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International audience; Biocide disinfectants are used in agro-food industries in order to limit the development of pathogens present in environment or on surfaces in contact with food for human or animal consumption. Biocide residues remaining on food surfaces may constitute a toxicological risk for the consumer. Very little feedback from the field on the rates of biocide residues in the industry exist due to a lack of simple, fast and responsive self-checking methods. The development of biosensors for the detection of biocide disinfectants represents a promising way to explore this, but most of this research remains limited.

International audience; Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCβ) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

International audience; Different types of contaminants (e.g., pesticides, veterinary drugs) can be found in food products intended for human consumption (e.g., water, food from animal or plant origin). The risks for the consumers' health are multiple (e.g., endocrine disruptor's effect, carcinogenic effect, allergic risks). The detection of contaminants in food products is the first critical step of the control. Conventional screening methods could be bioassays, immunological methods, or physicochemical methods. Biosensors are identified as innovative screening methods. Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Technological advances (e.g., nanomaterials, microfluidics) improved the methods' sensitivity.

Chapitre 29; International audience; The cell nucleus is a structurally complex and dynamic organelle ensuring key biological functions. Complex relationships between nuclear structure and functions require a better understanding of the three-dimensional organization of the genome and of the subnuclear compartments. Quantitative image analysis coupled with spatial statistics and modeling is a relevant approach to address these questions. In this chapter, we describe a step-by-step procedure to process images and to test a spatial random model for the distribution of nuclear objects using chromocenters as an example. More elaborate models can be designed on the basis of the random model by introducing additional and more complex constraints to better fit observations and to question determinants of these spatial organizations.

International audience; Screening methods are used as a first-line approach to detect the presence of antibiotic residues in food of animal origin. The validation process guarantees that the method is fit-for-purpose, suited to regulatory requirements, and provides evidence of its performance. This article is focused on intra-laboratory validation. The first step in validation is characterisation of performance, and the second step is the validation itself with regard to pre-established criteria. The validation approaches can be absolute (a single method) or relative (comparison of methods), overall (combination of several characteristics in one) or criterion-by-criterion. Various approaches to validation, in the form of regulations, guidelines or standards, are presented and discussed to draw conclusions on their potential application for different residue screening methods, and to determine whether or not they reach the same conclusions. The approach by comparison of methods is not suitable for screening methods for antibiotic residues. The overall approaches, such as probability of detection (POD) and accuracy profile, are increasingly used in other fields of application. They may be of interest for screening methods for antibiotic residues. Finally, the criterion-by-criterion approach (Decision 2002/657/EC and of European guideline for the validation of screening methods), usually applied to the screening methods for antibiotic residues, introduced a major characteristic and an improvement in the validation, i.e. the detection capability (CCbeta). In conclusion, screening methods are constantly evolving, thanks to the development of new biosensors or liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) methods. There have been clear changes in validation approaches these last 20 years. Continued progress is required and perspectives for future development of guidelines, regulations and standards for validation are presented here.

International audience; Efficient screening methods are needed to control antibiotic residues in eggs. A microbiological kit (Explorer® 2.0 test (Zeu Inmunotech, Spain)) and an immunobiosensor kit (Microarray II (AM® II) on Evidence Investigator™ system (Randox, UK)) have been evaluated and validated for screening of antibiotic residues in eggs, according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods. The e-reader™ system, a new automatic incubator/reading system, was coupled to the Explorer 2.0 test. The AM II kit can detect residues of six different families of antibiotics in different matrices including eggs. For both tests, a different liquid/liquid extraction of eggs had to be developed. Specificities of the Explorer 2.0 and AM II kit were equal to 8% and 0% respectively. The detection capabilities were determined for 19 antibiotics, with representatives from different families, for Explorer 2.0 and 12 antibiotics for the AM II kit. For the nine antibiotics having a maximum residue limit (MRL) in eggs, the detection capabilities CCβ of Explorer 2.0 were below the MRL for four antibiotics, equal to the MRL for two antibiotics and between 1 and 1.5 MRLs for the three remaining antibiotics (tetracyclines). For the antibiotics from other families, the detection capabilities were low for beta-lactams and sulfonamides and satisfactory for dihydrostreptomycin (DHS) and fluoroquinolones, which are usually difficult to detect with microbiological tests. The CCβ values of the AM II kit were much lower than the respective MRLs for three detected antibiotics (tetracycline, oxytetracycline, tylosin). Concerning the nine other antibiotics, the detection capabilities determined were low. The highest CCβ was obtained for streptomycin (100 µg kg–1).

In plants the adult organs are not determined during embryogenesis. Throughout their life cycle, plants undergo different developmental changes, which require deep alterations in the overall gene expression patterns. The timing at which these phase changes occur can have a strong impact on plant viability and fitness and therefore needs to be tightly regulated to assure they take place under optimal conditions. On the other hand, in Arabidopsis thaliana, once a transition is initiated, it is usually stable, independently of the surrounding environment, suggesting it should be memorized. Over the last years, several studies have shown that epigenetics plays an important role in the regulation of phase transitions in plants. For instance, Arabidopsis mutants where the repressive trimethylation of lysine 27 of histone 3 (H3K27me3) is absent were shown to germinate and further revert into calli-like structures bearing somatic embryos. Moreover, this histone mark has recently been shown to also regulate the transition from vegetative to reproductive development, as some mutants with reduced H3K27me3 levels are incapable of memorizing floral inducible stimuli, when transferred to suboptimal conditions. On the other hand, the epigenetic modifications need to be reset at the end of each generation, to ensure the normal development of the progeny. In fact, plants have developed an entire reprogramming machinery that culminates at gametogenesis, where a genome-wide resetting of gene programs takes place.

Advance Publication; International audience; LHP1-INTERACTING FACTOR2 (LIF2), a heterogeneous nuclear ribonucleoprotein involved in Arabidopsis thaliana cell fate and stress responses, interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a Polycomb Repressive Complex1 (PRC1) subunit. To investigate LIF2-LHP1 functional interplay, we mapped their genome-wide distributions in wild-type, lif2, and lhp1 backgrounds, under standard and stress conditions. Interestingly, LHP1-targeted regions form local clusters, suggesting an underlying functional organization of the plant genome. Regions targeted by both LIF2 and LHP1 were enriched in stress-responsive genes, the H2A.Z histone variant, and antagonistic histone marks. We identified specific motifs within the targeted regions, including a G-box-like motif, a GAGA motif, and a telo-box. LIF2 and LHP1 can operate both antagonistically and synergistically. In response to methyl jasmonate treatment, LIF2 was rapidly recruited to chromatin, where it mediated transcriptional gene activation. Thus, LIF2 and LHP1 participate in transcriptional switches in stress-response pathways.

International audience; The Evidence Investigator™ system (Randox, UK) is a biochip and semi-automated system. The microarray kit II (AM II) is capable of detecting several compounds belonging to different families of antibiotics: quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines. The performance of this innovative system was evaluated for the detection of antibiotic residues in new matrices, in muscle of different animal species and in aquaculture products. The method was validated according to the European Decision No. EC/2002/657 and the European guideline for the validation of screening methods, which represents a complete initial validation. The false-positive rate was equal to 0% in muscle and in aquaculture products. The detection capabilities CCβ for 12 validated antibiotics (enrofloxacin, difloxacin, ceftiofur, desfuroyl ceftiofur cysteine disulfide, thiamphenicol, florfenicol, tylosin, tilmicosin, streptomycin, dihydrostreptomycin, tetracycline, doxycycline) were all lower than the respective maximum residue limits (MRLs) in muscle from different animal origins (bovine, ovine, porcine, poultry). No cross-reactions were observed with other antibiotics, neither with the six detected families nor with other families of antibiotics. The AM II kit could be applied to aquaculture products but with higher detection capabilities from those in muscle. The detection capabilities CCβ in aquaculture products were respectively at 0.25, 0.10 and 0.5 of the respective MRL in aquaculture products for enrofloxacin, tylosin and oxytetracycline. The performance of the AM II kit has been compared with other screening methods and with the performance characteristics previously determined in honey.

