The medicinal plant St. John\u2019s wort (Hypericum perforatum L.) is an attractive model system
for the study of aposporous apomixis. The key biological features of apomixis in this species are the
by-passing of meiosis, the differentiation of aposporous initials into embryo sacs containing
unreduced egg cells, their autonomous development in functional embryos without fertilization, and
the formation of viable endosperm either via fertilization-independent means or following
fertilization with a sperm nucleus. The aim of this research is to define gene expression changes
occurring in the nucellar cell types of the ovules primarily involved in the differentiation of the
megaspore mother cells and aposporous initials. To this end, a laser-capture microdissection
approach was adopted in combination with the RNA-seq technology in order to restrict the frame of
our investigations to a specific portion of the ovule, i.e. the nucellus, at developmental stages
preceding the differentiation of aposporous initials. Overall, our gene expression analysis identified
270 and 81 unigenes that were found significantly up- and down-regulated between ovules collected
from sexual and apomictic accessions. Ontological annotation of differentially expressed genes
indicated that genes up-regulated in apomictic ovules were significantly enriched in ontological
terms related to the RNA-directed DNA polymerase activity and the RNA binding. Among these
genes, several actors of the RdDM pathway were found, suggesting that the phenotypic expression
of early events of aposporous apomixis is associated to changes in de novo DNA methylation
mediated by small RNAs. Furthermore, as deregulation of single components of the sexual
developmental pathway is believed to be a trigger of the apomictic reproductive program, genes
involved in sporogenesis, gametogenesis and response to hormonal stimuli were annotated and
investigated in great detail. The expression analysis of candidate genes was performed not only by
Real-Time qPCR but also by ISH assays in order to verify the temporal and spatial expression
patterns of selected transcripts in the ovule. Finally, the activity of specific genes in relation to
embryo sac and/or embryo formation was investigated by using A. thaliana knock-out lines.
Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with
the modulation of key genes involved in the sexual reproductive pathway, hormones and other
actors likely playing a crucial role in the RNA-directed DNA methylation pathway
