Skip to main content

Expression of Epitope-Tagged Proteins in Arabidopsis Leaf Mesophyll Protoplasts

  • Protocol
  • First Online:
Immunoelectron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 657))


Advances in genomic and proteomic platforms enable high-throughput studies for regulatory factors and interactors involved in signaling network at a molecular level. However, it has never been trivial to verify the omics data in vivo or functionally integrate the data in a cell signaling context. For plants, genetic approaches using knockout mutants and transgenic lines have been mainly used to characterize functions of gene products in vivo. In general, such approaches demand a longer time and a higher cost and have difficulties in understanding gene functions comprehensively in a high-throughput manner. Transient gene expression is a method of choice to examine the cellular functions of genetic components in vivo. The leaf mesophyll protoplasts (LMP) provide a perfect system to transiently express a gene encoding an epitope-tagged protein of interest and quickly and reliably trace subcellular locales of the protein in a near high-throughput manner. Here, a simple and straightforward method for isolating leaf meshophyll protoplasts from Arabidopsis has been described in detail to help beginners initiate their first cell-based functional genomic analysis.

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

Institutional subscriptions

Similar content being viewed by others


  1. Sheen, J. (2001) Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 127, 1466–1475.

    Article  PubMed  CAS  Google Scholar 

  2. Yoo, S. D., Cho, Y. H., and Sheen, J. (2007) A versatile experimental cell system, Arabidopsis leaf mesophyll protoplasts. Nat. Protoc. 2, 1565–1572.

    Article  PubMed  CAS  Google Scholar 

  3. Kovtun, Y., Chiu, W. L., Tena, G., and Sheen, J. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. USA 97, 2940–2945.

    Article  PubMed  CAS  Google Scholar 

  4. Sheen, J. (1993) Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO J. 12, 3497–3505.

    PubMed  CAS  Google Scholar 

  5. Jacobsen, J. V. and Beach, L. R. (1985) Control of transcription of alpha-amylase and rRNA genes in barley aleurone protoplasts by gibberellin and abscisic acid. Nature 316, 275–277.

    Article  CAS  Google Scholar 

  6. Assmann, S. M., Simoncini, L., and Schroeder, J. I. (1985) Blue light activates electrogenic ion pumping in guard cell protoplasts of Vicia faba. Nature 318, 285–287.

    Article  CAS  Google Scholar 

  7. Lee, Y., Kim, D., Kim, Y., and Hwang, I. (2001) Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo. Plant Cell 13, 2175–2190.

    PubMed  CAS  Google Scholar 

  8. Jin, J., Kim, Y., Kim, S., Lee, S., Kim, D., Cheong, G., and Hwang, I. (2001) A new dynamin-like protein, ADL6, is involved in trafficking from the trans-golgi network to the central vacuole in Arabidopsis. Plant Cell 13, 1511–1526.

    PubMed  CAS  Google Scholar 

  9. Sohn, E., Kim, E., Zhao, M., Kim, S., Kim, H., Kim, Y., Lee, Y., Hillmer, S., Sohn, U., Jiang, L., and Hwang, I. (2003) Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell 15, 1057–1070.

    Article  PubMed  CAS  Google Scholar 

  10. Kim, H., Park, M., Kim, S., and Hwang, I. (2005) Actin filaments play a critical role in vacuolar trafficking at the golgi complex in plant cells. Plant Cell 17, 888–902.

    Article  PubMed  CAS  Google Scholar 

  11. Cho, Y. H., Yoo S. D., and Sheen, J. (2006) Regulatory functions of nuclear hexokinase1 complex in glucose signalling. Cell 127, 579–589.

    Article  PubMed  CAS  Google Scholar 

  12. Chiu, W.-L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Curr.Biol. 6, 325–330.

    CAS  Google Scholar 

  13. Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi, S., Watanabe, M., Nishimura, M., and Hara-Nishimura, I. (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J. 35, 545–555.

    Article  PubMed  CAS  Google Scholar 

Download references


We thank Dr. Donald Hunter for his help in the preparation of this chapter. This work was supported by National Research Foundation of Korea Grant 2010-0007068 to Y.-H. Cho and grants 2009-0085565 and 2010-0016989 to S.-D. Yoo.

Author information

Authors and Affiliations


Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cho, YH., Yoo, SD. (2010). Expression of Epitope-Tagged Proteins in Arabidopsis Leaf Mesophyll Protoplasts. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-782-2

  • Online ISBN: 978-1-60761-783-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics