Regulation of Ovule Initiation in Arabidopsis thaliana by EPIDERMAL PATTERNING FACTOR-LIKE Proteins

Organ initiation and patterning in plants requires coordinated cell-to-cell communication to control cell growth and differentiation. In Arabidopsis thaliana, multiple ovules initiate asynchronously along the elongating placentae from stage 9 to stage 10 of floral development. Previous research has established a role for the phytohormone auxin in governing the site of ovule initiation, and for CUP-SHAPED COTYLEDON (CUC) transcription factors in regulation of ovule patterning. ERECTA family receptor-like kinase signaling via the EPIDERMAL PATTERNING FACTOR-LIKE 2 (EPFL2) secretory peptide ligand regulates ovule density and spacing during Arabidopsis fruit development, but the mechanisms underlying this regulation remain unclear. We further investigated the mechanism of ERECTA family signaling during ovule initiation in Arabidopsis. Our experiments established that EPFL1 and EPFL2 show distinct, cell-type specific expression throughout the process of ovule initiation. These two ligands positively regulate ovule number in a partially redundant manner without significantly affecting placenta length. To characterize the mechanisms of this regulation, hormone signaling, the expression of genes controlling ovule initiation, and genetic interactions were investigated using the epfl1 epfl2 mutant. While no clear defects were observed related to auxin, cytokinin, or gibberellins in the epfl1 epfl2 mutant, a synergistic reduction in ovule initiation was seen when the brassinosteroid related brassinazole resistant1-1D mutant or the boundary specific cup shaped cotyledon3 mutant were crossed with the epfl1 epfl2 mutant. The expression domain of DORNROSCHEN was expanded into boundary tissues in the epfl1 epfl2 mutant, and this suggests that EPFL1/2 may regulate early differentiation of cells during ovule initiation. In addition, synthetic biology was used to generate an inducible EPFL2 construct with expression is driven by the EPFL2 promoter as a future tool to probe the mechanisms and effects of EPFL2 signaling. Ultimately, this research further characterizes the mechanisms by which EPFL-mediated signaling positively regulates the initiation, spacing and outgrowth of ovules in Arabidopsis thaliana.