Embryo sac cellularization defects lead to supernumerary egg cells and twin embryos in Arabidopsis thaliana

Arabidopsis lines with loss-of-function mutation in Embryo sac-specific Pectin MethylEsterase Inhibitor (Atepmei) gene showed seed sterility with embryo sac cellularization defects. Examination of tissue-cleared mature ovules revealed irregularly positioned nuclei/embryos within the embryo sacs. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of ruthenium red and tannic acid-ferric chloride staining of developing Atepmei mutant ovules showed that cell wall formation and maintenance were altered around embryo sac nuclei, which also coincided with change in the gamete specification. This report implicates the role of cell walls in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.