Periodic Reporting for period 2 - MAD (Mechanisms of Apomictic Developments)
Berichtszeitraum: 2023-12-01 bis 2025-11-30
Numerous plants evolved the ability to form seeds harbouring maternal embryos, an intriguing phenomenon, known as apomixis. By allowing the development of true-breeding crops, harnessing apomixis would revolutionize agriculture. In particular, faster and cheaper plant breeding could yield locally adapted varieties and, therefore, help mitigate climate changes by making agrosystems more efficient for food security.
While apomixis has remained an enigma for plant biologists and a long-awaited tool by breeders and farmers, the recent advances in plant biotechnologies have provided valuable information to understand sexual reproduction in plants and, occasionally, to redirect the sexual development program toward apomixis-like reproductive modes. They also have opened the route for investigating apomictic species at unprecedented, cellular and molecular levels.
The MAD (Mechanisms of Apomictic Developments) project has established an international, research and training network aimed at contributing significantly to our understanding of key mechanisms involved in redirecting sexuality towards apomixis, and, consequently, at overcoming the barriers that prevent breeders and farmers from reaping the benefits of apomixis. In particular, the project has offered the junior and experienced participating scientists a transparent decision-making and gender balanced framework and provided them a wide range of opportunities to gain skills in rapidly growing technologies such as DNA/RNA sequencing, bioinformatics, live-cell imaging and image data analysis. The project also succeeded in promoting partner visibility through conferences and workshops organization; communicating results to the scientific community through articles and conference participation, and; reaching the general public through a regularly updated web site and social networks. Finally, the work has generated exploitable results for pursuing the characterization of apomictic developments and for providing resources for the on-going initiatives aiming at synthetic apomixis in major crops.
While apomixis has remained an enigma for plant biologists and a long-awaited tool by breeders and farmers, the recent advances in plant biotechnologies have provided valuable information to understand sexual reproduction in plants and, occasionally, to redirect the sexual development program toward apomixis-like reproductive modes. They also have opened the route for investigating apomictic species at unprecedented, cellular and molecular levels.
The MAD (Mechanisms of Apomictic Developments) project has established an international, research and training network aimed at contributing significantly to our understanding of key mechanisms involved in redirecting sexuality towards apomixis, and, consequently, at overcoming the barriers that prevent breeders and farmers from reaping the benefits of apomixis. In particular, the project has offered the junior and experienced participating scientists a transparent decision-making and gender balanced framework and provided them a wide range of opportunities to gain skills in rapidly growing technologies such as DNA/RNA sequencing, bioinformatics, live-cell imaging and image data analysis. The project also succeeded in promoting partner visibility through conferences and workshops organization; communicating results to the scientific community through articles and conference participation, and; reaching the general public through a regularly updated web site and social networks. Finally, the work has generated exploitable results for pursuing the characterization of apomictic developments and for providing resources for the on-going initiatives aiming at synthetic apomixis in major crops.
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.
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.
The project produced key biological resources such as plants carrying biosensors and/or mutations of interest; large datasets in several area including genome sequencing, DNA methylation and gene expression analyses, imaging, and; protocols and analytical tools in cellular biology, bioinformatics and 3D image analysis. Using these, MAD scientists generated new knowledge in the field of plant reproductive biology and, in particular, in explaining the molecular and cellular mechanisms capable of promoting novel reproductive pathways in sexual plants. In regards, the publications establish several landmarks in apomixis research, including: apomictic grass genomes characterization; candidate genes (e.g. ARF10, TGS1), and; 3D imaging and atlases of plant ovules.
Furthermore, we ezxpect that the dissemination to breeders and farmers of the MAD results, as well as the expected spin-offs over the next two years, will have an impact by providing genomic resources for molecular breeding in subtropical forages and, more broadly, by developing new tools for engineering apomixis in crops.
Furthermore, we ezxpect that the dissemination to breeders and farmers of the MAD results, as well as the expected spin-offs over the next two years, will have an impact by providing genomic resources for molecular breeding in subtropical forages and, more broadly, by developing new tools for engineering apomixis in crops.