In Arabidopsis thaliana, small cysteine-rich EGG CELL 1 (EC1) peptides secreted by the egg cell activate the arriving sperm cells, causing the gamete fusogen HAP2/GCS1 to relocate from internal vesicles to the sperm plasma membrane so that gamete fusion can occur. More than a decade after this discovery, the mechanism by which sperm cells perceive EC1 remains unknown: no EC1 receptor or sensor has been identified, the intracellular cascade linking EC1 contact to DMP8/9-dependent HAP2/GCS1 trafficking is undefined, and the native biochemistry of full-length EC1 (its three-dimensional structure, processing, and minimal active units) is uncharacterized because recombinant full-length EC1 has never been produced. This problem asks for the molecular definition of the EC1-triggered sperm activation pathway — perception, signal transduction, and fusogen mobilization — together with the identification of the EC1 form that constitutes the minimal active unit triggering sperm activation.

Background

Flowering plants (angiosperms) reproduce by double fertilization: the pollen tube delivers two non-motile sperm cells into the female gametophyte, where one sperm fuses with the egg cell to form the embryo and the other fuses with the central cell to form the endosperm. Sprunck et al. (Science, 2012) showed in Arabidopsis that this encounter is coordinated by mutual gamete activation. The egg cell accumulates small, cysteine-rich secreted peptides of the EGG CELL 1 (EC1) family (five paralogs, EC1.1–EC1.5, members of the ECA1 gametogenesis-related proteins with a prolamin-like domain containing six conserved cysteines and two short signature motifs shared by all EC1-like proteins) in storage vesicles; upon sperm arrival these vesicles are exocytosed. Applied EC1 peptides cause the sperm endomembrane system to redistribute HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) — an ancient, sperm-expressed class II fusogen (membrane-merge protein) required for gamete fusion in many eukaryotes — from internal compartments to discrete domains of the sperm plasma membrane. In ec1 quintuple mutants, sperm frequently fail to fuse with the female gametes and additional sperm-cell delivery (polytubey) occurs. Only synthetic peptides corresponding to the conserved EC1 motifs were tested in 2012, because full-length recombinant EC1 could not be produced; thus the biochemistry of the native protein is unknown. Subsequent work partially filled flanking gaps. On the sperm side, the DUF679-domain membrane proteins DMP8 and DMP9 (Cyprys et al., Nature Plants, 2019) were shown to be required for the EC1-induced translocation of HAP2/GCS1 to the sperm surface (Wang et al., PNAS, 2022), establishing the trafficking machinery downstream of EC1 but not how EC1 is perceived. On the female side, successful fertilization triggers secretion of the egg-cell endopeptidases ECS1/ECS2, which cleave the pollen-tube attractant LURE1 and thereby stop attraction of further pollen tubes (Yu et al., Nature, 2021; Mao et al., eLife, 2023), and RALF (rapid alkalinization factor) peptide signaling via FERONIA-class receptor kinases sets a complementary polytubey block (Zhong et al., Science, 2022) — largely resolving the post-fusion pollen-tube-attraction arm of the original question. EC1 was additionally implicated in egg-cell-dependent preferential fertilization (Wang et al., Nature Plants, 2024), and a 2026 preprint reports that EC1 forms extracellular amyloid-like assemblies ('EC1 bodies') that remodel the egg–central-cell interface into a sperm-landing 'gamete pit', adding a structural role for EC1 (Susaki et al., bioRxiv, 2026). A 2025 review states plainly that 'the receptor of EC1 remains a significant unresolved mystery' (Yan et al., Frontiers in Plant Science, 2025). What still stands open is the sperm side of this signaling event: how EC1 is detected, how detection is transduced to DMP8/9-dependent HAP2/GCS1 mobilization, and which EC1 form — the full-length protein or the conserved signature motifs — constitutes the minimal active unit that triggers sperm activation.

Problem Statement

Define the molecular mechanism by which flowering-plant sperm cells respond to egg-cell-secreted EC1 peptides. Specifically: (1) identify the sperm-side sensor(s)/receptor(s) — canonical or non-canonical, membrane-bound or other — that detect EC1 and initiate sperm activation; (2) establish the intracellular signal-transduction and membrane-trafficking cascade connecting EC1 detection to the DMP8/9-dependent redistribution of the fusogen HAP2/GCS1 from internal vesicles to discrete sperm plasma-membrane domains, including the causal order and regulation of each step; and (3) determine which EC1 form — the full-length protein or the conserved signature motifs — constitutes the minimal active unit that triggers sperm activation, given that only synthetic peptides corresponding to the conserved EC1 motifs have been assayed to date. Claims identifying the EC1 sensor and the perception-to-trafficking cascade must demonstrate mechanistic validity in vivo in Arabidopsis (loss- and gain-of-function evidence reproducing or blocking the EC1-induced HAP2/GCS1 relocalization and gamete-fusion phenotypes), not merely candidate correlation; biochemical evidence for the minimal active unit may include peptide-based and reconstitution assays, provided the activation claim itself is validated in planta. Whether additional egg- or sperm-derived molecules act cooperatively with EC1 in triggering activation and fusion competence is a related open context question rather than part of the completion criterion for this problem. The post-fusion cessation of pollen-tube attraction, originally raised alongside these questions, has been substantially resolved (ECS1/ECS2-mediated LURE1 degradation; RALF-dependent polytubey block) and is therefore part of the background rather than the open target.

References

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