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. <i>EPAD1</i>, 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 <i>EPAD1</i> in Poaceae, we performed an analysis on four <i>EPAD1</i> orthologs from two distinct sub-clades within the Poaceae family. The two functional redundant barley <i>EPAD1</i> genes (<i>HvEPAD1</i> and <i>HvEPAD2</i>) from the BOP clade, along with the single copy of sorghum (<i>SbEPAD1</i>) and millet (<i>SiEPAD1</i>) from the PACMAD clade were examined. The CRISPR-Cas9-generated mutants all exhibited defects in pollen wall formation, consistent with previous findings on <i>EPAD1</i> in rice and wheat. Interestingly, in barley, <i>hvepad2</i> single mutant also showed apical spikelets abortion, aligning with a decreased expression level of <i>HvEPAD1</i> and <i>HvEPAD2</i> from the apical to the bottom of the spike. Our finding provides evidence that <i>EPAD1</i> orthologs contribute to Poaceae specific pollen exine pattern formation via maintaining primexine integrity despite potential variations in copy numbers across different species.