A brassinosteroid receptor kinase is required for sex determination in the homosporous fern Ceratopteris richardii

Most ferns, unlike all seed plants, are homosporous and produce sexually undifferentiated spores. Sex in many homosporous ferns is environmentally regulated by the biosynthesis of antheridiogens. Secreted antheridiogen from female or hermaphrodite gametophytes is perceived by undetermined gametophytes which respond by developing as male. In the fern Ceratopteris richardii (Ceratopteris), several hermaphroditic (her) mutants develop as hermaphrodites even in the presence of antheridiogen. A set of five mutant alleles are tightly linked to her7, a locus of unknown molecular identity. The immense size of the Ceratopteris genome has made efficient identification of the underlying genes a challenge. Here, we used bulked segregant RNA-seq (BSR-Seq) to map two her mutants (her7-14 and her7-19) to a 16 Mbp interval on chromosome 29 of the Ceratopteris genome. A brassinosteroid receptor-like kinase (BRL) within this interval encoded a deletion mutation in her7-14 and a missense mutation in her7-19. Three other linked her mutants (her7-1, her7-11, and her7-15) encoded missense mutations in the same gene, which we name HER7, providing strong support for HER7 being required for antheridiogen-mediated sex determination in Ceratopteris. HER7-GFP fusion protein is localized in the plasma membrane and cytoplasm, consistent with its predicted function as a brassinosteroid receptor kinase. RNA-seq analysis of gene expression shows that numerous transcripts encoding enzymes, including those for brassinosteroid biosynthesis, accumulate in hermaphrodites compared to male gametophytes. The antheridiogen of Ceratopteris is currently unknown and our finding that a BRL mutant is insensitive to antheridiogen suggests that antheridiogen in Ceratopteris may be a brassinosteroid. Additional work is required to fully resolve the sex determination pathway and determine the chemical composition of antheridiogen in this species. Our work demonstrates we can identify the molecular basis of mutants in large genomes, such as those in homosporous ferns, which can be further applied to the extensive remaining collection of mutants in Ceratopteris.