Figure 5.

The zinc finger domain is required for the activity and function of HDA15 in Arabidopsis. A, Fluorometric HDAC activity assays of full-length and truncated GFP-HDA15. The schematic structure of HDA15 protein domains is also shown. Proteins were extracted from 10-d-old hda15-1 expressing full-length and truncated GFP-HDA15. Fluorometric HDAC activity assays were performed, and HDAC activity was indicated by AMC released (nanomolar). GFP alone was used as a negative control. Immunoblotting with the GFP antibody indicated the equal protein loading in HDAC activity assays. Data represent the mean (± sd) of three biological replicates compared with GFP alone. Asterisks indicate significant difference using Student’s t test (**P < 0.01). B and C, Relative fluorescence of protochlorophyllide in full emission wavelengths 600 to 800 nm in etiolated transgenic plants expressing full-length and truncated GFP-HDA15. Protochlorophyllide was extracted from 4-d-old etiolated seedlings. D, RT-qPCR analysis of GUN5 and PSBQ expression in 2-d-old etiolated seedlings of Col-0, hda15-1, and transgenic plants expressing full-length and truncated GFP-HDA15. UBQ10 was used as an internal control. E, ChIP-qPCR analysis of the enrichment of GFP-HDA15 and GFP-HDA15ΔZ at P and A regions of GUN5 in etiolated transgenic seedlings. Red box indicates the G-box element (CACGTG). The GFP antibody was used for immunoprecipitation. Data are shown as percentage of input. The values represent the mean (± sd) of three biological replicates. Asterisks indicate significant difference using Student’s t test (*P < 0.05). WB, Western blot.