PCP-B peptides and CrRLK1L receptor kinases control pollination via pH gating of aquaporins in Arabidopsis

During pollen-stigma interaction, pollen coat protein B-class peptides (PCP-Bs) compete with stigmatic rapid alkalinization factor (RALF) for interaction with FERONIA/ANJEA receptor kinases (FER/ANJ), stimulating pollen hydration and germination. However, the molecular mechanism underlying PCP-Bs-induced, FER/ANJ-regulated compatible responses remains largely unknown. Through PCP-Bγ-induced phosphoproteomic analysis, we characterized a series of pollination-related signaling pathways regulated by FER/ANJ. Interestingly, on stigmatic papillary cells, pollen PCP-Bγ induced an elevation in cytosolic pH near the plasma membrane (PM), sustained by stigmatic RALF23/33 through regulation of the autoinhibited H⁺-ATPase 1/2 (AHA1/2) activity. We further found that RALFs/PCP-Bs and FER/ANJ regulated the pH alterations via phosphorylation of AHA1/2 C terminus. Furthermore, RALF23/33-FER/ANJ maintained the protonation of H197 in plasma membrane intrinsic proteins (PIPs), whereas PCP-B relieved the protonation through AHA activity. Altogether, this study reveals that pollen PCP-Bs trigger FER/ANJ-controlled compatible responses, particularly the opening of aquaporins via AHA-mediated pH changes, thereby facilitating pollen hydration in Arabidopsis.