Final Report Summary - SEEDNET (REGULATORY GENE NETWORKS IN SEED COAT DEVELOPMENT) Angiosperm seeds are a unique biological model made of three genetically different tissues: the embryo which initiates the next sporophytic generation, the endosperm which has nourishing functions and the surrounding seed coat which interfaces with the environment. Briefly, seeds of the model angiosperm Arabidopsis thaliana develops from the ovule after a double fertilization event that leads to the formation of embryo and endosperm. The ovule integuments, that surround and protect the female gametophyte, do not take part in the fertilization process but give rise to the seed coat soon after. Cells in the five layers of the seed coat undergo a rapid phase of growth through division and expansion, and follow different fates. The endothelium, the innermost cell layer of the seed coat, lies in a critical position at the boundary of endosperm and seed coat and plays an essential role in the crosstalk among seed tissues. It also synthesizes proanthocyanidins (PA), flavonoid compounds that have a general protective function and impart the characteristic brown color to A. thaliana seeds. Despite the important role of the endothelium in seed formation and quality, we have a limited understanding of its development. The project aimed at characterizing endothelium development and its coordination with the surrounding tissues in A. thaliana seeds at the morphological and transcriptional level. In a genome-wide approach, we profiled the endothelium transcriptome through the INTACT method that allows affinity-based isolation of nuclei from individual cell types and mRNA profiling. Furthermore, we characterized the cell patterning of the endothelium and neighboring maternal tissues in wild-type and mutant seeds through tissue three-dimensional reconstruction imaging techniques. This approach allowed us to identify the presence of a sixth integument cell layer, previously uncharacterized. Finally, we assigned new functions to a handful of transcription factors that regulate seed coat development and characterized their downstream target genes. Most emphasis has been given to the study of the TRANSPARENT TESTA 16 (TT16) MADS box transcription, a master regulator of endothelium development. We showed that TT16 regulates endothelium cell orientation and together with SEEDSTICK, another MADS box transcription factor, its patterning of periclinal cell divisions. Furthermore, we characterized the role of TT16 and its paralog GORDITA in the perception of the fertilization signals that coordinate the degeneration of the nucellus maternal tissue and the production of PAs in the endothelium. Finally, we discovered that the sub-epidermal integument cell layer, which originates form periclinal cell divisions of the endothelium under the control of TT16, is insensitive to the fertilization signals that regulate all other integument cell layers. Overall, the SEEDNET project has led the foundations for a model of maternal tissue development in A. thaliana and might open new ways to bioengineering both seed development and the flavonoid biosynthetic pathway in angiosperm seeds. The molecular pathways underlying the development of the endothelium that we discovered in A. thaliana are now being translated to other species. Last but not least, the grant has benefited the applicant’s reintegration as he is now a permanent researcher at the host institution.
