Figure 8.

Effect of ZmHda1 Constructs on the Repression of Gene Transcription in Tobacco Protoplasts.

(A) Scheme of the reporter and expression plasmids used in the tobacco protoplast. The tobacco TAX line contains a reporter cassette that is integrated stably in the genome and allows for the expression of a reporter GUS controlled by a minimal promoter and four consecutive upstream TetR binding sites. Cotransfection of two plasmids with the 35S promoter driving the expression of the VP16 transcriptional activator and the protein of interest, respectively, both fused to the DNA binding domain of TetR, results in the competition of the two fusion proteins for the TET binding sites. The effect on transcription is monitored by determining GUS activity levels (Bohner et al., 1999).

(B) ZmHda1 constructs. The striped area represents the core catalytic histone deacetylase domain. The arrow depicts the position of the mutated His in the DAmut construct. aa, amino acids.

(C) Full-length ZmHda1 or ZmHda1 derivatives fused to the DNA binding domain of TetR were used together with plasmid expressing a TetR fusion of VP16 to transform protoplasts prepared from the tobacco TAX line. The effect on transcription was determined by measuring the activity of the GUS reporter gene. GUS activity is reported as pmol of 7-OH-4-methylcoumarin per milligram of protein per minute. “Untransformed” indicates protoplast samples transformed with the DNA carrier only. Error bars indicate the standard errors of assays performed on six independent transfections.