in numerous pathways, including tetrapyrrole biosynthesis and protein translocation. Chloroplasts need to import a large number of proteins from the cytosol because most are encoded in the nucleus. Here we report that a loss-of-function mutation in the outer plastid envelope 16-kDa protein ( oep16 ) gene causes a conditional seedling lethal phenotype related to defects in import and assembly of NADPH:protochlorophyllide (Pchlide) oxidoreductase A. In the isolated knockout mutant of Arabidopsis thaliana , excess Pchlide accumulated in the dark operated as photosensitizer and provoked cell death during greening. Our results highlight the essential role of the substrate-dependent plastid import pathway of precursor Pchlide oxidoreductase A for seedling survival and the avoidance of developmentally programmed porphyria in higher plants.

Gymnosperms, mosses and green algae are able to turn green even in the dark. In contrast, chlorophyll biosynthesis is blocked in angiosperms at its penultimate step, the reduction of protochlrophyllide into chlorophyllide. This reaction is catalyzed by the NADPH: protochlorophyllide oxidoreductase (POR), whose function is entirely light-dependent. Three POR isoforms (PORA, PORB and PORC) have been identified in Arabidopsis thaliana and their expression have been mainly studied at the transcript level. We have therefore decided to analyze both POR transcript and protein accumulation during plant development. We have confirmed that POR gene expression is tightly regulated by light: much like their transcripts, the PORA and PORB proteins are mainly accumulated in the dark while PORB and PORC are mainly observed in the photosynthetic tissues. However, weak levels of both PORA transcript and protein were detected in the light, raising the intriguing question of the possible role of PORA beyond skotomorphogenesis.Up to now, the studies concerning the role of the PORA enzyme are indirect. We have therefore decided to investigate the function of the protein more in details using independent porA mutants. Among them a knock out porA line presents a complex phenotype, in which defects in the photosynthetic process have been identified. We have also obtained a double mutant that is KO for both the PORA and PORB alleles. The comparison of the porA porB double mutant with the porA mutant has show that the PORA and PORB isoforms are functionally redundant in the establishment of the prolamellar bodies in cotyledons of etiolated plants. Our results also suggest that PORA may act as photoprotector., Si les gymnospermes, les mousses, ou encore les algues vertes sont capables de verdir à l'obscurité, ce n'est pas le cas des angiospermes. Chez ces derniers, la voie de biosynthèse de la chlorophylle est bloquée au niveau de la pénultième étape qui correspond à la réduction de la protochlorophyllide en chlorophyllide. Cette réaction est catalysée par la NADPH : protochlorophyllide oxydoréductase (POR) dont l'activité est strictement dépendante de la lumière. Chez Arabidopsis thaliana, trois isoformes, PORA, PORB et PORC, ont été identifiées. Nous avons décidé d'étudier de façon systématique l'expression de ces gènes au cours du développement d'Arabidopsis thaliana. Nos résultats confirment que l'expression des gènes POR est soumise à un contrôle par la lumière. Alors que les protéines PORA et PORB sont principalement exprimées à l'obscurité, PORB et PORC sont les formes majoritaires dans les tissus photosynthétiques à la lumière. Cependant un taux faible mais non nul de transcrit PORA ainsi que de la protéine correspondante a été détecté à la lumière au cours du développement de la plante. Cette donnée pose donc la question du rôle éventuel de la protéine PORA au delà de la skotomorphogenèse.Nous avons entrepris la caractérisation de mutants porA, le rôle de cette protéine n'ayant été déduit qu'à partir d'études indirectes jusqu'à ce jour. Un allèle nul a été identifié qui présente un phénotype complexe. Nous avons cherché à caractériser les anomalies photosynthétiques dont il est l'objet. Par ailleurs, nous avons construit un double mutant porA porB. Son étude parallèlement à celle du simple mutant porA, nous a permis de confirmer la redondance fonctionnelle des protéines PORA et PORB dans l'établissement des corps prolamellaires des cotylédons de la plante étiolée. Ces études suggèrent également un rôle photoprotecteur pour la protéine PORA.

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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.

