Figure 10.

OsGRF6 and OsGRF10 proteins have transactivation activity and can bind to the promoter of OsCR4 in vitro and in vivo. A, Schematic representation of the reporter and effector constructs used in the transcription activity assays. B, OsGRF6 and OsGRF10 function as transcription activators, whereas OsGIF1 protein may function as a coactivator. Arabidopsis leaf protoplasts were cotransfected with two reporter genes and one effector gene. Effector genes contain yeast Gal4 BD fused in frame with OsGRF6, OsGRF10, OsGIF1, OsGIF2, or Auxin Response Factor5M (ARF5M; positive control). Data are means of three independent experiments. C and D, EMSA showing that GRF10-GST and GRF6N-His fusion proteins can bind to the promoter of OsCR4. GARE cis-element 1,400 bp upstream of the transcription initiation site is shown in the black box in C. The OsCR4 P1 oligonucleotide was used as the biotin-labeled probe. Red arrowheads indicate the complexes composed of OsGRF10-GST, CR4 P1, and GST antibody or OsGRF6N-His, CR4 P1, and His antibody. E, ChIP assays of the binding of OsGRF10 to the promoter of OsCR4 tested using anti-OsGRF10 antibody. Regions in the promoter of OsCR4 analyzed by real-time PCR are shown by short lines marked with letters (a–c; c is the region containing the GARE). The binding ability of the GARE cis-element was reduced in the grf10 mutant; the promoter of Ubiquitin (UBQ) is the control. *, Significant difference at P ≤ 0.05 compared with DJ by Student’s t test. Data are means ± sds (n = 3). F, Transcriptional activation activity assays in Arabidopsis protoplasts. JMJ706 and CR4 indicate the promoters of OsJMJ706 and OsCR4 genes individually. Data are means of three independent experiments. LUC, Luciferase.