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Measurement of auxin transport in Arabidopsis thaliana

Nature Protocols volume 4, pages 437–451 (2009)Cite this article

Abstract

This protocol allows the measurement of auxin transport in roots, hypocotyls and inflorescences of Arabidopsis thaliana plants by examining transport of radiolabeled auxin or movement of an auxin-induced gene expression signal. The protocol contains four stages: seedling growth, auxin application, a transport period of variable length, and quantification of auxin movement or reporter expression. Beyond the time for plant growth, the transport assay can be completed within 4–18 h. Auxin is applied to seedlings in agar cylinders or droplets, which does not require specialized liquid-handling equipment or micromanipulators, in contrast with methods that apply auxin in liquid droplets. Spatial control of auxin application is reduced, but this method has the advantages of being technically more feasible for most laboratories and allowing agar containing radioactive auxin to be removed for pulse chase assays that determine transport rates. These methods allow investigation of genetic and environmental factors that control auxin transport.

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Figure 1: Polar IAA transport occurs in tissue-specific directional patterns in Arabidopsis.
Figure 2: Basipetal IAA transport is detected in the apical 10 mm of Arabidopsis roots.
Figure 3: IAA and IBA transport move unidirectionally in the hypocotyl and base of the root.
Figure 4: Auxin transport rates can be calculated in inflorescences and roots using a pulse chase assay.
Figure 5: Comparison of reporter-based and radioactive auxin transport assays.
Figure 6: Methods for the preparation of 3H-IAA containing lines and droplets.
Figure 7: Application of agar containing 3H-IAA to roots.

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References

  1. Blakeslee, J.J., Peer, W.A. & Murphy, A.S. Auxin transport. Curr. Opin. Plant Biol. 8, 494–500 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Sánchez-Bravo, J., Ortuño, A.M., Botía, J.M., Acosta, M. & Sabater, F. The decrease in auxin polar transport down the lupin hypocotyl could produce the indole-3-acetic acid distribution responsible for the elongation growth pattern. Plant Physiol. 99, 108–114 (1992).

    Article  Google Scholar 

  3. Bhalerao, R.P. & Bennett, M.J. The case for morphogens in plants. Nat. Cell Biol. 5, 939–943 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Casimiro, I. et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 13, 843–852 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Muday, G.K. & DeLong, A. Polar auxin transport: controlling where and how much. Trends Plant Sci. 6, 535–542 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Goldsmith, M.H.M. The polar transport of auxin. Ann. Rev. Plant Physiol. 28, 439–478 (1977).

    Article  CAS  Google Scholar 

  7. Okada, K., Ueda, J., Komaki, M.K., Bell, C.J. & Shimura, Y. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 3, 677–684 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Mitchell, E.K. & Davies, P.J. Evidence for three different systems of movement of indoleacetic acid in intact roots of Phaseolus coccineus . Physiol Plant. 33, 290–294 (1975).

    Article  CAS  Google Scholar 

  9. Tsurumi, S. & Ohwaki, Y. Transport of 14C-labeled indoleacetic acid in Vicia root segments. Plant Cell Physiol. 19, 1195–1206 (1978).

    CAS  Google Scholar 

  10. Rashotte, A.M., Poupart, J., Waddell, C.S. & Muday, G.K. Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis . Plant Physiol. 133, 761–772 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rashotte, A.M., Brady, S.R., Reed, R.C., Ante, S.J. & Muday, G.K. Basipetal auxin transport is required for gravitropism in roots of Arabidopsis . Plant Physiol. 122, 481–490 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rashotte, A.M., DeLong, A. & Muday, G.K. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth. Plant Cell 13, 1683–1697 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kramer, E.M. PIN and AUX/LAX proteins: their role in auxin accumulation. Trends Plant Sci. 9, 578–582 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Leyser, O. Dynamic integration of auxin transport and signalling. Curr. Biol. 16, R424–R433 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Friml, J. Auxin transport—shaping the plant. Curr. Opin. Plant Biol. 6, 7–12 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Chen, R.J. et al. The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier. Proc. Natl. Acad. Sci. USA 95, 15112–15117 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Müller, A. et al. AtPIN2 defines a locus of Arabidopsis for root gravitropism control. EMBO J. 17, 6903–6911 (1998).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Bennett, M.J. et al. Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273, 948–950 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. Marchant, A. et al. AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14, 589–597 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Marchant, A. et al. AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO J. 18, 2066–2073 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Negi, S., Ivanchenko, M.G. & Muday, G.K. Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana Plant J. 55, 175–187 (2008).

