Comprehensive Analysis of OsENTH Genes and Functional Characterization in Rice Pollen Germination and Pollen Tube Growth

Abstract Endocytosis and intracellular trafficking of plasma membrane (PM)-resident proteins are crucial for regulating their activity and polarized localization. However, the molecular mechanisms underlying these processes in plants remain largely unknown. In this study, we investigated two rice pollen-preferred epsin N-terminal homology (ENTH) domain-containing proteins, OsENTH3 and OsENTH6. Among the six rice ENTH proteins these two with high sequence similarity and short C-terminal regions were predominantly expressed in pollen and pollen tubes. CRISPR/Cas9-induced osenth3/6 double mutants exhibited reduced fertility due to decreased pollen germination and impaired pollen tube growth. Functional analyses demonstrated that OsENTH3 could contribute to clathrin-mediated trafficking from the trans-Golgi network/early endosome to the PM, while OsENTH6 may act redundantly based on overlapping expression and mutant phenotypes. Moreover, the ENTH domain of OsENTH3 interacted with the C-terminal tails of pollen-preferential cyclic nucleotide-gated channels, suggesting a cargo-specific mechanism regulating calcium dynamics at the tip. These findings highlight a novel role of short C-terminal ENTH proteins in rice reproduction and provide insights into plant endocytosis and vesicle trafficking.