Spatial organization and trafficking dynamics of ANX/BUPS-RALF-LLG signaling complexes during pollen tube growth

Recruitment of RALFs and LLGs for assembly at the apical plasma membrane of pollen tubes is orchestrated by ANXs/BUPSs through endomembrane trafficking. Pollen tube growth requires precise regulation of cell wall integrity, which is maintained by ANX/BUPS-RALF-LLG signaling complexes. While structural and biochemical studies have revealed physical interactions between these components, their spatial organization and assembly dynamics in growing pollen tubes remain unclear. Here, we systematically investigated the subcellular localization and endomembrane trafficking of ANX/BUPS-RALF-LLG complex components through transient expression studies in tobacco pollen tubes. We demonstrate that each component exhibits distinct subcellular distribution patterns: RALF4/19 peptide ligands predominantly localize after secretion to the cell wall, ANXs/BUPSs receptors are enriched at the apical plasma membrane (PM), while GPI-anchored LLG2/3 co-receptors exclusively co-localize to cytosolic vesicles. Through co-expression studies, we show that ANXs/BUPSs recruit both RALFs and LLGs that localize to the same vesicles to the PM, indicating their role as master organizers and scaffolds of signaling complex assembly during pollen tube growth. RALFs stabilize ANX/BUPS-LLG interactions at the PM. Disruption of endocytosis by Brefeldin A treatment severely disrupts PM localization of ANXs/BUPSs and causes cytoplasmic aggregation of LLGs, while Wortmannin leads to partial trapping of proteins in pre-vacuolar compartments. Altogether, these findings indicate that ANX/BUPS-RALF-LLG complex assembly is highly dynamic and depends on proper endomembrane trafficking to maintain pollen tube integrity during growth.