Figure 1. TTG1 has an effect on flowering time regulation in the A. thaliana Col-0 ecotype at long-day conditions.
(A–D) Flowering time of ttg1 mutants in Col-0 background. In (A–F, H, I), flowering time was recorded as the number of days until bolting (A, C, E, H) or the number of leaves at the time-point of bolting (B, D, F, I). Plants were grown at long-day conditions (16 h light, 8 h darkness) at 21 °C (“cold”) or 23 °C (“warm”). Black lines in the box plots represent the median, red dots are the mean for the shown representative experiment and crosses mark outliers. Asterisks indicate significant differences (*P < 0.05) between the mutants and the Col-0 wild type (orange) or between the two conditions (grey) in the shown representative experiment. Numbers in brackets indicate the number of experiments for which a significant difference was observed out of the total number of experiments. (G) Primer binding sites for the used primer pairs for qRT-PCR (arrow heads) relative to the TTG1 genomic, cDNA, CDS and overexpression construct sequence. The position of the point mutations in the ttg1-9 and ttg1-11 is indicated above. The T-DNA insertion for ttg1-21 and ttg1-22 are provided in Fig. S1 characterizing the mutants’ phenotypes. See Fig. S2 for an alignment of TTG1 with LWD1 and LWD2 in the primer binding region. The sense primers for the primer pairs “TTG both” and “TTG1 no LWD” are overlapping and the sequence of the latter deviates at its 3′ end from the sequence for LWD1 and LWD2. The name “TTG1 both” indicates that the CDS as well as the construct are amplified by this primer pair. (H, I) Flowering time of TTG1 overexpression lines as described above. (J–M) Characterization of transcript and protein levels in lines overexpressing YFP-TTG1 in Col-0 (OE01-03) and cop1-4 (OE19-21) background driven by the 35S promoter. (J–L) Highest levels of TTG1 transcript were observed in OE01-03 (Col-0) and OE20 (cop1-4) while the endogenous TTG1 transcript levels were in general not affected by overexpression despite in the OE19 (cop1-4) line. Note the elevated TTG1 levels in cop1-4 mutants. Transcript levels were determined by qRT-PCR relative to UBQ10 using 8-day-old seedlings and are normalized with Col-0 wild type values. Data represent the mean of three independent experiments. Asterisks indicate significant differences (#, P < 0.1; *, P < 0.05) between overexpression lines and the backgrounds Col-0 (orange) and cop1-4 (magenta), respectively. Please note that the scale in (K, L) differs from the scale in (J). The solid line equals 1. The y-axis is in log10-scaled. Error bars indicate the SD. (M) Western blot using 7d-old long-day grown seedlings form one of the repeats used in (J–L). In OE03 (Col-0), YFP-TTG1 levels varied between experiments never exceeding those in OE01 (Col-0). See also Fig. S3. Tables S2–S4 provide more details on the underlying data and statistics. OE01-03: Pro35s:YFP-TTG1 (Col-0), three independent insertion lines. OE19-21: Pro35s:YFP-TTG1 (cop1-4), three independent insertion lines.