The discovery that defensin-like cysteine-rich peptides (LUREs), secreted by the two synergid cells of the ovule, act as the last-step chemoattractants that guide pollen tubes into the micropyle (Okuda et al., Nature 2009) established the long-sought attractant for the final guidance step, but left the overall mechanism of pollen-tube guidance open on four linked fronts. Subsequent literature has resolved part of this picture and left other parts open: the pollen-tube receptors for Arabidopsis LURE1 were identified (PRK6 and the MDIS1-MIK1/2 heteromer, with structural basis known), while receptors for other LURE-type attractants remain unidentified, and the signal-transduction cascade from peptide perception to oriented tip growth, the molecular basis of pollen-tube competence, the identity of guidance cues acting at earlier stages and longer ranges, the biological roles of the many other synergid-expressed cysteine-rich peptides, and the sequence-level determinants of species-preferential recognition all remain open research problems.

Background

In flowering plants (angiosperms), fertilization is indirect: the immotile sperm cells are delivered by the pollen tube, a tubular cell that grows from the pollen grain through stigma and style, travels along the ovary's transmitting tract (a nutritive tissue), and finally targets an ovule, where it enters through a small opening called the micropyle and releases the two sperm cells for double fertilization of the egg cell and the central cell. How the pollen tube finds the correct ovule has been studied since the 19th century as a chemotaxis problem, and guidance is now described in steps: sporophytic guidance (control exerted by the diploid maternal tissues of the pistil, as distinct from signals from the haploid female gametophyte) through stigma and style, emergence from the transmitting tract, funicular guidance along the ovule stalk, and the final micropylar guidance into the ovule. Genetic and laser-ablation work showed that the synergid cells — two haploid accessory cells of the female gametophyte flanking the egg cell — emit the ovule's attraction signal for the last step (Higashiyama et al., Science 2001). Okuda et al. (Nature 2009) identified that signal in the plant Torenia fournieri, whose embryo sac protrudes from the ovule and is accessible to in vitro assays: two small, secreted, cysteine-rich polypeptides (CRPs) of the defensin-like subfamily, named LURE1 and LURE2, are abundantly and predominantly expressed in the synergid cells, are secreted at the egg-apparatus surface, and attract pollen tubes of the same species in vitro; knockdown of the LUREs by injected morpholino antisense oligomers impairs attraction. CRPs are short secreted peptides with several conserved cysteines forming disulfide bonds, a huge plant gene family whose members act as local intercellular signals. Follow-up work showed that Arabidopsis thaliana has its own species-specific cluster of LURE genes (AtLURE1/CRP810 family) with comparable attractant activity, and that attraction is species-preferential: pollen tubes respond more strongly to attractants of their own species than to those of closely related species, contributing to prezygotic reproductive isolation. Two further concepts frame the open questions. First, competence (or capacitation): a pollen tube can respond to LUREs only after growing through pistil tissue; in Torenia this competence is conferred by AMOR, an arabinogalactan sugar chain (terminal disaccharide 4-Me-GlcA-beta(1,6)-Gal) from the ovule, whose pollen-tube receptor is unidentified. Second, perception: the tip-localized pollen receptor kinase PRK6 and the MDIS1-MIK1/MIK2 receptor heteromer bind LURE peptides, and their crystallographic complex with LURE1.2 established the structural basis of recognition; transferring these receptors into related species can alter species recognition. What is still missing is a complete molecular account of how the female cues and male responsiveness together produce accurate, species-selective targeting of the ovule.

Problem Statement

The problem is to complete the molecular mechanism by which a pollen tube is guided to, and selectively enters, an ovule of its own species — the mechanism of which the synergid LURE peptides (Okuda et al. 2009) proved to be the last-step, species-specific signal. Building on the established LURE system, the following fronts of this mechanism remain open: (1) which chemoattractant or directional cues operate at the earlier guidance steps and longer ranges (sporophytic guidance in stigma/style and transmitting tract, pollen-tube emergence, funicular guidance), including the unknown long-distance integument-derived signal inferred from live imaging, and how those cues integrate with the LURE-mediated micropylar step; (2) the perception-and-response cascade in the pollen tube — the transduction pathway leading from LURE binding to the known receptors (PRK6, MDIS1-MIK1/2) through Ca2+ signals, Rho-of-plants (ROP) GTPases and vesicle-trafficking control to reoriented tip growth — together with the identity of receptors for other LURE-type attractants such as the XIUQIU/CRP810-family and TICKET peptides, and the mechanism by which pollen tubes acquire competence to respond, including the pollen-tube receptor for the AMOR arabinogalactan competence factor in Torenia and the corresponding mechanism in Arabidopsis; (3) the biological roles of the cysteine-rich peptides and small secreted proteins of the MYB98-dependent embryo-sac gene families in Arabidopsis (excluding the LURE-type attractants treated in front 2), for which functional or biochemical data are still lacking and whose possible defence-related or other roles in fertilization remain undetermined, this front being completely resolved once a role has been assigned and evidenced for each such family for which a loss-of-function allele exists; and (4) the molecular basis of species-preferential recognition: how the multiple attractant peptides and their pollen-tube receptors contribute to species preferentiality, which sequence and structural features of the rapidly evolving CRP attractants and of their pollen-tube receptors confer species specificity, and how ligand and receptor evolution interplay across species. These four fronts are jointly required and contribute to one overarching objective — a mechanistic, cellularly resolved account of accurate and species-selective pollen-tube targeting of the ovule; a complete resolution of any single front is accepted as an independent sub-result but does not by itself resolve the task. Established approaches in the field include the semi-in vivo guidance assay (in which a pollen tube first grows through living pistil tissue and is then presented with excised ovules on medium), in vivo observation within the pistil, genetic perturbation, and structural and comparative analysis, without restriction to a single species pair, peptide family, or method.

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