Activation Tag Screening for Cell Expansion Genes in Arabidopsis thaliana

  • Chaowen Xiao
  • Charles T. AndersonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1242)


Forward genetic screens for growth-deficient loss-of-function mutants have uncovered a wide array of genes involved in cell expansion. However, the centrality of cell growth to plant survival means that null mutations in many genes involved in this process are likely to be lethal early in development, making phenotypic analysis difficult. Additionally, the phenotypes of loss-of-function mutations in genes that are members of large gene families might be masked by functional redundancy with other family members. Activation tagging provides a method of screening for dominant overexpression phenotypes in an arbitrarily large collection of transgenic individuals, allowing for functional genomic identification of genes related to cell growth and expansion. In this chapter, we discuss the advantages and limitations of activation tag screening and describe a protocol for identifying activation tag lines with enhanced cell expansion, using dark-grown Arabidopsis thaliana seedlings as an experimental system. We also describe secondary screens to identify candidate genes for further cell biological and genetic characterization. These protocols can be adapted to any process or species of interest, as long as a suitable activation-tagged population and a genome sequence are available.

Key words

Activation tagging Forward genetics Genetic redundancy Overexpression Cell expansion Arabidopsis 



Thanks to Chris Somerville and members of the Somerville lab for helpful discussions on this topic, to Wenting Xi for technical assistance with primary and secondary screening, and to William Barnes for critical reading. The writing of this chapter was supported as part of the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences under Award # DE-SC0001090.


  1. 1.
    Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6(11):850–861PubMedCrossRefGoogle Scholar
  2. 2.
    Baskin TI (2005) Anisotropic expansion of the plant cell wall. Annu Rev Cell Dev Biol 21:203–222PubMedCrossRefGoogle Scholar
  3. 3.
    Dolan L, Davies J (2004) Cell expansion in roots. Curr Opin Plant Biol 7(1):33–39PubMedCrossRefGoogle Scholar
  4. 4.
    Weigel D et al (2000) Activation tagging in Arabidopsis. Plant Physiol 122(4):1003–1013PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Walden R et al (1994) Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development. Plant Mol Biol 26(5):1521–1528PubMedCrossRefGoogle Scholar
  6. 6.
    Jeong DH et al (2002) T-DNA insertional mutagenesis for activation tagging in rice. Plant Physiol 130(4):1636–1644PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Wan S et al (2009) Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Mol Biol 69(1–2):69–80PubMedCrossRefGoogle Scholar
  8. 8.
    Mur LA et al (2011) Exploiting the Brachypodium Tool Box in cereal and grass research. New Phytol 191(2):334–347PubMedCrossRefGoogle Scholar
  9. 9.
    Busov VB et al (2003) Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature. Plant Physiol 132(3):1283–1291PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8(3):457–463PubMedCrossRefGoogle Scholar
  11. 11.
    O’Malley RC, Alonso JM, Kim CJ, Leisse TJ, Ecker JR (2007) An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat Protoc 2(11):2910–2917PubMedCrossRefGoogle Scholar
  12. 12.
    Gendreau E et al (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol 114(1):295–305PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Boron AK, Vissenberg K (2014) The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion. Plant Cell Rep 33:697PubMedCrossRefGoogle Scholar
  14. 14.
    Xiao C, Somerville C, Anderson CT (2014) Polygalacturonase involved in cell expansion 1 functions in cell elongation and flower development in Arabidopsis thaliana. Plant Cell 26(3):1018–1035Google Scholar
  15. 15.
    Kardailsky I et al (1999) Activation tagging of the floral inducer FT. Science 286(5446):1962–1965PubMedCrossRefGoogle Scholar
  16. 16.
    van der Graaff E, Dulk-Ras AD, Hooykaas PJ, Keller B (2000) Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana. Development 127(22):4971–4980PubMedGoogle Scholar
  17. 17.
    van der Graaff E, Hooykaas PJ, Keller B (2002) Activation tagging of the two closely linked genes LEP and VAS independently affects vascular cell number. Plant J 32(5):819–830PubMedCrossRefGoogle Scholar
  18. 18.
    Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12(12):2383–2394PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Woodward C et al (2005) Interaction of auxin and ERECTA in elaborating Arabidopsis inflorescence architecture revealed by the activation tagging of a new member of the YUCCA family putative flavin monooxygenases. Plant Physiol 139(1):192–203PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Xiao C, Chen F, Yu X, Lin C, Fu YF (2009) Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana. Plant Mol Biol 71(1–2):39–50PubMedCrossRefGoogle Scholar
  21. 21.
    Mathews H et al (2003) Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15(8):1689–1703PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Zubko E et al (2002) Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants. Plant J 29(6):797–808PubMedCrossRefGoogle Scholar
  23. 23.
    Marsch-Martinez N (2011) A transposon-based activation tagging system for gene function discovery in Arabidopsis. Methods Mol Biol 754:67–83PubMedCrossRefGoogle Scholar
  24. 24.
    Harb A, Pereira A (2013) Activation tagging using the maize En-I transposon system for the identification of abiotic stress resistance genes in Arabidopsis. Methods Mol Biol 1057:193–204PubMedCrossRefGoogle Scholar
  25. 25.
    Fladung M, Ahuja MR (1997) Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus. Plant Mol Biol 33(6):1097–1103PubMedCrossRefGoogle Scholar
  26. 26.
    Spena A, Aalen RB, Schulze SC (1989) Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assay for transposon excision in transgenic plants. Plant Cell 1(12):1157–1164PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Waki T et al (2013) A GAL4-based targeted activation tagging system in Arabidopsis thaliana. Plant J 73(3):357–367PubMedCrossRefGoogle Scholar
  28. 28.
    Sedbrook JC, Ehrhardt DW, Fisher SE, Scheible WR, Somerville CR (2004) The Arabidopsis sku6/spiral1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion. Plant Cell 16(6):1506–1520PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Center for Lignocellulose Structure and FormationThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of BiologyThe Pennsylvania State UniversityUniversity ParkUSA

Personalised recommendations