Skip to main content
SpringerLink
Log in
Book cover

Plant Embryogenesis pp 127–139Cite as

Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2122))

Abstract

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Spencer MW, Casson SA, Lindsey K (2007) Transcriptional profiling of the Arabidopsis embryo. Plant Physiol 143:924–940

    Article  CAS  Google Scholar 

  2. Belmonte MF, Kirkbride RC, Stone SL et al (2013) Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc Natl Acad Sci U S A 110:E435–E444

    Article  CAS  Google Scholar 

  3. Slane D, Kong J, Berendzen KW et al (2014) Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo. Development 141:4831–4840

    Article  CAS  Google Scholar 

  4. Palovaara J, Saiga S, Wendrich JR et al (2017) Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo. Nat Plants 3:894–904

    Article  CAS  Google Scholar 

  5. Zhou X, Shi C, Zhao P, Sun M (2018) Isolation of living apical and basal cell lineages of early proembryos for transcriptome analysis. Plant Reprod 32(1):105–111. https://doi.org/10.1007/s00497-018-00353-6

    Article  CAS  PubMed  Google Scholar 

  6. Hofmann F, Schon MA, Nodine MD (2019) The embryonic transcriptome of Arabidopsis thaliana. Plant Reprod 32(1):77–91. https://doi.org/10.1007/s00497-018-00357-2

    Article  CAS  PubMed  Google Scholar 

  7. Gao P, Xiang D, Quilichini TD et al (2019) Gene expression atlas of embryo development in Arabidopsis. Plant Reprod 32(1):93–104. https://doi.org/10.1007/s00497-019-00364-x

    Article  CAS  PubMed  Google Scholar 

  8. Autran D, Baroux C, Raissig M et al (2011) Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 145:707–719

    Article  CAS  Google Scholar 

  9. Pignatta D, Gehring M, Missirian V, Henikoff S (2011) Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds. PLoS One 6:e23687

    Article  Google Scholar 

  10. Nodine MD, Bartel DP (2012) Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature 482:94–97

    Article  CAS  Google Scholar 

  11. Hsieh T, Shin J, Uzawa R et al (2011) Regulation of imprinted gene expression in Arabidopsis endosperm. Proc Natl Acad Sci U S A 108:1755–1762

    Article  CAS  Google Scholar 

  12. Pignatta D, Erdmann RM, Scheer E et al (2014) Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting. Elife 3:e03198

    Article  Google Scholar 

  13. Lu X, Chen D, Shu D et al (2013) The differential transcription network between embryo and endosperm in the early developing maize seed. Plant Physiol 162:440–455

    Article  CAS  Google Scholar 

  14. Chen J, Zeng B, Zhang M et al (2014) Dynamic transcriptome landscape of maize embryo and endosperm development. Plant Physiol 166:252–264

    Article  Google Scholar 

  15. Chen J, Strieder N, Krohn NG et al (2017) Zygotic genome activation occurs shortly after fertilization in maize. Plant Cell 29:2106–2125

    Article  CAS  Google Scholar 

  16. Itoh J, Sato Y, Sato Y et al (2016) Genome-wide analysis of spatiotemporal gene expression patterns during early embryogenesis in rice. Development 143:1217–1227

    Article  CAS  Google Scholar 

  17. Anderson SN, Johnson CS, Chesnut J et al (2017) The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes. Dev Cell 43:349–358

    Article  CAS  Google Scholar 

  18. Haig D (2013) Kin conflict in seed development: an interdependent but fractious collective. Annu Rev Cell Dev Biol 29:189–211

    Article  CAS  Google Scholar 

  19. Baroux C, Grossniklaus U (2019) Seeds-an evolutionary innovation underlying reproductive success in flowering plants. Curr Top Dev Biol 131:605–642

    Article  Google Scholar 

  20. Raissig MT, Gagliardini V, Jaenisch J et al (2013) Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds. J Vis Exp 76:e50371. https://doi.org/10.3791/50371

    Article  Google Scholar 

  21. Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In: Sanchez-Serrano JJ, Salinas J (eds) Arabidopsis Protocols. Humana Press, Totowa, NJ, pp 509–537

    Chapter  Google Scholar 

  22. Slane D, Kong J, Schmid M et al (2015) Profiling of embryonic nuclear vs. cellular RNA in Arabidopsis thaliana. Genom Data 4:96–98

    Article  Google Scholar 

  23. Deal RB, Henikoff S (2010) The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6:56–68

    Article  Google Scholar 

  24. Palovaara J, Weijers D (2018) Adapting INTACT to analyse cell-type-specific transcriptomes and nucleocytoplasmic mRNA dynamics in the Arabidopsis embryo. Plant Reprod 32(1):113–121. https://doi.org/10.1007/s00497-018-0347-0

    Article  CAS  PubMed  Google Scholar 

  25. Zanetti ME, Chang I-F, Gong F et al (2005) Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiol 138:624–635

    Article  CAS  Google Scholar 

  26. Basbouss-Serhal I, Soubigou-Taconnat L, Bailly C, Leymarie J (2015) Germination potential of dormant and nondormant Arabidopsis seeds is driven by distinct recruitment of messenger RNAs to polysomes. Plant Physiol 168:1049–1065

    Article  CAS  Google Scholar 

  27. Schön MA, Nodine MD (2017) Widespread contamination of Arabidopsis embryo and endosperm transcriptome data sets. Plant Cell 29:608–617

    Article  Google Scholar 

  28. Raissig MT, Bemer M, Baroux C, Grossniklaus U (2013) Genomic imprinting in the Arabidopsis embryo is partly regulated by PRC2. PLoS Genet 9:e1003862

    Article  Google Scholar 

  29. Wuest SE, Vijverberg K, Schmidt A et al (2010) Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes. Curr Biol 20:506–512

    Article  CAS  Google Scholar 

  30. Schmidt A, Wuest SE, Vijverberg K et al (2011) Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development. PLoS Biol 9:e1001155

    Article  CAS  Google Scholar 

  31. Schmid MW, Schmidt A, Klostermeier UC et al (2012) A powerful method for transcriptional profiling of specific sell types in eukaryotes: laser-assisted microdissection and RNA sequencing. PLoS One 7:e29685

    Article  CAS  Google Scholar 

  32. Florez-Rueda AM, Grossniklaus U, Schmidt A (2016) Laser-assisted microdissection (LAM) as a tool for transcriptional profiling of individual cell types. J Vis Exp 111:e53916. https://doi.org/10.3791/53916

    Article  CAS  Google Scholar 

  33. Hedhly A, Vogler H, Eichenberger C, Grossniklaus U (2018) Whole-mount clearing and staining of Arabidopsis flower organs and siliques. J Vis Exp 134:e56441. https://doi.org/10.3791/56441

    Article  CAS  Google Scholar 

  34. Picelli S, Faridani OR, Björklund AK et al (2014) Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9:171–181

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Christof Eichenberger, Daniela Guthörl, and Arturo Bolaños for taking care of the LCM and continuous support on LAM procedures. We acknowledge Samuel Wuest, Kitty Vijverberg, Marc Schmid, and Anja Schmidt for establishing and standardizing LAM procedures in Arabidopsis and Boechera, and Anja Schmidt for training on LAM. Work using LAM in the Grossniklaus laboratory was supported over the years by the University of Zurich and grants from the Swiss National Science Foundation, the European Research Council and, as a subgrantee, a grant from the Bill and Melinda Gates Foundation to CSIRO.

Author information

Authors and Affiliations

  1. Department of Plant and Microbial Biology and Zurich–Basel Plant Science Center, University of Zurich, Zurich, Switzerland

    Ana Marcela Florez-Rueda, Lucas Waser & Ueli Grossniklaus

Authors
  1. Ana Marcela Florez-Rueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lucas Waser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ueli Grossniklaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ueli Grossniklaus .

Editor information

Editors and Affiliations

  1. Department of Cell Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany

    Martin Bayer

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Florez-Rueda, A.M., Waser, L., Grossniklaus, U. (2020). Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling. In: Bayer, M. (eds) Plant Embryogenesis. Methods in Molecular Biology, vol 2122. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0342-0_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0342-0_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0341-3

  • Online ISBN: 978-1-0716-0342-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation