Rozprawy doktorskie na temat „Flower development biology”

Flower shape, color and size are extensively studied to both identify and classify different angiosperm taxa. The availability of well-supported molecular phylogenies produced using complex models of sequence evolution, coupled with an understanding of the genes that regulate morphological form in model organisms, and new methods to infer gene expression patterns in diverse species now allow us to understand the genetic basis of morphological differences among closely related species. Studies in Plantaginaceae, Gesneriaceae, Fabaceae and Brassicaceae show the importance of CYCLOIDEA (CYC), RADIALIS (RAD) and DIVARICATA (DIV) in regulating flower shape, but also show divergence in gene function within flowering plants. Previous studies in the zygomorphic model species Antirrhinum majus (snapdragon) have shown that AmCYC is expressed in the adaxial (dorsal) petals of flowers where it activates AmRAD . This expression of AmRAD within adaxial petals represses AmDIV expression causing AmDIV to be restricted to abaxial (ventral) and lateral petals. Like Antirrhinum , traditional Diplacus flowers have distinct dorsal, ventral and lateral petal identities. However, within the clade actinomorphic flowers have evolved independently on two occasions: once in D. pictus and once in D. mohaviensis. mRNA reveal DIV expression to be conserved between D. pictus and snapdragon, whereas CYC and RAD expression, and presumably function, differ between the two species. DpCYC is expressed in a narrow portion on the upper lip of abaxial petals, whereas DpRAD is expressed within both lateral and abaxial petals. D. pictus flowers are characterized by a novel upturned abaxial petal which may be linked to localized CYC expression along the upper surface of the structure. This study sheds new light on the mechanisms regulating flower shape in an endemic Californian monkey flower and shows the importance of testing hypotheses from model species such as Arabidopsis and snapdragon in non-model taxa such as D. pictus to undercover the true variety of mechanisms driving morphological evolution.

The reproductive organs of conifers and angiosperms differ in morphology in several fundamental respects. The conifer Norway spruce (Picea abies) form pollen and seed cones from separate meristems whereas angiosperms bear bipartite flowers with sepals and petals surrounding two inner whorls of stamens and carpels. Despite these differences in morphology this thesis present data to suggest that reproductive development in conifers and angiosperms is regulated by a similar molecular mechanism. This implies an evolutionary conservation of the major mechanism for reproductive development since the origin of seed plants.

Flower organ identity in angiosperms is determined by regulatory genes belonging to the MADS-box gene family of transcription factors. This thesis presents the cloning and characterisation of four novel MADS-box genes from Norway spruce. Three of these genes DAL11, DAL12 and DAL13 are most closely related to angiosperm B function genes i.e. genes required for petal and stamen development. DAL11, 12 and 13 all are specifically active in developing pollen cones, with different temporal and spatial expression pattern. Functional analysis in transgenic Arabidopsis and yeast suggest that the reproductive aspect of the B-function is conserved between conifers and angiosperms. The results also suggest that the B-function in conifers is separated into one shoot identity and one organ identity determinant.

A fourth gene presented; DAL10, is specifically expressed in vegetative parts of pollen- and seed cones. Phylogenetically DAL10 is not closely related to any of the known angiosperm clades, but rather forms a separate clade with other gymnosperm genes, suggesting a gymnosperm specific function. We suggest that the DAL10 activity reflects a function in the determination of the reproductive shoot.

Dans le domaine de la biologie développementale, un des principaux défis est de comprendre comment des tissus multicellulaires, à l'origine indifférenciés, peuvent engendrer des formes aussi complexes que celles d'une fleur. De part son implication dans l'organogenèse florale, l'auxine est une phytohormone majeure. Nous avons donc déterminé son réseau binaire potentiel, puis y avons appliqué des modèles de clustering de graphes s'appuyant sur les profils de connexion présentés par ces 52 facteurs de transcription (FT). Nous avons ainsi pu identifier trois groupes, proches des groupes biologiques putatifs: les facteurs de réponse à l'auxine activateurs (ARF+), répresseurs (ARF-) et les Aux/IAAs. Nous avons détecté l'auto-interaction des ARF+ et des Aux/IAA, ainsi que leur interaction, alors que les ARF- en présentent un nombre restreint. Ainsi, nous proposons un mode de compétition auxine indépendent entre ARF+ et ARF- pour la régulation transcriptionelle. Deuxièmement, nous avons modélisé l'influence des séquences de dimérisation des FT sur la structure de l'interactome en utilisant des modèles de mélange Gaussien pour graphes aléatoires. Les groupes obtenus sont proches des précédents, et les paramètres estimés nous on conduit à conclure que chaque sous-domaine peut jouer un rôle différent en fonction de leur proximité phylogénétique.Enfin, nous sommes passés à l'échelle multi-cellulaire ou, par un graphe spatio-temporel, nous avons modélisé les premiers stades du développement floral d'A. thaliana. Nous avons pu extraire des caractéristiques cellulaires (3D+t) de reconstruction d'imagerie confocale, et avons démontré la possibilité de caractériser l'identité cellulaire en utilisant des méthodes de classification hiérarchique et des arbres de Markov cachés
A striking aspect of flowering plants is that, although they seem to display a great diversity of size and shape, they are made of the same basics constituents, that is the cells. The major challenge is then to understand how multicellular tissues, originally undifferentiated, can give rise to such complex shapes. We first investigated the uncharacterised signalling network of auxin since it is a major phytohormone involved in flower organogenesis.We started by determining the potential binary network, then applied model-based graph clustering methods relying on connectivity profiles. We demonstrated that it could be summarise in three groups, closely related to putative biological groups. The characterisation of the network function was made using ordinary differential equation modelling, which was later confirmed by experimental observations.In a second time, we modelled the influence of the protein dimerisation sequences on the auxin interactome structure using mixture of linear models for random graphs. This model lead us to conclude that these groups behave differently, depending on their dimerisation sequence similarities, and that each dimerisation domains might play different roles.Finally, we changed scale to represent the observed early stages of A. thaliana flower development as a spatio-temporal property graph. Using recent improvements in imaging techniques, we could extract 3D+t cellular features, and demonstrated the possibility of identifying and characterising cellular identity on this basis. In that respect, hierarchical clustering methods and hidden Markov tree have proven successful in grouping cell depending on their feature similarities

Titre du projet: Recherche de marqueurs moléculaires précoces de l’anomalie florale mantled du palmier à huile Objectifs : - identifier des marqueurs d’expression de la variation somaclonale mantled par comparaison entre les transcriptomes conformes et variants.- valider la capacité de discrimination des marqueurs sélectionnés lors des stades précoces du processus in vitro. Stratégie et Méthode: Analyse transcriptomique de l’inflorescence normale de palmier à huile et construction d’un transcriptome de référence. Technique : RNAseq, séquençage Illumina.Identification des séquences et voies de régulation d’intérêt. Technique: analyse bioinformatique des données de séquençage.Comparaison entre les trancriptomes issus d’inflorescences normales vs. mantled par re-séquençage de banques obtenues ) partir de différents génotypes clonaux. Technique : Illumina.Identification des séquences présentant de manière cohérente des profils d’expression dépendant du phénotype. Technique : analyse bioinformatique des données de séquençage, analyse statistique des profils d’expression. Validation des marqueurs candidats sur des paires de régénérants normal/mantled issus de lignées clonales variées, ainsi que sur des cultures in vitro à différents stades du processus de régénération. Technique : PCR quantitative (q-PCR)
Project title : Search for early molecular markers of the mantled floral variation of oil palmObjectives : - identifying expression markers of the mantled somaclonal variation through the comparison between the true-to-type and the variant transcriptome. - assessing the discriminating power of the selected markers at early stages of the in vitro process.Strategy and Methods : Transcriptomic analysis of the normal oil palm inflorescence, construction of a reference transcriptome. Technique : RNAseq, Illumina sequencing.Identification of sequences and pathways of interest. Technique : bioinformatic analysis of sequencing data.Comparison between the normal and the mantled inflorescence transcriptome through the re-sequencing of libraries generated from several different clonal lines. Technique : Illumina. Identification of sequences displaying consistently a phenotype-dependent differential expression pattern. Technique : bioinformatic analysis of sequencing data, statistical analysis of expression patterns. Validation of candidate markers on normal/mantled regenerant palm pairs from different clonal lines and on normal-/mantled-derived in vitro cultures at various stages of the industrial regeneration process. Technique : quantitative PCR (q-PCR)

Flowering time regulation has a strong impact on plant life cycle, since it allows plants to flower and to reproduce under environmental permissive conditions. Several genes are involved in the regulatory pathways that determine the floral transition step, i.e. the switch from the plant vegetative phase to the reproductive phase and the consequent flower formation and fruit set. Among those genes, SHORT VEGETATIVE PHASE (SVP), a MADS box transcription factor, acts as strong repressor of the so called florigen promoting genes, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). Moreover, SVP has been also reported to act as a repressor of flower homeotic gene expression, thus ensuring the correct maintenance of floral meristem identity. Due to the relevance of SVP in both such important plant developmental stages, during my Ph.D. research program I tried to elucidate the molecular mechanisms at the basis of SVP activities. That has been done through different and complementary strategies that had the dual aim to identify SVP protein partners and to move the first steps towards the comprehension of the role of chloroplasts and chloroplast-nucleus signaling pathways in SVP functions. Co-immunoprecipitation assays followed by Mass Spectrometry analyses have allowed to draw up a list of Arabidopsis putative robust SVP interactors involved, at different levels, in chromatin organization and histone modification. Interestingly, the detailed characterization of the major Arabidopsis trimethyltransferase enzyme, SET DOMAIN GROUP 2 (SDG2), has revealed the existence of an SVP-SDG2 containing protein complex able to regulate the expression of SVP gene at the vegetative and reproductive meristems, by affecting the H3K4 methylation pattern within the first exon of SVP. Furthermore, our interests on the role of chloroplast-nucleus communication and its possible interactions with the flowering time regulation, have been met through the detailed characterization of two chloroplast-located PENTATRICO-PEPTIDE-REPEAT (PPR) containing proteins, which share three main features: i) they are part of the chloroplast gene expression machinery, ii) they are involved in chloroplast-nucleus communication, iii) they have been reported to be target genes of SVP by ChiP-seq assays. The detailed characterization of the Arabidopsis PPR proteins, GENOME UNCOUPLED 1 (GUN1) and CHLOROPLAST RNA PROCESSING 1 (AtCRP1), has provided the first preliminary insights into how chloroplast-nucleus signaling mechanisms may enable higher plants to more effectively adapt to the ever-changing internal and external conditions and mitigate detrimental effects to fitness.

LEAFY (LFY) est un facteur de transcription central pour le développement des plantes, en particulier pour la floraison chez les angiospermes. LFY est très conservée, même chez les espèces ne portant pas de fleurs. On dispose de nombreuses données génétiques sur LFY et son réseau de régulation chez la plante modèle Arabidopsis thaliana, mais les mécanismes moléculaires impliqués dans son fonctionnement ne sont pas entièrement élucidés. LFY possède deux domaines conservés : un domaine de liaison à l'ADN et un domaine de fonction inconnue en position N-terminal. L'objectif a été de comprendre le rôle du domaine N-terminal et d'étudier l'évolution de la spécificité de liaison à l'ADN de LFY. Nous avons obtenu la structure cristallographique du domaine N-terminal de LFY et découvert qu'il s'agissait d'un domaine SAM (Sterile Alpha Motif) permettant l'oligomérisation de la protéine. Nous avons validé l'importance de cette propriété pour la fonction florale de LFY chez A. thaliana. Nous avons ensuite montré, par des analyses in vitro et in vivo en ChIP-seq que l'oligomérisation influençait la liaison à l'ADN en permettant une liaison coopérative sur plusieurs sites de liaison, en assurant la sélectivité de la protéine vis-à-vis de l'ADN et en permettant l'accès de la protéine à des régions génomiques où la conformation de la chromatine est normalement défavorable à la liaison. Cette étude intégrative a permis de mieux comprendre le fonctionnement de LFY. Des modifications dans les réseaux de régulation de l'expression des gènes sont source de nouveauté et d'évolution. LFY étant très conservée et ne faisant pas partie d'une famille multigénique, nous nous sommes demandé si sa spécificité de liaison à l'ADN avait évoluée. Nous avons montré que LFY était apparue chez les algues multicellulaires et que sa spécificité avait connue au moins deux changements majeurs au cours de l'évolution. Nous avons expliqué ces modifications au niveau moléculaire par des approches de biologie structurale et de biochimie. Nous avons identifié une espèce chez qui LFY a une spécificité relâchée et nous proposons qu'une telle forme ait pu permettre les transitions d'une spécificité à une autre.

During the production of flowers in Arabidopsis thaliana many key decisions are taken in a short lapse of time. The floral primordium has to be positioned correctly on the inflorescence meristem and it has to grow to the required dimension before flower organs are themselves positioned and differentiate. All these tasks are strictly controlled at a molecular level and the genetic networks that underlies them have been intensively studied in the last 30 years. Nevertheless we are far from having a comprehensive knowledge on this process and the genetic mechanism controlling the arise, identity of the floral primordium and the timing of its developmental phases are widely unknown. We have identified new genes potentially involved in early flower development with two approaches: (i) Analysis of the specific transcriptome of the earliest stages of flower development and (ii) Co-expression analysis using APETALA1 and LEAFY, two genes that determine the identity of the floral meristem, which is the earliest stage of flower development. We have observed that multiple REM transcription factors are co-expressed with APETALA1 and LEAFY. Characterizing insertional mutants for genes potentially involved in early flower development and REM transcription factors, we have rarely observed a phenotype in the stages under study. This is consistent with the hypothesis that genes controlling early flower development are often functionally redundant. We are implementing various methods to overcome functional redundancy implementing analysis of gene families, multiple RNA interference and gene targeting strategies.

Specialization on insect and animal pollinators is thought to be the driving force for the evolution of floral traits. Specifically in the New World (NW), the oil-bee pollination syndrome has led to the convergence of floral characters in two distantly related families of core eudicots, Malpighiaceae and Krameriaceae. Both families display a flag-like structure that establishes a zygomorphic flower and floral oil rewards in epithelial elaiophores. These traits work concomitantly to attract and reward female oil-bees that help fertilize these flowers and in return receive oils. The underlying genetics of floral zygomorphy were studied in several clades of core eudicots, which implicated CYCLOIDEA2-(CYC2-)like genes of the TCP gene family to play a role in the establishment and maintenance of this trait. In Malpighiaceae, previous work demonstrated that two CYC2-like genes, CYC2A and CYC2B, are expressed during development correlated with establishing zygomorphy in flowers of NW Malpighiaceae. In this thesis work, I investigated the underlying developmental and genetic basis for the establishment of non-homologous and yet functionally similar traits in the oil-bee pollinated flowers of Malpighiaceae and Krameriaceae. In Chapter 1, I investigated the modification of a conserved CYC2 genetic program in the Old World (OW) acridocarpoids of Malpighiaceae following a major biogeographic disjunction. And in Chapter 2, I studied the floral ontogeny and genetic basis of floral zygomorphy development in Krameria lanceolata Torrey of Krameriaceae. This thesis work sheds light on the significance of the interdisciplinary study of floral symmetry evolution through comparative pollination ecology, development, and genetics.

Despite the sustainable development goals to increase access to improved water there are still 884 million people in the world without access to an improved water source (WHO, 2017). One method to improve access to water in rural, mountainous areas, is through construction of gravity fed water distribution systems. These systems should be designed based upon fundamental principles of hydraulics. One method of doing so in a time efficient manner with minimal engineering knowledge is to utilize a downloadable computer program such as Neatwork, which aids in design of rural, gravity fed water distribution systems and has been used by volunteers in Peace Corps Panama for years. It was the goal of this research to validate the results of the Neatwork program by comparing the flow results produced in the simulation program with flow results measured at tap stands of a rural gravity fed water distribution system in the community of Alto Nube, Comarca Ngöbe Bugle, Panama. The author measured flow under default Neatwork conditions of 40% faucets open in the system (in the field an equivalent of 8 taps) to have an initial basis as to whether the Neatwork program and field conditions yielded corresponding flows. The second objective would be to vary the number of taps open if the default condition did not produce comparable results between the field and the simulation, to pinpoint if under a certain condition of open faucets in the system the two methods would agree. The author did this by measuring flow at varying combinations from 10-100% of the open taps in the system (2-20 taps). Lastly the author observed the flow differences in the Neatwork program against the field flows, when the elevation of water in the water reservoir is set to the Neatwork default, where elevation of water is the tank outlet (at the bottom of the tank) versus when the elevation is established at the overflow at the tank (at the top of the tank) for the case of two taps open. The author used paired t-tests to test for statistical difference between Neatwork and field produced flows. She found that for the default condition of 40% taps open and all other combinations executed between 30-80% taps open, the field and Neatwork flows did not produce statistically similar results and, in fact, had the tendency to overestimate flows. The author also found that the change in water elevation in the storage tank from outlet to overflow increased the flow at the two taps measured by 0.140 l/s and 0.145 l/s and in this case, did not change whether the flows at these taps were within desired range (0.1 -0.3 l/s). Changing the elevation of the water level in the tank in the Neatwork program to correspond to a “full” tank condition is not recommended, as assuming an empty tank will account for seasonal changes or other imperfections in topographical surveying that could reduce available head at each tap. The author also found that the orifice coefficients, θ, of 0.62 and 0.68, did not demonstrate more or less accurate results that coincided with field measurements, but rather showed the tendency of particular faucets to prefer one coefficient over the other, regardless of combination of other taps open in the system. This study demonstrates a consistent overestimation in flow using the computer program Neatwork. Further analysis on comparisons made show that between field and flow results across each individual faucet, variations between Neatwork and the field were a result of variables dependent upon the tap, such as flow reducers or errors in surveying. Flow reducers are installed before taps to distribute flow equally amongst homes over varying distances and elevations and are fabricated using different diameter orifices depending on the location of the tap. While Neatwork allows the user to simulate the effect of these flow reducers on tap flow, it may not account for the imperfect orifices made by the simple methods used in the field to make such flow reducers. The author recommends further investigation to be done on the results of field flow versus Neatwork simulated flow using other methods of flow reducer fabrication which produce varying degrees of accuracy in orifice sizing. The author also recommends executing these field measurements over a greater sample size of faucets and more randomized combination of open/closed taps to verify the results of this research. More work should be done to come up with a practical solution for poor and rural communities to fabricate and/or obtain more precisely sized flow reducers. A full sensitivity analysis of the input variables into the Neatwork program should be performed to understand the sensitivity of varying each input.

Most embryonic development and tissue self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types, which are integrated with the signaling networks in these 3D environments. Although the role of cell mechanics in tissue self-assembly has been demonstrated, little is known about the mechanical responses of 3D multi-layer tissues to chemical cues. To investigate the collective movements within multilayered tissues, I developed a novel microfluidic technique capable of removing desired height or width of tissue from a composite tissue. I call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. I used a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues microsurgically isolated from embryos of the African Clawtoed frog, Xenopus laevis. Xenopus embryos and explanted tissues have long been used to elucidate signaling and other cellular processes during development and here provide an ideal model 3D tissue etching. Long exposure to a narrow etchant stream cuts completely through cell-cell layers to expose the substrate. By reducing the exposure time a single layer may be removed. By controlling the width of the etchant and the exposure time a broader swath of the surface layer may be removed. For more refined etching, after removal of a broad swath the resistance circuits can be switched and a second narrow stream can remove only a single narrow band within the swath exposed cells. I developed tissue-etching techniques that allow me to shape complex multi-layered embryonic tissues. The ability to control 3D stimulation and the form of multicellular tissues will provide extend the tools of tissue engineering to synthesize highly complex 3D integrated multicellular biosystems. Integration of tissue etching in my custom microfluidic system provides a "test-bed" where a range of hypotheses concerning the control and regulation of development and cell differentiation can be implemented and tested.

Crayfish exhibit innate immune responses via hemocytes and their products. There are 3 hemocyte populations: hyaline cells, granular cells, and semigranular cells. Hemocytes from laboratory housed, untreated crayfish (normal crayfish) have been quantified on the basis of cell type, cell size, and cell granularity using Flow Cytometry. These data present the first overall picture of normal hemocytes from Red Swamp Crayfish with regard to cell type, cell size, and cell granularity and will serve as a baseline for all future studies in our lab. Experiments using crayfish injected with Pseudomonas aeruginosa, Staphylococcus aureus, or crayfish saline alone showed significant and consistent changes in cell type in cells from crayfish injected with bacteria with a decrease in hyaline cells and an increase in granular cells. This effect was greater in crayfish injected with Gram - bacteria. In addition, crayfish injected with Pseudomonas aeruginosa showed a significant difference in Granular cell size with a shift to larger cells and a significant decrease in granularity in the Granular cell population. Cells from crayfish treated with Staphylococcus aureus did not show these changes.

This thesis describes the development of a computer controlled on-line system for fermentation monitoring and control. The entire system consists of a laboratory fermenter, flow injection system (four channels), a newly designed on-line filter, biomass analysis channel, pH and oxygen controllers as well as a spectrophotometer. A new design of gas driven flow injection analysis (FIA) allows a large number of reagents to be handled. The computer-controlled four channel PIA system is well suited for sequential analysis, which is important for fermentation on-line mOnitoring. The system can change the wavelength of the spectrophotometer automatically for each PIA channel, which makes the system powerful and flexible. A high frequency, low energy ultrasonic filter was modified and applied to the system for on-line mammalian cell culture sampling without breaking the sterile barrier. The results show that this novel application of ultrasonic filter technology results in higher efficiency and reliability and a longer life cycle than other types of filter. All the operations of the analytical system are controlled by a Macintosh computer (Quadra 950). The control program was written in LabVIEW which is a graphical programming language and well applicable to fermentation control. The software communicates with detectors, data acquisition, data analysis and presentation. The system can programmatically control up to 50 devices. Mammalian cell batch culture was used as an example of the application of the system. The system consists of a laboratory fermenter with a continuous sample withdrawal filter and an analysis system where glucose, lactate and ammonia, lactate dehydrogenase and biomass were measured. Cell viability was estimated by microscopic assay with trypan blue. pH and Oxygen were also measured. The system response was fast and yields a large number of reliable and precise analytical results which can be of great importance in the monitoring and control of mammalian cell culture conditions.

Flowering is the most important process in a plant’s life as it is the essential step for its reproduction. Flower development starts with a tightly regulated process called the floral transition in which different regulatory pathways, which are regulated by environmental and internal signals, culminate in the transition from vegetative to reproductive growth. Subsequently, flowers develop instead of leaves and the formation of these flowers is controlled by complex regulatory pathways. In model organism Arabidopsis thaliana there are at least five different pathways that regulate the floral transition to guarantee that it occurs under the best possible conditions. The signals derived from these pathways are than integrated at the level of the floral pathway integrators which are LEAFY (LFY), FLOWERING LOCUS T (FT), SOPPRESSOR OF OVEREXPRESSION OF CO (SOC1). These genes are responsible for the switch from the shoot apical meristem (SAM) to the inflorescence meristem (IM) and are involved in the activation of the floral meristem identity (FMI) genes: LFY, LATE MERISTEM IDENTITY1 (LMI1), APETALA1 (AP1), CAULIFLOWER (CAL), SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE 24 (AGL24). In fact, after the floral transition, the inflorescence meristem (IM) starts to produce floral meristems from its flanks. These meristems remain undifferentiated until stage 3 of flower development, thanks to the action of the FMI genes; afterwards, when some of these genes become repressed, the floral organs start to differentiate. In these first undifferentiated stages, the floral meristem grows and produces enough cells to support the subsequent differentiation of all the floral organs. Many of the genes involved in these two processes, floral transition and floral meristem determination, are MADS-box transcription factors. The MADS-box family is one of the best-characterized gene families in Arabidopsis and the its members represent key regulators of developmental processes. MADS-box factors are combinatorial proteins that act via multimerization and interact with other regulatory proteins in complexes to regulate the transcription of target genes. The aim of this thesis is the analysis of the genetic interactions of MADS-box transcription factors playing key roles during the floral transition and early stages of flower development. The floral pathways integrator SOC1 is a MADS-box gene that integrates at least four pathways that control flowering (photoperiod, vernalization, autonomous and gibberellin pathways), giving rise to the activation of the floral meristem identity genes (Parcy, 2005). In chapter 2, , we show that AGAMOUS-LIKE 42 (AGL42), AGAMOUS-LIKE 71 (AGL71) and AGAMOUS-LIKE 72 (AGL72) that are phylogenetically related to SOC1, are also involved in the floral transition of both the shoot apical meristem and axillary meristems and moreover, are involved in the gibberellin pathway. The soc1 agl42 ami::agl71-72 mutant shows an aerial rosettes bearing nodes phenotype. Our findings suggest that the SOC1-like genes are involved in the floral transition especially in the axillary meristem and the GA pathway is the main player controlling flowering in these axillary meristems both under short day and long day conditions. Furthermore SOC1 is able to directly control the expression of AGL42, AGL71 and AGL72 to maintain a proper expression level of SOC1-like genes. In chapter 3, the interactions between the floral meristem identity genes SVP, AGL24, AP1, CAL, which are all MADS-box transcription factors, and LFY is described. The lfy mutant shows partial reversions of flowers in inflorescence shoot-like structures and this phenotype is enhanced in the lfy ap1 double mutant. Here we show that combining the lfy mutant with agl24, svp single or agl4 svp double mutant enhances the lfy phenotype and that the agl24 svp lfy triple mutant phenocopies the ap1 lfy double mutant. Analysis of the molecular interactions between LFY and AGL24 and SVP showed that LFY is, together with AP1, a repressor of AGL24 and SVP whereas AGL24 and SVP positively regulate AP1 and LFY by direct binding to their regulatory regions. Since all genes are important to establish floral meristem identity this regulatory loop is probably important to maintain the correct relative expression levels of these genes. In chapter 4, we focalize our attention on SVP, a MADS-box gene involved in floral repression, before the floral transition, and in floral meristem (FM) identity determination, after the floral transition. An interesting feature of SVP is that it is the only Floral Meristem Identity Gene identified so far that is expressed exclusively in the undifferentiated FM. To date some transcription factors that are able to bind the SVP genomic region have already been identified by ChIP experiments, but it is still not clear how this gene is regulated. To understand this as a first step we are interested in the identification of the SVP minimal promoter region that fully comprises all its regulatory elements. We use, for our studies, lines that contain different SVP promoter fragments, that are cloned as transcriptional or translational fusions to the uidA gene, that encodes the beta-glucuronidase enzyme. This studies show that at least two regions are necessary for normal SVP expression: a 1 Kb fragment, located from 3000 to 2000 bp upstream of the start codon, and the first intron. In fact constructs lacking one of these two regions aren’t able to express GUS in the flower primordia. In conclusion this work contributes to get a better understanding of what exactly happens during the floral transition and, afterwards, in undifferentiated flower meristems.

Genomweite Bemühungen haben eine große Anzahl langer nichtkodierender RNAs (lncRNAs) identifiziert, obwohl ihre möglichen Funktionen weitgehend rätselhaft bleiben. Hier verwendeten wir ein System zur synchronisierten Blüteninduktion in Arabidopsis, um 4106 blütenbezogene lange intergene RNAs (lincRNAs) zu identifizieren. Blütenbezogene lincRNAs sind typischerweise mit funktionellen Enhancern assoziiert, die bidirektional transkribiert werden und mit verschiedenen funktionellen Genmodulen assoziiert sind, die mit der Entwicklung von Blütenorganen zusammenhängen, die durch Koexpressionsnetzwerkanalyse aufgedeckt wurden. Die Master-regulatorischen Transkriptionsfaktoren (TFs) APETALA1 (AP1) und SEPALLATA3 (SEP3) binden an lincRNA-assoziierte Enhancer. Die Bindung dieser TFs korreliert mit der Zunahme der lincRNA-Transkription und fördert möglicherweise die Zugänglichkeit von Chromatin an Enhancern, gefolgt von der Aktivierung einer Untergruppe von Zielgenen. Darüber hinaus ist die Evolutionsdynamik von lincRNAs in Pflanzen, einschließlich nicht blühender Pflanzen, noch nicht bekannt, und das Expressionsmuster in verschiedenen Pflanzenarten war ziemlich unbekannt. Hier identifizierten wir Tausende von lincRNAs in 26 Pflanzenarten, einschließlich nicht blühender Pflanzen. Ein direkter Vergleich von lincRNAs zeigt, dass die meisten lincRNAs speziesspezifisch sind und das Expressionsmuster von lincRNAs einen hohen Transkriptionsumsatz nahe legt. Darüber hinaus zeigen konservierte lincRNAs eine aktive Regulation durch Transkriptionsfaktoren wie AP1 und SEP3. Konservierte lincRNAs zeigen eine konservierte blütenbezogene Funktionalität sowohl in der Brassicaceae- als auch in der Grasfamilie. Die Evolutionslandschaft von lincRNAs in Pflanzen liefert wichtige Einblicke in die Erhaltung und Funktionalität von lincRNAs.
Genome-wide efforts have identified a large number of long non-coding RNAs (lncRNAs), although their potential functions remain largely enigmatic. Here, we used a system for synchronized floral induction in Arabidopsis to identify 4106 flower-related long intergenic RNAs (lincRNAs). Flower-related lincRNAs are typically associated with functional enhancers which are bi-directionally transcribed and are associated with diverse functional gene modules related to floral organ development revealed by co-expression network analysis. The master regulatory transcription factors (TFs) APETALA1 (AP1) and SEPALLATA3 (SEP3) bind to lincRNA-associated enhancers. The binding of these TFs is correlated with the increase in lincRNA transcription and potentially promotes chromatin accessibility at enhancers, followed by activation of a subset of target genes. Furthermore, the evolutionary dynamics of lincRNAs in plants including non-flowering plants still remain to be elusive and the expression pattern in different plant species was quite unknown. Here, we identified thousands of lincRNAs in 26 plant species including non-flowering plants, and allow us to infer sequence conserved and synteny based homolog lincRNAs, and explore conserved characteristics of lincRNAs during plants evolution. Direct comparison of lincRNAs reveals most lincRNAs are species-specific and the expression pattern of lincRNAs suggests their high evolutionary gain and loss. Moreover, conserved lincRNAs show active regulation by transcriptional factors such as AP1 and SEP3. Conserved lincRNAs demonstrate conserved flower related functionality in both the Brassicaceae and grass family. The evolutionary landscape of lincRNAs in plants provide important insights into the conservation and functionality of lincRNAs.

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.

La Polarité Cellulaire Planaire (PCP) est une voie de signalisation originellement identifiée chez les invertébrés pour son rôle dans l’établissement d’une asymétrie cellulaire perpendiculaire à l’axe apico‐basal. Elle définit une polarité dans le plan d’un épithélium et coordonne cette polarité dans tout l'épithélium. L'activation de la voie PCP conduit à une réorganisation ducyto squelette en passant par une modulation des zones d'adhésion, régulant ainsi la forme et les mouvements des cellules. La voie de signalisation de la PCP est conservée tout au long de l'évolution jusqu'au mammifères, et contrôle la morphogénèse de divers tissus dont les tissus épithéliaux et mésenchymateux, ainsi que pour les tissues cardiaques, osseux, pulmonaire ou encore rénaux, mais aussi le système nerveux pour n'en citer que quelques‐uns.Afin d'identifier le rôle de vangl2, un des gènes centraux de la PCP, dans la mise en place de la circuiterie hippocampale, nous avons créé un modèle murin où vangl2 est supprimé de façon conditionnelle (cKO) dans le télencéphale à des stades précoces de l’embryogénèse. J’ai d'abord montré que Vangl2 est enrichi dans les neurones immatures de la zone sous granulaire du DG, ainsi que dans l’arborisation des neurites (axones et dendrites) des cellules granulaires (CG) du gyrus denté (DG) de l’hippocampe. Ainsi, Vangl2 est enrichi dans le stratum lucidum (sl), une région dense en contacts synaptiques entre le DG et le CA3. Dans cette région a lieu une synapse très particulière entre l'axone des CG, la fibre moussue (Mf) qui forme des boutons géants (MfB) et les excroissances épineuse (TE) issues de la partie proximale des dendrites apicaux. L'analyse structurale et ultra structurale de ces épines démontre que l'élargissement et la complexification de la synapse MfB/TE est bloquée dans nos mutants, alors que les zones actives (PSD) des épines sont présentes, mais réorganisées. De façon intéressante,dans une zone plus distale des dendrites des neurones du CA3 (sl), les épines sont, elles, plus grosses, suggérant un remodelage complexe du réseau en l'absence de vangl2. Enfin, j’ai pu montrer que ces défauts morphologiques étaient corrélés à des problèmes de mémoire complexe (mémoire déclarative) qui dépendent de l’hippocampe mais aussi du cortex. Cette étude montre pour la première fois l’importance du signal PCP dans maturation in vivo d’un circuit hippocampique spécifique ainsi que ces conséquences cognitives. D'autres résultats in vitro montrent que la suppression de vangl2 augmente la vitesse de déplacement des cônes de croissance sur des substrats de N‐cadhérine. J’ai utilisé la microscopie en super résolution spt‐PALM‐TIRF pour montrer que cette augmentation de croissance est inversement proportionnelle à la vitesse du flux rétrograde d’actine. Des expériences de FRAP permettent de suggérer que les molécules de N‐cadhérine engagées dans des interactions hémophiliques (adhésion) est plus importante dans les mutants vangl2 Je propose que Vangl2 contrôle le recyclage et la stabilité des protéines N‐cadhérine dans les sites d’adhésion afin de réguler localement les dynamiques d’actine et par conséquent la croissance neuronale
Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico‐basal axis, in the plane of an epithelium. PCPsignaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization. Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop‐tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant‐negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported inhomozygotes Looptail mice. To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high‐density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses. I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal‐dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences. Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on Ncadherin substrate. I used spt‐PALM‐TIRF super‐resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flowand to a decrease in the number of directed actin trajectories. These results strongly suggest that N‐cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstratedthat in Vangl2 cKO neurons the recovery of N‐cadherin molecules engaged in homophilicbindings (adhesion) was decreased, suggesting that the turnover of N‐cadherin involved inadhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of Ncadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth

L’épithélium digestif est recouvert d’une couche protectrice de mucus, qui est un hydrogel perméable et viscoélastique. La couche de mucus est formée d'un réseau de fibres de mucines. Ces dernières sont des glycoprotéines de haut poids moléculaire avec un squelette protéique riche en sérine et thréonine, lié à une grande variété de O-glycanes qui représentent une source nutritionnelle pour les bactéries et/ou des ligands potentiels pour les adhésines bactériennes, contribuant ainsi probablement à la sélection et l'implantation d'un microbiote régio-spécifique. Nous nous sommes intéressés aux capacités muco-adhésives de L. lactis TIL448 par le couplage de (i) la microscopie à force atomique (AFM), à l'échelle de la cellule unique et en mode statique et (ii) la méthode hydrodynamique en chambre à écoulement cisaillé, à l'échelle de l’ensemble de la population bactérienne. Dans l'optique d'identifier la nature et le rôle fonctionnel des déterminants de surface mis en jeu, nous avons testé, outre la souche sauvage, la souche curée de plasmides TIL1230 et deux mutants TIL1289 et TIL1290, altérés dans la synthèse de pili et d'une protéine "mucus-binding", respectivement. Pour relier les propriétés muco-adhésives et diffusives de L. lactis, les capacités de migration de la souche TIL448 et de ses dérivés ont ensuite été évaluées dans des suspensions de PGM à concentration variable (0,5% et 5% (m/v)), en mettant en œuvre une nouvelle méthode "Diffusion Front Tracking" (DFT). Cette méthode consiste à suivre le front de diffusion de la suspension bactérienne au cours du temps au sein du réseau de PGM, dans une chambre de Hele-Shaw, couplée à une caméra CCD. Les bactéries L. lactis sont préalablement marquées avec la fuschine pour mieux visualiser le front de diffusion. Par ailleurs, nous avons démontré que les bactéries L. lactis ont tendance à être plus diffusives dans PGM 0,5% (m/v) que dans PGM 5% (m/v). La microstructure du réseau de mucines a donc été caractérisée par des approches de microrhéométrie 1 point (1P) et 2 points (2P) et de suivi de particules fluorescentes
The digestive epithelium is covered with a protective mucus layer, regarded as a viscoelastic and permeable hydrogel. Mucins are large glycoproteins with a serine and threonine-rich protein backbone, linked to a wide variety of O-linked oligosaccharide side chains arranged in a bottle-brush configuration. Such O-glycans are nutritive sources for bacteria and/or potential ligands for bacterial adhesins, probably contributing in this way to the selection of the species-specific microbiota. In this thesis, we focused on unraveling multi-scale interactions between a vegetal L. lactis subsp. lactis isolate, TIL448 and a model mucin, Pig Gastric Mucin (PGM). Our study, based on the combination of different biophysical approaches and tools, has allowed dissecting the muco-adhesive and diffusive phenotype of L. lactis TIL448, in relation with the nature of the bacterial surface determinants and the structural, mechanical and rheological properties of the PGM network. Firstly, the muco-adhesion of TIL448 were examined using the single-cell scale AFM measurements with dedicated lacto-probes and shear stress flow chamber experiments at the bacterial population level, under laminar flow conditions. We also tested the plasmid-cured strain and two mutants, obtained by disruption of the genes encoding the major pilin and the mucus-binding protein. Then, the diffusion ability of L. lactis was determined by implementing a novel method, named Diffusion Front Tracking (DFT). It consists of tracking the diffusion front of stained cell suspensions over time within the PGM network. In a second part, in order to have a more thorough understanding of the L. lactis muco-adhesive and diffusive ability, the microstructure and mechanical properties of PGM were determined. Gel microstructure for varying PGM concentration was probed by the analysis of diffusivities of 200-nm and 500-nm fluorescent nanoparticles with different surface properties (carboxyl-terminated, amine-terminated and neutral charged tracers), using fluorescence Multiple-Particle Tracking

Le laboratoire d’Embryologie Moléculaire étudie les mécanismes moléculaires contrôlant le développement embryonnaire et utilise comme système expérimental l’embryon de xénope. En collaboration le laboratoire du Dr Daniel Christophe, nous avons abordé l’étude du gène XHRT1 chez le xénope. Ce gène est l’orthologue du gène HRT-1/Hey1/Hesr-1/HERP2/CHF2 de souris. Celui-ci, avec deux autres protéines apparentées (HRT2 et HRT3) forme une sous-famille de facteurs de transcription de type bHLH-O qui se différencient des autres facteurs bHLH-O par l’absence d’un motif carboxy-terminal de séquence « WRPW » et par la présence d’un nouveau motif carboxy-terminal conservé de séquence « TEI/VGAF ». Le rôle de ces facteurs HRT dans le développement est encore actuellement mal connu.

Dans un premier temps, nous avons déterminé le profil d’expression de XHRT1 au cours de l’embryogenèse. Nous avons observé que ce gène est fortement exprimé au stade neurula dans le plancher du tube neural, et que plus tardivement celui-ci est exprimé dans différentes régions du système nerveux, dans les somites et le dans le pronéphros. Comme attendu pour un membre de la famille des facteurs bHLH-O, nous avons également observé que l’expression précoce de HRT1 au niveau du plancher du tube neural est bien régulée par la voie de signalisation Notch.

Dans un deuxième temps, nous nous sommes intéressés au rôle et au mode d’action du facteur XHRT1 dans le développement du plancher du tube neural. Nous avons pu montrer que XHRT1 agit comme répresseur transcriptionnel et que cette répression nécessite la présence du domaine bHLH et de séquences en aval de celui-ci. Nous avons montré en embryon que la surexpression précoce de XHRT1 induit un blocage de l’expression des marqueurs du mésoderme et une augmentation de marqueur du plancher du tube neural, ce qui est en accord avec le modèle selon lequel la voie de signalisation Notch interviendrait dans le choix de la destinée des cellules de la région médiane en inhibant la différenciation des cellules en notocorde et en favorisant leur différenciation en cellules du plancher du tube neural. XHRT1 n’étant cependant activé qu’à partir du stade neurula, nous avons conclu que les effets observés n’étaient probablement pas dus à XHRT1 mais à un autre facteur bHLH-O apparenté exprimé plus précocement dans les cellules de la ligne mediane de l’embryon. Afin d’éviter ces effets non spécifiques précoces, nous avons utilisé un vecteur d’expression de XHRT1 permettant un contrôle temporel de l’activité de la protéine. Nous avons ainsi montré que l’activation de XHRT1 au stade neurula dans l’ectoderme inhibe la différenciation des cellules précurseurs neurales en neurones et qu’il pourrait ainsi jouer un rôle important dans le développement du plancher du tube neural. Nos résultats ont montré également que XHRT1 est capable d’homo- et hétérodimériser in vivo avec les facteurs Xhairy1 et Xhairy2b coexprimés avec XHRT1 dans le plancher du tube neural. Enfin, nous avons montré que les propriétés de dimérisation de XHRT1 sont dépendantes non seulement du domaine bHLH, mais aussi du domaine Orange et des séquences situées en aval, séquences jouant un rôle important dans le choix du partenaire.

Des travaux récents ayant montré que la voie de signalisation Notch joue un rôle important dans le développement du rein, nous avons voulu déterminer l’importance de XHRT1 dans le développement du pronéphros. Nos résultats ont montré que XHRT1 ainsi que d’autres facteurs bHLH-O sont exprimés de manière dynamique, d’abord dans le glomus puis dans la partie dorso-antérieure de l’ébauche du pronéphros à l’origine des tubules proximaux, et que leur expression est régulée positivement par Notch. La surexpression de XHRT1 à la fin de la neurulation inhibe la formation du canal et du tubule distal, tandis que l’inhibition de la traduction de la protéine entraîne une réduction de l’expression de marqueurs spécifiques des tubules proximaux et du glomus. Ces résultats démontrent que XHRT1 joue un rôle important comme médiateur de la voie de signalisation Notch dans le pronéphros.
Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

Cystine-knot peptides are members of the large family of Cysteine-rich proteins with a dimension typically less than 50 amino acids in their mature form, characterized by the presence of six conserved cysteine (Cys) residues linked by three disulfide bonds in a knotted arrangement (Rees & Lipscomb, 1980). Peptides containing the knot motif are widespread in various species such as fungi, insects, mollusks, and mammals, where they mainly play a defense role against microorganisms, acting as toxins, or as signals in cell defense pathways (Craik et al., 2001; Iyer & Acharya, 2011; Schwarz, 2017; Vitt et al., 2001). In plants, cystine-knot peptides (also referred to as cysteine-knot miniproteins) are often involved in resistance to pathogens with the function of protease inhibitors, namely metallocarboxypeptidases and serine proteases (Daly & Craik, 2011; Molesini et al., 2017). A class of cystine-knot protease inhibitors specific to the Solanaceae family was described for the first time in the 1980s (Hass & Hermodson, 1981a; Rees & Lipscomb, 1980). This class includes the tomato cystine-knot miniproteins 1 and 2 (TCMP-1 and TCMP-2), of 37 and 44 amino acids, respectively. TCMP-1 and TCMP-2 display a sequential expression pattern, which is highly modulated during flower and fruit development. TCMP-1 is expressed at a very high level in flower buds before anthesis, then its expression decreases rapidly after anthesis and increases again during fruit development (Cavallini et al., 2011). TCMP-1 mRNA level is very low in leaves, although its expression is induced by wounding and elicitors of responses to biotic stress (Díez-Díaz et al., 2004; Martineau et al., 1991). The expression of TCMP-2 is low in flower buds before anthesis, and gradually increases after fertilization, reaching a maximum in green and ripe fruits, whereas it is apparently absent in leaves, roots, and stems (Cavallini et al., 2011; Pear et al., 1989; Treggiari et al., 2015). Indeed, the TCMP-2 promoter (also named 2A11; X13743; [Pear et al., 1989]) has been successfully used to improve qualitative trait in tomato fruit (Davuluri et al., 2005). Although the biological activity of metallocarboxypeptidase inhibitors supports a role for Solanaceous cystine-knot proteins in plant defense, it has recently been demonstrated that tomato TCMPs are implicated in fruit development (Molesini et al., 2018). In the paper by Molesini et al. (2018), tomato plants transformed with a chimeric gene containing the entire TCMP-1 coding region under the control of the TCMP-2 promoter (pTCMP-2::TCMP-1) showed a marked increase in the expression of TCMP-2 before anthesis, associated with anticipated fruit production. This evidence suggests that TCMPs are regulators of fruiting time and that the maintenance of a correct TCMP-1/TCMP-2 ratio is required for proper initial fruit growth. The mode of action of TCMPs remains largely unexplored also due to the absence of homologous genes in other model species, including Arabidopsis thaliana. In several cases, Cysteine-rich peptides act as signaling molecules in plant development, by interfering with receptors or modifying the activity of multimeric complexes (de Coninck & de Smet, 2016; Tavormina et al., 2015). The general aim of this thesis was a further investigation of the functional role played by TCMP-2 during the first phases of reproductive development. Specific aims were: 1) phenotypic and molecular analyses of pTCMP-2::TCMP-1 and 35S::TCMP-2 plants, during the transition from vegetative growth to reproductive development; 2) identification of TCMP-2 interacting partners by Yeast Two-Hybrid (Y2H) screening using a tomato cDNA library; 3) functional study of one of these interactors (i.e., a B-BOX motif-containing protein) in both Arabidopsis and tomato. A detailed analysis of pTCMP-2::TCMP-1 plants during the transition from the vegetative to the reproductive stage showed an anticipated termination of the sympodial units linked with an induced expression of the florigen gene SINGLE FLOWER TRUSS (SFT), which is the main inducer of flowering. Moreover, MicroTom plants over-expressing TCMP-2 under the control of the CaMV35S constitutive promoter exhibited a reduction of the primary shoot length, very often accompanied by a decreased number of leaves before the first inflorescence, which are indicators of early flowering. The Y2H screen permitted the identification of 47 potential interactors of TCMP-2. Among them, we focused on the B-Box domain-containing protein 16 (SlBBX16). The interaction between TCMP-2 and SlBBX16 was validated in vivo in plant cells by bimolecular fluorescence complementation (BIFC) analysis. We demonstrated that TCMP-2 is also able to interact with SlBBX17, which is the closest tomato homolog of SlBBX16, and with the SlBBX16 Arabidopsis homolog, AtBBX31. A recent study showed the involvement of AtBBX30 and AtBBX31 microproteins (also referred to as miP1b and miP1a) in a multiprotein complex which regulates flowering time in Arabidopsis (Graeff et al., 2016). These proteins interact with the flowering regulator CONSTANS (AtBBX1) and additionally engage in a larger protein complex involving the co-repressor protein TOPLESS through a specific amino acid motif (PFVFL). These interactions suppress the CO-mediated induction of FT expression, causing the severe late flowering phenotype observed in plants over-expressing AtBBX30/31. The implication of AtBBX30/31 in flowering control may indicate that the TCMP-2-SlBBX16/SlBBX17 interaction could play a role in the regulation of flowering. To test this hypothesis, we ectopically overexpressed TCMP-2, SlBBX16 and SlBBX17 in Arabidopsis. The overexpression of TCMP-2 led to an anticipated flowering phenotype linked to an increased FT mRNA level, whereas the overexpression of SlBBX16 and SlBBX17 in Arabidopsis WT and AtBBX30/31 KO mutant resulted in a slight delay in flowering time, suggesting that tomato BBXs were unable to fully phenocopy AtBBX30/31 overexpression. One of the possible reasons for the weak phenotype displayed by A. thaliana over-expressing SlBBX16 and SlBBX17 could be attributed to their inability to interact with AtCO and AtTPL, since the interaction between AtBBX30/31 with AtCO and AtTPL is due to the PFVLF motif (Graeff et al., 2016), which is absent in SlBBX16 and SlBBX17. Indeed, the Y2H experiments revealed no interaction between the tomato BBXs and AtCO and AtTPL, even when using a mutated version of SlBBXs containing the PFVLF motif. The functional study in tomato was conducted on SlBBX17, which presents a peculiar expression pattern in the floral organs. MicroTom plants over-expressing SlBBX17 showed a number of leaves at the first inflorescence similar to that of WT plants, but a delay in the time to reach the flower anthesis stage and a reduced number of ripe fruits. To investigate whether in tomato TCMP-2 and SlBBX17 may participate in a multiprotein complex similar to that described in Arabidopsis (AtBBX30/31-CO-TPL), we carried out ad hoc Y2H analyses to test the interaction between TCMP-2 and SlCO1 and SlTPL1 and the interaction between SlBBX17 and SlCO1 and SlTPL1. Our data indicate that neither TCMP-2 nor SlBBX17 can directly bind to SlCO1 and SlTPL1. Further investigation of the role in flowering and fruiting of SlBBX16, the homologue of SlBBX17, may provide additional insight into the function of BBXs microproteins in tomato.

Title: Investigating Partners of OsMADS1 Transcription Factor and functions for some associated factors for roles in rice inflorescence and floral development The classic ABCDE model for floral organ identity show that the combinatorial action of MADS domain transcription factors directs floral organ identity and their normal development. While floral MADS-box transcription factors are in general conserved among diverse flowering plants, several studies suggest emergence of distinct functions for some of these conserved factors. Rice E- or SEP-class genes, when compared to Arabidopsis SEP-class, are only partly conserved with rice homologous genes which show some distinct functional diversifications. Rice genes of this class form two clades: LOFSEP- (OsMADS1, OsMADS34, OsMADS5) and SEP3 (OsMADS7, OsMADS8) clades. Unlike Arabidopsis SEP-class genes that are redundant, just loss of function of OsMADS1 alone leads to severe defects in the development of all floral organs. Extensive molecular genetic studies, including from our laboratory, showed OsMADS1 is a master transcription regulator for floral meristem determinacy, floral organ specification and identity. The gene regulatory network controlled by OsMADS1 including other transcription factor targets, and phytohormone auxin and cytokinin pathways. Here we probed for the interacting partners of OsMADS1 to get a better understanding of its function in higher protein complexes during the broad program of floral development. In addition, we took up functional genomic studies of OsMADS1 associated factors for studies on roles for downstream factors controlling rice inflorescence and floral development. Part I: Investigating interacting partners of OsMADS1 transcription factor While interactions between MADS transcription factors are well described, interactions with other classes of factors that can influence its regulatory functions in higher protein complexes are not studied yet. We adopted two methods to identify OsMADS1 interacting factors. First, we predicted interactors of OsMADS1 based on the previous report from our group that identified the co-occurrence of cis DNA binding motifs in the genome-wide OsMADS1ChIP-Seq dataset (Khanday et al., 2016). This was combined with co-expression analysis of transcription factors that could bind these cis-elements based using publicly available rice transcriptome databases. A small set of putative deduced from the above approaches were examined by yeast-two hybrid (Y2H) assays and the interactions noted were further validated by the in-planta Bimolecular fluorescence complementation (BiFC) assay. Y2H assay suggests OsbZIP47 and ERF68 are strong interactors of OsMADS1. Further, we note RFL as a weak interactor, whereas the eudicot Arabidopsis homologs LEAFY and SEP3 have a strong association. In the second approach, we carried out pulldown of endogenous OsMADS1 associated protein complexes from maturing 3-5cm inflorescence tissue lysates using OsMADS1specific antibodies. By mass spectroscopy analysis we identify several classes of proteins that demonstrate association of OsMADS1 function with transcriptional corepressors, meristem transition regulators of floral meristem determinacy, organ identity and also indicate interactions with factors in auxin and Brassinosteroid signaling pathways. Part II: Functional studies on the predicted meristem regulator OsbZIP47, a downstream target and a partner of OsMADS1 OsbZIP47 is a predicted rice homolog for the Arabidopsis and maize factors, PERIANTHIA (PAN) and FASCIATED EAR4 (FEA4) respectively that regulate meristem and floral organs in those model species. Here we aimed to understand functional relevance and pathways controlled by OsbZIP47 for normal rice inflorescence development. Transgenic plants with RNAi based knockdown of OsbZIP47 were raised and they showed pleiotropic effects on both vegetative and reproductive phases of growth including meristem and floral organ defects. Whereas transgenics with overexpression (Ox) of OsbZIP47 did not show any phenotypic defects. Importantly, similar to the PAN protein, we observe that oligomerization of the OsbZIP47 protein is sensitive to redox reagents (diamide, S-glutathionylation modifier and DTT). We also detect robust transcriptional activity for a reporter gene in yeast despite the fact that OsbZIP47 lacks the extended N-ter protein domain seen in PAN which is essential for PAN functions in Arabidopsis. We also show that OsMADS1 can positively regulate expression levels of OsbZIP47 and that of the gene encoding its potential glutaredoxin enzyme, OsGRX19 or MICROSPORELESS1 (OsMIL1). This co-ordinated effect may be to maintain homeostasis and enforce their interaction. To reveal OsbZIP47 downstream pathways, global transcriptome profiling (RNA-Seq) of 0.1-0.5cm panicle tissues from OsbZIP47 KD line was compared with similarly staged wild-type tissues and the data suggest ~2800 deregulated genes. Most important inference is that OsbZIP47 regulates meristem characteristics by controlling the canonical WUS-CLV pathway for stem cell homeostasis and the cytokinin-mediated WUS-KNOX (OSH1/STM/KN1) pathway. Floral defects (particularly of stamens and lodicules) seen in OsbZIP47 KD lines can to an extent be explained by the deregulation of OsMADS16 (B-class gene), OsDL (DROOPING LEAF), APO1, OsMADS68, TGA10, Osnop (Oryza sativa no pollen) that are known regulators for proper development of these organs. Taking leads from Arabidopsis and Maize reports we also tested interaction of OsbZIP47 with auxin response factors ETTIN1 and ETTIN 2, and with meristem regulators, KNOXI-OSH1, RFL using the Y2H assay. We report strong interaction of OsbZIP47 with OSH1 and RFL which further add a layer of complexity in molecular mechanisms by which OsbZIP47 can contribute to meristem characteristics. Part III: B-class OsMADS2 gene, a partner and a downstream factor of OsMADS1 controls gene expression for normal lodicule and stamen development In rice, and all grass florets, lodicules are modified organs equivalent to petal and they have a crucial role for efficient fertilization. Rice floret stamens have functions and structure with greater similarity to their eudicot counterparts. OsMADS1 regulates the normal development and identity of all floral organs; its partnership with B-class factors AP3-like OsMADS16, PI-like OsMADS2, and OsMADS4 is known and is critical for lodicule and stamen developmental control. Noteworthy is that B-class genes are expressed in the lodicule and stamen floral primordia but OsMADS1 transcripts are not detected in these primordia. Prior work from our group showed that OsMADS2, a PI-like factor has evolved to have a greater role in lodicule identity and lesser redundant role for stamen identity. Here, using immunolocalization we detect the presence of OsMADS1 protein in lodicule and stamen primordia, hinting at possibility of either intercellular trafficking or other non-cell-autonomous manner of OsMADS1 action in lodicule and stamen primordia. Further, to delineate gene sets that are coregulated by OsMADS1 and OsMADS2 we determined OsMADS2 genome-wide occupancy by ChIP-Seq using 0.3-2cm panicle tissues and OsMADS2 affinity-purified antibodies. After comparing data acquired in this study with the OsMADS1ChIP-Seq dataset (Khanday et al., 2016) we identify a common set of 280 gene targets. Also, OsMADS2 ChIP-Seq shows that homologs of Arabidopsis AP1 and AP2 class genes are enriched. Thus, as known in Arabidopsis the latter factors can explain OsMADS2 functions in lodicule development. Interestingly, OsMADS2 ChIP-PCR and qRT-PCR transcript measurements in transgenic with partial knockdown of OsMADS2 show that OsMADS1 and OsMADS2 are linked by a positive feedback loop. We confirm HECATE/OsbHLH120 as a common target of OsMADS1 (Dr. Grace Lhainekim Thesis, UVR lab) and OsMADS2 (this study). The homolog of OsbHLH120 in Arabidopsis is a meristem and carpel development regulator. Here we perform functional characterization of OsbHLH120 by RNA interference-mediated (dsRNAi) knockdown (KD). The transgenics had majority of their florets with elongated and deformed lodicules, and a minority showed increased stigma/pistil number. The spikelet organ called the sterile lemma was elongated resembling a lemma-like organ. These phenotypes suggest OsbHLH120 regulates spikelet and floral organ development.
IISc

Rice have highly derived florets borne on a short branch called ‘spikelet’ comprised of a pair of rudimentary glumes and sterile lemma (empty glumes) that subtends a single fertile floret. The floral organs consist of a pair of lodicules, six stamens and a central carpel that are enclosed by a pair of bract-like organs, called lemma and palea. A progressive reprogramming of meristem identity during the floral development of flowers, on branches on the inflorescence, is correlated with changes in transcriptional status of regulatory genes that execute cascades of distinct developmental events. On the other hand phytohormones such as auxin and cytokinin that are critical in predetermining the sites of new organ primordia emergence and in maintaining the size or populations of meristems. Molecular genetic analyses of mutants have expanded the repository of genes regulating floral organ specification and identity, yet the finer mechanistic details on process downstream to these regulatory genes and co-ordination with phytohormone signalling pathways needs further investigation. One aim of the study presented in this thesis is to develop a tool that would display of spatial description of dynamic auxin or cytokinin accumulation in developing rice inflorescence and floral meristems and to evaluate auxin distribution defects of OsMADS1-RNAi florets using this tool. Additionally, we aim to understand the regulatory effects on OsMADS1 on candidate floral organ and meristem fate determining genes during two temporal phases of flower development to decipher other regulatory cascades controlled by OsMADS1. Spatial distribution profile of phytohormones in young and developing meristems of rice Cytokinin promotes meristem activity (Su et al., 2011) while auxin accumulation, directed by auxin efflux transport PIN proteins predicts sites of new organ initiation (Reinhardt et al., 2003; van Mourik et al., 2012). Previous studies in the lab deciphered that OsMADS1 exerts positive regulatory effects on genes in auxin pathways and repressive effects on cytokinin signaling and biosynthetic genes (Khanday et al., 2013). Thus, the need for a reliable system to understand auxin and cytokinin activity in live inflorescence and floral meristems of rice motivated us to raise promoter: reporter tools to map the spatial and temporal phytohormone distribution. Confocal live imaging conditions in primary roots of IR4DR-GFP and DR5-CyPet lines was performed and responsiveness of the DR5 elements to auxin was authenticated. Auxin maxima were distinctly seen in the epidermal and sub-epidermal cells of inflorescence branch primordia anlagen and apices of newly emerged branch primordia. As floral organs were being initiated, on the floret meristem, we discerned the sequential appearance of auxin accumulation at sites of organ primordia while apices of early floral meristems (FM) showed low auxin content. We clearly detect canalization of auxin streams marking regions of vascular inception. Using this live imaging system we probed auxin patterns and levels in malformed and indeterminate OsMADS1-RNAi florets and we observed a significant reduction in the levels of auxin. Two oppositely positioned peaks of auxin were noted in the persistent FM of OsMADS1-RNAi florets, a pattern similar to auxin dynamics at sites of rudimentary glume primordia on the wild-type (WT) spikelet meristem. These studies were followed up with immunohistochemistry (IHC) on fixed tissues for “PIN” transport proteins that suggest PIN convergence towards organ initiation sites, regions where auxin accumulation was clearly visualized by the IR4DR5-GFP and DR5-CyPet reporters. IHC experiments that detected GFP, in fixed tissues of TCSn-mGFP ER (WT) and TCSn-mGFP ER;OsMADS1-RNAi (OsMADS1-RNAi) inflorescence and florets showed an ectopic increase in the domain of cells with cytokinin response in OsMADS1-RNAi florets, compared to that of WT. Intriguingly, cytokinin responsive cells persisted in the central FM of OsMADS1-RNAi florets that might partially account for some of the FM indeterminacy defects seen in these florets. A correlative observation of these different imaging data hint at some exclusive patterns of the IR4DR5/DR5 and TCSn reporters that in turn lead us to speculate that a cross talk between auxin and cytokinin distribution may contribute to the precise phyllotaxy of lateral organs in rice inflorescence. Studies on novel targets of OsMADS1 in floral organ identity and meristem determinacy Loss of OsMADS1 function results in rice florets with miss specified floral organs and an indeterminate carpel produces new abnormal florets. Despite having several mutants in OsMADS1, mechanisms of how OsMADS1 regulates meristem maintenance and termination is not well understood. Global expression profile in OsMADS1-RNAi vs. WT tissues encompassing a wide range of developing florets (0.2 to 2cm panicles), gave an overview of OsMADS1 functions in many aspects of floret development. Here, a gene-targeted knockout of OsMADS1 named - osmads1ko (generated in a collaborative study) was characterized and found to display extreme defects in floral organs and an indeterminate FM. Strikingly, in addition to loss of determinacy, FM reverts to a prior developmental fate of inflorescence on whose new rachis are leaf-like malformed florets. We suggest these phenotypes reflect the null phenotype of OsMADS1 and its role in meristem fate maintenance. We tested gene expression levels for some proven targets of OsMADS1 (Khanday et al., 2013) and utilized panicles in two developmental phases- young early FMs (panicles of 0.2 to 0.5 cm) and older florets with organ differentiation (panicles of 0.5 to 1cm). We observed temporally different effects on the regulation of OsMADS34 that together with histology of young osmads1ko inflorescences suggest that the mutant is impeded for spikelet to floral meristem transition. In addition, OsMADS1 had a positive regulatory effect on genes implicated for lemma and palea organ identity such as OsIDS1, OsDH1, OsYABBY1, OsMADS15, OsMADS32, OsDP1 and OsSPL16 in both young and old panicles while OsIG1 was negatively regulated in both phases of development. MADS-box genes important for carpel and ovule development - OsMADS13 and OsMADS58 were had significantly reduced expression in florets undergoing organ differentiation. OsMADS1 positively regulated several other non MADS-box developmental genes - OsSPT, OsHEC2 and OsULT1, whose Arabidopsis homologs control carpel development and FM determinacy. These genes are de-regulated in later stages of osmads1ko floret development and are unaffected in younger panicles. Finally, OsMADS1 continually activated meristem maintenance genes - OsBAM2-like and OsMADS6 while the activation of OSH1 in early floral meristems was later altered to a repressive effect in developing florets. Perhaps such dynamic temporal effects on meristem genes are instrumental in the timely termination of the floral meristem after floral organ differentiation. More importantly, we show that regulation of many of these genes is directly affected by OsMADS1, through our studies on expression levels before and after chemical induction of OsMADS1-GR protein in amiRNAOsMADS1 florets. Further, some key downstream targets were re-affirmed by studying expression status in transgenic lines, with the OsMADS1-EAR repressive protein variant. These results provide new insights into the developmentally phased roles of OsMADS1 on floral meristem regulators and determinants of organ identity to form a determinate rice floret. Gene networks regulated by OsMADS1 during early flower development To identify global targets in early floret meristems, we determined the differential RNA transcriptome in osmads1ko tissues as compared to wild-type tissues. These data revealed regulators of inflorescence architecture, floral organ identity including MADS-box floral homeotic factors, factors for meristem maintenance, auxin response, transport and biosynthesis as some of the important functional classes amongst the 2725 differentially expressed genes (DEGs). Integrating DEGs with OsMADS1 ChIP-seq data (prior studies from our lab) we deciphered direct vs. indirect and positive vs. negatively regulated targets of OsMADS1. These datasets reveal an enrichment for functional categories such as metabolic processes, signaling, RNA transcription and processing, hormone metabolism and protein modification. Using Bio-Tapestry plot as a tool we present a visualization of a floral stage-specific regulatory network for genes with likely functional roles in meristem specification and in organ development. Further, to examine if indirect targets regulated by OsMADS1 could be mediated through transcription factors (that are themselves direct targets), we constructed a small network with the transcription factors OSH1, OSH15 and OsYABBY1 as key nodal genes and we predicted their downstream effects. Taken together, these analyses provide examples of the complex networks that OsMADS1 controls during the process of rice floret development. In summary, we surmise that defect in phytohormone distribution in OsMADS1 knockdown florets results in irregular patterns of lateral organ primordia emergence. In addition, the derangements in the developmentally stage specific expression of floral meristems identity and organ identity genes culminates in miss-specified and irregularly patterned abnormal organs in Osmads1 florets. Thus, our study highlights the versatility of OsMADS1 in regulating components of hormone signaling and response, and its effects on various floral development regulators results in the formation of a single determinate floret on the spikelet. References: Khanday I, Yadav S.R, and Vijayraghavan U. (2013). Plant Physiol 161, 1970–1983. van Mourik S , Kaufmann K, van Dijk AD, Angenent G.C, Merks R.M.H, Molenaar J. (2012). PLOS One 1, e28762 Reinhardt D, Pesce E, Stieger P, Mandel T, Baltensperger K, Bennett M, Traas J, Friml J and Kuhlemeier C. (2003). Nature 426, 255-260 Su Y, Liu Y and Zhang X. (2011) Mol Plant 4, 616–625

Rice have highly derived florets borne on a short branch called ‘spikelet’ comprised of a pair of rudimentary glumes and sterile lemma (empty glumes) that subtends a single fertile floret. The floral organs consist of a pair of lodicules, six stamens and a central carpel that are enclosed by a pair of bract-like organs, called lemma and palea. A progressive reprogramming of meristem identity during the floral development of flowers, on branches on the inflorescence, is correlated with changes in transcriptional status of regulatory genes that execute cascades of distinct developmental events. On the other hand phytohormones such as auxin and cytokinin that are critical in predetermining the sites of new organ primordia emergence and in maintaining the size or populations of meristems. Molecular genetic analyses of mutants have expanded the repository of genes regulating floral organ specification and identity, yet the finer mechanistic details on process downstream to these regulatory genes and co-ordination with phytohormone signalling pathways needs further investigation. One aim of the study presented in this thesis is to develop a tool that would display of spatial description of dynamic auxin or cytokinin accumulation in developing rice inflorescence and floral meristems and to evaluate auxin distribution defects of OsMADS1-RNAi florets using this tool. Additionally, we aim to understand the regulatory effects on OsMADS1 on candidate floral organ and meristem fate determining genes during two temporal phases of flower development to decipher other regulatory cascades controlled by OsMADS1. Spatial distribution profile of phytohormones in young and developing meristems of rice Cytokinin promotes meristem activity (Su et al., 2011) while auxin accumulation, directed by auxin efflux transport PIN proteins predicts sites of new organ initiation (Reinhardt et al., 2003; van Mourik et al., 2012). Previous studies in the lab deciphered that OsMADS1 exerts positive regulatory effects on genes in auxin pathways and repressive effects on cytokinin signaling and biosynthetic genes (Khanday et al., 2013). Thus, the need for a reliable system to understand auxin and cytokinin activity in live inflorescence and floral meristems of rice motivated us to raise promoter: reporter tools to map the spatial and temporal phytohormone distribution. Confocal live imaging conditions in primary roots of IR4DR-GFP and DR5-CyPet lines was performed and responsiveness of the DR5 elements to auxin was authenticated. Auxin maxima were distinctly seen in the epidermal and sub-epidermal cells of inflorescence branch primordia anlagen and apices of newly emerged branch primordia. As floral organs were being initiated, on the floret meristem, we discerned the sequential appearance of auxin accumulation at sites of organ primordia while apices of early floral meristems (FM) showed low auxin content. We clearly detect canalization of auxin streams marking regions of vascular inception. Using this live imaging system we probed auxin patterns and levels in malformed and indeterminate OsMADS1-RNAi florets and we observed a significant reduction in the levels of auxin. Two oppositely positioned peaks of auxin were noted in the persistent FM of OsMADS1-RNAi florets, a pattern similar to auxin dynamics at sites of rudimentary glume primordia on the wild-type (WT) spikelet meristem. These studies were followed up with immunohistochemistry (IHC) on fixed tissues for “PIN” transport proteins that suggest PIN convergence towards organ initiation sites, regions where auxin accumulation was clearly visualized by the IR4DR5-GFP and DR5-CyPet reporters. IHC experiments that detected GFP, in fixed tissues of TCSn-mGFP ER (WT) and TCSn-mGFP ER;OsMADS1-RNAi (OsMADS1-RNAi) inflorescence and florets showed an ectopic increase in the domain of cells with cytokinin response in OsMADS1-RNAi florets, compared to that of WT. Intriguingly, cytokinin responsive cells persisted in the central FM of OsMADS1-RNAi florets that might partially account for some of the FM indeterminacy defects seen in these florets. A correlative observation of these different imaging data hint at some exclusive patterns of the IR4DR5/DR5 and TCSn reporters that in turn lead us to speculate that a cross talk between auxin and cytokinin distribution may contribute to the precise phyllotaxy of lateral organs in rice inflorescence. Studies on novel targets of OsMADS1 in floral organ identity and meristem determinacy Loss of OsMADS1 function results in rice florets with miss specified floral organs and an indeterminate carpel produces new abnormal florets. Despite having several mutants in OsMADS1, mechanisms of how OsMADS1 regulates meristem maintenance and termination is not well understood. Global expression profile in OsMADS1-RNAi vs. WT tissues encompassing a wide range of developing florets (0.2 to 2cm panicles), gave an overview of OsMADS1 functions in many aspects of floret development. Here, a gene-targeted knockout of OsMADS1 named - osmads1ko (generated in a collaborative study) was characterized and found to display extreme defects in floral organs and an indeterminate FM. Strikingly, in addition to loss of determinacy, FM reverts to a prior developmental fate of inflorescence on whose new rachis are leaf-like malformed florets. We suggest these phenotypes reflect the null phenotype of OsMADS1 and its role in meristem fate maintenance. We tested gene expression levels for some proven targets of OsMADS1 (Khanday et al., 2013) and utilized panicles in two developmental phases- young early FMs (panicles of 0.2 to 0.5 cm) and older florets with organ differentiation (panicles of 0.5 to 1cm). We observed temporally different effects on the regulation of OsMADS34 that together with histology of young osmads1ko inflorescences suggest that the mutant is impeded for spikelet to floral meristem transition. In addition, OsMADS1 had a positive regulatory effect on genes implicated for lemma and palea organ identity such as OsIDS1, OsDH1, OsYABBY1, OsMADS15, OsMADS32, OsDP1 and OsSPL16 in both young and old panicles while OsIG1 was negatively regulated in both phases of development. MADS-box genes important for carpel and ovule development - OsMADS13 and OsMADS58 were had significantly reduced expression in florets undergoing organ differentiation. OsMADS1 positively regulated several other non MADS-box developmental genes - OsSPT, OsHEC2 and OsULT1, whose Arabidopsis homologs control carpel development and FM determinacy. These genes are de-regulated in later stages of osmads1ko floret development and are unaffected in younger panicles. Finally, OsMADS1 continually activated meristem maintenance genes - OsBAM2-like and OsMADS6 while the activation of OSH1 in early floral meristems was later altered to a repressive effect in developing florets. Perhaps such dynamic temporal effects on meristem genes are instrumental in the timely termination of the floral meristem after floral organ differentiation. More importantly, we show that regulation of many of these genes is directly affected by OsMADS1, through our studies on expression levels before and after chemical induction of OsMADS1-GR protein in amiRNAOsMADS1 florets. Further, some key downstream targets were re-affirmed by studying expression status in transgenic lines, with the OsMADS1-EAR repressive protein variant. These results provide new insights into the developmentally phased roles of OsMADS1 on floral meristem regulators and determinants of organ identity to form a determinate rice floret. Gene networks regulated by OsMADS1 during early flower development To identify global targets in early floret meristems, we determined the differential RNA transcriptome in osmads1ko tissues as compared to wild-type tissues. These data revealed regulators of inflorescence architecture, floral organ identity including MADS-box floral homeotic factors, factors for meristem maintenance, auxin response, transport and biosynthesis as some of the important functional classes amongst the 2725 differentially expressed genes (DEGs). Integrating DEGs with OsMADS1 ChIP-seq data (prior studies from our lab) we deciphered direct vs. indirect and positive vs. negatively regulated targets of OsMADS1. These datasets reveal an enrichment for functional categories such as metabolic processes, signaling, RNA transcription and processing, hormone metabolism and protein modification. Using Bio-Tapestry plot as a tool we present a visualization of a floral stage-specific regulatory network for genes with likely functional roles in meristem specification and in organ development. Further, to examine if indirect targets regulated by OsMADS1 could be mediated through transcription factors (that are themselves direct targets), we constructed a small network with the transcription factors OSH1, OSH15 and OsYABBY1 as key nodal genes and we predicted their downstream effects. Taken together, these analyses provide examples of the complex networks that OsMADS1 controls during the process of rice floret development. In summary, we surmise that defect in phytohormone distribution in OsMADS1 knockdown florets results in irregular patterns of lateral organ primordia emergence. In addition, the derangements in the developmentally stage specific expression of floral meristems identity and organ identity genes culminates in miss-specified and irregularly patterned abnormal organs in Osmads1 florets. Thus, our study highlights the versatility of OsMADS1 in regulating components of hormone signaling and response, and its effects on various floral development regulators results in the formation of a single determinate floret on the spikelet. References: Khanday I, Yadav S.R, and Vijayraghavan U. (2013). Plant Physiol 161, 1970–1983. van Mourik S , Kaufmann K, van Dijk AD, Angenent G.C, Merks R.M.H, Molenaar J. (2012). PLOS One 1, e28762 Reinhardt D, Pesce E, Stieger P, Mandel T, Baltensperger K, Bennett M, Traas J, Friml J and Kuhlemeier C. (2003). Nature 426, 255-260 Su Y, Liu Y and Zhang X. (2011) Mol Plant 4, 616–625

In angiosperms, specialized reproductive structures are borne in flowers to ensure their reproductive success. After the vegetative growth, plants undergo reproductive phase change to produce flowers. Floral meristems (FMs) are generated on the flanks of inflorescence and groups of specialized stem cells in the FM differentiate into four whorls of organs of a flower. In dicots, floral meristem successively gives rise to sepals, petals, stamens and carpels; after which it terminates. The fate of organs formed on FM is under the control of genetic regulators, key among which are members of MADS box transcription factor family. Their individual and combined act confers distinct identities to floral organs. Grass flowers are highly modified in structure. Rice flower, a model for grasses, is borne on a short branch called spikelet and they together from the basic structural units of the rice infloresences known as panicle. The outer whorl organs of a grass floret are bract-like structures known as lemma and palea to dicot sepals is highly dibated (see Chapter 1). In grass florets, petal homologs are a pair of highly reduced, fleshy bracts known as lodicules, while stamen and carpel homologs occupy the same position and share the same functions as their dicot counterparts. Aside from these distinct outer whorl organs, the florets are subtended by two pairs of bracts known as empty glumes and rudimentary glumes. The genetic regulators that control their unique identities and those that perform conserved functions are very intriguing and central questions in plant developmental biology. Using various contemporary and complementary technologies, we have analysed the molecular functions and downstream pathways of a MADS box transcription factor, OsMADSI during the rice floret meristem specification and organ development. Further by reverse genetics and overexpression studies, we have also functionally characterized two target genes of OsMADSI, OsETTINI and OsETTINI2 to understand their roles downstream to OsMADSI during the rice floret development.

In angiosperms, specialized reproductive structures are borne in flowers to ensure their reproductive success. After the vegetative growth, plants undergo reproductive phase change to produce flowers. Floral meristems (FMs) are generated on the flanks of inflorescence and groups of specialized stem cells in the FM differentiate into four whorls of organs of a flower. In dicots, floral meristem successively gives rise to sepals, petals, stamens and carpels; after which it terminates. The fate of organs formed on FM is under the control of genetic regulators, key among which are members of MADS box transcription factor family. Their individual and combined act confers distinct identities to floral organs. Grass flowers are highly modified in structure. Rice flower, a model for grasses, is borne on a short branch called spikelet and they together from the basic structural units of the rice infloresences known as panicle. The outer whorl organs of a grass floret are bract-like structures known as lemma and palea to dicot sepals is highly dibated (see Chapter 1). In grass florets, petal homologs are a pair of highly reduced, fleshy bracts known as lodicules, while stamen and carpel homologs occupy the same position and share the same functions as their dicot counterparts. Aside from these distinct outer whorl organs, the florets are subtended by two pairs of bracts known as empty glumes and rudimentary glumes. The genetic regulators that control their unique identities and those that perform conserved functions are very intriguing and central questions in plant developmental biology. Using various contemporary and complementary technologies, we have analysed the molecular functions and downstream pathways of a MADS box transcription factor, OsMADSI during the rice floret meristem specification and organ development. Further by reverse genetics and overexpression studies, we have also functionally characterized two target genes of OsMADSI, OsETTINI and OsETTINI2 to understand their roles downstream to OsMADSI during the rice floret development.

Durante il mio dottorato di ricerca, abbiamo dimostrato, attraverso l’alterazione del gene biosintetico della prolina P5CS1, che la modulazione della prolina influenza, in piante di Arabidopsis, la transizione fiorale e la proliferazione delle gemme ascellari. Inoltre abbiamo investigato il ruolo relativo del gene P5CS2, un gene omologo al P5CS1 e abbiamo scoperto un ruolo non ridondante di questi due geni nell’embriogenesi, mentre un ruolo ridondante nella transizione fiorale. Durante l’ultimo anno abbiamo cercato di mettere in relazione la prolina con una o alcune delle diverse vie di segnalazione fiorale. Dati preliminari di RT-PCR relativa e quantitativa hanno rivelato un significativo aumento dei livelli di espressione del gene FLC, suggerendo un interazione della prolina con il pathway autonomo.
During my PhD we showed, by the alteration of proline biosynthetic gene P5CS1, that the modulation of proline affects, in Arabidopsis plants, the flower transition and axillary bud proliferation. Morover we investigated the role and the relative contribution in flowering and development of P5CS2, a gene homolog to P5CS1 and we discovered a non redundant role of these two genes in embryo development while a their redundant role in flower transition. In the last year of my PhD thesis we are investigated the floral pathway proline is involved in. Preliminary data, from relative and quantitative RT-PCR, revealed a significative upregulation of the floral integrator gene FLC, strongly suggesting that proline interacts with the autonomous pathway.

Le microbiote intestinal équin joue un rôle important dans le maintien de la santé de l'hôte. Le microbiote intestinal est composé de nombreux micro-organismes tels que les bactéries, les virus, les champignons et les archées. Cependant, la majorité de ces cellules microbiennes sont bactériennes, et par conséquent, de nombreuses études, y compris la présente, se concentrent sur l'exploration des communautés bactériennes dans l'intestin. Un déséquilibre du microbiote intestinal, appelé dysbiose, a été observé dans plusieurs conditions, telles que la colite, après l’administration d'antibiotiques ou la modification du régime alimentaire. La restauration du microbiote peut être effectuée par la transplantation de microbiote fécal (FMT). Des études utilisant les recommandations actuelles pour la FMT ont montré une récupération clinique chez les chevaux souffrant de diarrhée, mais le microbiote reste largement inchangé après la FMT et aucune étude randomisée avec contrôle placébo n'a été réalisée. Les hypothèses de ce projet étaient que le traitement avec une FMT concentrée corrigera la dysbiose plus rapidement qu’une FMT conventionnelle et le véhicule, et que le microbiote intestinal des chevaux traités avec une FMT concentrée ressemblera au microbiote intestinal du cheval donneur. L'objectif de ce projet était de développer un protocole pour améliorer la FMT chez les chevaux, en augmentant la concentration de bactéries présentes dans les selles du donneur par centrifugation, et de le tester chez les chevaux atteints de dysbiose intestinale induite par les antibiotiques. L'antibiotique triméthoprime sulfadiazine (TMS) a été administré à neuf chevaux pour induire une dysbiose intestinale. Les chevaux ont été séparés en trois groupes: les chevaux recevant une FMT concentrée (cFMT, n = 3); les chevaux recevant la FMT fraîche (fFMT), selon les recommandations actuelles (n = 3); et les chevaux recevant un véhicule (VEH) avec 10% de glycérol dans une solution saline à 0,9% (n=3). Des échantillons fécaux ont été prélevés avant et après l'administration du TMS, ainsi qu'avant, pendant et après la transplantation. Le séquençage a été réalisé à l'aide de la plateforme Illumina MiSeq et les données analysées à l'aide du logiciel Mothur. Tel qu’attendu, l'antibiotique TMS a significativement diminué la richesse microbienne chez tous les chevaux. De manière inattendue, la composition des suspensions fécales des donneurs cFMT et fFMT était significativement différente de la composition de base des receveurs cFMT et fFMT, respectivement. La composition du microbiote des chevaux ayant reçu une transplantation fécale (concentrée ou non) était significativement différente après la transplantation, alors que ce n’était pas le cas chez les chevaux ayant reçu le véhicule. En outre, l’abondance relative de Escherichia était significativement plus élevée dans les suspensions fécales du donneur cFMT par rapport aux suspensions fécales du donneur fFMT. Les principales limites de ce projet sont la petite taille des groupes et l'exposition des selles des donneurs à l'oxygène et à la congélation-décongélation. En outre, le modèle de dysbiose peut ne pas être optimal pour tester l'efficacité de la FMT, et des études réalisant la FMT chez les chevaux souffrant de diarrhée sont nécessaire. Cette étude a contribué à la recherche de nouvelles approches pour améliorer la FMT chez les chevaux. Le faible effet mesuré avec les deux protocoles de FMT et l’augmentation de Escherichia démontre que les protocoles actuels doivent être optimisés avant de pouvoir recommander la FMT pour traiter et prévenir la dysbiose chez les chevaux.
The equine gut microbiota plays an important role in maintaining the health of the host. The gut microbiota is composed of many microorganisms such as bacteria, viruses, fungi, and archaea. However, the majority of these microbial cells are bacterial cells, and consequently, many studies, including the present one, focus on exploring bacterial communities in the gut. An imbalance of the gut microbiota, termed dysbiosis, has been observed in several conditions such as colitis, colic, after antibiotic administration, or diet modification. Restoration of the gut to a healthy state can be performed through fecal microbiota transplantation (FMT). Studies using current recommendations for FMT have shown clinical recovery in horses with diarrhea, but the microbiota remains largely unchanged after FMT and no controlled studies have been performed. The hypotheses of this project were that treatment with concentrated FMT will correct dysbiosis faster than conventional FMT and the vehicle, and that the gut microbiota of horses treated with concentrated FMT will resemble the gut microbiota of the donor. The objective of this project was to develop an improved protocol for FMT in horses, by increasing the concentration of bacteria found in the donor stool using centrifugation, and to test it in horses with antibiotic-induced intestinal dysbiosis. The antibiotic trimethoprim sulfadiazine (TMS) was administered to nine horses to induce intestinal dysbiosis. Horses were separated into three groups: horses receiving concentrated FMT (cFMT) (n=3); horses receiving fresh FMT (fFMT), as per current recommendations (n=3); horses receiving a vehicle (VEH) with 10% glycerol in 0.9% saline (n=3). Fecal samples were collected before and after antibiotic administration, as well as before, during, and after transplantation. Sequencing was performed using the Illumina MiSeq platform and data analysed using the software Mothur. As expected, the antibiotic TMS significantly decreased the richness in all horses (P < 0.05). Unexpectedly, the membership of the cFMT and fFMT donor fecal suspensions was significantly different from cFMT and fFMT recipients’ baseline membership, respectively. The membership of the cFMT and fFMT recipient horses was significantly different after transplantation, while the vehicle recipients were not. In addition, the Escherichia genus was found in significantly higher relative abundances in the cFMT donor fecal suspensions when compared to the fFMT donor fecal suspensions. The main limitations of this study are the small sample size and exposure of cFMT donor stool to oxygen and freeze-thawing. In addition, the dysbiosis model may not be optimal to test the efficacy of FMT, and studies performing FMT in horses with diarrhea are warranted. This study contributed to the search for novel approaches to improve FMT in horses. The weak effect of both FMT protocols on the gut microbiota and the increase in Escherichia suggest that further clinical studies are needed before FMT can be recommended to treat and prevent dysbiosis in horses.