Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable processes. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The database generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.
During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri.
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.
Sexual reproduction in flowering plants requires pollen-tube guidance, which is thought to be mediated by chemoattractants derived from target ovules. To date, however, no convincing evidence has been reported of a particular molecule being the true attractant. Emerging data indicate that two synergid cells, which are on either side of the egg cell, emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen-tube guidance. Recently, it was demonstrated that LUREs (LURE1 and LURE2), cysteine-rich polypeptides secreted from the synergid cell, are the key molecules in pollen-tube guidance. In this review, we summarize the mechanism of pollen-tube guidance, with special focus on gametophytic guidance and the attractants.
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.
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