The phage-like RNA polymerases, encoded in thenucleus (NEP ; Nuclear Encoded RNA Polymerase) ensure partially plastidial genome transcription in higher plants. This work underlined the existence of a new protein family potentially able to interact with NEP in dicotyledon plants: NIP proteins (NEP Interacting Protein). NIP proteins are only present in higher plants and their synthesis is light dependant. Their C-terminal region presents a RING finger protein-protein interacting domain. Using immundetection, we show the first time that NIP proteins are integrated into thylakoid membranes, keeping probably NEP close to the membrane on the stroma side. This association to membranes offers new insights into NEP activity in chloroplasts of dicotyledon plants., Les ARN polymérases de type phagique, codées dans le noyau (NEP ; Nuclear Encoded RNA Polymerase), assurent une partie de la transcription du génome plastidial des végétaux supérieurs. Ces travaux mettent en évidence l'existence d'une nouvelle famille de protéines d'A. thaliana susceptibles d'interagir avec les NEP des plantes dicotylédones : les protéines NIP (NEP Interacting Protein). Les protéines NIP sont retrouvées uniquement dans les végétaux supérieurs et leur synthèse est dépendante de la lumière. Elles présentent un domaine d'interaction protéine-protéine RING finger en C-terminal. Nous avons montré pour la première fois par immunodétection que ces protéines sont intégrées aux membranes thylacoïdiennes et qu'elles retiennent probablement les NEP plastidiales à la surface de la membrane. Cette interaction avec les membranes pourrait apporter une nouvelle vision du fonctionnement des NEP dans le chloroplaste des plantes dicotylédones.

Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production.

Fibrillin is one of the major proteins in fibrillar carotenoid storage structures of bell pepper chromoplasts. In order to understand better the function of this protein, we studied the fibrillin homologues in the Arabidopsis thaliana plant model. We have identified 13 genes encoding members of this family in Arabidopsis thaliana which form 10 groups. As proteins of the fibrillin type are also present in other plants and in cyanobacteria, we propose that during evolution, plants multiplied their fibrillin genes from an ancestral gene that still has a relative in some cyanobacteria. In Arabidopsis thaliana, the 13 FIB genes show differential expression when stress- or organ-related expression is examined. We focused on the FIB1-2 subfamily which is the closest to the bell pepper fibrillin that was first characterised. FIB1-2 subfamily genes show a developmental regulation with a good induction in leaves and in flowers. The FIB1b protein accumulates in these organs, suggesting a transcriptional regulation. FIB1a and FIB1b are also induced under stress conditions like the bell pepper FIB gene, whereas FIB2 is more constitutively expressed. The involvement of the fibrillin gene in stress response has been studied in fibrillin RNAi plants which repress several members of this gene family. These plants showed a growth delay, but no drastic modification in response to stress conditions. Finally, we have developed a mutagenesis approach in order to dissect the regulation pathway leading to the bell pepper FIB gene expression. We have produced and screened an EMS mutagenised Arabidopsis thaliana population carrying a double reporter gene construct (LUC, GUS) driven by the bell pepper fibrillin promoter. We isolated mutants altered in the regulation of the fibrillin promoter in flowers., La fibrilline est une protéine majeure des structures fibrillaires de stockage des caroténoïdes dans les chromoplastes de poivron et elle semble être impliquée lors de réponses de la plante à des stress environnementaux. Afin de mieux comprendre la fonction de cette protéine, nous avons étudié ses homologues chez la plante modèle Arabidopsis thaliana. Nous avons identifié 13 gènes codant des membres de cette famille de protéines et nous les avons classés en 10 sous-familles. Sachant que des protéines de type fibrilline sont également présentes chez d'autres plantes et chez des cyanobactéries, nous proposons un schéma de l'évolution de cette famille de gènes dont les membres présents chez les plantes seraient issus de la multiplication de gènes ancestraux qui présentent des homologues chez les cyanobactéries. L'expression des 13 gènes de type fibrilline d'Arabidopsis thaliana est régulée différemment suivant les organes ou les conditions environnementales. Nous avons concentré notre étude sur la sous-famille 1-2 qui est la plus proche de la fibrilline de poivron identifiée à l'origine. Les 3 gènes présentent une régulation développementale avec une expression conséquente dans les feuilles et les fleurs. L'accumulation de la protéine FIB1b dans ces organes suggère une régulation transcriptionnelle de FIB1b. Les gènes FIB1a et FIB1b sont également induits lors de stress environnementaux, comme l'est le gène de la fibrilline de poivron, alors que le gène FIB2 est constitutivement exprimé. L'implication des fibrillines dans la réponse au stress a été étudiée par l'analyse de plantes ARNi réprimant l'expression des gènes de la sous-famille FIB1-2. Ces plantes montrent une croissance retardée, mais peu de modifications en réponse à un stress. Afin de mieux comprendre la voie de régulation qui conduit à l'induction du promoteur du gène de la fibrilline de poivron en réponse à un stress, nous avons développé une approche de mutagenèse. Pour cela, nous avons produit et criblé une population de mutant EMS d'Arabidopsis thaliana comportant une construction dans laquelle deux gènes rapporteurs (LUC, GUS) sont sous le contrôle du promoteur de la fibrilline de poivron. Des mutants, dont l'activité des gènes rapporteurs est altérée, ont été isolés.