International audience; Antibiotic residues may be found in food of animal origin, since veterinary drugs are used for preventive and curative purposes to treat animals. The control of veterinary drug residues in food is necessary to ensure consumer safety. Screening methods are the first step in the control of antibiotic residues in food of animal origin. Conventional screening methods are based on different technologies, microbiological methods, immunological methods or physico-chemical methods (e.g. thin-layer chromatography, HPLC, LC-MS/MS). Screening methods should be simple, quick, inexpensive and specific, with low detection limits and high sample throughput. Biosensors can meet some of these requirements. Therefore, the development of biosensors for the screening of antibiotic residues has been increasing since the 1980s. The present review provides extensive and up-to-date findings on biosensors for the screening of antibiotic residues in food products of animal origin. Biosensors are constituted of a bioreceptor and a transducer. In the detection of antibiotic residues, even though antibodies were the first bioreceptors to be used, new kinds of bioreceptors are being developed more and more (enzymes, aptamers, MIPs); their advantages and drawbacks are discussed in this review. The different categories of transducers (electrochemical, mass-based biosensors, optical and thermal) and their potential applications for the screening of antibiotic residues in food are presented. Moreover, the advantages and drawbacks of the different types of transducers are discussed. Lastly, outlook and the future development of biosensors for the control of antibiotic residues in food are highlighted.

L'article original est publié par The American Society of Plant Biologists; The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Antibiotics are mixed with the food of bees to fight against diseases. No maximum residue limits have been set for honey. Recommended concentrations (RCs) have been published by European Union Reference Laboratories for tylosin and streptomycin. The objective of this project was to select and validate enzyme-linked immunosorbent assay (ELISA) kits for the screening of tylosin and streptomycin/dihydrostreptomycin residues to be implemented in the French honey control plan. Four ELISA kits for tylosin and five ELISA kits for streptomycin were evaluated. At the end, one kit each was selected and validated for tylosin (TECNA AB620) and streptomycin (Europroxima). Both ELISA kits for tylosin and streptomycin are specific, robust, fast and easy-to-use tests. The detection capability CC β of tylosin A was less than or equal to 10 µg kg(-1) (half the RC). The CC β of desmycosin (the hydrolysed product of tylosin A in acidic conditions) is approximately 200 µg kg(-1), which is five times the RC for tylosin (20 µg kg(-1)). Thus, this kit is fit for the screening of tylosin A but is unsuitable to detect desmycosin. The detection capability CC β of streptomycin was less than or equal to 10 µg kg(-1) (one fourth the RC). The cross-reactivity with dihydrostreptomycin was equal to 136%. Both ELISA kits were applicable to a wide variety of honey (single flower and multiflower, different floral origins, different geographic origins, different consistencies [liquid or solid] and different colours).

International audience; The Sulfasensor® Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the CRL guideline for the validation of screening methods European regulation to achieve the French control plan in honey. The test is robust, quick (90 minutes for 40 samples), easy to perform and easy to read. The false positive rate was estimated to 12.5 %. The detection capabilities CC of the Sulfasensor® Honey kit were lower than or equal to 25 µg kg-1 for sulfamethazineSMZ, sulfamerazine, sulfathiazole, sulfapyridine, and between 25 to 50 µg kg-1 for sulfadiazine and sulfadimethoxine, 150 µg kg-1 for sulfaquinoxaline and 1000 µg kg-1 for sulfamethoxazole and sulfamethizole. Sulfanilamide is not detected by the kit. The kit is applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was implementing for the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011, the Sulfasensor® Honey kit has been replaced by a LC-MS/MS method for the screening and the confirmation of sulfonamide residues in honey, which detect all the sulfonamides of interest.

A rapid receptor test dedicated to the screening of tetracycline (TTC) residues, the Tetrasensor? kit in different matrices, has been evaluated and validated according to the European regulation to achieve the French control plan in honey. The detection capabilities (CC?s) were included between 5 and 15 µg kg?1 for chlortetracycline (CTC), oxytetracycline (OTC), TTC and doxycycline (DOXY). The cross-reactions of both kits towards other families of antibiotics were negligible and there was no masking effect of these antibiotics on the detection of tetracyclines. The false positive rate was equal to 9.4%. This kit was applicable to a wide variety of honeys [monofloral and multifloral, different floral origins, different geographical origins and different consistencies (liquid or solid), different colours]. Finally, the Tetrasensor? test is a robust method, quick, easy to perform and easy to read which is now applied to implement the French control plan for the detection of tetracyclines in honey. A rapid receptor test dedicated to the screening of tetracycline (TTC) residues, the Tetrasensor? kit in different matrices, has been evaluated and validated according to the European regulation to achieve the French control plan in honey. The detection capabilities (CC?s) were included between 5 and 15 µg kg?1 for chlortetracycline (CTC), oxytetracycline (OTC), TTC and doxycycline (DOXY). The cross-reactions of both kits towards other families of antibiotics were negligible and there was no masking effect of these antibiotics on the detection of tetracyclines. The false positive rate was equal to 9.4%. This kit was applicable to a wide variety of honeys [monofloral and multifloral, different floral origins, different geographical origins and different consistencies (liquid or solid), different colours]. Finally, the Tetrasensor? test is a robust method, quick, easy to perform and easy to read which is now applied to implement the French control plan for the detection of tetracyclines in honey.

International audience; DamID (DNA adenine methylation identification) is an adenine methylation-based tagging method designed to map protein-DNA interactions in vivo. DamID, an alternative method to chromatin immunoprecipitation (ChIP), is based on the covalent linking of a "fingerprint" in the vicinity of the DNA-binding sites of the protein of interest. The fingerprints can be further mapped by simple molecular approaches. First developed by van Steensel's group in Drosophila melanogaster, DamID was successfully adapted to Arabidopsis thaliana, and its feasibility demonstrated by using the well-known yeast GAL4 transcription factor. The method was further used to establish a genome-wide map of the target sites of LHP1, a regulatory chromatin protein in A. thaliana.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear “compartments”. Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types., Author Summary Several reports suggest functional relationships within the spatial organization of the nucleus, gene regulation and cell differentiation. However, it still remains difficult to extract common rules, mostly because i) most data have been gathered on limited sets of nuclear elements and in nuclei outside their normal physiological environment, and ii) few three-dimensional (3D) quantitative measures have been performed. Thus, we questioned whether common nuclear organization principles exist in the animal and plant kingdoms. For that purpose, we investigated the 3D distribution of centromeres/chromocenters in five populations of animal and plant nuclei: rabbit embryos at 8-cell and blastocyst stages, rabbit mammary gland epithelial cells and Arabidopsis thaliana plantlets. We set up adapted procedures to segment confocal images and developed a new analytical methodology based on distances between positions within the nucleus and centromeres/chromocenters. We showed that in all systems, despite large differences in chromosome number (44 in rabbit; 10 in A. thaliana) and genome size (rabbit estimated size 2.77 Gbp; A. thaliana 125 Mbp), centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation. This suggests that, whatever their specific features, conserved rules govern the spatial distribution of genomes in nuclei of differentiated cells.

International audience; A multiresidue method was developed for monitoring antibiotic residues in milk using liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS). Two very short extractions followed by two LC/MS-MS acquisitions allow the screening of 58 antibiotics belonging to 8 different families (penicillins, cephalosporins, sulfonamides, macrolides, lincosamides, aminoglycosides, tetracyclines and quinolones). This method is currently implemented in the laboratory in a qualitative way: i.e. monitoring the presence or absence of residue in a sample and identification of the analyte before the step of confirmation. In order to assess the performance of this method, a validation strategy described in an internal guideline for the validation of screening methods was applied. The aim of the validation was to prove the sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest (MRL) at least. According to the EU commission decision 2002/657/EC, the suiTable sensitivity of a screening method can be demonstrated when the CCbeta is below or equal to the MRL level and so the false compliant rate below or equal to 5 % at the MRL level. The validation scheme was established in order to take into account various variability factors: the apparatus response, the interday repeatability, the matrix effect, etc ... The results of the validation clearly demonstrate the suitability of this method for the detection and identification of more than 50 antibiotics and are in agreement with the results obtained in routine analysis.

Explorer® is a simple and fast new kit for the detection of inhibitory substances in raw meat. The test, a 96-well microtitre plate, is based on the inhibition of microbial growth (Geobacillus stearothermophilus spores). It was validated in accordance with European Commission Decision 2002/657/EC (2002). The specificity and detection capabilities for five compounds from major antimicrobial families and robustness were studied. The specificity of the test was assessed with four different animal species and was found to be very satisfactory (false-positive rates lower than 10%). The detection capabilities for amoxicillin (10 μg kg-1) and tylosin (100 μg kg-1) were at the maximum residue limit (MRL) level (50 and 100 μg kg-1, respectively) for both, for doxycycline (200 μg kg-1) and sulfathiazole (200 μg kg-1) at twice the MRL (100 μg kg-1 for each) and for cefalexin (500 μg kg-1) at 2.5 times the MRL (200 μg kg-1). Twenty-one samples were analysed in parallel with the Four Plate Test, the STAR protocol and the Explorer® test. One false-positive result and two false-negative results (samples containing oxytetracycline) were reported with the Explorer® test. In conclusion, the Explorer® test was shown to be robust and easily automated. Photometric reading allows informatic data storage and objective readings between technicians and days. The test can be used as a wide screening test because it enables detection of most of the antimicrobial families (penicillins, cephalosporins, tetracyclines, sulphonamides, and macrolides) in muscles from different animal species (porcine, bovine, ovine, poultry). © 2009 Taylor & Francis.

A semi-quantitative immunological method (BIACORE method) for detecting sulfamethazine in bovine milk was developed and validated using the total error approach. The acceptance limits were set at ±40% and the risk of procedure of (1−β) proportion measurements falling outside the acceptance limits was chosen at 5%. Different response functions were tested on the basis of the accuracy index (IA). The best model was a weighted (1/X2) quadratic regression and the simplest one was an unweighted quadratic regression. This approach identified the weak point of the method, which was precision. Finally this BIACORE method was able to detect positive samples containing sulfamethazine in the dosing range between 50 and 150 ng/ml.

Background Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill. Results We report a rapid protocol for fluorescent in situ hybridization (FISH) performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure. Conclusion Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization.

The HP1 family proteins are involved in several aspects of chromatin function and regulation in Drosophila, mammals and the fission yeast. Here we investigate the localization of LHP1, the unique Arabidopsis thaliana HP1 homolog known at present time, to approach its function. A functional LHP1-GFP fusion protein, able to restore the wild-type phenotype in the lhp1 mutant, was used to analyze the subnuclear distribution of LHP1 in both A. thaliana and Nicotiana tabacum. In A. thaliana interphase nuclei, LHP1 was predominantly located outside the heterochromatic chromocenters. No major aberrations were observed in heterochromatin content or chromocenter organization in lhp1 plants. These data indicate that LHP1 is mainly involved in euchromatin organization in A. thaliana. In tobacco BY-2 cells, the LHP1 distribution, although in foci, slightly differed suggesting that LHP1 localization is determined by the underlying genome organization of plant species. Truncated LHP1 proteins expressed in vivo allowed us to determine the function of the different segments in the localization. The in foci distribution is dependent on the presence of the two chromo domains, whereas the hinge region has some nucleolus-targeting properties. Furthermore, like the animal HP1beta and HP1gamma subtypes, LHP1 dissociates from chromosomes during mitosis. In transgenic plants expressing the LHP1-GFP fusion protein, two major localization patterns were observed according to cell types suggesting that localization evolves with age or differentiation states. Our results show conversed characteristics of the A. thaliana HP1 homolog with the mammal HP1gamma isoform, besides specific plant properties.

