Figure 8.

Trimer Comprising NF-YA2, NF-YB3, and DPB3-1 Enhanced Reporter Activity in Cooperation with DREB2A.

(A) Schematic diagram of the effector and reporter constructs for the transactivation experiment. The HsfA3 1-kb promoter:GUS fusion gene was used as the reporter. The plasmids containing the CaMV 35S promoter and the tobacco mosaic virus Ω sequence fused to NF-YA2, NF-YB3, DPB3-1, full-length DREB2A, or DREB2A CA coding sequence were cotransfected into protoplasts. Nos-T indicates the terminator sequence of the gene for nopaline synthetase.

(B) Transactivation of the HsfA3 1-kb promoter:GUS reporter gene by various combinations of full-length DREB2A, NF-YA2, NF-YB3, and DPB3-1. The effector plasmids cotransfected are indicated as plus signs (+) in the table, and minus signs (-) indicate the cotransfection of empty effector plasmids. Bars indicate the mean values from assays performed in triplicate and the sd, and asterisks indicate significant differences between the reporter activities (P < 0.05 according to Bonferroni-corrected Student’s t test). The 35S:luciferase (LUC) plasmid was also cotransfected in each experiment as an internal control.

(C) Transactivation of the HsfA3 1-kb promoter:GUS reporter gene by DREB2A CA and the trimer of NF-YA2, NF-YB3, and DPB3-1. Bars indicate the mean values from assays performed in triplicate and the sd, and 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.

(D) Yeast three-hybrid assay to identify candidate trimers containing DPB3-1. The growth of yeast cells harboring NF-YB family proteins fused to the GAL4 DNA binding domain (BD), NF-YA family proteins fused to the GAL4 activation domain (AD), and DPB3-1 fused to a nuclear localization signal (NLS) on selective medium (SD/-L-W-H-M-Ade) are shown.

(E) Transactivation of the HsfA3 1-kb promoter:GUS reporter gene by various combinations of trimers and DREB2A CA. The effector plasmids cotransfected are indicated with the plus signs (+) or numbers in the table; for example, the number “2” in the “NF-YA” column of the table indicates that NF-YA2 was cotransfected. The minus signs (−) indicate the cotransfection of empty effector plasmids. Values represent means from assays performed in triplicate, and the bars indicate the sd. The asterisks indicate significant high (*) or low (**) differences between the reporter activities (P < 0.05 according to Bonferroni-corrected Student’s t test). The 35S:LUC plasmid was also cotransfected in each experiment as an internal control.