The Medusa Mutant Disrupts Ovule Integument Identity Specification and Female Gametophyte Development in Mimulus nudatus

Here we identify and characterize a spontaneous mutant allele that disrupts the initiation and specification of the ovule integument in Mimulus nudatus. The mutant phenotype is characterized by the early arrest of the ovule integument or alternatively, the delayed development of a carpel-like structure in place of the integument. Additionally, medusa mutant ovule primordia fail to develop female gametophytic structures. Morphological analyses indicate that a nucellar domain is specified within the ovule and a megaspore-like cell is often specified, but that cell aborts before generating the female gametophyte. We mapped the position of the mutation to a location on Chromosome1. Mapping revealed a primary candidate gene that was a member of a family of MADS-domain containing transcription factors with sequence similarity to the Arabidopsis BEL1 gene (At5G41410) and is an orthologue of the Mimulus guttatus gene MgTOL.A0789.1. Analysis of the sequence of the Mimulus nudatus homolog (referred hereafter as MnBEL1) locus, revealed the insertion of a mitochondrial DNA genomic fragment within the MnBEL1 locus that segregated with the medusa mutant phenotype. The mitochondrial insertion sequence is predicted to disrupt the coding sequence of MnBEL1. RNA expression analysis revealed dramatically reduced levels of the expression of the MnBEL1 in gynoecia from medusa mutant plants relative to wild type sisters. RNA seq analysis revealed that the medusa mutant seedpods failed to express MnBEL1 at wildtype levels. RNA sequence data comparisons between wildtype and medusa mutant seedpods also identified a set of genes, that are expressed in wildtype seedpods, but that are expressed at very low levels or that are not detected in the medusa seed pods. Homologues of many of these genes have been previously shown to be strongly expressed in female gametophyte in Arabidopsis. This RNA seq analysis further indicates a strong developmental disruption and identifies sets of genes with expected roles in female gametophyte development in Mimulus species for further investigation. Our evidence strongly suggests that MnBEL1 is a true functional orthologue of AtBEL1. To the best of our knowledge, these studies provide the first mutational analysis of a BEL1 orthologue outside of Arabidopsis and indicate the functional conservation of this gene between Mimulus and Arabidopsis. This work also presents the first identification of a mutational event in Mimulus that is linked to an insertion of a mitochondrial sequence in the nuclear genome. An inbreed line (form from ≥ 6 generations of self-fertilization) of Mimulus nudatus (GMR2) was grown in a chamber at 18-hour days, 21°C, 30% relative humidity. Mutant plants showing carpeloid ovules were serendipitously discovered among the population and were selected as pollen donors for further genetic crosses with wild-type (Wt) plants for the generation of F1, and F2 plants. Since the carpeloid ovule phenotype is recessive, and it is only manifested in the F2 generation, 150 F2 plants were screened phenotypic- and genotypically to identify heterozygous/homozygous Wt and mutant lines. Because the carpeloid ovules are difficult to detached from the septum, we isolated total RNA as follows: three biological replicates of homozygous Wt and mutant plants were selected for harvesting 6 gynoeciums/plant from opened flowers, and then the valves were removed to collect the ovules attached to the septum. Ovules/septum were immediately collected in cold TRIzol LS (Ambion) for further total RNA isolation. We used the RNeasy Micro Kit (Qiagen) with the following modifications: ovules/septum in cold TRIzol were ground gently with a pellet pestle in a 1.7 mL microcentrifuge tube for 2 min then mixed with 220 uL of chloroform. The solution was then shaken vigorously by hand for 15 s, then incubated for 2 min at room temperature followed by a centrifugation at 12,000g for 15 min at 4 °C to separate phases. Total RNA was isolated according to the manufacturer’s instructions, which includes in column Dnase-I treatment for removal of contaminated DNA. We generated and sequenced a total of 6 RNA sequencing libraries. The RNA integrity number for all samples >7.3, as determined by an Agilent 2100 Bioanalyzer. Six strand-specific complementary DNA (cDNA) libraries were prepared from approximately 100 ng of total RNA by the North Carolina State University Genomic Science Laboratory (GSL) using a NEB Ultra Directional Library Prep Kit for Illumina then sequenced on an Illumina NovaSeq 6000. Average size of the cDNA fragment was approximately 350 bp.