Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes

Nature Protocols volume 6, pages 419–426 (2011)Cite this article

Subjects

Abstract

The pollen tube is an excellent single-cell model system for studying cellular processes in plant cell biology. This protocol describes a detailed step-by-step procedure with optimized conditions for introducing various fluorescent reporter proteins into lily, tobacco and Arabidopsis pollen grains by means of biolistics for their transient expression and subsequent analysis in germinating pollen tubes. The whole experiment consists of four major stages: coating gold microcarriers with DNA constructs, preparation of pollen grains, transformation of plasmid DNA into pollen grains by particle delivery system and germination of bombarded pollen grains in optimized germination media to obtain pollen tubes for protein trafficking, protein localization, drug treatment and organelle dynamics analysis. This protocol takes about 4–12 h from pollen preparation to protein detection.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Transient expression of fluorescent reporter proteins in growing lily pollen tubes.
Figure 2: Transient expression of fluorescent reporter proteins in growing tobacco and Arabidopsis pollen tubes.

Similar content being viewed by others

References

  1. Krichevsky, A. et al. How pollen tubes grow. Dev. Biol. 303, 405–420 (2007).

    Article  CAS  Google Scholar 

  2. Samaj, J., Muller, J., Beck, M., Bohm, N. & Menzel, D. Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes. Trends Plant Sci. 11, 594–600 (2006).

    Article  CAS  Google Scholar 

  3. Hepler, P.K., Vidali, L. & Cheung, A.Y. Polarized cell growth in higher plants. Annu. Rev. Cell Dev. Biol. 17, 159–187 (2001).

    Article  CAS  Google Scholar 

  4. Taylor, L.P. & Hepler, P.K. Pollen germination and tube growth. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 461–491 (1997).

    Article  CAS  Google Scholar 

  5. Raghavan, V. Molecular Embryology of Flowering Plants, 525–531 (Cambridge University Press, 1997).

  6. Nishimura, A., Aichi, I. & Matsuoka, M. A protocol for Agrobacterium-mediated transformation in rice. Nat. Protoc. 1, 2796–2802 (2006).

    Article  CAS  Google Scholar 

  7. Sparkes, I.A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).

    Article  CAS  Google Scholar 

  8. Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W. & Chua, N.H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646 (2006).

    Article  CAS  Google Scholar 

  9. Ishida, Y., Hiei, Y. & Komari, T. Agrobacterium-mediated transformation of maize. Nat. Protoc. 2, 1614–1621 (2007).

    Article  CAS  Google Scholar 

  10. Cheung, A.Y. et al. The dynamic pollen tube cytoskeleton: live cell studies using actin-binding and microtubule-binding reporter proteins. Mol. Plant 1, 686–702 (2008).

    Article  CAS  Google Scholar 

  11. de Graaf, B.H. et al. Rab11 GTPase-regulated membrane trafficking is crucial for tip-focused pollen tube growth in tobacco. Plant Cell 17, 2564–2579 (2005).

    Article  CAS  Google Scholar 

  12. Helling, D., Possart, A., Cottier, S., Klahre, U. & Kost, B. Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling. Plant Cell 18, 3519–3534 (2006).

    Article  CAS  Google Scholar 

  13. Lee, Y.J., Szumlanski, A., Nielsen, E. & Yang, Z. Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J. Cell Biol. 181, 1155–1168 (2008).

    Article  CAS  Google Scholar 

  14. Twell, D., Klein, T.M., Fromm, M.E. & McCormick, S. Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol. 91, 1270–1274 (1989).

    Article  CAS  Google Scholar 

  15. Vidali, L., Rounds, C.M., Hepler, P.K. & Bezanilla, M. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells. PLoS One 4, e5744 (2009).

    Article  Google Scholar 

  16. Xiang, Y. et al. ACTIN BINDING PROTEIN 29 from Lilium pollen plays an important role in dynamic actin remodeling. Plant Cell 19, 1930–1946 (2007).

    Article  CAS  Google Scholar 

  17. Zhang, Y., He, J., Lee, D. & McCormick, S. Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes. Plant Physiol. 152, 2200–2210 (2010).

    Article  CAS  Google Scholar 

  18. Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456 (2001).

    Article  CAS  Google Scholar 

  19. Miao, Y. & Jiang, L. Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat. Protoc. 2, 2348–2353 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Yoo, S.D., Cho, Y.H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  Google Scholar 

  22. Cole, R.A., Synek, L., Zarsky, V. & Fowler, J.E. SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol. 138, 2005–2018 (2005).

    Article  CAS  Google Scholar 

  23. Fu, Y., Wu, G. & Yang, Z. Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes. J. Cell Biol. 152, 1019–1032 (2001).

    Article  CAS  Google Scholar 

  24. Hwang, J.U., Vernoud, V., Szumlanski, A., Nielsen, E. & Yang, Z. A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex. Curr. Biol. 18, 1907–1916 (2008).

    Article  CAS  Google Scholar 

  25. Kost, B., Spielhofer, P. & Chua, N.H. A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393–401 (1998).

    Article  CAS  Google Scholar 

  26. Okada, T., Bhalla, P.L. & Singh, M.B. Transcriptional activity of male gamete-specific histone gcH3 promoter in sperm cells of Lilium longiflorum. Plant Cell Physiol. 46, 797–802 (2005).

    Article  CAS  Google Scholar 

  27. Twell, D., Klein, T.M. & McCormick, S. Transformation of pollen by particle bombardment. In Plant Tissue Culture Manual, Fundamentals and Applications Vol. D1 (ed. Lindsey, K.) 1–12 (Kluwer Academic Publishers, 1991).

