For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the β isomer of methyl-glucuronosyl galactose (4-Me-GlcA-β-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

ORCID IDs: 0000-0003-3726-3798 (J.J.); 0000-0002-0398-2113 (S.S.); 0000-0002-7346-360X (T.H.).Successful fertilization in flowering plants depends on the precise directional growth control of pollen tube through the female pistil tissue toward the female gametophyte contained in the ovule for delivery of nonmotile sperm cells. Cys-rich peptides LUREs secreted from the synergid cells on either side of the egg cell act as ovular attractants of pollen tubes. Competency control by the pistil is crucial for the response of pollen tubes to these ovular attractants. We recently reported that ovular 4-O-methylglucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. The beta isomer of the terminal disaccharide 4-O-methyl-glucuronosyl galactose was essential and sufficient for the competency induction. However, critical and noncritical structures in the disaccharide have not been dissected deeply. Herein, we report the synthesis of new AMOR analogs and the structure-activity relationships for AMOR activity in the presence of these synthesized analogs. Removal of 4-O-methyl group or -COOH from the glucuronosyl residue of the disaccharide dramatically reduces AMOR activity. The pyranose backbone of the second sugar of disaccharide is essential for the activity but not hydroxy groups. The role of beta isomer of the disaccharide 4-Me-GlcA-b(1,6)-Gal is very specific for competency control, as there was no difference in effect among the sugar analogs tested for pollen germination. This study represents the first structure-activity relationship study, to our knowledge, of a sugar molecule involved in plant reproduction, which opens a way for modification of the molecule without loss of activity.

The pollen tube attractant peptide LUREs of Torenia fournieri are diffusible peptides that attract pollen tubes in vitro. Here, we report a method enabling the direct visualization of a LURE peptide without inhibiting its attraction activity by conjugating it with the Alexa Fluor 488 fluorescent dye. After purifying and refolding the recombinant LURE2 with a polyhistidine tag, its amino groups were targeted for conjugation with the Alexa Fluor dye. Labeling of LURE2 was confirmed by its fluorescence and mass spectrometry. In our in vitro assay using gelatin beads, Alexa Fluor 488-labeled LURE2 appeared to have the same activity as unlabeled LURE2. Using the labeled LURE2, the relationship between the spatiotemporal change of distribution and activity of LURE2 was examined. LURE2 attracted pollen tubes when embedded in gelatin beads, but hardly at all when in agarose beads. Direct visualization suggested that the significant difference between these conditions was the retention of LURE2 in the gelatin bead, which might delay diffusion of LURE2 from the bead. Direct visualization of LURE peptide may open the way to studying the spatiotemporal dynamics of LURE in pollen tube attraction.