Fig. 1. Overexpression of HA:FLAG:FVE in acg1. (A) RNA-gel blot analysis of acg1 overexpressing HA: FLAG:FVE. Total RNAs isolated from 10-day-old plants, Col-0, acg1, and two lines [FVE (N1-11) and FVE (N3- 3)] of Pro35S:HA:FLAG:FVE/acg1, were subjected to RNA-gel blot analysis with FVE or ACTIN7 DNA probe. (B) Immunoblot analysis of acg1 overexpressing HA: FLAG:FVE. Total proteins extracted from 10-day-old plants, Col-0, acg1, and two l ines of Pro35S: HA:FLAG: FVE/acg1, were subjected to immunoblot analysis with anti-HA or anti-FLAG antibody. The rbcL protein encoding for the large subunit of rubisco is shown as a loading control. (C) Eight-week-old Col-0, acg1, and acg1 overexpressing HA:FLAG:FVE. Plants were grown for eight weeks under long-day conditions and photographed. (DE) Bolting day (D) and the rosette leaf numbers (E) of Col-0, acg1, and acg1 overexpressing HA:FLAG:FVE. n ≥ 16. (F) Real-time RT-PCR analysis of FLC, SOC1, and FT from Col-0, acg1, and acg1 overexpressing HA:FLAG:FVE. Total RNAs isolated from 10-day-old plants, Col-0, acg1, and Pro35S:HA:FLAG:FVE/acg1 (N1-11), were quantified by real-time RT-PCR. Copies of the transcripts were plotted per ng of total RNA after the normalization of ACTIN7 RNA. Multiplication of the number in the brackets inside the graph by the number in the y-axis generates copy numbers of the corresponding transcripts. The mean values and standard errors from biological triplicate experiments were plotted. (G) Fluorometric quantification of the GUS activities of 4CRT/ DRE-GUS, acg1, and acg1 overexpressing HA:FLAG: FVE. Ten-day-old plants were used for fluorometric quantification of GUS activities. Specific activities (units) are expressed as the pmol reaction product (4-methylumbelliferone) generated per min per mg of total protein. The data are expressed as the means ± standard errors of three independent treatments. The open column and closed column indicate the sample treated at 4℃ for 2 days and the sample treated at 23℃, respectively.
