Epi-genetic regulation throughout zygotic embryogenesis in European chestnut (Castanea sativa Miller)

Resumo

European chestnut (Castanea sativa Miller) is a multipurpose species distributed within the Mediterranean area and extensive areas in France. It possesses paramount value due to the quality of its wood and nuts. Many cultivars highly appreciated have been obtained by traditional breeding in the past by crosses between individuals regarding production of nuts in terms of quality and yield, but cloning due to the recalcitrant character of adult individuals represents a bottleneck for vegetative reproduction. Chestnut usually produces monoembrionic nuts due to the development of a unique ovule, dominant, from all the ovules contained in a given ovary; those which do not enter in the embryogenic program are called companion ovules and abort in parallel with the development of the dominant ovule. On the contrary, polyembryonic seeds are generated when two or more ovules develop a zygotic embryo, which diminishes their commercial value. On the basis of the expounded knowledge, the aim of this thesis is to get insight the understanding of the possible mechanisms affecting the establishment of a dominant ovule that gives rise to the mature embryo and the associated cell death of companion ovules by the study of physiological and (epi)genetic variables and analyze their possible interactions throughout chestnut sexual reproduction as well as evaluate the best explants produced during the consecutive stages of development for the induction of somatic embryogenesis from flowering to the establishment of the mature embryo. Our results show that specific levels of selected hormones, epigenetic marks and gene expression are necessary throughout in order to achieve the mature seed. The destiny of the ovules within an ovary has been discovered to be fixed in the early step of pollination given that the provenance of the pollen tags the ovules for abortion if autopollination takes place whereas cross-pollination triggers the normal zygotic embryo development and companion ovules death. This bifurcation in ovules¿ fate seems to be related with specific dynamics in ABA and JA contents. Fertilization of the ovule induces a transient demethylation status and specific ratios of CKs and IAA, known for their vital role in the determination of the polarity of the embryo while posterior embryo development and also the abortion of companion ovules concurs with hypermethylation of the DNA in the tissues until reaching maturing and death. In addition, companion ovules degeneration process coincides with increases of the epigenetic marks associated with genetic silencing status of the chromatin H3K9me3 and H3K27me3. Maturation of the embryo implies morphological changes at the tissue level that are reflected in different dynamics and distribution of epigenetic marks and hormones. A strong increase of H4ac marks the beginning of maturation together with dramatic changes in the ratio ABA:GA4 that varies towards the end of development. In addition, differential gene expression is likely to support the quantification of hormones and epigenetic marks studied such as the relation between ABA levels respect to its repressive role in the expression of histone deacetylases. The specific levels of the studied variables at a given developmental stage has been found to be related with the observation of a developmental window leading to somatic embryogenesis induction from post-fertilization embryogenic stages until the mature embryo. From the first stimulus consistent on pollination to the establishment of the mature embryo, it can be concluded that the transition between developmental stages and the destiny of the ovules are controlled in a cross-talk fashion by the integration of external and internal cues. Such control is exerted through the orchestrated action of different physiolocial and (epi)genetic actors that interact in order to generate a global, major response reflected in the establishment of the mature embryo.