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.
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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.
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