Figure 7. MG132-induced accumulation of GFP-DREB2A is not sufficient for the induction of DREB2A target genes under normal conditions.

(A) The effects of various protease and proteasome inhibitors on the degradation of the DREB2A protein in transgenic Arabidopsis seedlings. Total protein was extracted from 10-day-old DREB2A or WT seedlings treated with water, 1% (v/v) DMSO, proteasome inhibitors (20 µM epoxomicin, 200 µM MG115 or 200 µM MG132) or protease inhibitors (200 µM leupeptin or 200 µM PMSF) under normal growth conditions for 10 h. A protein extract was prepared from non-treated seedlings (Before) to serve as a control. The protein extracts corresponded to 4 mg seedling FW and were loaded onto an SDS-PAGE gel. Immunoblot analysis was performed using the anti-DREB2A antibody. The arrowhead indicates the major band of DREB2A. rbcL bands visualized by Ponceau S are shown as loading controls. (B) Accumulation levels of GFP-DREB2A. Ten-day-old seedlings of GFP/dreb2a and two independent lines of GFP-DREB2A/dreb2a were treated with or without 200 µM MG132 under normal (22°C) conditions or conditions of heat stress (37°C). The arrowhead indicates the major band of GFP-DREB2A. (C) The effects of MG132 treatment on the expression of DREB2A target genes. The mRNA levels of two heat-inducible DREB2A target genes were determined using quantitative RT-PCR. The amounts of the transcripts were normalized to those of ACT8. The values represent the means of triplicate technical repeats and the error bars indicate SDs. The highest expression level was set to 100 for each gene. Similar results were obtained in two independent experiments.