This problem concerns a question left open by Iwano et al. (2012, Development 139(22):4202–4209), who imaged cytosolic Ca2+^{2+} in Arabidopsis pollen tubes and synergid cells during semi-in vivo fertilization and observed that the receptive synergid starts to oscillate in Ca2+^{2+} upon pollen tube arrival, with a peak at pollen tube rupture. Four of the five questions raised there have since been resolved by direct external evidence: the Arabidopsis micropylar attractants were identified (AtLURE1 and XIUQIU peptides), the source of the synergid Ca2+^{2+} rise was established as plasma-membrane influx through the NORTIA channel, the FERONIA pathway was shown to transduce through Ca2+^{2+} (pollen-tube RALF4/19 → FER–LORELEI → NORTIA), and the causal role of ACA9 in burst was defined through the DEB–ACA9 axis. What remains open is the downstream half of the pathway: the identity of the Ca2+^{2+} sensors/decoders and their effector targets that execute the two terminal cell-death events of reception (pollen-tube rupture with sperm discharge, and degeneration of the receptive synergid), the in-vivo causal ordering of Ca2+^{2+} influx relative to reactive oxygen species production and to each death event, and which features of the Ca2+^{2+} oscillation dynamics are necessary or sufficient for rupture and synergid death.

Background

In flowering plants, the haploid pollen grain germinates a pollen tube — a single, rapidly elongating cell that carries two immotile sperm cells through the pistil toward an ovule. The pollen tube enters the ovule through a small opening called the micropyle and arrives at one of the two synergid cells, specialized secretory cells that flank the egg cell of the female gametophyte (the embryo sac). Each synergid bears a filiform apparatus, an elaborate wall invagination at its micropylar pole that enlarges the plasma-membrane surface and is the site of attractant secretion. Contact between the pollen tube and this receptive synergid triggers a stereotyped sequence: the tube stops growing, ruptures, and discharges its cytoplasm and the two sperm cells into the degenerating synergid; the sperm then fuse with the egg and the central cell (double fertilization).

Cytosolic free Ca2+^{2+} ([Ca2+]cyt[\mathrm{Ca}^{2+}]_{cyt}) is a ubiquitous second messenger in plants. Iwano et al. (2012) monitored it with yellow cameleon 3.60 (YC3.60), a genetically encoded Ca2+^{2+} sensor based on Förster resonance energy transfer (FRET) — the distance-dependent transfer of excitation energy between two fluorescent proteins, reported here as a fluorescence-emission ratio that rises with Ca2+^{2+} concentration — expressed in pollen tubes and synergid cells of Arabidopsis thaliana under semi-in vivo conditions (an assay in which pollen tubes grow through an excised pistil onto cultured ovules, where fertilization can be imaged directly). They found that synergid [Ca2+]cyt[\mathrm{Ca}^{2+}]_{cyt} is quiet while the tube approaches, begins to oscillate at the micropylar pole upon arrival, propagates toward the chalazal pole (the end of the synergid opposite the micropyle), and peaks at rupture. Their discussion posed five open questions: the identity of the Arabidopsis micropylar attractants (in Torenia fournieri, defensin-like LURE peptides — small secreted cysteine-rich peptides related to antimicrobial defensins — secreted from synergids were the known attractants, Okuda et al. 2009); whether the synergid Ca2+^{2+} rise reflects extracellular influx or release from internal stores such as the endoplasmic reticulum; whether receptor-like kinase pathways preventing premature rupture — the pollen-tube-expressed ANXUR1/2 and the synergid-expressed FERONIA — transduce via Ca2+^{2+}; the causal roles of specific Ca2+^{2+} transporters and pumps (for example ACA9, a pollen plasma-membrane Ca2+^{2+}-ATPase whose loss blocks pollen-tube burst; Schiøtt et al. 2004); and the cascade, including possible reactive oxygen species (ROS) involvement, that links the Ca2+^{2+} oscillation to synergid programmed cell death (PCD).

Subsequent work resolved the upstream signaling architecture. FERONIA (FER) is a synergid plasma-membrane receptor kinase of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) subfamily, required for pollen tube reception; LORELEI (LRE) is its co-receptor, a glycosylphosphatidylinositol (GPI)-anchored protein, tethered to the outer face of the plasma membrane by a lipid anchor; and NORTIA (NTA/AtMLO7) is a synergid-expressed member of the MLO family of heptahelical transmembrane proteins that bind calmodulin (CaM), a small ubiquitous eukaryotic Ca2+^{2+}-binding sensor protein. Gao et al. (2022) showed that pollen-tube-derived RALF4 and RALF19 peptides (RALF, rapid alkalinization factor, ~5 kDa cysteine-rich peptides) act as ligands for the FER–LRE co-receptor, which recruits NTA to the plasma membrane; NTA functions as a calmodulin-gated Ca2+^{2+} channel required for synergid Ca2+^{2+} spiking, with Ca2+^{2+}–calmodulin feedback inhibition driving the oscillations — establishing plasma-membrane influx, not ER-store release, as the necessary Ca2+^{2+} route, and answering the FERONIA–Ca2+^{2+} question affirmatively. On the male side, pollen-expressed receptor-like cytoplasmic kinases DEB1/2/3 phosphorylate ACA9 to govern pollen-tube Ca2+^{2+} dynamics and timely burst (Xu et al. 2024). Duan et al. (2014) established that FERONIA controls high ROS levels at the entrance to the female gametophyte and that hydroxyl-radical-type ROS induce pollen-tube rupture in a Ca2+^{2+}-dependent process.

What is still missing is the decoding layer: which Ca2+^{2+}-binding proteins or other sensors in the synergid read the NTA-mediated spiking, what their effector targets are, how Ca2+^{2+}, ROS, and the two death events are causally ordered in vivo, and what the oscillation dynamics themselves are functionally required for. Pollen tube reception is the terminal, irreversible step of fertilization and a key node of the block against polyspermy.

Problem Statement

Identify the molecular components and causal chain, together with the existing experimental evidence supporting each step, by which pollen-tube-contact-induced, RALF4/19–FER–LORELEI–NORTIA-dependent Ca2+^{2+} influx and oscillation in the receptive synergid of Arabidopsis thaliana is decoded to execute the two terminal events of pollen tube reception — rupture of the pollen tube with discharge of its cytoplasm and sperm cells, and degeneration (programmed cell death) of the receptive synergid. A complete answer must specify (i) the Ca2+^{2+} sensors/decoders (for example calmodulin-like proteins or other Ca2+^{2+}-binding effectors) and their downstream targets in the synergid that transduce the NORTIA-mediated Ca2+^{2+} signal into each death event; (ii) the in-vivo causal ordering of synergid Ca2+^{2+} influx relative to ROS production (including which ROS species and their enzymatic sources) and to pollen-tube rupture and synergid death; and (iii) which of the Ca2+^{2+} oscillation-dynamics features — occurrence, amplitude, frequency, and spatial propagation pattern (an illustrative, non-exhaustive set) — are necessary and which are sufficient for rupture and for synergid cell death, stating each modality only where the evidence supports it. Of the five mechanistic questions posed in the discussion of Iwano et al. (2012), this is the one that remains open; the other four (identity of the Arabidopsis micropylar attractants, plasma-membrane-versus-internal-store source of the Ca2+^{2+} rise, whether FERONIA-related receptor-kinase signaling acts through Ca2+^{2+}, and the causal role of specific Ca2+^{2+} pumps such as ACA9 in burst) have been substantially resolved by later literature.

References

  1. Iwano M, Ngo QA, Entani T, Shiba H, Nagai T, Miyawaki A, Isogai A, Grossniklaus U, Takayama S. Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells. Development 139(22):4202–4209, 2012. doi:10.1242/dev.081208, https://doi.org/10.1242/dev.081208
  2. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458(7236):357–361, 2009. doi:10.1038/nature07882, https://doi.org/10.1038/nature07882
  3. Schiøtt M, Romanowsky SM, Bækgaard L, Jakobsen MK, Palmgren MG, Harper JF. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proceedings of the National Academy of Sciences USA 101(25):9502–9507, 2004. doi:10.1073/pnas.0401542101, https://doi.org/10.1073/pnas.0401542101
  4. Takeuchi H, Higashiyama T. A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biology 10(12):e1001449, 2012. doi:10.1371/journal.pbio.1001449, https://doi.org/10.1371/journal.pbio.1001449
  5. Takeuchi H, Higashiyama T. Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis. Nature 531(7593):245–248, 2016. doi:10.1038/nature17413, https://doi.org/10.1038/nature17413
  6. Ngo QA, Vogler H, Lituiev DS, Nestorova A, Grossniklaus U. A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Developmental Cell 29(4):491–500, 2014. doi:10.1016/j.devcel.2014.04.008, https://doi.org/10.1016/j.devcel.2014.04.008
  7. Denninger P, Bleckmann A, Lausser A, Vogler F, Ott T, Ehrhardt DW, Frommer WB, Sprunck S, Dresselhaus T, Grossmann G. Male–female communication triggers calcium signatures during fertilization in Arabidopsis. Nature Communications 5:4645, 2014. doi:10.1038/ncomms5645, https://doi.org/10.1038/ncomms5645
  8. Hamamura Y, Nishimaki M, Takeuchi H, Geitmann A, Kurihara D, Higashiyama T. Live imaging of calcium spikes during double fertilization in Arabidopsis. Nature Communications 5:4722, 2014. doi:10.1038/ncomms5722, https://doi.org/10.1038/ncomms5722
  9. Duan Q, Kita D, Johnson EA, Aggarwal M, Gates L, Wu HM, Cheung AY. Reactive oxygen species mediate pollen tube rupture to release sperm for fertilization in Arabidopsis. Nature Communications 5:3129, 2014. doi:10.1038/ncomms4129, https://doi.org/10.1038/ncomms4129
  10. Leydon AR, Tsukamoto T, Dunatunga D, Qin Y, Johnson MA, Palanivelu R. Pollen tube discharge completes the process of synergid degeneration that is initiated by pollen tube–synergid interaction in Arabidopsis. Plant Physiology 169(1):485–496, 2015. doi:10.1104/pp.15.00528, https://doi.org/10.1104/pp.15.00528
  11. Zhong S, Liu M, Wang Z, Huang Q, Hou S, Xu YC, Ge Z, Song Z, Huang J, Qiu X, Shi Y, Xiao J, Liu P, Guo YL, Dong J, Dresselhaus T, Gu H, Qu LJ. Cysteine-rich peptides promote interspecific genetic isolation in Arabidopsis. Science 364(6443):eaau9564, 2019. doi:10.1126/science.aau9564, https://doi.org/10.1126/science.aau9564
  12. Liu M, Wang Z, Hou S, Wang L, Huang Q, Gu H, Dresselhaus T, Zhong S, Qu LJ. AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance. Plant Physiology 186(2):865–873, 2021. doi:10.1093/plphys/kiab105, https://doi.org/10.1093/plphys/kiab105
  13. Gao Q, Wang C, Xi Y, Shao Q, Li L, Luan S. A receptor–channel trio conducts Ca2+ signalling for pollen tube reception. Nature 607(7919):534–539, 2022. doi:10.1038/s41586-022-04923-7, https://doi.org/10.1038/s41586-022-04923-7
  14. Völz R, Harris W, Hirt H, Lee YH. ROS homeostasis mediated by MPK4 and SUMM2 determines synergid cell death. Nature Communications 13:1746, 2022. doi:10.1038/s41467-022-29373-7, https://doi.org/10.1038/s41467-022-29373-7
  15. Ponvert N, Johnson MA. Synergid cell calcium oscillations refine understanding of FERONIA/LORELEI signaling during interspecific hybridization. Plant Reproduction 37(1):57–68, 2024 (published online 7 November 2023). doi:10.1007/s00497-023-00483-6, https://doi.org/10.1007/s00497-023-00483-6
  16. Susaki D, Izumi R, Oi T, Takeuchi H, Shin JM, Sugi N, Kinoshita T, Higashiyama T, Kawashima T, Maruyama D. F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells. The Plant Cell 35(4):1222–1240, 2023. doi:10.1093/plcell/koac371, https://doi.org/10.1093/plcell/koac371
  17. Xu YJ, Luo T, Zhou PM, Wang WQ, Yang WC, Li HJ. Pollen-expressed RLCKs control pollen tube burst. Plant Communications 5(8):100934, 2024. doi:10.1016/j.xplc.2024.100934, https://doi.org/10.1016/j.xplc.2024.100934
  18. Baillie AL, Sloan J, Qu LJ, Smith LM. Signalling between the sexes during pollen tube reception. Trends in Plant Science 29(3):343–354, 2024. doi:10.1016/j.tplants.2023.07.011, https://doi.org/10.1016/j.tplants.2023.07.011
  19. Chen J, Wang H, Wang J, Zheng X, Qu W, Fang H, Wang S, He L, Hao S, Dresselhaus T. Fertilization-induced synergid cell death by RALF12-triggered ROS production and ethylene signaling. Nature Communications 16:3059, 2025. doi:10.1038/s41467-025-58246-y, https://doi.org/10.1038/s41467-025-58246-y
  20. Zhong S, Lan Z, Qu LJ. Ingenious male–female communication ensures successful double fertilization in angiosperms. Annual Review of Plant Biology 76:401–431, 2025. doi:10.1146/annurev-arplant-083123-071512, https://doi.org/10.1146/annurev-arplant-083123-071512
  21. Kato C, Goromaru T, Nagae TT, Mizuta Y, Kurihara D, Sato Y, Okuda S, Higashiyama T. Calcium dynamics during pollen tube reception in Arabidopsis ovules. bioRxiv preprint, 2026. doi:10.64898/2026.03.30.715275, https://doi.org/10.64898/2026.03.30.715275 (not peer-reviewed)