The KLU–PRE module provides positional cues that maintain somatic cell identity around the megasporocyte cell in Arabidopsis

In most flowering plants, a single hypodermal cell from the ovule primordium differentiates into the megaspore mother cell (MMC) to initiate the female germline. However, how positional cues maintain this cell identity remains unclear. Here, we report that in Arabidopsis (Arabidopsis thaliana), the basic helix-loop-helix transcription factor genes PACLOBUTRAZOL RESISTANCE 1 (PRE1) and PRE2/3/5/6 (referred to as PREs), are expressed in the distal nucellus domain of ovule primordia. PRE proteins accumulate near the chalaza region of the ovule primordia, where the cytochrome P450 gene KLU (KLUH/CYP78A5)is also expressed. Ectopic expression of PREs driven by the KLU promoter or misexpression of KLU in the distal end of the ovule primordia caused the development of extra MMC-like cells. In the klu mutant, PRE protein localization extended toward the inner integument primordia and chalaza region. KLU physically interacts with PREs depending on the PRE M8 motif. The transcription factor BRASSINAZOLE-RESISTANT1 (BZR1) directly targets the PRE and KLU promoters and is expressed in all ovule primordia cells except the MMC. BZR1 physically interacts with SWR1 COMPLEX 6 (SWC6), and together they affect the chromatin state at PRE loci. In summary, the KLU-PRE module integrates brassinosteroid signaling with chromatin remodeling to establish positional cues that restrict MMC differentiation and female germline initiation to a single cell in Arabidopsis.