In flowering plants, two sperm cells delivered by a pollen tube fuse with the egg cell and the central cell in a double fertilization event that is driven by the conserved sperm membrane fusogen GCS1/HAP2. How the fusogen is deployed and activated selectively at each female gamete remains unresolved: the female-side proteins that interact with GCS1/HAP2 have not been identified, the egg-cell-specific role of the sperm proteins DMP8/DMP9 (overcoming a blocker versus enabling partner interaction) lacks direct biochemical evidence, GCS1/HAP2 trimerization has never been observed in vivo in flowering plant fertilization, and the quantitative relationship between sperm-surface GCS1/HAP2 abundance and fusion competence is unmeasured. This problem asks for the molecular mechanism that renders GCS1/HAP2-mediated membrane fusion competent and selective toward the two female gametes, combining identification of the female-side interactors, temporal resolution of DMP8/DMP9-dependent trafficking, in vivo readout of fusogen oligomerization, and quantification of fusogen dosage versus fusion outcome.

Background

Angiosperms (flowering plants) perform double fertilization: the pollen tube releases two sperm cells into the embryo sac (the female gametophyte), and one sperm fuses with the egg cell to form the embryo while the other fuses with the central cell to form the endosperm of the seed. Gamete fusion is mediated on the sperm side by GCS1/HAP2 (GENERATIVE CELL SPECIFIC 1, also called HAPLESS 2), a single-pass sperm membrane protein of the fusexin family, structurally homologous to class II viral fusion proteins: like viral fusogens, it is thought to insert an extracellular hydrophobic fusion loop into the target membrane and refold into a trimeric post-fusion hairpin that pulls the two membranes together. Trimeric post-fusion structures of HAP2 have been solved by X-ray crystallography in the green alga Chlamydomonas, and Arabidopsis GCS1/HAP2 ectodomains form trimers in liposome assays (Fedry et al. 2018); however, in flowering plants the fusion event occurs deep inside ovular tissue, and HAP2 oligomerization has never been observed in vivo. Several regulators act before fusion: GEX2 (GAMETE EXPRESSED 2), a sperm membrane protein with immunoglobulin-like ectodomains, is required for gamete attachment; EC1 (EGG CELL 1), a cysteine-rich peptide secreted by the egg cell upon sperm arrival, activates the sperm; and DMP8/DMP9, two redundant sperm-expressed multipass membrane proteins of the DUF679 (domain of unknown function 679) family, are more important for egg-cell fertilization than for central-cell fertilization. Wang et al. (2022) showed that DMP8/DMP9 directly interact with GCS1/HAP2 and are required for EC1-induced translocation of GCS1/HAP2 from internal storage vesicles to the sperm plasma membrane. Against this model, Shiba et al. (2023), using a functional GCS1-mNeonGreen reporter, observed GCS1/HAP2 on the plasma membrane of arrested sperm cells even in dmp8 dmp9 mutants, and proposed two non-exclusive mechanistic scenarios for the egg-cell-specific step: either the female-side GCS1/HAP2 partner is not exposed on the egg surface until DMP8/DMP9-mediated recognition occurs, or the fusogen is masked by a blocker protein that is removed upon that recognition. Their ectopic-expression assay with GAH (a secreted, transmembrane-domain-lacking GCS1/HAP2 variant expressed from the egg cell) blocked fertilization competitively and in a species-specific manner (the rice ortholog did not block), supporting the existence of an unidentified GCS1/HAP2-interacting protein on the female gametes, whose identification via GAH-scheme proteomics was ongoing at the time of writing.

Problem Statement

Determine the molecular mechanism by which the sperm fusogen GCS1/HAP2 is selectively deployed and activated at the two female gametes during Arabidopsis double fertilization, as posed by Shiba et al. (2023). This objective comprises four components, each raised as an open question by the source; the first two form the jointly required core of the objective and the last two are independently-standing sub-questions: (1) identify the GCS1/HAP2-interacting protein(s) on the egg cell and the central cell — GAH-scheme female membrane proteomics is one route being pursued — and validate their participation in fertilization; (2) establish the egg-cell-specific regulatory step performed by the sperm proteins DMP8/DMP9 — whether they act by removing a masking blocker or by enabling interaction with the female partner — with direct biochemical evidence, and reconcile the conflicting observations of DMP8/DMP9-dependent (Wang et al. 2022) and DMP8/DMP9-independent (Shiba et al. 2023) appearance of GCS1/HAP2 on the sperm surface, for example through analysis of GCS1/HAP2 dynamics in vivo at defined times after pollination; (3) determine whether GCS1/HAP2 undergoes trimerization in vivo during flowering plant fertilization and whether that trimerization, as in Chlamydomonas, requires prior gamete attachment; and (4) measure the quantitative relationship between the amount of GCS1/HAP2 on the sperm surface and fusion competence toward each female gamete, using analytical tools capable of capturing weak membrane signals in embedded tissue (a tool class exemplified by blue-light-induced pollen tube rupture imaging).

Completion of this objective requires a decisive result on both core components: for (1), identifying the female-side GCS1/HAP2-interacting protein(s) and demonstrating their participation in fertilization; for (2), direct biochemical evidence that establishes whether DMP8/DMP9 remove a masking blocker or enable interaction with the female partner, and that reconciles the conflicting DMP8/DMP9-dependent (Wang et al. 2022) and DMP8/DMP9-independent (Shiba et al. 2023) observations of surface GCS1/HAP2. Components (3) and (4) stand alone as independently-standing sub-questions — in vivo trimerization, and the quantitative sperm-surface GCS1/HAP2-versus-competence relationship, respectively — but neither is required for completion of the objective.

