The synergids appear to be specialized secretory cells (Higashiyama and Inat-sugi, this volume). This feature led to the hypothesis that the synergids were a source for a chemotropic factor directing the pollen tube into the micropyle. This was directly tested using a semi-in vitro system developed for Torenia fournieri (Higashiyama et al. 1998; Higashiyama and Inatsugi, this volume) in which tubes grow through an excised style and out on to the surface of a culture medium before targeting the ovules placed on the surface of the medium. The FG of Torenia fournieri protrudes from the micropyle facilitating experimental manipulation and making it possible to analyze the role of the FG in tube guidance without intervening sporophytic cells. Laser ablation studies showed that at least one intact synergid was required for pollen tubes to target the FG (Higashiyama et al. 2001). The synergids were found to be the only FG cell type that is essential for attraction; ablation of the egg, central cell, or antipodals had no effect (Higashiyama et al. 2001).
Which phase of tube guidance as defined above for Arabidopsis is being analyzed in the Torenia system? In the Torenia in vitro system, tubes
Fig. 2 Arabidopsis FG mutants define phases 3-6 of pollen tube guidance. Wild type ovules attract a single tube that enters the micropyle and bursts within one of the two synergids. Ovules that contain no FG do not attract pollen tubes to the micropyle and also show defects in the patterns of tube exit from the transmitting tissue. Maa mutants attract multiple tubes but these fail to enter the micropyle. Myb98 mutant attracts multiple tubes to the funiculus but fails to attract them to the micropyle. Feronia and sirene attract tubes that fail to burst within the synergids
Fig. 2 Arabidopsis FG mutants define phases 3-6 of pollen tube guidance. Wild type ovules attract a single tube that enters the micropyle and bursts within one of the two synergids. Ovules that contain no FG do not attract pollen tubes to the micropyle and also show defects in the patterns of tube exit from the transmitting tissue. Maa mutants attract multiple tubes but these fail to enter the micropyle. Myb98 mutant attracts multiple tubes to the funiculus but fails to attract them to the micropyle. Feronia and sirene attract tubes that fail to burst within the synergids grow directly to the filiform apparatus, a specialized area of cell wall at the basal junction of the two synergids; they do not contact sporophytic cells. In vivo, Torenia pollen tubes grow directly from the placental surface to the filiform apparatus without contacting the funiculus (Higashiyama et al. 1998). Thus, Torenia appears not to have a phase analogous to funicular guidance in Arabidopsis. It is likely that the phase of guidance being interrogated in the Torenia system is analogous to the micropylar guidance phase in Arabidopsis.
Support for this idea comes from the recent analysis of the Arabidopsis FG mutant, myb98 (Kasahara et al. 2005). All cells of myb98 FGs develop normally except the synergids, which have a subtle and specific defect in the ultrastructure of the filiform apparatus. MYB98 is expressed specifically in the synergid cells and possibly functions as a transcription factor that regulates development of the filiform apparatus. Myb98 FGs attract pollen tubes to the funiculus, but have defects in micropylar targeting. These data strongly suggest that the synergids are the source of the micropylar guidance cue in Arabidopsis and that the filiform apparatus is required for production and/or secretion of this chemotropic guidance cue (Kasahara et al. 2005). Biochemical purification of the active factor in Torenia, further analysis of Arabidopsis FG mutants, and analysis of genes that are specifically expressed in the synergids are promising approaches that could lead to identification of this molecule in the near future. Large-scale genetic analysis of the Arabidopsis FG has yielded a very interesting group of 18 une mutants that have apparently normal FG development but remain unfertilized (Pagnussat et al. 2005). Six une mutants were identified that failed to attract pollen tubes; further analysis of the genes disrupted in these mutants, which include genes of unknown function (UNE1, UNE4), a calcium binding protein (UNE14) and a small LEA protein (UNE15) will be an exciting area for future studies. Additionally, analysis of FG-specific gene expression has lead to the identification of a candidate micropylar guidance factor in maize, EA1 (Marton et al. 2005; Sect. 5.4).
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