    CAS  PubMed  Google Scholar 

  22. Geisler, M. & Murphy, A.S. The ABC of auxin transport: the role of p-glycoproteins in plant development. FEBS Lett. 580, 1094–1102 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Geisler, M. et al. Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1. Plant J. 44, 179–194 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Lewis, D.R., Miller, N.D., Splitt, B.L., Wu, G. & Spalding, E.P. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes. Plant Cell 19, 1838–1850 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wu, G., Lewis, D.R. & Spalding, E.P. Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development. Plant Cell 19, 1826–1837 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Terasaka, K. et al. PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots. Plant Cell 17, 2922–2939 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cho, M., Lee, S. & Cho, H.-T. P-glycoprotein4 displays auxin efflux transporter-like action in Arabidopsis root hair cells and tobacco cells. Plant Cell 19, 3930–3943 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lin, R. & Wang, H. Two homologous ATP-binding cassette transporter proteins, AtMDR1 and AtPGP1, regulate Arabidopsis photomorphogenesis and root development by mediating polar auxin transport. Plant Physiol. 138, 949–964 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Murphy, A., Peer, W.A. & Taiz, L. Regulation of auxin transport by aminopeptidases and endogenous flavonoids. Planta. 211, 315–324 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Brown, D.E. et al. Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis . Plant Physiol. 126, 524–535 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Noh, B., Bandyopadhyay, A., Peer, W.A., Spalding, E.P. & Murphy, A.S. Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1. Nature. 424, 999–1002 (2003).

    Article  Google Scholar 

  32. Muday, G.K. et al. RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling. Plant Physiol. 141, 1617–1629 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yang, Y., Hammes, U.Z., Taylor, C.G., Schachtman, D.P. & Nielsen, E. High-affinity auxin transport by the AUX1 influx carrier protein. Curr. Biol. 16, 1123–1127 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Titapiwatanakun, B. et al. ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis . Plant J. 57, 27–44 (2008).

    Article  PubMed  Google Scholar 

  35. Petrasek, J. et al. PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312, 914–918 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Blakeslee, J.J. et al. Interactions among PIN-FORMED and P-glycoprotein auxin transporters in Arabidopsis . Plant Cell. 19, 131–147 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Swarup, K. et al. The auxin influx carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 10, 946–954 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Stasinopoulos, T.C. & Hangarter, R.P. Preventing photochemistry in culture media by long-pass light filters alters growth of cultured tissues. Plant Physiol. 93, 1365–1369 (1989).

    Article  Google Scholar 

  39. Ludwig-Muller, J., Sass, S., Sutter, E., Wodner, M. & Epstein, E. Indole-3-butyric acid in Arabidopsis thaliana Plant Growth Reg. 13, 179–187 (1993).

    Article  Google Scholar 

  40. Woodward, A.W. & Bartel, B. Auxin: regulation, action, and interaction. Ann. Bot. (Lond). 95, 707–735 (2005).

    Article  CAS  Google Scholar 

  41. Poupart, J., Rashotte, A.M., Muday, G.K. & Waddell, C.S. The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture. Plant Physiol. 139, 1460–1471 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Delbarre, A., Muller, P., Imhoff, V. & Guern, J. Comparison of mechanisms controlling uptake and accumulation of 2,3-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta. 198, 532–541 (1996).

    Article  CAS  PubMed  Google Scholar 

  43. Yamamoto, M. & Yamamoto, K.T. Differential effects of 1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on the gravitropic response of roots in an auxin-resistant mutant of Arabidopsis, aux1 . Plant Cell Physiol. 39, 660–664 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Parry, G. et al. Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1 . Plant J. 25, 399–406 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Nadella, V., Shipp, M.J., Muday, G.K. & Wyatt, S.E. Evidence for altered polar and lateral auxin transport in the gravity persistent signal (gps) mutants of Arabidopsis . Plant Cell Environ. 29, 682–690 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Sabatini, S. et al. An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell. 99, 463–472 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Ottenschläger, I. et al. Gravity-regulated differential auxin transport from columella to lateral root cap cells. Proc. Natl. Acad. Sci. USA 100, 2987–2991 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Buer, C.S. & Muday, G.K. The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light. Plant Cell 16, 1191–1205 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Peer, W.A. et al. Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana . Plant Cell 16, 1898–1911 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Peer, W.A. & Murphy, A.S. Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci. 12, 556–563 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Swarup, R., Parry, G., Graham, N., Allen, T. & Bennett, M. Auxin cross-talk: integration of signalling pathways to control plant development. Plant Mol Biol. 49, 411–426 (2002).

    Article  CAS  PubMed  Google Scholar 

  52. Jensen, P.J., Hangarter, R.P. & Estelle, M. Auxin transport is required for hypocotyl elongation in light-grown but not dark-grown Arabidopsis . Plant Physiol. 116, 455–462 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Laxmi, A., Pan, J., Morsy, M. & Chen, R. Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana PLoS ONE 3, e1510 (2008).

    Article  PubMed  Google Scholar 

  54. Noh, B., Murphy, A.S. & Spalding, E.P. Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell 13, 2441–2454 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Jain, A. et al. Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis . Plant Physiol. 144, 232–247 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Buer, C.S., Sukumar, P. & Muday, G.K. Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis . Plant Physiol. 140, 1384–1396 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Buer, C.S., Wasteneys, G.O. & Masle, J. Ethylene modulates root-wave responses in Arabidopsis . Plant Physiol. 132, 1085–1096 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Muday, G.K. & Rahman, A. In Auxin Transport and the Integration of Gravitropic Growth (eds. Gilroy, S. & Masson, P.) 47–78 (Blackwell Publishing, Oxford, 2007).

    Google Scholar 

  59. Thimann, K.V. A History of the Knowledge of Auxin. In Physiology and Biochemistry of Auxins in Plants (eds. Kutacke, M., Bandurski, R. S. & Krekule, J.) 3–20 (SPB Academic Publishing, Prague, 1988).

    Google Scholar 

  60. Reed, R.C., Brady, S.R. & Muday, G.K. Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis . Plant Physiol. 118, 1369–1378 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Vieten, A. et al. Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression. Development. 132, 4521–4531 (2005).

    Article  CAS  PubMed  Google Scholar 

  62. Paciorek, T. et al. Auxin inhibits endocytosis and promotes its own efflux from cells. Nature 435, 1251–1256 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Sieburth, L.E. et al. SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis . Plant Cell 18, 1396–1411 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Butler, J.H., Hu, S., Brady, S.R., Dixon, M.W. & Muday, G.K. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls. Plant J. 13, 291–301 (1998).

    Article  CAS  PubMed  Google Scholar 

  65. Imhoff, V., Muller, P., Guern, J. & Delbarre, A. Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells. Planta 210, 580–588 (2000).

    Article  CAS  PubMed  Google Scholar 

  66. McCourt, P. & Keith, K. Sterile techniques in Arabidopsis . Methods Mol. Biol. 82, 13–17 (1998).

    CAS  PubMed  Google Scholar 

  67. Buer, C.S., Masle, J. & Wasteneys, G.O. Growth conditions modulate root-wave phenotypes in Arabidopsis . Plant Cell Physiol. 41, 1164–1170 (2000).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We appreciate the capture of the images used to illustrate these protocols as provided by Kevin Cooper with the assistance of Poornima Sukumar and Anita McCauley. We appreciate the helpful comments on the text of this procedure from Poornima Sukumar, Sangeeta Negi and Cassie Mattox. We acknowledge the important role of Aaron Rashotte, Shari Brady and Charles Buer in the initial development of these Arabidopsis methods during their tenure in the Muday research laboratory. This work was supported by the USDA Cooperative State Research, Education and Extension Service (grant 2006-35304-17311 to G.K.M.) and microscopic images were possible by purchase of a Leica stereomicroscope with the support of the National Science Foundation Major Research Instrumentation grant (#05-00702).

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  1. Department of Biology, Wake Forest University, Winston-Salem, North Carolina, USA

    Daniel R Lewis & Gloria K Muday

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Correspondence to Gloria K Muday.

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Lewis, D., Muday, G. Measurement of auxin transport in Arabidopsis thaliana. Nat Protoc 4, 437–451 (2009). https://doi.org/10.1038/nprot.2009.1

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