Species preferentiality of the pollen tube attractant derived from the synergid cell of Torenia fournieri

Abstract

The synergid cell of Torenia fournieri attracts pollen tubes by a diffusible but yet unknown chemical attractant. Here we investigated the species difference of the attractant using five closely related species in two genera, namely T. fournieri, Torenia baillonii, Torenia concolor, Lindernia (Vandellia) crustacea, and Lindernia micrantha. These five species have an exserted embryo sac, and ablation experiments confirmed that their synergid cells attracted the pollen tube. When ovules of T. fournieri and one of the other species were cultivated together with pollen tubes of each species, pollen tubes were significantly more attracted to synergid cells of the corresponding species. The attraction was not affected by the close proximity of embryo sacs of different species. This suggests that the attractant is a species-preferential molecule that is likely synthesized in the synergid cell. The calcium ion, long considered a potential attractant, could not serve as the sole attractant in these species, because elevation of the calcium ion concentration did not affect the observed attraction. In vivo crossing experiments also showed that the attraction of the pollen tube to the embryo sac was impaired when pollen tubes of different species arrived around the embryo sac, suggesting that the species preferentiality of the attractant may serve as a reproductive barrier in the final step of directional control of the pollen tube.

Figure 1.

The five Scrophulariaceae species used in this study with exserted embryo sacs. Flowers (A, D, G, J, and M), ovules (B, E, H, K, and N), and exserted embryo sacs (C, F, I, L, and O) of T. fournieri (A–C), T. baillonii (D–F), T. concolor (G–I), L. crustacea (J–L), and L. micrantha (M–O) are shown. Arrowheads indicate an exserted embryo sac. Bars in A, D, G, J, and M indicate 1 cm; the bar in B indicates 100 μm for B, E, H, K, and N; and the bar in C indicates 30 μm for C, F, I, L, and O.

Figure 2.

Phylogenetic reconstruction based on MP analysis of rbcL nucleotide sequences. From an analysis of 41 Scrophulariaceae species, only the branch that contained Torenia and Lindernia is shown. L. antipoda and L. setulosa, which do not produce exserted embryo sacs, are also included. Bootstrap values in percentages for the MP and NJ methods and the posterior probability (for Bayes' theorem) are indicated.

Figure 3.

Pollen tube attraction by synergid cell of each plant species in vitro. A, DIC images of pollen tube attraction. The species of both the ovule and pollen tube are indicated at the top left of each segment. Arrowheads indicate an exserted embryo sac, and arrows indicate attracted pollen tubes. B, Frequencies of pollen tube attraction are shown for complete embryo sacs (SYs+) and embryo sacs with two ablated synergid cells (SYs−). Each column indicates the mean value with the sd of three replications. Bar = 50 μm.

Figure 4.

Species preferentiality of the pollen tube attraction signal derived from the synergid cell. Each column indicates the mean value with the sd of three replications. OV, Ovule; PT, pollen tube; ST, style; Tf, T. fournieri; Tb, T. baillonii; Tc, T. concolor; Lc, L. crustacea; Lm, L. micrantha.

Figure 5.

Species preferentiality assay following micromanipulation of ovules to place embryo sacs of different genera opposite each other. A, DIC images of T. fournieri (left) and L. micrantha (right) pollen tube attraction in the presence of a heterogeneous embryo sac. Arrows indicate attracted pollen tubes. B, Frequency of pollen tube attraction following micromanipulation of ovules to place embryo sacs of different genera opposite each other. Ten sets of ovules were used in each experiment, and each column indicates the mean value with the sd of three replications. ES, Embryo sac; OV, ovule; PT, pollen tube; ST, style; Tf, T. fournieri; Lc, Lindernia crustacea; Lm, L. micrantha. Bar = 50 μm.

Figure 6.

Species preferentiality of pollen tube attraction with stigma and style tissues of different species. Each column indicates the mean value with the sd of three replications. Lindernia pollen tubes did not come out of the T. fournieri styles, and Lindernia stigma/style tissues poorly supported growth of Torenia pollen tubes semi-in vitro. OV, Ovule; PT, pollen tube; ST, style; Tf, T. fournieri; Tb, T. baillonii; Tc, T. concolor; Lc, L. crustacea; Lm, L. micrantha.

Figure 7.

Pollen tube attraction in the presence of high concentrations of Ca²⁺ and GABA in the medium. A, Relationship between the concentrations of Ca²⁺ and GABA and pollen tube length. GABA was tested in the presence of 2 mm Ca²⁺. Arrows indicate concentrations of Ca²⁺ or GABA tested in B. B, Frequencies of pollen tube attraction in the presence of high concentrations of Ca²⁺ and GABA. The mean value with the sd of three replications is shown.

Figure 8.

In vivo crossing analysis. A, DIC images show ovules with or without pollen tube penetration, and aniline-blue-stained images show pollen tubes whose growth has stopped in the style (Lc × Tf; Lm × Tf). The ovules and pollen tubes were excised and observed at 1 d after pollination, as described previously (Higashiyama et al., 1997). Labels indicate the species of pollen (former) and pistil (latter) used in each cross. Arrowheads indicate an exserted embryo sac, and arrows indicate pollen tubes attracted to the sacs. B, Frequencies of penetration of the embryo sac by the pollen tube at 1 d after pollination. Each column indicates the mean value with the sd of three replications. PI, Pistil; PT, pollen tube; Tf, T. fournieri; Tb, T. baillonii; Tc, T. concolor; Lc, L. crustacea; Lm, L. micrantha. Bar = 50 μm.