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
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.


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.


Egg 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).

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)


  1. Amborella Genome Project (2013) The Amborella genome and the evolution of flowering plants. Science 342:1241089.
  2. Amien S, Kliwer I, Márton ML, Debener T, Geiger D, Becker D, Dresselhaus T (2010) Defensin-like ZmES4 mediates pollen tube burst in maize via opening of the potassium channel KZM1. PLoS Biol 8:e1000388CrossRefGoogle Scholar
  3. Anderson SN, Johnson CS, Jones DS, Conrad LJ, Gou X, Russell SD, Sundaresan V (2013) Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. Plant J 76:729–741. CrossRefGoogle Scholar
  4. Bell CD, Soltis DE, Soltis PS (2005) The age of the angiosperms: a molecular timescale without a clock. Evolution 59:1245–1258. CrossRefGoogle Scholar
  5. Bray NL, Pimentel H, Melsted P, Pachter L (2016) Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34:525. CrossRefGoogle Scholar
  6. Campbell L, Turner SR (2017) A comprehensive analysis of RALF proteins in green plants suggests there are two distinct functional groups. Front Plant Sci 8:37. CrossRefGoogle Scholar
  7. Cao Y, Russell SD (1997) Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica. Sex Plant Reprod 10:368–373. CrossRefGoogle Scholar
  8. Chen SH, Yang YH, Liao JP, Kuang AX, Tian HQ (2008) Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge. Zygote 16:179–186. CrossRefGoogle Scholar
  9. Chen J, Strieder N, Krohn NG, Cyprys P, Sprunck S, Engelmann JC, Dresselhaus T (2017) Zygotic genome activation occurs shortly after fertilization in maize. Plant Cell 29:2106–2125CrossRefGoogle Scholar
  10. Conesa A et al (2016) A survey of best practices for RNA-seq data analysis. Genome Biol 17:1–13. CrossRefGoogle Scholar
  11. Doyle JA (2012) Molecular and fossil evidence on the origin of angiosperms. In: Jeanloz R (ed) Annual review of earth and planetary sciences, vol 40. Department of Evolution and Ecology, University of California, Davis, pp 301–326Google Scholar
  12. Dresselhaus T, Sprunck S, Wessel GM (2016) Fertilization mechanisms in flowering plants. Curr Biol 26:R125–R139. CrossRefGoogle Scholar
  13. Edstam MM, Viitanen L, Salminen TA, Edqvist J (2011) Evolutionary history of the non-specific lipid transfer proteins. Mol Plant 4:947–964. CrossRefGoogle Scholar
  14. Englhart M, Šoljić L, Sprunck S (2017) Manual isolation of living cells from the Arabidopsis thaliana female gametophyte by micromanipulation. In: Schmidt A (ed) Plant germline development: methods and protocols. Springer, New York, pp 221–234. CrossRefGoogle Scholar
  15. Friedman WE (2006) Embryological evidence for developmental lability during early angiosperm evolution. Nature 441:337–340CrossRefGoogle Scholar
  16. Friedman WE (2008) Hydatellaceae are water lilies with gymnospermous tendencies. Nature 453:94–97CrossRefGoogle Scholar
  17. Friedman WE, Ryerson KC (2009) Reconstructing the ancestral female gametophyte of angiosperms: insights from Amborella and other ancient lineages of flowering plants. Am J Bot 96:129–143. CrossRefGoogle Scholar
  18. Ge Z, Bergonci T, Zhao Y, Zou Y, Du S, Liu MC, Luo X, Ruan H, García-Valencia LE, Zhong S, Hou S, Huang Q, Lai L, Moura DS, Gu H, Dong J, Wu HM, Dresselhaus T, Xiao J, Cheung AY, Qu LJ (2017) Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling. Science 358:1596–1600. CrossRefGoogle Scholar
  19. Haruta M, Sabat G, Stecker K, Minkoff BB, Sussman MR (2014) A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 343:408–411CrossRefGoogle Scholar
  20. He E-M, Wang Y-Y, Liu H-H, Zhu X-Y, Tian H (2012) Egg cell isolation in Datura stramonium (Solanaceae). Ann Bot Fenn 49:7–12. CrossRefGoogle Scholar
  21. Higashiyama T, Takeuchi H (2015) The mechanism and key molecules involved in pollen tube guidance. Annu Rev Plant Biol 66:393–413CrossRefGoogle Scholar
  22. Higashiyama T, Yang W-c (2017) Gametophytic pollen tube guidance: attractant peptides, gametic controls, and receptors. Plant Physiol 173:112–121CrossRefGoogle Scholar
  23. Holm PB, Knudsen S, Mouritzen P, Negri D, Olsen FL, Roue C (1994) Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6:531–543. CrossRefGoogle Scholar
  24. Hoshino Y, Murata N, Shinoda K (2006) Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump. Ann Bot 97:1139–1144. CrossRefGoogle Scholar
  25. Hu S-Y, L-g Li, Zhu C (1985) Isolation of viable embryo sacs and their protoplasts of Nicotiana tabacum. Acta Bot Sin 27:343–347Google Scholar
  26. Huang B-Q, Russell SD (1992) Female germ unit: organization, isolation, and function. Int Rev Cytol 140:233–293CrossRefGoogle Scholar
  27. Huang B-Q, Pierson ES, Russell SD, Tiezzi A, Cresti M (1992) Video microscopic observations of living, isolated embryo sacs of Nicotiana and their component cells. Sex Plant Rep 5:156–162. Google Scholar
  28. Jones-Rhoades MW, Borevitz JO, Preuss D (2007) Genome-wide expression profiling of the Arabidopsis female gametophyte identifies families of small, secreted proteins. PLoS Genet 3:1848–1861CrossRefGoogle Scholar
  29. Katoh N, Lorz H, Kranz E (1997) Isolation of viable egg cells of rape (Brassica napus L.). Zygote 5:31–33CrossRefGoogle Scholar
  30. Kersey PJ et al (2016) Ensembl genomes 2016: more genomes, more complexity. Nucleic Acids Res 44:D574–D580. CrossRefGoogle Scholar
  31. Kovács M, Barnabás B, Kranz E (1994) The isolation of viable egg cells of wheat (Triticum aestivum L.). Sex Plant Reprod 7:311–312. CrossRefGoogle Scholar
  32. Kranz E, Bautor J, Lörz H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4:12–16. Google Scholar
  33. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  34. Leljak-Levanić D, Juranić M, Sprunck S (2013) De novo zygotic transcription in wheat (Triticum aestivum L.) includes genes encoding small putative secreted peptides and a protein involved in proteasomal degradation. Plant Reprod 26:267–285. CrossRefGoogle Scholar
  35. Lin M-Z, Chen L, Zhu X-Y, Tian H-Q, Teixeira da Silva JA (2012) Isolation of eggs and synergids in Ceiba speciosa. Ann Bot Fenn 49:229–233. CrossRefGoogle Scholar
  36. Maheshwari P (1950) An introduction to the embryology of angiosperms. McGraw-Hill, New YorkCrossRefGoogle Scholar
  37. Márton ML, Cordts S, Broadhvest J, Dresselhaus T (2005) Micropylar pollen tube guidance by egg apparatus 1 of maize. Science 307:573. CrossRefGoogle Scholar
  38. Márton ML, Fastner A, Uebler S, Dresselhaus T (2012) Overcoming hybridization barriers by the secretion of the maize pollen tube attractant ZmEA1 from Arabidopsis ovules. Curr Biol 22:1194–1198CrossRefGoogle Scholar
  39. Mól R (1986) Isolation of protoplasts from female gametophytes of Torenia fournieri. Plant Cell Rep 5:202–206. CrossRefGoogle Scholar
  40. Ohnishi T, Takanashi H, Mogi M, Takahashi H, Kikuchi S, Yano K, Okamoto T, Fujita M, Kurata N, Tsutsumi N (2011) Distinct gene expression profiles in egg and synergid cells of rice as revealed by cell type-specific microarrays. Plant Physiol 155:881–891. CrossRefGoogle Scholar
  41. Ohshika K, Ikeda H (1994) Isolation and preservation of the living embryo sac of Crinum asiaticum L. var. japonicum baker. J Plant Res 107:17–21. CrossRefGoogle Scholar
  42. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458:357–361. CrossRefGoogle Scholar
  43. Proost S, Mutwil M (2018) CoNekT: an open-source framework for comparative genomic and transcriptomic network analyses. Nucleic Acids Res 46:W133–W140. CrossRefGoogle Scholar
  44. Rademacher S, Sprunck S (2013) Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey. Plant Signal Behav 8:e27377CrossRefGoogle Scholar
  45. Resentini F et al (2017) SUPPRESSOR OF FRIGIDA (SUF4) supports gamete fusion via regulating Arabidopsis EC1 gene expression. Plant Physiol 173:155–166. CrossRefGoogle Scholar
  46. Rudall PJ, Remizowa MV, Beer AS, Bradshaw E, Stevenson DW, Macfarlane TD, Tuckett RE et al (2008) Comparative ovule and megagametophyte development in Hydatellaceae and water lilies reveal a mosaic of features among the earliest angiosperms. Ann Bot 101:941–956CrossRefGoogle Scholar
  47. Scutt CP (2018) The origin of angiosperms. In: Nuño de la Rosa L, Müller GB (eds) Evolutionary developmental biology. Springer, Berlin. Google Scholar
  48. Sprunck S, Groß-Hardt R (2011) Nuclear behavior, cell polarity and cell specification in the female gametophyte. Sex Plant Reprod 24:123–136CrossRefGoogle Scholar
  49. Sprunck S, Baumann U, Edwards K, Langridge P, Dresselhaus T (2005) The transcript composition of egg cells changes significantly following fertilization in wheat (Triticum aestivum L.). Plant J 41:660–672. CrossRefGoogle Scholar
  50. Sprunck S, Rademacher S, Vogler F, Gheyselinck J, Grossniklaus U, Dresselhaus T (2012) Egg cell-secreted EC1 triggers sperm cell activation during double fertilization. Science 338:1093–1097CrossRefGoogle Scholar
  51. Sprunck S, Hackenberg T, Englhart M, Vogler F (2014) Same same but different: sperm-activating EC1 and ECA1 gametogenesis-related family proteins. Biochem Soc Trans 42:401–407. CrossRefGoogle Scholar
  52. Steffen JG, Kang IH, Macfarlane J, Drews GN (2007) Identification of genes expressed in the Arabidopsis female gametophyte. Plant J 51:281–292. CrossRefGoogle Scholar
  53. Strasburger E (1879) Die Angiospermen und die Gymnospermen. Fischer, Jena, p 1879CrossRefGoogle Scholar
  54. Takeuchi H, Higashiyama T (2012) A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol 10:e1001449. CrossRefGoogle Scholar
  55. Tekleyohans DG, Nakel T, Groß-Hardt R (2017) Patterning the female gametophyte of flowering plants. Plant Physiol 173:122–129. CrossRefGoogle Scholar
  56. Tian HQ, Russell SD (1997) Micromanipulation of male and female gametes of Nicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture. Plant Cell Rep 16:657–661. CrossRefGoogle Scholar
  57. Tobe H, Kimoto Y, Prakash N (2007) Development and structure of the female gametophyte in Austrobaileya scandens (Austrobaileyaceae). J Plant Res 120:431–436CrossRefGoogle Scholar
  58. Uchiumi T, Komatsu S, Koshiba T, Okamoto T (2006) Isolation of gametes and central cells from Oryza sativa L. Sex Plant Rep 19:37–45. CrossRefGoogle Scholar
  59. van der Maas HM, Zaal MACM, de Jong ER, Krens FA, Van Went JL (1993) Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.). Protoplasma 173:86–89. CrossRefGoogle Scholar
  60. Van Went JL, Kwee H-S (1990) Enzymatic isolation of living embryo sacs of Petunia. Sex Plant Rep 3:257–262. CrossRefGoogle Scholar
  61. Williams JH, Friedman WE (2004) The four-celled female gametophyte of Illicium (Illiciaceae; Austrobaileyales): implications for understanding the origin and early evolution of monocots, eumagnoliids, and eudicots. Am J Bot 91:332–351CrossRefGoogle Scholar
  62. Willis K, McElwain J (2013) The evolution of plants, 2nd edn. Oxford University Press, Oxford, ISBN: 9780199292233Google Scholar
  63. Wuest SE et al (2010) Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr Biol 20:506–512. CrossRefGoogle Scholar
  64. Yang SJ, Mei Wei D, Tian H (2015) Isolation of sperm cells, egg cells, synergids and central cells from Solanum verbascifolium L. J Plant Biochem Biot 24:400–407. CrossRefGoogle Scholar
  65. Zhang D (2009) Homology between DUF784, DUF1278 domains and the plant prolamin superfamily typifies evolutionary changes of disulfide bonding patterns. Cell Cycle 8:3428–3430. CrossRefGoogle Scholar
  66. Zhou L-Z, Juranic M, Dresselhaus T (2017) Germline development and fertilization mechanisms in maize. Mol Plant 10:389–401CrossRefGoogle Scholar

Copyright information

© 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

Personalised recommendations