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Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells

  • María Flores-Tornero
  • Sebastian Proost
  • Marek Mutwil
  • Charles P. Scutt
  • Thomas Dresselhaus
  • Stefanie SprunckEmail author
Methods Paper
First Online:
Part of the following topical collections:
  1. Cellular Omics Methods in Plant Reproduction Research

Key message

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.

Abstract

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.

Keywords

Egg cell Synergid cell Microdissection EC1 RALF RNA-seq Amborella 
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Notes

Acknowledgements

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).

Supplementary material

497_2019_361_MOESM1_ESM.pdf (1.1 mb)
Supplementary Figure 1 Electropherograms of total RNA samples isolated from tepals, leaves and roots. (PDF 1115 kb)
497_2019_361_MOESM2_ESM.pdf (810 kb)
Supplementary Figure 2 Principal Component Analysis (PCA). (PDF 809 kb)
497_2019_361_MOESM3_ESM.pdf (306 kb)
Supplementary Figure 3 (PDF 305 kb)
497_2019_361_MOESM4_ESM.xlsx (7.4 mb)
Supplementary Table 1 Normalized gene expression values in egg apparatus cells of large (L)-, medium (M)- and small (S)-size categories in comparison with tepals, leaves and roots. (XLSX 7581 kb)
497_2019_361_MOESM5_ESM.pdf (2.2 mb)
Supplementary Table 2 Genes expressed in large (L), medium (M) and small (S) egg apparatus cell size categories (TPM ≥ 1) and their overlap. (PDF 2206 kb)
497_2019_361_MOESM6_ESM.xlsx (23 kb)
Supplementary Table 3 Top ten list of A. trichopoda genes enriched in large (L), medium (M), or small (S) cell size categories, respectively, but not expressed in sepals, leaves and roots (average TPM < 3). (XLSX 22 kb)
497_2019_361_MOESM7_ESM.xlsx (2.7 mb)
Supplementary Table 4Amborella trichopoda expressed genes (TPM ≥ 1) in large (L), medium (M), and small (S) egg apparatus cell size categories and average expression values in the control tissues. (XLSX 2753 kb)

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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • María Flores-Tornero
    • 1
  • Sebastian Proost
    • 2
    • 5
  • Marek Mutwil
    • 2
    • 3
  • Charles P. Scutt
    • 4
  • Thomas Dresselhaus
    • 1
  • Stefanie Sprunck
    • 1
    Email author
  1. 1.Cell Biology and Plant Biochemistry, Biochemie-Zentrum RegensburgUniversity of RegensburgRegensburgGermany
  2. 2.Max-Planck Institute for Molecular Plant PhysiologyPotsdamGermany
  3. 3.School of Biological SciencesNanyang Technological UniversitySingaporeSingapore
  4. 4.Laboratoire Reproduction et Développement des PlantesÉcole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, CNRS, INRA, Université de LyonLyonFrance
  5. 5.Laboratory of Molecular Bacteriology (Rega Institute)KU LeuvenLouvainBelgium

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