Apomixis defines a plant reproductive strategy that, unlike sexual reproduction, permits the
inheritance of the maternal genome without genetic recombination and syngamy. Earliest
phenotypical symptoms of the aposporous developmental program in ovules are the avoidance of
meiosis (i.e., apomeiosis) and the differentiation of functional unreduced embryo sacs from somatic
cells of the nucellus (i.e., aposporous initials). Apospory deviates from sexuality as, in this latter
case, the commitment to develop an embryo sac is strictly restricted to the reduced functional
megaspore (FM) and failure of the meiotic program is typically not accompanied by the initiation of
embryo sac development from cell lineages of the ovule other than the FM.
Our research goal is a better understanding on gene expression changes accompanying the
onset of aposporous apomixis in the ovule of the model species H. perforatum L. To this purpose,
gene expression analyses were performed by adopting the RNA-seq technology on Laser-Capture
Microdissected (LCM) ovule cells collected from sexual and apomictic genotypes at pre-meiotic
developmental stages. We identified 402 differentially expressed genes (DEGs) (Bonferroni p-value
64 0.05) between ovules belonging to sexual and apomictic genotypes. Among these, 97 transcripts
were only found in apomictic libraries, suggesting apomictic-specific expression. At the same time,
25 transcripts were only detected in sexual libraries. Differential expression was validated by RealTime qPCR and in-situ hybridization assay. Among identified DEGs, we found several RNAs
whose products are related to biological processes modulated in other aposporous apomictic model
species. Ontological annotation revealed an enrichment of the following biological processes in
apomictic ovules: RNA binding, RNA splicing and RNA-directed DNA polymerase activity, this
latter being associated to putative non-LTR retroelements. The massive expression of TEs in
apomictic ovules suggested that DNA methylation is compromised in these cells. To address this
question, we investigated the promoter and gene body DNA methylation level of a subset of DEGs
by chop-PCR assays. Gene body methylation level of DEGs annotated as putative non-LTR
retroelements supports the idea that transcriptomic changes for these genes might be epigenetically
controlled. Furthermore, several genes involved in auxin and cytokinin (CK) homeostasis and
signalling were found differentially expressed, implying that apomictic ovules might be subjected to
alternative hormonal interplays. This let us hypothesise that hormonal response and DNA
methylation might be connected to the transcriptional changes observed in apomictic ovules. To
address this question, gene expression and promoter methylation studies were performed on flowers
treated with synthetic CK and its antagonist PI-55. Gene expression and DNA methylation data will
be presented and critically discussed in the frame of ovule and gamete development. Overall, our
data suggest that phenotypic expression of early events of aposporous apomixis in H. perforatum is
concomitant with the modulation of key genes involved in hormonal homeostasis, DNA
methylation and cell cycle progression
