RALF signaling pathway activates MLO calcium channels to maintain pollen tube integrity

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.