Functional characterization reveals the importance of Arabidopsis ECA4 and EPSIN3 in clathrin mediated endocytosis and wall structure in apical growing cells

Localized clathrin mediated endocytosis is vital for secretion and wall deposition in apical growing plant cells. Adaptor and signalling proteins, along with phosphoinositides, are known to play a regulatory, yet poorly defined role in this process. Here we investigated the function of Arabidopsis ECA4 and EPSIN3, putative mediators of the process, in pollen tubes and root hairs. Homozygous eca4 and epsin3 plants exhibited altered pollen tube morphology (in vitro) and self-pollination led to fewer seeds and shorter siliques. These effects were augmented in eca4/epsin3 double mutant and quantitative polymerase chain reaction data revealed changes in phosphoinositide metabolism and flowering genes suggestive of a synergistic action. No visible changes were observed in root morphology, but atomic force microscopy in mutant root hairs showed altered structural stiffness. Imaging and FRET-FLIM analysis of ECA4 and EPSIN3 X-FP constructs revealed that both proteins interact at the plasma membrane but exhibit slightly different intracellular localization. FT-ICR-MS metabolomic analysis of mutant cells showed changes in lipids, amino acids and carbohydrate composition consistent with a role in secretion and growth. Characterization of double mutants of eca4 and epsin3 with phospholipase C genes (plc5, plc7) indicates that phosphoinositides (e.g. PtdIns(4,5)P₂) are fundamental for a combined and complementary role of ECA4-EPSIN3 in cell secretion.