Pollen tube tip growth requires intricate Ca²⁺ signaling. Recent studies have also identified rapid alkalization factor (RALF)-family peptides and their receptors as critical components for pollen tube tip growth and integrity. The functional relationship of RALF and calcium signaling modules remains largely unclear. Here we report that disruption of RALF signaling pathway abolished the cytosolic Ca²⁺ gradient in the pollen tube, indicating that Ca²⁺ signaling is downstream of the RALF signaling pathway. We identified MILDEW RESISTANCE LOCUS O (MLO) family proteins MLO1, 5, 9, 15, as Ca²⁺ channels required for Ca²⁺ influx and pollen tube integrity. We further reconstituted the biochemical pathway in which signaling via RALF and RALF receptors activated MLO1/5/9/15 calcium channels. Together, we conclude that RALF peptides derived from pollen tube bind to their receptors to establish pollen tube Ca²⁺ gradient through activation of the MLO channels. Our finding has thus provided a mechanistic link between the RALF signaling pathway and Ca²⁺ signaling in controlling pollen tube integrity and growth.

Precise signalling between pollen tubes and synergid cells in the ovule initiates fertilization in flowering plants¹. Contact of the pollen tube with the ovule triggers calcium spiking in the synergids^2,3 that induces pollen tube rupture and sperm release. This process, termed pollen tube reception, entails the action of three synergid-expressed proteins in Arabidopsis: FERONIA (FER), a receptor-like kinase; LORELEI (LRE), a glycosylphosphatidylinositol-anchored protein; and NORTIA (NTA), a transmembrane protein of unknown function^4-6. Genetic analyses have placed these three proteins in the same pathway; however, it remains unknown how they work together to enable synergid-pollen tube communication. Here we identify two pollen-tube-derived small peptides⁷ that belong to the rapid alkalinization factor (RALF) family⁸ as ligands for the FER-LRE co-receptor, which in turn recruits NTA to the plasma membrane. NTA functions as a calmodulin-gated calcium channel required for calcium spiking in the synergid. We also reconstitute the biochemical pathway in which FER-LRE perceives pollen-tube-derived peptides to activate the NTA calcium channel and initiate calcium spiking, a second messenger for pollen tube reception. The FER-LRE-NTA trio therefore forms a previously unanticipated receptor-channel complex in the female cell to recognize male signals and trigger the fertilization process.