Three-dimensional ultrastructural analysis of fertilization-defective Arabidopsis mutants using STEM and array tomography.

Double fertilization in angiosperms is completed by the fusion of two sperm cells with the egg and central cells. While sperm membrane proteins such as GEX2, DMP8/9, and GCS1/HAP2 have been identified as key regulators of gamete attachment and fusion, their specific ultrastructural roles within the embryo sac have remained unclear owing to the technical limitations of live-cell imaging based on fluorescent markers. Here, we optimized high-pressure freezing and freeze-substitution protocols for Arabidopsis pistils and performed three-dimensional reconstructions using scanning transmission electron microscopy (STEM) and array tomography. We identified an electron-dense, partially thickened structure at the apical region of the egg cell before pollination. This structure disappeared following wild-type fertilization, suggesting it serves as a specialized "docking site" to accommodate pollen tube contents and facilitate subsequent gamete interactions. Comparative analysis of fertilization mutants revealed distinct functional stages in the gamete fusion process. In gex2 mutants, sperm cells failed to establish robust connections when positioned at the lateral side of the egg cell, confirming the requirement of GEX2 for initial attachment. In contrast, dmp8 dmp9 double mutant sperm cells facing each other were observed, exhibiting a more distant position from the egg surface, suggesting that DMP8/9 is involved in maintaining stable membrane adhesion before fusion. Furthermore, although gcs1 sperm cells maintained intimate contact and displayed membrane thinning, they failed to undergo final fusion. Our findings demonstrate that STEM and array tomography are powerful tools for visualizing gamete dynamics, providing an integrated model of the transition from stable adhesion to membrane fusion.