Skip to main content

Microscopic and Biochemical Visualization of Auxins in Plant Tissues

  • Protocol
  • First Online:
Environmental Responses in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1398))

Abstract

Auxins are a particularly notable class of phytohormones in that they regulate plant growth and development at sites of synthesis, and via a regulated polar transport system comprising PIN, ABCB, and AUX/LAX transport proteins. In order to fully understand auxin-regulated physiological processes, it is therefore essential to be able to determine where indole-3-acetic acid and related compounds are being synthesized, where they are transported to, and how much IAA is accumulating in any given tissue. Auxin may be visualized either indirectly, through the use of auxin responsive promoters; directly, through the use of radiolabelled auxin or fluorescent auxin analogs; or biochemically through extraction and mass-spectrometric quantification of auxin and auxin metabolites from target cells or tissues. Here we focus on the use of the DR5::GUS synthetic auxin promoter reporter construct, fluorescent auxin analogs, and confirmatory biochemical (high-pressure liquid chromatography tandem mass-spectrometry) visualization of auxin and auxin metabolites.

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

Access this chapter

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

Institutional subscriptions

References

  1. Soeno K, Goda H, Ishii T, Ogura T, Tachikawa T, Sasaki E, Yoshida S, Fujioka S, Asami T, Shimada Y (2010) Auxin biosynthesis inhibitors, identified by a genomics-based approach, provide insights into auxin biosynthesis. Plant Cell Physiol 51(4):524–536

    Article  CAS  PubMed  Google Scholar 

  2. Mano Y, Nemoto K (2012) The pathway of auxin biosynthesis in plants. J Exp Bot 63(8):2853–2872

    Article  CAS  PubMed  Google Scholar 

  3. Blakeslee JJ, Peer WA, Murphy AS (2005) Auxin transport. Curr Opin Plant Biol 8(5):494–500

    Article  CAS  PubMed  Google Scholar 

  4. Peer WA, Blakeslee JJ, Yang HB, Murphy AS (2011) Seven things we think we know about auxin transport. Mol Plant 4(3):487–504

    Article  CAS  PubMed  Google Scholar 

  5. Multani DS, Briggs SP, Chamberlin MA, Blakeslee JJ, Murphy AS, Johal GS (2003) Loss of an MDR transporter in compact stalks of maize br2 and sorghum dw3 mutants. Science 302(5642):81–84

    Article  CAS  PubMed  Google Scholar 

  6. Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433(7021):39–44

    Article  CAS  PubMed  Google Scholar 

  7. Grunewald W, Friml J (2010) The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells. EMBO J 29(16):2700–2714

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Krecek P, Skupa P, Libus J, Naramoto S, Tejos R, Friml J, Zazimalova E (2009) The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biol 10(12):249

    Article  PubMed Central  PubMed  Google Scholar 

  9. Friml J (2010) Subcellular trafficking of PIN auxin efflux carriers in auxin transport. Eur J Cell Biol 89(2-3):231–235

    Article  CAS  PubMed  Google Scholar 

  10. Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K (2002) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415(6873):806–809

    Article  PubMed  Google Scholar 

  11. Mravec J, Kubes M, Bielach A, Gaykova V, Petrasek J, Skupa P, Chand S, Benkova E, Zazimalova E, Friml J (2008) Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development. Development 135(20):3345–3354

    Article  CAS  PubMed  Google Scholar 

  12. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9(11):1963–1971

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Brunoud G, Wells DM, Oliva M, Larrieu A, Mirabet V, Burrow AH, Beeckman T, Kepinski S, Traas J, Bennett MJ et al (2012) A novel sensor to map auxin response and distribution at high spatio-temporal resolution. Nature 482(7383):103–U132

    Article  CAS  PubMed  Google Scholar 

  14. Blakeslee JJ, Zhou HW, Heath JT, Skottke KR, Barrios JAR, Liu SY, DeLong A (2008) Specificity of RCN1-mediated protein phosphatase 2A regulation in meristem organization and stress response in roots. Plant Physiol 146(2):539–553

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Pattison RJ, Catala C (2012) Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families. Plant J 70(4):585–598

    Google Scholar 

  16. Zhou J, Yu FB, Wang XM, Yang Y, Yu CL, Liu HJ, Cheng Y, Yan CQ, Chen JP (2014) Specific expression of DR5 promoter in rice roots using a tCUP derived promoter-reporter system. PLoS One 9(1):e87008

    Article  PubMed Central  PubMed  Google Scholar 

  17. Chen YR, Yordanov YS, Ma C, Strauss S, Busov VB (2013) DR5 as a reporter system to study auxin response in Populus. Plant Cell Rep 32(3):453–463

