In order to become activated, some TFs require post-translational modifications. We have observed that overexpression of DREB2A and AREB1 did not activate the expression of downstream genes (Liu et al., 1998; Uno et al., 2000). These peculiar data suggested that these TFs require post-translational modification for their activation. The protoplast transactivation assay can serve as an effective approach to identify the regulatory regions of TFs that require modification. By this method, Sakuma et al. (2006) and Fujita et al. (2005) have shown that the (136-165 amino acid) region of DREB2A and the QT region of AREB1 are regulatory domains of
DREB2A and AREB1, respectively. Furihata et al. (2006) used both a protoplast transactivation and an in-gel kinase assay to further demonstrate that the ABA-dependent phosphorylation of Ser/Thr residues in the conserved regions of AREB1 is required for the activation of AREB1. Methods for in-gel kinase assay can be obtained from Furihata et al. (2006).
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