Figure 9.

The Importance of an Upstream CCAAT Motif for the Enhancement of DREB2A-Dependent Reporter Activation by the Trimer of NF-YA2, NF-YB3, and DPB3-1 and a Model of the Function of the Identified Trimer under Heat Stress Conditions.

(A) Schematic diagram of the promoter fragments used in the reporter constructs. Each HsfA3 promoter fragment was fused to the GUS reporter gene. The positions of the DREs, CCAAT boxes, and an ABRE-like motif on the promoter are indicated. Crosses (×) indicate the mutation at the CCAAT motif. The effector plasmids used in this analysis are described in Figure 8A.

(B) Transactivation of each HsfA3 promoter:GUS reporter gene indicated in (A) by DREB2A CA and the trimer of NF-YA2, NF-YB3, and DPB3-1. Bars indicate mean values obtained from assays performed in triplicate and sd, and the asterisks indicate significant differences between the reporter activities (P < 0.05 according to Student’s t test). The 35S:LUC plasmid was also cotransfected in each experiment as an internal control.

(C) A model of the function of NF-YA2, NF-YB3, and DPB3-1 under heat stress conditions. The DREB2A, DPB3-1, and NF-YB3 genes are induced by heat stress. The DREB2A protein is stabilized under heat stress, and the NF-YB3 protein is translocated into the nucleus. NF-YA2, NF-YB3, and DPB3-1 form a trimer in the nucleus; thereafter, the trimer forms a transcriptional complex with DREB2A at the promoters of heat stress-inducible DREB2A target genes. Finally, the trimer enhances the efficiency of transcription by DREB2A. The effects of DPB3-1 levels on HsfA2 expression and the direct binding of DPB3-1 to the HsfA2 promoter under heat stress imply that DPB3-1 is involved in the transcription of HsfA2 by HsfA1s.