The Pollen Tube Penetrates the Synergid Cell by Formation of a Peritubular Membrane

In flowering plants, successful reproduction relies on an exchange of signals between synergids and pollen tubes (PTs), mediating the invasion of a synergid by the PT, which then ruptures and releases two sperm cells to effect double fertilization. However, how exactly the PT invades the receptive synergid is unknown as the spatial relationship between these two cells is unclear. To better understand this process we performed 3D live imaging of PT reception in Arabidopsis thaliana. Upon arrival at the filiform apparatus (FA), a region rich in membrane folds at the micropylar pole of the synergids, the PT gradually deforms the FA before it rapidly grows into the receptive synergid. Upon penetration, the membrane of the receptive synergid invaginates and envelopes the PT. We termed this newly discovered structure the peri-tubular membrane (PRM). We show that, in feronia mutants disrupting PT reception, the PT still enters the receptive synergid, forming a normal PRM. This results in extensive invagination of the synergid membrane without sperm release. We show that PRM formation is associated with a cytosolic calcium ([Ca2+]cyt) spike of high amplitude in the PT and flooding of [Ca2+]cyt in the synergids. In PTs lacking AUTOINHIBITED Ca2+ ATPASE9 activity, PTs have lower amplitude [Ca2+]cyt spiking and the PTs frequently fail to penetrate the synergid. Our findings suggest that synergid penetration and the non-cell autonomous control of PT rupture are distinct regulated processes required for fertilization in flowering plants.