In flowering plants, double fertilization requires that of the two sperm cells delivered by a pollen tube, one fuses with the egg cell (forming the embryo) and the other with the central cell (forming the endosperm). Expressing diphtheria toxin A chain (DTA) from the HAP2(GCS1) promoter in Arabidopsis blocks the second pollen mitosis and yields pollen tubes carrying a single sperm-like cell (SSLC) that preferentially fertilized the central cell (Frank and Johnson, 2009), whereas three other SSLC-producing genotypes each preferentially fertilize the egg cell via an egg-cell- and EGG CELL 1 (EC1)-dependent mechanism (Wang et al., 2024). What molecular mechanism implements gamete fusion preference, why the two SSLC systems show opposite biases, and which male germline translation products are required to complete generative-cell mitosis and full fusion competence remain unresolved.

Background

Angiosperm reproduction involves double fertilization: the pollen tube releases two immotile sperm cells into the female gametophyte (embryo sac), where one sperm fuses with the egg cell to form the zygote and the other fuses with the diploid central cell to form the triploid endosperm, the nourishing tissue of the seed. The two sperm are isomorphic (morphologically similar) siblings of the generative cell, itself produced by the first pollen mitosis; the second pollen mitosis divides the generative cell into the two sperm. HAP2 (also called GCS1) is a sperm-specific transmembrane fusogen — the protein that drives gamete plasma-membrane fusion — conserved across plants, algae, and protozoan parasites (von Besser et al. 2006; Mori et al. 2006). Frank and Johnson (2009) expressed the A subunit of diphtheria toxin (DTA), a potent inhibitor of translation, from the HAP2(GCS1) promoter. This arrested sperm development before the second pollen mitosis, producing pollen tubes that carry a single sperm-like cell (SSLC) instead of two sperm. These SSLCs could still fertilize: ovules received either an embryo or endosperm alone, and endosperm-only seeds significantly outnumbered embryo-only seeds, indicating that HAP2(GCS1):DTA SSLCs preferentially fuse with the central cell. Frank & Johnson proposed that blocking late male-germline translation may prevent production of a factor that directs or enables sperm-egg fusion, and that comparing SSLC systems could identify such factors, but these proposals were not tested. The paper left open why this bias arises, which translationally produced male germline factors (candidates included the cyclin AtCycB1;1) are needed to complete generative-cell mitosis and egg-targeted fusion, whether the egg or the central cell controls pairing, and how normal double fertilization guarantees exactly one sperm per female gamete.

Subsequent work transformed this landscape. Live imaging of photolabeled sperm (Hamamura et al. 2011) showed the two wild-type sperm are functionally equivalent and fuse with the two female gametes in no particular order, arguing against intrinsic sperm pre-programming. The egg cell was shown to actively prepare sperm for fusion: egg-cell-secreted EC1 peptides trigger sperm activation (Sprunck et al. 2012), and sperm DUF679-family membrane proteins DMP8 and DMP9 are required for the EC1-induced translocation of HAP2/GCS1 from internal storage vesicles to the sperm plasma membrane, thereby timing the acquisition of fusion competence (Wang et al. 2022). Female-side safeguards were identified: the fertilized egg secretes endopeptidases to block additional pollen tubes (Yu et al. 2021), and unfertilized central cells secrete SALVAGER1/2 peptides that attract new pollen tubes to rescue seed production (Meng et al. 2023). Imaging of sperm discharge further showed that the two sperm are initially deposited at an inopportune position, and that a 'one-step rectification' — release and re-adhesion of one sperm while the other remains stagnant — aligns each sperm with a female gamete before fusion (Huang et al. 2015). Most directly, Wang et al. (2024) re-examined fusion preference using three SSLC-producing genotypes (NTL11ΔC, pDMP9::KRP1, and fbl17-3) plus egg-cell-eliminated lines: all three SSLC types proved functionally equivalent, and each fused predominantly with the egg cell; only when the egg cell was eliminated did SSLCs fuse with the central cell, and EC1-family peptides were required for the preference. This establishes fusion preference as egg-cell-dependent rather than sperm-intrinsic — the opposite direction from the central-cell bias reported for HAP2(GCS1):DTA SSLCs, a conflict the authors explicitly note remains unresolved. Most recently, a preprint (Susaki et al. 2026, bioRxiv) reported that extracellular EC1-peptide-enriched protein assemblies ('EC1 bodies') drive localized dissociation of the egg-cell–central-cell interface to form a transient 'gamete pit' that serves as the sperm landing site — structural progress on how EC1 acts, though as a non-peer-reviewed preprint it does not settle how the choice between the two female gametes is implemented, nor the SSLC bias conflict.

Problem Statement

Determine the molecular mechanism that controls which female gamete a sperm fuses with during double fertilization in Arabidopsis thaliana. All three of the following parts are required for a complete answer, and addressing one part does not settle the others. (1) Establish how the egg cell, through EGG CELL 1 (EC1)-family secreted peptides and their sperm-side partners such as the DMP8/DMP9-HAP2/GCS1 trafficking axis, implements preferential sperm-egg fusion at the molecular level. (2) Explain the opposite fusion biases reported for different single sperm-like cell (SSLC) systems — preferential fertilization of the central cell by HAP2(GCS1):DTA SSLCs (Frank & Johnson 2009) versus preferential fertilization of the egg by SSLCs produced by NTL11ΔC (a C-terminally truncated form of the NAC transcription factor NTL11), pDMP9::KRP1 (the cyclin-dependent-kinase inhibitor KRP1 expressed from the sperm-expressed DMP9 promoter), and fbl17-3 (a mutant of the F-box protein F-BOX-LIKE17, which normally degrades cell-cycle inhibitors during male germline proliferation) (Wang et al. 2024) — and identify the male germline gene products whose de novo translation is required to complete generative-cell mitosis and to endow sperm with full fusion competence (candidates such as AtCycB1;1). (3) Determine how this egg-cell-dependent preference operates in the normal paired-sperm context — in relation to the polyspermy blocks and fertilization recovery that ensure exactly one sperm-egg and one sperm-central-cell fusion. Where an explanation rests on seed/ovule outcome scoring, it must address the known confounders of that assay — differential abortion, fertilization recovery and polytubey.

References

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