  28. Wang, H. et al. Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth. Plant J. 61, 826–838 (2010).

    Article  CAS  Google Scholar 

  29. Wang, H.J., Wan, A.R. & Jauh, G.Y. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes. Plant Physiol. 147, 1619–1636 (2008).

    Article  CAS  Google Scholar 

  30. Jiang, L. & Rogers, J.C. Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways. J. Cell Biol. 143, 1183–1199 (1998).

    Article  CAS  Google Scholar 

  31. Lam, S.K., Cai, Y., Hillmer, S., Robinson, D.G. & Jiang, L. SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells. Plant Physiol. 147, 1637–1645 (2008).

    Article  CAS  Google Scholar 

  32. Lam, S.K. et al. Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells. Plant Cell 19, 296–319 (2007).

    Article  CAS  Google Scholar 

  33. Lam, S.K., Tse, Y.C., Robinson, D.G. & Jiang, L. Tracking down the elusive early endosome. Trends Plant Sci. 12, 497–505 (2007).

    Article  CAS  Google Scholar 

  34. Miao, Y., Li, K.Y., Li, H.Y., Yao, X. & Jiang, L. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells. Plant J. 56, 824–839 (2008).

    Article  CAS  Google Scholar 

  35. Miao, Y., Yan, P.K., Kim, H., Hwang, I. & Jiang, L. Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells. Plant Physiol. 142, 945–962 (2006).

    Article  CAS  Google Scholar 

  36. Tse, Y.C. et al. Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells. Plant Cell 16, 672–693 (2004).

    Article  CAS  Google Scholar 

  37. Hicks, G.R., Rojo, E., Hong, S., Carter, D.G. & Raikhel, N.V. Geminating pollen has tubular vacuoles, displays highly dynamic vacuole biogenesis, and requires VACUOLESS1 for proper function. Plant Physiol. 134, 1227–1239 (2004).

    Article  CAS  Google Scholar 

  38. Yang, Z. Cell polarity signaling in Arabidopsis. Annu. Rev. Cell Dev. Biol. 24, 551–575 (2008).

    Article  Google Scholar 

  39. Cheung, A.Y. & Wu, H.M. Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu. Rev. Plant Biol. 59, 547–572 (2008).

    Article  CAS  Google Scholar 

  40. Hamilton, D.A. et al. Dissection of a pollen-specific promoter from maize by transient transformation assays. Plant Mol. Biol. 18, 211–218 (1992).

    Article  CAS  Google Scholar 

  41. Hamilton, D.A., Schwarz, Y.H. & Mascarenhas, J.P. A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements. Plant Mol. Biol. 38, 663–669 (1998).

    Article  CAS  Google Scholar 

  42. Keller, N.L. & Hamilton, D.A. Transient expression of the green fluorescent protein in pollen. Sex. Plant Reprod. 11, 163–165 (1998).

    Article  CAS  Google Scholar 

  43. Muschietti, J., Dircks, L., Vancanneyt, G. & McCormick, S. LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization. Plant J. 6, 321–338 (1994).

    Article  CAS  Google Scholar 

  44. Twell, D., Yamaguchi, J., Wing, R.A., Ushiba, J. & McCormick, S. Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev. 5, 496–507 (1991).

    Article  CAS  Google Scholar 

  45. Sambrook, J. & Russell, D.W. Preparation of plasmid DNA by alkaline lysis with SDS: maxipreparation. Cold Spring Harbor Protocols. doi:10.1101/pdb.prot4084 (2006).

    Google Scholar 

  46. Kim, S.H. et al. Growth and development of Lilium longiflorum 'Nellie White' during bulb production under controlled environments I. Effects of constant, variable and greenhouse day/night temperature regimes on scale and stem bulblets. Sci. Hort. 112, 95–98 (2007).

    Article  Google Scholar 

  47. Weigel, D. & Glazebrook, J. Arabidopsis: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2002).

  48. Boavida, L.C. & McCormick, S. Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J. 52, 570–582 (2007).

    Article  CAS  Google Scholar 

  49. Lovy-Wheeler, A., Kunkel, J.G., Allwood, E.G., Hussey, P.J. & Hepler, P.K. Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily. Plant Cell 18, 2182–2193 (2006).

    Article  CAS  Google Scholar 

  50. Parton, R.M., Fischer-Parton, S., Watahiki, M.K. & Trewavas, A.J. Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes. J. Cell Sci. 114, 2685–2695 (2001).

    CAS  PubMed  Google Scholar 

  51. Hala, M. et al. An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell 20, 1330–1345 (2008).

    Article  CAS  Google Scholar 

  52. Molendijk, A.J. et al. Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth. EMBO J. 20, 2779–2788 (2001).

    Article  CAS  Google Scholar 

  53. Fan, L.M., Wang, Y.F., Wang, H. & Wu, W.H. In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. J. Exp. Bot. 52, 1603–1614 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Research Grants Council of Hong Kong (CUHK488707, CUHK465708, CUHK466309 and CUHK466610), Chinese University of Hong Kong (CUHK) Schemes B and C, University Grants Committee/Area of Excellence (to L.J.).

Author information

Authors and Affiliations

  1. School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Hong Kong, China

    Hao Wang & Liwen Jiang

Authors
  1. Hao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Liwen Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.W. executed the experiments and wrote the manuscript. L.J. supervised the study and edited the final version of the manuscript.

Corresponding author

Correspondence to Liwen Jiang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Dynamics of GFP-BP-80 in growing lily pollen tube. (MOV 249 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, H., Jiang, L. Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes. Nat Protoc 6, 419–426 (2011). https://doi.org/10.1038/nprot.2011.309

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.309

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing