EPAD1 Orthologs Play a Conserved Role in Pollen Exine Patterning.

The pollen wall protects pollen during dispersal and is critical for pollination recognition. In the Poaceae family, the pollen exine stereostructure exhibits a high degree of conservation with similar patterns across species. However, there remains controversy regarding the conservation of key factors involved in its formation among various Poaceae species. , as a gene specific to the Poaceae family, and its orthologous genes play a conserved role in pollen wall formation in wheat and rice. However, they do not appear to have significant functions in maize. To further confirm the conserved function of in Poaceae, we performed an analysis on four orthologs from two distinct sub-clades within the Poaceae family. The two functional redundant barley genes ( and ) from the BOP clade, along with the single copy of sorghum () and millet () from the PACMAD clade were examined. The CRISPR-Cas9-generated mutants all exhibited defects in pollen wall formation, consistent with previous findings on in rice and wheat. Interestingly, in barley, single mutant also showed apical spikelets abortion, aligning with a decreased expression level of and from the apical to the bottom of the spike. Our finding provides evidence that orthologs contribute to Poaceae specific pollen exine pattern formation via maintaining primexine integrity despite potential variations in copy numbers across different species.