To achieve its goal, the project relied on an interconnected portfolio of work packages, including coordination (WP1), communication (WP2) and research (WP3-to-6).
WP1 ensured transparent and effective governance and compliance with scientific and education objectives through regular coordination meetings and the organisation of three general meetings and through the set up and collect of documents for material and data transfer and ethical rules.
WP2 ensured wide visibility of the project and of its objectives and achievements by: producing a logo and two videos for presenting the project to a broad audience; setting up and maintaining a project website; publishing a on-line course on Plant Reproductive Biology; organizing three workshops, and; contributing to the organization of the IV and the V international conferences on apomixis in Rosario, Argentina (Dec 3-7, 2023) and Montpellier, France (Sept 16-19, 2025). Finally, participants contributed to 15 scientific publications and to communications (talks and posters) at national (13) and international (64) conferences.
WP3 - What is the functional role of the genomic regions specific of apomicts genomes? The structure of the genomic region responsible for apomixis in three grass species (Paspalum notatum, Paspalum simplex, and Eragrostis curvula) was resolved using Next-Generation Sequencing approaches and their gene content was determined. Several of them were selected as candidate genes for apomixis for further characterization. Functional analyses have been carried out in P. simplex for one of the genes (ORIGIN REPLICATION COMPLEX3), and are currently underway in Arabidopsis thaliana for several E. curvula candidate genes. Finally, epigenome characterization was performed using the MCSeEd technique in E. curvula and P. simplex, yielding gene sets exhibiting differential DNA methylation in reproductive tissues between sexual and apomictic plants.
WP4 - Are the transcriptional patterns promoting apomixis shaped transcriptionally or post-transcriptionally? This work package was mainly dedicated to the functional analysis of candidate genes for apomixis either from previous works or identified in the course of this project in conjunction with WP3 and WP5. Functional analysis was performed in grass models (rice and P. notatum) and in A. thaliana. This work has revealed mis-expression of several genes leads to phenotypes reminiscent of apomixis, therefore suggesting a key role in the emergence of apomixis in sexual plants and involved in different pathways, such as the role of miRNAs in the regulation of ARF genes (e.g. miR160/ARF10) and of genes regulating alternative splicing (e.g. BUD13 and TGS1).
WP5 - How do phytohormones interact to establish and control reproductive cell fate during plant reproduction? To understand the role of auxin and cytokinin signalling on MMC cell fate and female gametophyte development in Paspalum notatum, we generated transgenic plants carrying auxin and cytokinin fluorescent reporters for direct imaging in growing ovules and also used using mass spectrometry to quantify auxins, cytokinins and jasmonate hormones in ovules of sexual and apomictic Paspalum plants. In addition, we identified candidate genes affected in both AUX and CK pathways in A. thaliana and generated mutant lines in rice or maize using CRISPR/Cas9 genome editing.
WP6: does ovule architecture exert control over female germ line fate? Through the assembly and analysis of a collection of >1000 3D images, we obtained the first 3D cellular atlas of ovule primordia of sexual (maize and P. simplex) and apomitic (P. simplex) plants. Using these resources, we quantified cell proliferation, cell growth anisotropy and cell wall changes in growing ovules of sexual plants and apomicts. Finally, we were successful in obtaining a marker gene for germline identity in P. rufum ovules, a critical result for establishing the relationships between cell division, growth and germline differentiation in the plant ovule.
