ARF2–ARF4 and ARF5 are Essential for Female and Male Gametophyte Development in Arabidopsis

The plant hormone auxin plays critical roles in plant growth and development. Auxin response factors (ARFs) are a class of transcription factors which regulate auxin-mediated gene expression. While the functions of ARFs in sporophytic development have been well characterized, their functions specific to gametophytic development have not been studied extensively. In this study, Arabidopsis ARF genes were selectively down-regulated in gametophytes by misexpression of targeted microRNAs (amiRARF234, amiRARFMP and MIR167a) to silence AtARF2-AtAEF4, AtARF5, AtARF6 and AtARF8. Embryo sacs in amiRARF234- and amiRARFMP-expressing plants exhibited identity defects in cells at the micropylar pole, such as formation of two cells with egg cell-like morphology, concomitant with loss of synergid marker expression and seed abortion. The pollen grains of the transgenic plants were morphologically aberrant and unviable, and the inclusions and nuclei were lost in the abnormal pollen grains. However, plants misexpressing MIR167a showed no obvious abnormal phenotypes in the embryo sacs and pollen grains. Overall, these results provide evidence that AtARF2-AtARF4 and AtARF5 play significant roles in regulating both female and male gametophyte development in Arabidopsis.