    Article  CAS  PubMed  Google Scholar 

  18. Jefferson RA, Burgess SM, Hirsh D (1986) Beta-glucuronidase from Escherichia-coli as a gene-fusion marker. Proc Natl Acad Sci U S A 83(22):8447–8451

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Pengelly W, Meins F (1977) Specific radioimmunoassay for nanogram quantities of auxin, indole-3-acetic-acid. Planta 136(2):173–180

    Article  CAS  PubMed  Google Scholar 

  20. Takahashi N (1986) Chemistry of plant hormones. CRC Press, Boca Raton, FL

    Google Scholar 

  21. Marcussen J, Ulvskov P, Olsen CE, Rajagopal R (1989) Preparation and properties of antibodies against indoleacetic-acid (Iaa)-C5-Bsa, a novel ring-coupled Iaa antigen, as compared to 2 other types of Iaa-specific antibodies. Plant Physiol 89(4):1071–1078

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Fernandez B, Centeno ML, Feito I, Sancheztames R, Rodriguez A (1995) Simultaneous analysis of cytokinins, auxins and abscisic-acid by combined immunoaffinity chromatography, high-performance liquid-chromatography and immunoassay. Phytochem Anal 6(1):49–54

    Article  CAS  Google Scholar 

  23. Jiraskova D, Poulickova A, Novak O, Sedlakova K, Hradecka V, Strnad M (2009) High-throughput screening technology for monitoring phytohormone production in microalgae. J Phycol 45(1):108–118

    Article  CAS  Google Scholar 

  24. Hayashi KI, Nakamura S, Fukunaga S, Nishimura T, Jenness MK, Murphy AS, Motose H, Nozaki H, Furutani M, Aoyama T (2014) Auxin transport sites are visualized in planta using fluorescent auxin analogs. Proc Natl Acad Sci U S A 111(31):11557–11562

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Ohwaki Y (1966) Thin-layer chromatography of diffusible auxin of corn coleoptiles. Bot Mag Tokyo 79(934):200

    Article  Google Scholar 

  26. Bayer MH, Ahuja MR (1968) Tumor formation in Nicotiana—auxin levels and auxin inhibitors in normal and tumor-prone genotypes. Planta 79(4):292–8

    Article  CAS  PubMed  Google Scholar 

  27. Raj RK (1970) Indoles and Auxins.8. partition chromatography of naturally occurring indoles on cellulose thin layers and sephadex columns. Anal Biochem 33(2):471–4

    Article  CAS  PubMed  Google Scholar 

  28. Akiyoshi DE, Morris RO, Hinz R, Mischke BS, Kosuge T, Garfinkel DJ, Gordon MP, Nester EW (1983) Cytokinin auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. P Natl Acad Sci-Biol 80(2):407–411

    Article  CAS  Google Scholar 

  29. Morgan PW, Durham JI (1983) Strategies for extracting, purifying, and assaying auxins from plant-tissues. Bot Gaz 144(1):20–31

    Article  CAS  Google Scholar 

  30. Goswami D, Thakker JN, Dhandhukia PC (2015) Simultaneous detection and quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) produced by rhizobacteria from L-tryptophan (Trp) using HPTLC. J Microbiol Methods 110:7–14

    Article  CAS  PubMed  Google Scholar 

  31. Cohen JD, Lilly N (1984) Changes in (45)calcium concentration following auxin treatment of protoplasts isolated from etiolated soybean hypocotyls. Plant Physiol 75:109–109

    Google Scholar 

  32. Rivier L (1986) GC-MS of auxins. In: Linskens HF, Jackson JF (eds) Modern methods of plant analysis, volume 3: gas chromatography/mass spectrometry. Springer-Verlag, Berlin Heidelberg, pp 146–188

    Chapter  Google Scholar 

  33. Barkawi LS, Tam YY, Tillman JA, Normanly J, Cohen JD (2010) A high-throughput method for the quantitative analysis of auxins. Nat Protoc 5(10):1609–1618

    Article  CAS  PubMed  Google Scholar 

  34. Novak O, Henykova E, Sairanen I, Kowalczyk M, Pospisil T, Ljung K (2012) Tissue-specific profiling of the Arabidopsis thaliana auxin metabolome. Plant J 72(3):523–536

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Joshua J. Blakeslee .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Blakeslee, J.J., Murphy, A.S. (2016). Microscopic and Biochemical Visualization of Auxins in Plant Tissues. In: Duque, P. (eds) Environmental Responses in Plants. Methods in Molecular Biology, vol 1398. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3356-3_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3356-3_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3354-9

  • Online ISBN: 978-1-4939-3356-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics