Abstract
Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b alpha-amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the alpha-amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley aleurone cells.
Highlights
Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering
We showed that HvWRKY38 suppressed the GA-induced expression of Amy32bGUS (Xie et al, 2007)
When the relative amounts of effector were higher than 25%, GUS expression was reduced to 2-fold or less. These data indicate that the effect of HvWRKY38 on the repression of GA induction of the Amy32b a-amylase gene expression is dosage dependent, similar to what was reported for OsWRKY71 (Xie et al, 2006)
Summary
Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. In the low-pI a-amylase promoter, Amy32b, five elements, namely, O2S/W-box, pyrimidine box, GA response element (GARE), amylase box (Amy), and downstream amylase element, are essential for the high level of GA-induced expression (Lanahan et al, 1992; Rogers and Rogers, 1992; Rogers et al, 1994; Gomez-Cadenas et al, 2001) Each of these elements may be bound by one or more transcription factor(s) of R2R3 MYB, R1 MYB, DOF, and zinc finger protein families (Gubler et al, 1995; Raventos et al, 1998; Diaz et al, 2002; Isabel-LaMoneda et al, 2003; Washio, 2003; Mare et al, 2004; Peng et al, 2004; Zhang et al, 2004; Rubio-Somoza et al, 2006; Xie et al, 2006; Moreno-Risueno et al, 2007). It still remains unclear how these repressors and activators interact with each other in regulating gene expression
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