Immunohistochemistry Relative to Gravity: A Simple Method to Retain Information About Gravity for Immunolocalization and Histochemistry

  • Benjamin R. Harrison
  • Patrick H. Masson
Part of the Methods in Molecular Biology book series (MIMB, volume 1309)


We describe a simple method to preserve information about a plant organ’s orientation relative to the direction of the gravity vector during sample processing for immunolocalization or histochemical analysis of cell biological processes. This approach has been used in gravity stimulated roots of Arabidopsis thaliana and Zea mays to study PIN3 relocalization, study the asymmetrical remodeling of the actin network and the cortical microtubule array, and to reveal the asymmetrical expression of the auxin signaling reporter DR5::GUS. This method enables the rapid analysis of a large number of samples from a variety of genotypes, as well as from tissue that may be too thick for microscopy in live plants.

Key words

Gravity Gravitropism Plants Immunolocalization Immunohistochemistry Cell Biology Histochemistry Signal Transduction Hormones Auxin 



The authors would like to thank K. Rawlins for critical reading and helpful suggestions.


  1. 1.
    Hashiguchi Y, Tasaka M, Morita MT (2013) Mechanism of higher plant gravity sensing. Am J Bot 100:91–100CrossRefPubMedGoogle Scholar
  2. 2.
    Baldwin KL, Strohm AK, Masson PH (2013) Gravity sensing and signal transduction in vascular plant primary roots. Am J Bot 100:126–142CrossRefPubMedGoogle Scholar
  3. 3.
    Bridges IG, Wilkins MB (1974) The role of reducing sugars in the geotropic response of the wheat node. Planta 117:243–250CrossRefPubMedGoogle Scholar
  4. 4.
    Momonoki YS (1988) Asymmetric distribution of glucose and indole-3-acetyl-myo-inositol in geostimulated Zea mays seedlings. Plant Physiol 87:751–756CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Parker KE, Briggs WR (1990) Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles. Plant Physiol 94:1763–1769CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Young LM, Evans ML, Hertel R (1990) Correlations between gravitropic curvature and auxin movement across gravistimulated roots of Zea mays. Plant Physiol 92:792–796CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Blancaflor EB, Hasenstein KH (1993) Organization of cortical microtubules in graviresponding maize roots. Planta 191:231–237PubMedGoogle Scholar
  8. 8.
    Perera IY, Heilmann I, Boss WF (1999) Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini. Proc Natl Acad Sci U S A 96:5838–5843CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Heilmann I, Shin J, Huang J et al (2001) Transient dissociation of polyribosomes and concurrent recruitment of calreticulin and calmodulin transcripts in gravistimulated maize pulvini. Plant Physiol 127:1193–1203CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Long JC, Zhao W, Rashotte AM et al (2002) Gravity-stimulated changes in auxin and invertase gene expression in maize pulvinal cells. Plant Physiol 128:591–602CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Zhang Q, Pettolino FA, Dhugga KS et al (2011) Cell wall modifications in maize pulvini in response to gravitational stress. Plant Physiol 156:2155–2171CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Blancaflor EB, Hasenstein KH (1995) Time course and auxin sensitivity of cortical microtubule reorientation in maize roots. Protoplasma 185:72–82CrossRefPubMedGoogle Scholar
  13. 13.
    Young LS, Harrison BR, Narayana Murthy UM et al (2006) Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis. Plant Physiol 142:564–573CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Harrison BR, Masson PH (2008) ARL2, ARG1 and PIN3 define a gravity signal transduction pathway in root statocytes. Plant J 53:380–392CrossRefPubMedGoogle Scholar
  15. 15.
    Blancaflor EB, Hasenstein KH (1997) The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize. Plant Physiol 113:1447–1455PubMedCentralPubMedGoogle Scholar
  16. 16.
    Larkin PJ, Gibson JM, Mathesius U et al (1996) Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development. Transgenic Res 5:325–335CrossRefPubMedGoogle Scholar
  17. 17.
    Rashotte AM, DeLong A, Muday GK (2001) Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth. Plant Cell 13:1683–1697CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Wyatt RE, Ainley WM, Nagao RT et al (1993) Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants. Plant Mol Biol 22:731–749CrossRefPubMedGoogle Scholar
  19. 19.
    Friml J, Wiśniewska J, Benková E et al (2002) Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature 415:806–809CrossRefPubMedGoogle Scholar
  20. 20.
    Li Y, Hagen G, Guilfoyle TJ (1991) An auxin-responsive promoter is differentially induced by auxin gradients during tropisms. Plant Cell 3:1167–1175CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Aloni R, Langhans M, Aloni E et al (2004) Role of cytokinin in the regulation of root gravitropism. Planta 220:177–182CrossRefPubMedGoogle Scholar
  22. 22.
    Perera IY, Hung CY, Brady S et al (2006) A universal role for inositol 1,4,5-trisphosphate-mediated signaling in plant gravitropism. Plant Physiol 140:746–760CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Villani TS, Koroch AR, Simon JE (2013) An improved clearing and mounting solution to replace chloral hydrate in microscopic applications. Appl Plant Sci. doi: 10.3732/apps.1300016 Google Scholar
  24. 24.
    Buer CS, Wasteneys GO, Masle J (2003) Ethylene modulates root-wave responses in Arabidopsis. Plant Physiol 132:1085–1096CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Barker R, Cox B, Mackie TR et al (2013) Vacuum seed sowing manifold: a novel device for high-throughput sowing of Arabidopsis seeds. Plant Methods 9:41CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Rutherford R, Masson PH (1996) Arabidopsis thaliana sku mutant seedlings show exaggerated surface-dependent alteration in root growth vector. Plant Physiol 111:987–998CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Brooks TL, Miller ND, Spalding EP (2010) Plasticity of Arabidopsis root gravitropism throughout a multidimensional condition space quantified by automated image analysis. Plant Physiol 152:206–216CrossRefPubMedGoogle Scholar
  28. 28.
    Massa GD, Gilroy S (2003) Touch modulates gravity sensing to regulate the growth of primary roots of Arabidopsis thaliana. Plant J 33:435–445CrossRefPubMedGoogle Scholar
  29. 29.
    Boonsirichai K, Sedbrook JC, Chen R et al (2003) ALTERED RESPONSE TO GRAVITY is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes. Plant Cell 15:2612–2625CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124:33–44PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Biological SciencesUniversity of Alaska AnchorageAnchorageUSA
  2. 2.Genetics DepartmentUniversity of WisconsinMadisonUSA

Personalised recommendations