In Arabidopsis thaliana, each ovule is normally fertilized by exactly one pollen tube. Two-photon deep imaging of intact living pistils (Mizuta et al. 2024) revealed that this one-to-one guidance rests on female signals whose molecular identities are all still unknown: a long-range directional cue from ovular sporophytic tissue (particularly the outer integument) that promotes pollen tube emergence from the transmitting tract and adhesion to the septum and funiculus surfaces; adhesion molecules mediating attachment to these maternal surfaces; a gametophytic FERONIA/LORELEI-dependent polytubey block established when the first pollen tube enters the funiculus, which implies long-distance communication between the first tube and the synergid cell and a two-stage kinetic (permissive within the first ~45 minutes, stricter afterwards); and a FERONIA/LORELEI-independent mechanism that repels excess pollen tubes from the funiculus back toward the septum. The problem is to identify these signaling molecules and to characterize mechanistically and spatiotemporally how they jointly achieve one-to-one guidance.

Background

Flowering plants (angiosperms) perform double fertilization: the pollen tube, a tubular cell grown from the pollen grain, carries two immotile sperm cells from the stigma through the style and the transmitting tract — the strand of extracellular-matrix-rich tissue that lines the interior of the ovary — until it reaches an ovule and releases the sperm cells into the female gametophyte (embryo sac). An ovule consists of a stalk, the funiculus, bearing integuments (sporophytic seed-coat precursors) that enclose the embryo sac and leave a narrow opening, the micropyle; the embryo sac contains the egg cell, the central cell, and two synergid cells at its micropylar end. Because a pollinated ovary typically contains many pollen tubes and many ovules, each ovule must attract one tube and reject the rest; failure of this 'one-to-one' control causes polytubey (multiple pollen tubes on one ovule) and polyspermy (fertilization by more than one sperm), which usually aborts the seed.

The chemistry of the final guidance step is partly known. Synergid cells secrete LUREs — small cysteine-rich defensin-like peptides that act as species-specific, short-range attractants at the micropyle. Pollen tubes acquire the competence to respond to ovular cues ('capacitation') during growth through the pistil, a process promoted by ovule-derived sugar chains such as the AMOR arabinogalactan and by brassinosteroid hormones. Several polytubey blocks at later stages are also characterized: at the septum (the tissue wall between the ovary's two locules), pollen-tube-derived RALF peptides, perceived by the FERONIA (FER) receptor kinase together with the ANJEA and HERK1 receptors, suppress emergence of further tubes; at the micropyle, FERONIA-dependent production of nitric oxide inactivates LUREs; the fertilized egg cell secretes ECS endopeptidases that degrade attractants; and the persistent synergid is eliminated after fertilization. FERONIA is a CrRLK1L receptor-like kinase at the cell surface, and LORELEI (LRE) is a glycosylphosphatidylinositol-anchored co-receptor protein; both act in the synergid during pollen tube reception.

What remained almost unobserved was the earlier, pre-micropylar phase deep inside the ovary, because it could not be imaged in vivo. Mizuta et al. (2024) developed two-photon microscopy — a fluorescence technique whose infrared excitation penetrates deep into intact, light-scattering tissue — into a live-imaging method for pollen tubes inside intact Arabidopsis pistils, and discovered four regulatory phenomena for which no molecules exist: (1) ovular sporophytic tissue emits a long-range signal, dependent on the outer integument, that reaches the inner surface of the septum epidermis, promotes the emergence of pollen tubes out of the transmitting tract, and enhances their adhesion to the maternal surfaces (septum epidermis and funiculus) — attached tubes slow from about 1.4 to about 0.5 micrometers per minute before emerging; (2) no molecule mediating this adhesion of pollen tubes to female tissue surfaces has been described in Arabidopsis (the best precedent, the lily stigma/stylar cysteine-rich adhesin SCA, belongs to a different species and organ); (3) when the first pollen tube enters a funiculus, a polytubey block is rapidly established that is gametophytic and FERONIA/LORELEI-dependent — it begins even before the first tube reaches the micropyle, implying long-distance communication between the first pollen tube and the distant synergid cell, and it has two stages: it is permissive during roughly the first 45 minutes (which explains genetically observed polyspermy) and becomes strict afterwards; and (4) excess pollen tubes that fail to enter the micropyle can be actively repelled from the funiculus back to the septum, a FERONIA/LORELEI-independent behavior most visible in fer, lre, and dif1 mutant ovaries.

Problem Statement

Identify the female signaling molecules and mechanisms that implement the pre-micropylar stages of one-to-one pollen tube guidance in Arabidopsis thaliana, and characterize their spatiotemporal dynamics, as framed by the two-photon live-imaging analysis of Mizuta et al. (2024). The requested results, which jointly constitute one scientific objective — the molecular-mechanistic account of how female tissues make pollen tube guidance one-to-one before the tube reaches the micropyle — are: (i) the identity and mechanism of the long-range directional cue(s) emitted by ovular sporophytic tissue, particularly the outer integument, that reach the inner surface of the septum epidermis, promote pollen tube emergence from the transmitting tract, and enhance adhesion of pollen tubes to the maternal surfaces (septum epidermis and funiculus); (ii) the molecule(s) that directly mediate the adhesion of pollen tubes to these female tissue surfaces; (iii) the molecular mechanism of the gametophytic FERONIA/LORELEI-dependent polytubey block that is established when the first pollen tube enters the funiculus — including the identity of the long-distance communication between the first pollen tube and the synergid cell (the block begins before the first tube reaches the micropyle) and how the signal(s) transmitted over this distance and their spatial spread produce the observed two-stage kinetics, in which the block is permissive during roughly the first 45 minutes after first-tube entry onto the funiculus and stricter thereafter; and (iv) the molecular basis of the FERONIA/LORELEI-independent repulsion that turns excess pollen tubes away from the funiculus back toward the septum. Parts (i)–(iv) are independent sub-questions, each addressable on its own, and all four together constitute the complete molecular-mechanistic account. For each part, name the molecule(s), signal, or mechanism responsible and give the grounds for the proposal — evidence drawn from the cited literature, or a falsifiable model tied to the mutant phenotypes described below — together with the discriminating observation, genetic test, or experiment that would confirm or refute it. These questions concern the dynamics recorded by that imaging analysis in the wild type and in mutant backgrounds that perturb the relevant tissues (dif1, in which ovules lack a female gametophyte; ant and ino, mutants with defective outer integuments; fer and lre, mutants of the synergid-expressed FERONIA receptor kinase and its LORELEI co-receptor): emergence counts, the adhesion-associated growth-rate reduction, the 45-minute block kinetics, and repulsion events, the last of which were observed in the dif1, fer and lre mutant ovaries.

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