Summary 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.

中文翻译：

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功能表征揭示了拟南芥 ECA4 和 EPSIN3 在网格蛋白介导的顶端生长细胞内吞作用和壁结构中的重要性


摘要 局部网格蛋白介导的内吞作用对于顶端生长植物细胞的分泌和壁沉积至关重要。众所周知，衔接蛋白和信号蛋白以及磷酸肌醇在此过程中起着调节作用，但定义不明确。在这里，我们研究了拟南芥 ECA4 和 EPSIN3 在花粉管和根毛中的功能，它们是该过程的推定介质。纯合子 eca4 和 epsin3 植物表现出花粉管形态改变（体外）和自花授粉导致种子减少和角果短。这些影响在 eca4/epsin3 双突变体中得到增强，定量聚合酶链反应数据揭示了磷酸肌醇代谢和开花基因的变化，表明协同作用。在根形态中没有观察到明显的变化，但突变根毛中的原子力显微镜显示结构刚度发生了变化。ECA4 和 EPSIN3 X-FP 构建体的成像和 FRET-FLIM 分析显示，两种蛋白质在质膜上相互作用，但表现出略有不同的细胞内定位。突变细胞的 FT-ICR-MS 代谢组学分析显示脂质、氨基酸和碳水化合物组成的变化与在分泌和生长中的作用一致。用磷脂酶 C 基因 （plc5， plc7） 对 eca4 和 epsin3 的双突变体的表征表明，磷酸肌醇（例如 PtdIns（4,5）P2）是 ECA4-EPSIN3 在细胞分泌中联合和互补作用的基础。