Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance.

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.

Quantification of Species-Preferential Micropylar Chemoattraction in Arabidopsis by Fluorescein Diacetate Staining of Pollen Tubes.

Sexual reproduction between males and females of the same species is essential for species maintenance. Ovular micropylar guidance, the last step of pollen tube guidance in angiosperms, contributes to species-preferential reproduction. Previous studies using semi-in vivo attraction assays showed that species-preferential attractant peptides are secreted from the ovule through its micropyle. However, conventional semi-in vivo assays usually depend on transgenic pollen tubes expressing a fluorescent protein to determine whether the tubes are attracted to the ovule to precisely penetrate the micropyle. Here, we found that fluorescein diacetate (FDA) staining was suitable for evaluating the micropylar guidance rate of non-transgenic pollen tubes in semi-in vivo conditions. Micropylar guidance was quantified for ovules and pollen tubes of Arabidopsis thaliana and Arabidopsis lyrata by combining FDA staining with modified semi-in vivo assays. Our results using the simple staining method showed that the ovules of each species secrete species-preferential attractants, and that pollen tubes respond more strongly to attractants of their own species compared with those of closely related species. LURE-type CRP810 attractant peptides were shown to be responsible for micropylar attraction of A. thaliana in the semi-in vivo assay. The POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 6 (PRK6) receptor for LURE1, as well as an unidentified receptor for other LURE-type attractants, are involved in the species-preferential response of these two Arabidopsis species.

Structural basis for receptor recognition of pollen tube attraction peptides.

Transportation of the immobile sperms directed by pollen tubes to the ovule-enclosed female gametophytes is important for plant sexual reproduction. The defensin-like (DEFL) cysteine-rich peptides (CRPs) LUREs play an essential role in pollen tube attraction to the ovule, though their receptors still remain controversial. Here we provide several lines of biochemical evidence showing that the extracellular domain of the leucine-rich repeat receptor kinase (LRR-RK) PRK6 from Arabidopsis thaliana directly interacts with AtLURE1 peptides. Structural study reveals that a C-terminal loop of the LRR domain (AtPRK6LRR) is responsible for recognition of AtLURE1.2, mediated by a set of residues largely conserved among PRK6 homologs from Arabidopsis lyrata and Capsella rubella, supported by in vitro mutagenesis and semi-in-vivo pollen tube growth assays. Our study provides evidence showing that PRK6 functions as a receptor of the LURE peptides in A. thaliana and reveals a unique ligand recognition mechanism of LRR-RKs.