Year : 2009 Disruption of the pollen-expressed FERONIA homologs ANXUR 1 and ANXUR 2 triggers pollen tube discharge

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization. 3279 RESEARCH ARTICLE INTRODUCTION In eukaryotes, complex and specialized mechanisms have evolved to achieve the union of male and female gametes at fertilization by regulating attraction, signal exchange and recognition between the gametes (Boavida et al., 2005; Dresselhaus, 2006; Hirohashi et al., 2008; Hiscock and Allen, 2008; Kothe, 2008; Lord and Russell, 2002). In plants, the gametes are not produced directly from the meiotic products as in animals, but rather from multicellular, haploid gametophytes. Sperm cells of flowering plants are non-motile and need to be delivered via the haploid male gametophyte (pollen) (McCormick, 2004) to the female gametophytes (embryo sacs). The latter are enclosed by the diploid tissues of the ovules that are themselves deeply embedded in maternal tissues of the pistil. In compatible interactions, the pollen grain germinates on the stigma of the pistil producing a pollen tube (PT) that grows through the style into the transmitting tissue, from where it emerges and is guided to the micropylar opening of the ovule (Crawford and Yanofsky, 2008; Dumas and Rogowsky, 2008). In the plant model Arabidopsis, typically a single PT is guided to each ovule, enters the micropyle and penetrates the female gametophyte. In Arabidopsis, the female gametophyte is a seven-celled haploid structure that contains an egg cell flanked by two synergid cells, a central cell and three antipodals (Kägi and Gross-Hardt, 2007; Yadegari and Drews, 2004). The PT penetrates one of the two synergid cells, where it terminates its growth and ruptures to deliver the two sperm cells – processes collectively referred to as PT reception (Weterings and Russell, 2004). Subsequently, double fertilization initiates seed development: one of the sperm cells is transported to, and fuses with, the egg cell to form the zygote, whereas the second sperm fertilizes the central cell, thereby initiating endosperm development (Berger et al., 2008). The precise guidance of the PT from the stigma to the female gametophyte and successful PT reception followed by gamete fusions require several complex interactions between the pollen and the female tissues (Johnson and Lord, 2006). The identification of a few sporophytic factors that provide guidance cues at early stages of PT growth has been reported (e.g. Dong et al., 2005; Palanivelu et al., 2003; Wolters-Arts et al., 1998). However, the signals produced by the female gametophyte itself, which controls the late stages of PT guidance, were unknown (Chen et al., 2007; Higashiyama et al., 2001; Marton et al., 2005) until the recent identification of the defensin-like LURE polypeptides, which serve as the synergidsecreted PT attractants in Torenia fournieri (Okuda et al., 2009). As the PT reaches the micropyle, several interactions between the PT and the female gametophyte ensue. However, our knowledge about the molecular and genetic mechanisms that mediate the interactions between the gametophytes is very limited. In the female gametophytic Arabidopsis mutants feronia (fer) and sirène (srn), PTs reach the micropyle but do not arrest their growth and are unable to rupture (PT overgrowth phenotype), demonstrating that the female gametophyte controls PT reception (Huck et al., 2003; Rotman et al., 2003). This indicates that successful PT reception requires that female and male partners recognize each other (Huck et al., 2003; Rotman et al., 2003). Interestingly, FER, which is allelic to SRN, encodes a plasma membrane-localized, synergid-expressed receptor-like kinase (Escobar-Restrepo et al., 2007). Receptor-like serine-threonine kinases (RLKs) are transmembrane proteins that typically receive signals through their extracellular domain and subsequently activate signaling cascades via their intracellular Disruption of the pollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge Aurélien Boisson-Dernier1,2,*, Sucharita Roy1, Konstantinos Kritsas1, Monica A. Grobei1, Miloslawa Jaciubek1, Julian I. Schroeder2 and Ueli Grossniklaus1,* The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 doublemutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANXRLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.