Abstract
Key messageA protocol for the isolation of egg apparatus cells from the basal angiosperm Amborella trichopoda to generate RNA-seq data for evolutionary studies of fertilization-associated genes.Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm Amborella trichopoda is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate Amborella egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed Amborella-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in Amborella egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.
Highlights
Molecular and fossil evidence indicates that flowering plants arose abruptly in the late Jurassic/early Cretaceous from a not yet identified ancestral lineage
We report here an isolation procedure for Amborella egg apparatus cells by manually dissecting carpels of female flowers, without treating ovules with cell wall-degrading enzymes
A similar function for Amborella EGG CELL 1 (EC1) during double fertilization remains to be experimentally confirmed, our findings strongly suggest that the molecular mechanism of EC1-mediated sperm activation by the egg cell, as previously reported for Arabidopsis EC1 (Sprunck et al 2012; Rademacher and Sprunck 2013), existed already in the most recent common ancestor of all extant flowering plants
Summary
Molecular and fossil evidence indicates that flowering plants (angiosperms) arose abruptly in the late Jurassic/early Cretaceous from a not yet identified ancestral lineage. The highly reduced female gametophyte (embryo sac) develops within the ovule and contains the egg cell and central cell. Both cells become fertilized and give rise to the embryo and the embryo-nourishing endosperm of the seed, respectively. A large majority of extant angiosperms produce the monosporic Polygonum-type embryo sac, which was first described in Polygonum divaricatum (Strasburger 1879). This originates from a single haploid spore (the functional megaspore), which undergoes three incomplete mitotic division cycles to develop into an eight-nucleate syncytium. Three antipodal cells form at the opposite chalazal end of the embryo sac, but little is known about their function (Sprunck and Groß-Hardt 2011)
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