Michel Herzog, Professeur, Université Joseph Fourrier, Président du jury Pierre Carol, Professeur, Université Pierre et Marie Curie Rapporteur Pascal Rey, Ingénieur CEA, Cadarache Rapporteur; Fibrillin is one of the major proteins in fibrillar carotenoid storage structures of bell pepper chromoplasts. In order to understand better the function of this protein, we studied the fibrillin homologues in the Arabidopsis thaliana plant model. We have identified 13 genes encoding members of this family in Arabidopsis thaliana which form 10 groups. As proteins of the fibrillin type are also present in other plants and in cyanobacteria, we propose that during evolution, plants multiplied their fibrillin genes from an ancestral gene that still has a relative in some cyanobacteria. In Arabidopsis thaliana, the 13 FIB genes show differential expression when stress- or organ-related expression is examined. We focused on the FIB1-2 subfamily which is the closest to the bell pepper fibrillin that was first characterised. FIB1-2 subfamily genes show a developmental regulation with a good induction in leaves and in flowers. The FIB1b protein accumulates in these organs, suggesting a transcriptional regulation. FIB1a and FIB1b are also induced under stress conditions like the bell pepper FIB gene, whereas FIB2 is more constitutively expressed. The involvement of the fibrillin gene in stress response has been studied in fibrillin RNAi plants which repress several members of this gene family. These plants showed a growth delay, but no drastic modification in response to stress conditions. Finally, we have developed a mutagenesis approach in order to dissect the regulation pathway leading to the bell pepper FIB gene expression. We have produced and screened an EMS mutagenised Arabidopsis thaliana population carrying a double reporter gene construct (LUC, GUS) driven by the bell pepper fibrillin promoter. We isolated mutants altered in the regulation of the fibrillin promoter in flowers.; La fibrilline est une protéine majeure des structures fibrillaires de stockage des caroténoïdes dans les chromoplastes de poivron et elle semble être impliquée lors de réponses de la plante à des stress environnementaux. Afin de mieux comprendre la fonction de cette protéine, nous avons étudié ses homologues chez la plante modèle Arabidopsis thaliana. Nous avons identifié 13 gènes codant des membres de cette famille de protéines et nous les avons classés en 10 sous-familles. Sachant que des protéines de type fibrilline sont également présentes chez d'autres plantes et chez des cyanobactéries, nous proposons un schéma de l'évolution de cette famille de gènes dont les membres présents chez les plantes seraient issus de la multiplication de gènes ancestraux qui présentent des homologues chez les cyanobactéries. L'expression des 13 gènes de type fibrilline d'Arabidopsis thaliana est régulée différemment suivant les organes ou les conditions environnementales. Nous avons concentré notre étude sur la sous-famille 1-2 qui est la plus proche de la fibrilline de poivron identifiée à l'origine. Les 3 gènes présentent une régulation développementale avec une expression conséquente dans les feuilles et les fleurs. L'accumulation de la protéine FIB1b dans ces organes suggère une régulation transcriptionnelle de FIB1b. Les gènes FIB1a et FIB1b sont également induits lors de stress environnementaux, comme l'est le gène de la fibrilline de poivron, alors que le gène FIB2 est constitutivement exprimé. L'implication des fibrillines dans la réponse au stress a été étudiée par l'analyse de plantes ARNi réprimant l'expression des gènes de la sous-famille FIB1-2. Ces plantes montrent une croissance retardée, mais peu de modifications en réponse à un stress. Afin de mieux comprendre la voie de régulation qui conduit à l'induction du promoteur du gène de la fibrilline de poivron en réponse à un stress, nous avons développé une approche de mutagenèse. Pour cela, nous avons produit et criblé une population de mutant EMS d'Arabidopsis thaliana comportant une construction dans laquelle deux gènes rapporteurs (LUC, GUS) sont sous le contrôle du promoteur de la fibrilline de poivron. Des mutants, dont l'activité des gènes rapporteurs est altérée, ont été isolés.

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.

International audience

International audience; Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot.Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot.