Extracellular diffusion barriers are central to plant survival and stress resistance: Large parts of the terrestrial surface of our planet have been successfully colonised by plants. The exit of photo-synthetic organisms from their original, aqueous environment and their transition to the fully dry-land adapted life cycles seen in extant higher plants required numerous evolutionary innovations. Key among these innovations was the development of effective, tissue-spanning diffusion barriers that prevent loss of water and allow maintenance of steep concentration gradients between the organism and its environment. Centrally important diffusion barriers in extant plants are the epidermal cuticle of aerial tissues, the Casparian strip and suberin lamellae of the root endodermis and the sporopollenin layer of pollen coats. These barriers often cover very large surface areas and even small defects in them can lead to strong functional impairments, leading to chronic loss of water, nutrients or entry of pathogens. How plants are able to ensure the effective sealing of their diffusion barriers and monitor their integrity is not understood and – to our knowledge –few attempts have been made to address this question. Recently, work on Arabidopsis mutants with impaired endodermal diffusion barrier has defined the outlines of a signalling pathway whose role might be to allow diffusion barrier surveillance during Casparian strip formation. Intriguingly, central components of this pathway have been implicated not only in Casparian strip formation, but also in forming an intact embryonic cuticle and - based on our recent, unpublished observations – might also have a role in the formation of pollen coats. Does a common system of barrier surveillance exist that ensures integrity of the major plant diffusion barriers? These findings lead us to speculate that a common signalling module might exist that is repeatedly employed by the plant in very different circumstances in order to ensure the integrity of its different, extracellular diffusion barriers and to stimulate their formation. Here, we propose a number of coordinated experiments that will explore and substantiate this idea and establish the extent to which identical or homologous components are employed in these very different developmental processes. Our aim is to work out whether a common regulatory logic underlies these very diverse barrier formation events. The great fundamental interest of this proposal resides in the opportunity to contextualise a plant signalling pathway and study its modularity and the mechanism underlying its functional adaptation to different developmental circumstances. A more applied interest of our proposal lies in the identification of agonists and antagonists that regulate crucially important diffusion barrier formation in plants and might open avenues for the inducible alteration of these barriers in order to enhance plant resistance to diverse environmental insults.

The Arabidopsis zygotic embryo, like that of almost all angiosperms, develops buried deep within the seed, nourished and protected by the maternally derived seed-coat and a second zygotic tissue, the endosperm. Although the endosperm plays important roles in ferrying nutrients from the maternal tissues to the developing embryo, and in determining the final size of the seed, recent work from our laboratory suggests that its presence may also generate developmental problems, which have had to be surmounted during angiosperm evolution. The angiosperm endosperm is thought to be a sexualized homologue of the gymnosperm female gametophyte, which also serves a nutritive role, but which develops before fertilization. In angiosperms seeds the simultaneous and closely juxtaposed development of the embryo and endosperm following fertilization may make it difficult for the embryo to establish a well-defined boundary, in the form of a properly cuticularized epidermal cell wall. This hypothesis is supported by recent published and unpublished data from our laboratory showing that a signalling pathway involving an endosperm specific protease (ALE1), and two embryo specific receptor kinases (GSO1 and GSO2) is necessary for the formation of a functional embryonic cuticle, and indeed for the stabilization of embryonic epidermal cell fate. This signalling pathway is not required for cuticle formation during post-germination growth, indicating that it is necessary only in the context of zygotic embryo development. Our recent transcriptional analyses of mutants defective in this signalling pathway, have provided tantalizing and novel clues that at least part of this cuticle reinforcing signalling pathway is closely allied to signalling pathways involved in innate immune responses, and particularly to systemic acquired resistance mechanisms in plants. The hypothesis that a form of “autoimmune” response is necessary for the formation of the boundary between the embryo and the endosperm in plants is entirely novel, and is basis for the proposed project. In this work we will develop novel tools allowing biochemical characterization of the seedling cuticle in Arabidopsis, a structure which remains almost entirely unstudied despite its important role in seedling survival. These tools will subsequently be applied to the analysis of mutant lines studied in this project. Using transcriptomic analysis, combined with spatial analysis of gene expression, we will determine where, both spatially and genetically, the ALE1-dependent innate immune response fits into the cuticle reinforcing signalling pathway which we have established. A candidate gene approach, combined with the analysis of a novel protein which we have recently shown is essential for the ALE1/GSO1/GSO2 signalling pathway, will permit the identification and characterization of further pathway components. Finally, we will establish the identities and biochemical functions of the downstream targets of the ALE1/GSO1/GSO2 signalling pathway, in order to understand more about the mechanisms underlying the biogenesis of the embryonic cuticle. In summary, our multidisciplinary approach will allow us not only to dissect in detail an entirely new and very striking example of how immune responses may have been harnessed by plants in order to fulfil developmental roles during the evolution of increasingly complex organs, but also provide novel and much needed understanding both of the composition of the embryonic cuticle, and of the effectors necessary for its biogenesis.

Les hormones contrôlent le développement et la physiologie des plantes toute au long de leur vie. Des combinaisons d’hormones intègrent les signaux environnementaux au programme génétique pour réguler de façon coordonnée la biologie de la plante. De nombreuses interactions protéine-protéine entre des effecteurs de voies de signalisation hormonale différentes ont été identifiées, fournissant un mécanisme putatif pour coupler l'activité de deux ou plusieurs voies hormonales. Comment ces interactions protéine-protéine modulent le traitement simultané de plusieurs signaux hormonaux est une question qui n'a pas été explorée jusqu'à présent. Nous aborderons cette question dans ce projet en nous concentrant sur auxine, cytokinines et gibbérellines, trois hormones végétales majeures. Partant de travaux publiés et non publiés où nous avons reconstruit des voies de signalisation d'hormones végétales fonctionnelles en cellules de mammifères, nous utiliserons une approche de biologie synthétique pour explorer quantitativement comment les interactions protéine-protéine connues entre effecteurs de différentes voies de signalisation régulent l'activité transcriptionnelle en réponse à de multiples hormones. Nous effectuerons également une analyse exhaustive des interactions protéine-protéine inter-voies et analyserons la signification fonctionnelle d'une sélection de ces interactions. Nous génèrerons des mutations pour affaiblir ou renforcer les interactions identifiées, et pour créer de nouvelles interactions entre effecteurs, ceci afin de perturber le couplage entre voies hormonales. Nous testerons ensuite in planta les connaissances générées en cellules de mammifères pour analyser comment les interactions protéine-protéine régulent le traitement conjoint de multiples signaux hormonaux, et impactent la dynamique du développement des plantes. Notre analyse fournira une vision unique de la façon dont les plantes traitent des signaux multiples pour coordonner leur développement.

Seeds play a key role in plant dissemination and evolution, as well as in agriculture and food production. Understanding the core mechanisms underpinning seed development and the accumulation of storage compounds therefore constitutes a fundamental goal of plant biology. The transitions between the successive phases of seed development (i.e embryogenesis, maturation, and germination) and the successful completion of maturation are controlled by the LAFL transcriptional regulators, which are well conserved among angiosperms, namely LEC1, ABI3, FUS3, and LEC2 in Arabidopsis. These regulators can form different protein complexes that control the expression of both structural and regulatory genes involved in various pathways, triggering and maintaining embryo development and maturation, while repressing seed germination. Hence, the LAFL network controls seed development, quality and yield. Consistent with these key functions, the expression of the LAFL gene is strictly controlled by numerous transcriptional and chromatin-based mechanisms involving, in particular, the PRC complexes. PRC2 is an evolutionary conserved histone H3 lysine 27 methyltransferase (H3K27me3) with a key role in terminating seed development. Moreover, it has been recently hypothesized that the LAFL could themselves modify the chromatin landscape during seed development, acting as “pioneer factors”, by recruiting different chromatin regulators that remain to be characterized. Despite their critical role, we still ignore how the LAFL and chromatin modifiers interact to control both, phase transitions and seed maturation. We believe that the chromatin-based regulations of LAFL expression, the pleiotropic effects of LAFL mutations, and the difficulty of accessing specific tissues inside the seed (i.e. the embryo surrounded by the endosperm and the testa) have prevented deciphering the mechanisms involved and hidden the fact that the LAFL may themselves impinge on chromatin state. Another hurdle resides in the small size of the seed and the tricky access to the embryonic tissues within seeds. To discover possible yet unknown players and elucidate the molecular mechanisms involved, it is now crucial to apply novel approaches to monitor chromatin and transcriptome dynamics within the seed in a tissue-specific manner. Moreover, to unravel the epistatic relationships between the LAFL and chromatin regulators, we need to develop new inducible alleles allowing to precisely activate or repress these regulators or/and to uncouple LAFL expression from chromatin regulations. Therefore, the ambition of the project is to develop original approaches and tools to identify novel regulators and elucidate how the LAFL interact with chromatin-based mechanisms to control proper seed development and maturation. The short-term outputs of this project will be both scientific and technical. We will indeed need to use cutting-edge techniques recently adapted in our labs, generate original inducible, and/or tissue-specific alleles, and will edit the epigenome of the LAFL using a new CRISPR/dCas9 approach. Using Arabidopsis will be essential and an asset to perform all these molecular and genetic analyses in the frame of the project. The main scientific outcome of this project will consist in knowledge on the genetic and molecular mechanisms that control seed development and maturation. Beyond seeds, the project will contribute to decipher fundamental mechanisms of phase transitions during plant development and will exemplify if and/or to which extent chromatin modifications and transcriptional regulation are impinging on each other. Last, because the LAFL and chromatin regulators are conserved among angiosperms, including crop species, this project may deeply impact agriculture on the long term. In conclusion, this project may therefore provide major scientific, societal and environmental benefits

Significant loss in agricultural products is caused by increasingly variable climate and vulnerability of crops to pathogen attacks or extreme environmental conditions. In addition, the agro-industry and the final market often require highly homogeneous crops. This raises the issue of resilience: How to produce robust and homogeneous crops? Here we address the corresponding key question in developmental biology: How do organs form with consistent sizes and shapes in the face of internal and external perturbations? We have assembled an interdisciplinary team to resolve the apparent dichotomy between highly variable cells and robust organs. Previous research has focused on mutants and conditions that affect the global size and shape of organs, enabling the discovery of a large number of regulators. However, most analyses have considered only average cell behaviours, overlooking local heterogeneity and stochastic variation. Here we adopt an orthogonal approach: We screen for mutants with enhanced variability in organ size or shape. Plants produce many flowers, allowing us to detect variability within an individual organism. We have chosen the abaxial sepal, the outermost leaf-like floral organ, for its accessibility for imaging and micromanipulation: the whole process of organogenesis from a primordium to the mature organ can be observed with a confocal microscope. Variability of sepal size and shape can be assessed within an individual plant. Based on our previous work, we propose that tissue mechanics and mechanosensing are key regulators of organ variability, because morphogenesis directly depends on the mechanical control of growth and because mechanical stress is largely prescribed by organ size and shape. We will test this hypothesis in Arabidopsis thaliana, chosen for the availability of genetic and molecular resources. Our main objectives are (i) to analyse spatial and temporal variability in sepal morphogenesis at all scales, considering growth and its effectors, (ii) to identify and characterise genes that regulate variability, by performing a directed screen among mutants affecting the cell wall, plant hydraulics, and mechanosensing, and (iii) to integrate the corresponding mechanisms in mechanical models of growth and test these models experimentally, notably by local mechanical and genetic perturbations to sepals. Overall, our project addresses a central question in developmental biology and is relevant to food security, especially in the context of increasing climatic fluctuations. On the one hand, we will isolate and characterise molecular regulators of the robustness of flower organs. We will overexpress these regulators in plant lines and examine if these lines have more homogeneous organs (e.g. homogenous siliques). On the other hand, we will develop biophysical measurements in the context of plant sciences, which may apply to a broad range of R&D problems, from fruit firmness to biomaterial properties or biomechanical resilience.