International audience; The plant life cycle involves a series of developmental phase transitions. These transitions require the regulation and highly co-ordinated expression of many genes. Epigenetic controls have now been shown to be a key element of this mechanism of regulation. In the model plant Arabidopsis, recent genetic and molecular studies on chromatin have begun to dissect the molecular basis of these epigenetic controls. Chromatin dynamics represent the emerging and exciting field of gene regulation notably involved in plant developmental transitions. By comparing plant and animal systems, new insights into the molecular complexes and mechanisms governing development can be delineated. We are now beginning to identify the components of chromatin complexes and their functions.

Agrobacterium tumefaciens and some Agrobacterium rhizogenes strains possess auxin biosynthesis genes (tms and aux genes respectively), responsible for a de novo auxin biosynthetic pathway in transformed plant cells. A comparison is presented of the potential expression of these genes in a monocotyledonous (barley) and a dicotyledonous plant (tobacco). The promoters of the genes were translationally fused to the β-glucuronidase reporter gene and analysed in transient expression experiments. The tms and aux fusions were highly expressed in tobacco, but not in barley. However, the aux enhancer active in tobacco, conferred low β-glucuronidase expression in barley when fused to a truncated cauliflower mosaic virus 35S promoter. The results are discussed in relation to the differential responses to Agrobacterium infection in monocots and dicots.

The transition to flowering results from a complex interplay between endogenous and environmental cues. The leaves play a key role in this process, by perceiving the light changes and producing photosynthates, which participate to the floral signalling. However, our knowledge on the changes occurring in leaves during floral transition is still limited. We characterised the morphological, molecular and transcriptional events related to floral transition in mature leaves in Arabidopsis, using a short-day to long-day shift to induce a synchronized flowering. We identified the temporal window of the floral transition, monitored the leaf growth and observed an increase in their ploidy level during the process. By RNA-seq we studied the transcriptional dynamics of the leaf gene network, and compared with events occurring in roots and meristems to get an integrated view of floral transition in the whole plant. Furthermore, we investigated the mode of action of LIKE HETEROPROTEIN 1 (LHP1), a PRC1 subunit, by exploiting transgenic lines with conditional alterations of LHP1 dosage and analysing the effects on chromatin and transcription of flowering genes. A short-term modulation of LHP1 dosage altered the deposition of H3K27me3 and H3K4me3, showing a functional interaction between LHP1 and PRC2, and also suggesting a new role in the formation of bivalent chromatin regions. Finally, since nuclear organisation plays a key role in gene regulation, we searched and identified determinants of the nuclear architecture by using innovative spatial statistical tools.

La dynamique chromatinienne joue un rôle central dans les contrôles développementaux, la différenciation cellulaire ou les réponses des organismes à l’environnement. Chez les animaux, les protéines du groupe Polycomb sont impliquées dans l’établissement d’états chromatiniens silencieux. Chez les plantes, des données récentes suggèrent que la protéine LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) participerait à un complexe de type Polycomb. Nous nous sommes intéressés aux complexes LHP1 en étudiant un de ses partenaires, LHP1 INTERACTING FACTOR 2 (LIF2). L’objectif de ce travail de thèse a été de poursuivre la caractérisation de LIF2. LIF2 se caractérise par la présence de domaines de liaison à l’ARN, suggérant la participation d’une composante ARN dans les complexes LHP1. Nous avons recherché des ARN ligands de LIF2 et étudié les interactions LIF2/ARN par différentes approches dont la technologie Biacore. En analysant le transcriptome du mutant lif2, nous avons remarqué un enrichissement pour des gènes impliqués dans la réponse aux stress biotiques et abiotiques. Nous avons étudié la fonction de LIF2 dans la réponse aux pathogènes et avons pu mettre en évidence que LIF2 joue un rôle dans l’immunité innée des plantes et est essentiel pour réguler négativement les réactions de défenses en l’absence de pathogènes.