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 This hypothesis is supported by recent published and unpublished data from our laboratory showing that a signalling pathway involving an endosperm specific protease (ALE1), and two embryo specific receptor kinases (GSO1 and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.