In vivo gynogenesis in maize: novel insights into plant double fertilization – NOT-LIKE-DAD

In this project, we propose a number of coordinated activities to explore and elucidate these fascinating aspects of plant reproduction. Imaging of double fertilization with nuclear and plasma membrane marker lines will be used to investigate fertilization defects in nld mutant lines. We will assess the timing, extent and specificity of male genome fragmentation in sperm cells and gynogenetic embryos and endosperms. The biochemical characterization of NLD phospholipase activities in vitro coupled with comparative in vivo pollen lipid composition analyses will shed light on NLD substrates/products and will clarify its biological function. Finally, based partly on an existing interactome, and partly on genetic approaches, new molecular players associating with NLD will be identified and functionally characterized.

A first main result of this project is the new sub-cellular localization of NLD outside the sperm cells on a unique membrane. Indeed, contrary to previous reports, we demonstrated that NLD does not localize to cytosol and plasma membrane of sperm cells but on a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells: the endo-PM. We additionally describe NLD trafficking, and we demonstrated that lipid anchoring and electrostatic interactions target NLD to pollen endo-PM. The use of lipid biosensors allows us to decipher the lipid signature of this pollen endo-PM (https://doi.org/10.1083/jcb.202010077).

At this stage, an important step of the project is now the isolation and characterization of new genes involved in haploid induction. Candidate genes have been selected and both CRISPR and insertion mutants are underway and will be analyzed during the remaining time of the project.

1. Gilles LM, Calhau A, La Padula V, Jacquier NMA, Lionnet C, Martinant J-P, Rogowsky P, Widiez T. (2021)
Lipid anchoring and electrostatic interactions target NOT-LIKE-DAD to pollen endo-plasma membrane. Journal of Cell Biology, doi.org/10.1083/jcb.202010077

2. Jacquier NMA, Gilles LM, Pyott DE, Martinant JP, Rogowsky PM, Widiez T. (2020).
Puzzling out plant reproduction by haploid induction for innovations in plant breeding. Nature Plants, 6(6):610-619. doi.org/10.1038/s41477-020-0664-9

3. Seguí-Simarro JM, Jacquier NMA and Widiez T. (2021)
Overview of in vitro and in vivo doubled haploid technologies. Methods in Molecular Biology (Springer), doi.org/10.1007/978-1-0716-1315-3_1

4. Jacquier NMA, Gilles LM, Martinant JP, Rogowsky PM, and Widiez T. (2021)
The maize in planta haploid induction lines, a corner stone for doubled haploid technology. Methods in Molecular Biology (Springer), doi.org/10.1007/978-1-0716-1335-1_2

Sexual reproduction is key to the evolutionary success of higher eukaryotes. In all flowering plants, sexual reproduction is characterized by a unique biological process called double fertilization. Double fertilization consists of two separate, parallel fusion events between the two sperm cells and two female gametes, egg cell and central cell, giving rise to the embryo and the nourishing endosperm tissue, respectively. A maize line, called the haploid inducer line, deviates from this classical double fertilization process: instead of producing normal diploid embryos (2n), it induces the formation of haploid embryos (1n) containing solely the mother’s chromosomes. This process is known as in vivo gynogenesis. Derivatives of this maize haploid inducer line have become the preferred tool of numerous maize breeding companies, because they can produce perfectly homozygous plants in only 2 generations instead of 5 to 8 in classical breeding schemes.


Fifty years after the discovery of the first haploid inducer line, our recent work identified the major gene responsible for haploid induction (Gilles et al., 2017, EMBO Journal; Gilles et al., 2017, Current Biology; Martinant et al., 2016, patent WO/2016/177887). The gene product codes for a type A2 phospholipase, which we named NOT LIKE DAD (NLD), since the male genome is absent in haploid embryos. Using a combination of genetics, genomics and cell biology, we demonstrated that an intact sperm-localized phospholipase is required for successful sexual reproduction. This project aims to solve the question of why the sperm cell membrane binding NLD protein is needed to maintain paternal genome integrity in embryos. We would like to understand how paternal genome integrity is maintained during normal double fertilization, how the biochemical function of NLD relates to paternal genome integrity and, more generally, how double fertilization is coordinated.


Building on recent results, we propose a number of coordinated activities that will explore and elucidate these fascinating aspects of plant reproduction. Real-time imaging of double fertilization with nuclear and plasma membrane marker lines as well as in vitro fertilization assays will be used to investigate fertilization defects in nld mutant lines. We will assess the timing, extent and specificity of male genome fragmentation in sperm cells and gynogenetic embryos and endosperms. The biochemical characterization of NLD phospholipase activities in vitro coupled with comparative in vivo pollen lipid composition analyses will shed light on NLD substrates/products and will clarify its biological function. Finally, based partly on an existing interactome, and partly on genetic approaches, new molecular players associating with NLD will be identified and functionally characterized. Together, these independent and complementary experiments will both provide new insights into the maize haploid induction mechanism, and more generally answer fundamental questions about how plant double fertilization is coordinated.