Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells
A 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.
KeywordsEgg cell Synergid cell Microdissection EC1 RALF RNA-seq Amborella
We are grateful to Maximilian Weigend, Cornelia Löhne and Bernhard Reinken (Botanical Garden of the University of Bonn, Germany) for providing Amborella plant material. We thank Maria Lindemeier for her support in single-cell collection. Illumina deep sequencing was carried out at a genomics core facility: Center of Excellence for Fluorescent Bioanalytics (KFB, University of Regensburg, Germany). This work was supported by the ERA-CAPS Grant EVOREPRO (DR 334/12-1) to SS and TD, funded by the Deutsche Forschungsgemeinschaft (DFG).
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