Identification and characterization of TcCRP1, a pollen tube attractant from Torenia concolor

Abstract

Background and aims: 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.

Methods: A candidate pollen tube attractant protein in Torenia concolor, a related species of T. fournieri, was isolated and the attractant abilities between them were compared.

Key results: TcCRP1, an orthologous gene of TfCRP1 from T. concolor, is expressed predominantly in the synergid cell. The gene product attracted pollen tubes in a concentration-dependent manner, but attracted fewer pollen tubes from the other species.

Conclusions: The results indicated that this class of CRP proteins is a common pollen tube attractant in Torenia species. The sequence diversity of these proteins is important for species-specific pollen tube attraction.

Fig. 1.

Flower morphology and pollen tube attraction in Torenia species: (A, B) flowers of (A) T. fournieri and (B) T. concolor; (C, D) protruding embryo sac of (C) T. fournieri and (D) T. concolor; (E–H) pollen tubes emerging from the cut end of the style (arrowheads). The cross combinations (female × male) are indicated: Tf = Torenia fournieri; Tc = T.concolor. Scale bars: (C, D) = 50 µm; (E–H) = 200 µm.

Fig. 2.

In vivo pollen tube growth and attraction to the ovule: aniline blue staining of (A–D) pollen tubes in the ovary and (E–H) pollen tubes attracted to the ovules (arrowheads). (I–L) DIC images of ovules attracting pollen tubes. The cross combinations (female × male) are indicated above each column of images: Tf = Torenia fournieri; Tc = T.concolor. Scale bars: (A–D) = 1 mm; (E–H) = 100 µm; (I–L) = 20 µm.

Fig. 3.

Amino acid sequence of TcCRP1 and TcCRP1 expression. (A) Multiple alignments of TcCRP1, TfCRP1 and TfCRP3 proteins. Sequences in the top row represent signal sequences, and those in the middle and bottom rows represent mature proteins. Conserved amino acids in all three sequences are shown in black boxes, and in two of the three are shaded. Conserved cysteine residues have asterisks. (B) Phylogenetic analysis of group 1 (LURE-like) CRPs. Bootstrap values are shown on each branch. (C) RT-PCR of TcCRP1. GAPDH served as a control. Abbreviations: st, style; le, leaf; pe, petal; oy, ovary; ov, ovules; sc, synergid cells; ec, egg cells; g, genomic DNA.

Fig. 4.

Pollen tube attraction by TcCRP1: (A, B) T. concolor pollen tube just after (A) and 20 min after (B) applying assay buffer only (green); (C, D) T. concolor pollen tube just after (C) and 20 min after (D) applying TcCRP1 protein (green). Arrowheads indicate the pollen tube tip. (E) Concentration-dependent pollen tube attraction by TcCRP1. Data are means ± s.d. (n > 3 with >10 pollen tubes per replicate). Scale bar = 50 µm.

Fig. 5.

Interspecies pollen tube attraction by CRP1: (A, B) T. fournieri pollen tube (A) just after and (B) 20 min after applying the TcCRP1 protein (red); (C) comparison of the T. fournieri pollen tube attraction rate using buffer, 40 nm of TfCRP1 and 40 nm of TcCRP1; (D, E) T. concolor pollen tube (D) just after and (E) 20 min after applying the TfCRP1 protein (green); (F) comparison of the T. concolor pollen tube attraction rate using buffer, 40 nm of TcCRP1 and 40 nm of TfCRP1. (C, F) Data are means ± s.d. (n > 3 with >10 pollen tubes per replicate). The asterisk in (F) indicates a significant difference between TcCRP1 and TfCRP1 (Fisher's exact test, P < 0·05). Scale bars = 50 µm.