Skip to main content
SpringerLink
Log in
Book cover

Pollen and Pollen Tube Biology pp 41–72Cite as

Isolation of the Pistil-Stimulated Pollen Tube Secretome

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2160))

Abstract

Detection of secreted proteins and peptides during pollen tube guidance has been impeded due to lack of techniques to capture the pollen tube secretome without contamination from the female secreted proteins. Here we present a protocol to detect tobacco pollen tube secreted proteins, semi-in vivo pollen tube secretome assay (SIV-PS), following pollen tube crosstalk with the female reproductive tissues. This method combines the advantages of in vivo pollen tube–pistil interaction and filter-aided sample preparation (FASP) techniques to obtain an in-depth proteome coverage. The SIV-PS method is rapid, efficient, inexpensive, does not require specialized equipment or expertise, and provides a snapshot of the ongoing molecular interplay. We show that the secretome obtained is of greater purity (<1.4% ADH activities) and that pollen tubes are physiologically and cytologically unaffected. A compendium of quality controls is described and a rough guide on downstream bioinformatics analysis is outlined. The SIV-PS method is applicable to all studies of protein secretion using pollen tube as a model and can be easily adapted to other flowering species with modification. The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis.

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   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
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. Frantz C, Stewart KM, Weaver VM (2010) The extracellular matrix at a glance. J Cell Sci 123(Pt 24):4195–4200

    Article  CAS  Google Scholar 

  2. Johnson MA, Harper J, Palanivelu R (2019) A fruitful journey: pollen tube navigation from germination to fertilization. Annu Rev Plant Biol 70:809–837

    Article  CAS  Google Scholar 

  3. Dresselhaus T, Franklin-Tong N (2013) Male-female crosstalk during pollen germination, tube growth and guidance, and double fertilization. Mol Plant 6(4):1018–1036

    Article  CAS  Google Scholar 

  4. Kessler SA, Shimosato-Asano H, Keinath NF, Wuest SE, Ingram G, Panstruga R, Grossniklaus U (2010) Conserved molecular components for pollen tube reception and fungal invasion. Science 330(6006):968–971

    Article  CAS  Google Scholar 

  5. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D et al (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458(7236):357–361

    Article  CAS  Google Scholar 

  6. 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(6110):1093–1097

    Article  CAS  Google Scholar 

  7. Higashiyama T (2015) The mechanism and key molecules involved in pollen tube guidance. Annu Rev Plant Biol 66:393–413

    Article  CAS  Google Scholar 

  8. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T (1998) Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri. Plant Cell 10(12):2019–2032

    Article  CAS  Google Scholar 

  9. Palanivelu R, Preuss D (2006) Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol 6:7

    Article  Google Scholar 

  10. Hafidh S, Potesil D, Fila J, Capkova V, Zdrahal Z, Honys D (2016) Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization. Genome Biol 17(1):81

    Article  Google Scholar 

  11. Hafidh S, Potesil D, Fila J, Fecikova J, Capkova V, Zdrahal Z, Honys D (2014) In search of ligands and receptors of the pollen tube: the missing link in pollen tube perception. Biochem Soc Trans 42(2):388–394

    Article  CAS  Google Scholar 

  12. Leydon AR, Beale KM, Woroniecka K, Castner E, Chen J, Horgan C, Palanivelu R, Johnson MA (2013) Three MYB transcription factors control pollen tube differentiation required for sperm release. Curr Biol 23(13):1209–1214

    Article  CAS  Google Scholar 

  13. Qin Y, Leydon AR, Manziello A, Pandey R, Mount D, Denic S, Vasic B, Johnson MA, Palanivelu R (2009) Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PLoS Genet 5(8):e1000621

    Article  Google Scholar 

  14. Wiśniewski JR, Ostasiewicz P, Mann M (2011) High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J Proteome Res 10:3040–3049

    Article  Google Scholar 

  15. Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860

    Article  CAS  Google Scholar 

  16. Wiśniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362

    Article  Google Scholar 

  17. Terry ME, Bonner BA (1980) An examination of centrifugation as a method of extracting an extracellular solution from peas, and Its use for the study of indoleacetic acid-induced growth. Plant Physiol 66(2):321

    Article  CAS  Google Scholar 

  18. Jung Y-H, Jeong S-H, Kim SH, Singh R, Lee J-E, Cho Y-S, Agrawal GK, Rakwal R, Jwa N-S (2008) Systematic secretome analyses of rice leaf and seed callus suspension-cultured cells: workflow development and establishment of high-density two-dimensional gel reference maps. J Proteome Res 7(12):5187–5210

    Article  CAS  Google Scholar 

  19. Agrawal GK, Jwa N-S, Lebrun M-H, Job D, Rakwal R (2010) Plant secretome: unlocking secrets of the secreted proteins. Proteomics 10(4):799–827

    Article  CAS  Google Scholar 

  20. Alexandersson E, Ali A, Resjo S, Andreasson E (2013) Plant secretome proteomics. Front Plant Sci 4:9

    Article  Google Scholar 

  21. Liu Y, Joly V, Dorion S, Rivoal J, Matton DP (2015) The plant ovule secretome: a different view toward pollen-pistil interactions. J Proteome Res 14(11):4763–4775

    Article  CAS  Google Scholar 

  22. Tupý J, Süss J, Hrabětová E, Řihová L (1983) Developmental changes in gene expression during pollen differentiation and maturation in Nicotiana tabacum L. Biol Plant 25(3):231–237

    Article  Google Scholar 

  23. Taylor SC, Posch A (2014) The design of a quantitative western blot experiment. Biomed Res Int 2014:361590

    Article  Google Scholar 

Download references

Acknowledgments

We thank Jana Feciková for her help with flowers emasculation and secretome concentration and Anna J. Wiese for help with imaging. This work was supported by the Czech Science Foundation grants 17-23203S, 18-07027S and 18-02448S, and by Ministry of Education, Youth and Sport CR from European Regional Development Fund-Project “Centre for Experimental Plant Biology” (No. CZ.02.1.01/0.0/0.0/16_019/0000738). We acknowledge the Imaging Facility of the Institute of Experimental Botany, Czech Acad. Sci. supported by the MEYS CR (LM2018129 Czech-BioImaging and OPPK CZ.2.16/3.1.00/21519 Projects). Author contributions: S.H. designed and wrote the protocol. D.H. edited the manuscript.

Author information

Authors and Affiliations

  1. Laboratory of Pollen Biology, Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic

    Said Hafidh & David Honys

Authors
  1. Said Hafidh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Honys
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Said Hafidh or David Honys .

Editor information

Editors and Affiliations

  1. Department of Plant Science, McGill University, Montreal, QC, Canada

    Anja Geitmann

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

Hafidh, S., Honys, D. (2020). Isolation of the Pistil-Stimulated Pollen Tube Secretome. In: Geitmann, A. (eds) Pollen and Pollen Tube Biology. Methods in Molecular Biology, vol 2160. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0672-8_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0672-8_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0671-1

  • Online ISBN: 978-1-0716-0672-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation