FIGURE 3.

Gibberellic acid (GA)-induced expression of the α-amylase gene is attenuated in the presence of TOR kinase inhibitors. (A) Concentration-dependent influence of rapamycin or torin1 on GA-induced production of α-amylase. The wheat embryos dissected from 1-DAG seeds were incubated for 24 h in the presence 10 mM CaCl₂ (control) with or without 10 μM abscisic acid (ABA), or 1 μM GA, or 1 μM GA with different concentrations of rapamycin or torin1. The embryos were then lysed and subjected to immunoblotting. Rap, rapamycin. (B) Concentration-dependent effects of rapamycin and torin1 on GA-induced accumulation of α-amylase mRNA. The wheat embryos were incubated as described in panel (A). A 409-bp fragment of α-amylase cDNA was amplified by RT-PCR with gene-specific primers. α-Tubulin expression served as an internal loading control. (C) Detection of α-amylase activity by staining. The wheat embryos were incubated for 24 h in the presence of 1 μM GA and 10 mM CaCl₂ with or without rapamycin, torin1, or pp242. The embryos were then lysed and subjected to staining for the activity of α-amylases following partition in a polyacrylamide gel electrophoresis (native PAGE).