References

  1. Shiba Y, Takahashi T, Ohashi Y, Ueda M, Mimuro A, Sugimoto J, Noguchi Y, Igawa T (2023). Behavior of Male Gamete Fusogen GCS1/HAP2 and the Regulation in Arabidopsis Double Fertilization. Biomolecules 13(2):208. DOI 10.3390/biom13020208. https://doi.org/10.3390/biom13020208
  2. Wang W, Xiong H, Cyprys P, Malka R, Flores-Tornero M, Zhao P, Peng X, Sprunck S, Sun MX (2022). DMP8 and 9 regulate HAP2/GCS1 trafficking for the timely acquisition of sperm fusion competence. Proceedings of the National Academy of Sciences USA 119(45):e2207608119. DOI 10.1073/pnas.2207608119. https://doi.org/10.1073/pnas.2207608119
  3. Cyprys P, Lindemeier M, Sprunck S (2019). Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins. Nature Plants 5:253-257. DOI 10.1038/s41477-019-0382-3. https://doi.org/10.1038/s41477-019-0382-3
  4. Sprunck S, Rademacher S, Vogler F, Gheyselinck J, Grossniklaus U, Dresselhaus T (2012). Egg Cell-Secreted EC1 Triggers Sperm Cell Activation During Double Fertilization. Science 338(6110):1093-1097. DOI 10.1126/science.1223944. https://doi.org/10.1126/science.1223944
  5. Mori T, Igawa T, Tamiya G, Miyagishima SY, Berger F (2014). Gamete Attachment Requires GEX2 for Successful Fertilization in Arabidopsis. Current Biology 24(2):170-175. DOI 10.1016/j.cub.2013.11.030. https://doi.org/10.1016/j.cub.2013.11.030
  6. Fedry J, Forcina J, Legrand P, Péhau-Arnaudet G, Haouz A, Johnson M, Rey FA, Krey T (2018). Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes. PLOS Biology 16(8):e2006357. DOI 10.1371/journal.pbio.2006357. https://doi.org/10.1371/journal.pbio.2006357
  7. Zhang J, Pinello JF, Fernández I, Baquero E, Fedry J, Rey FA, Snell WJ (2021). Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2. Nature Communications 12:4380. DOI 10.1038/s41467-021-24613-8. https://doi.org/10.1038/s41467-021-24613-8
  8. Pinello JF, Liu Y, Snell WJ (2021). MAR1 links membrane adhesion to membrane merger during cell-cell fusion in Chlamydomonas. Developmental Cell 56(24):3380-3392. DOI 10.1016/j.devcel.2021.10.023. https://doi.org/10.1016/j.devcel.2021.10.023
  9. Wang W, Malka R, Lindemeier M, Cyprys P, Tiedemann S, Sun K, Zhang X, Xiong H, Sprunck S, Sun MX (2024). EGG CELL 1 contributes to egg-cell-dependent preferential fertilization in Arabidopsis. Nature Plants 10(2):268-282. DOI 10.1038/s41477-023-01616-5. https://doi.org/10.1038/s41477-023-01616-5
  10. Becker A, Chen X, Dresselhaus T, Gutsche N, Müller-Schüssele SJ, Sprunck S, Theißen G, de Vries S, Zachgo S (2025). Sexual reproduction in land plants: an evolutionary perspective. Plant Reproduction. DOI 10.1007/s00497-025-00522-4. https://doi.org/10.1007/s00497-025-00522-4
  11. Wang L, Zhou PM, Chen SY, Wang J, Wang XF, Yang WC, Li HJ (2026). The sperm cell-surface protein GEX3 coordinates with GEX2 and DMP8/9 to mediate sperm-egg adhesion and fusion in Arabidopsis. Plant Communications (in press). DOI 10.1016/j.xplc.2026.101883. https://doi.org/10.1016/j.xplc.2026.101883
  12. Wang Y, Liu Y, Xu YM, Liu T, Lan Z, Li L, Yang J, Chen X, Lu H, Quan X, Gu H, Qu LJ, Zhong S (2026). GEX3 interacts with GEX2 to function in gamete attachment and plasma membrane fusion in Arabidopsis. Plant Communications. DOI 10.1016/j.xplc.2026.101885. https://doi.org/10.1016/j.xplc.2026.101885
  13. Yoshimura A, Igawa T (2026). Intracellular domain of Arabidopsis GENERATIVE CELL SPECIFIC 1/HAPLESS 2 is involved in double fertilization control. Plant and Cell Physiology pcag118 (online ahead of print). DOI 10.1093/pcp/pcag118. https://doi.org/10.1093/pcp/pcag118
  14. Yoshimura A, Seo Y, Kobayashi S, Igawa T (2025). Production of homozygous deletion mutants targeting fertilization regulator genes through multiplex genome editing. bioRxiv 2025.02.28.640930. DOI 10.1101/2025.02.28.640930. https://doi.org/10.1101/2025.02.28.640930
  15. Sugi N, Susaki D, Mizuta Y, Kinoshita T, Maruyama D (2024). Letter to the Editor: Blue Light Irradiation Induces Pollen Tube Rupture in Various Flowering Plants. Plant and Cell Physiology 65(5):704-707. DOI 10.1093/pcp/pcae018. https://doi.org/10.1093/pcp/pcae018 (preprint: bioRxiv 2023.12.06.570347, DOI 10.1101/2023.12.06.570347)
  16. Pinello JF, Ferriño A, Hussain H, Legrand P, Manikumar S, Demissie R, Rey FA, Snell WJ, Baquero E (2025). Molecular determinants of species-specific cell-cell recognition activating the class II gamete fusogen HAP2. bioRxiv 2025.05.09.653094. DOI 10.1101/2025.05.09.653094. https://doi.org/10.1101/2025.05.09.653094
  17. Susaki D, Oshirabe H, Nagahara S, Goto Y, Oi T, Takeuchi H, Sato M, Toyooka K, Kakizaki H, Sugi N, Yoshimura A, Tonosaki K, Kinoshita T, Maruyama D (2026). EC1 body controls sperm cell reception via gamete pit formation. bioRxiv 2026.07.14.738444. DOI 10.64898/2026.07.14.738444. https://doi.org/10.64898/2026.07.14.738444
  18. Igawa T, Yamada L, Sawada H, Mori T (2017). Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells. Plant Biotechnology 34:119-123. DOI 10.5511/plantbiotechnology.17.0522a. https://doi.org/10.5511/plantbiotechnology.17.0522a