Figure 6. FTIP1 does not regulate FT mRNA or protein stability.

(A) Schematic diagrams showing native FT and transgenic SUC2:FT:GFP transcripts. The fragments labeled with a, b, and c indicate amplicons in real-time PCR analyses shown in (B). Fragments a, b, and c were amplified with primers FT-F and FT-R, FT(UTR)-F and FT(UTR)-R, and GFP-F1 and GFP-R1 (Table S1), respectively. (B) Examination of steady-state levels of FT or FT:GFP mRNA in wild-type and ftip1-1 backgrounds. Amplification of fragment a, which detects the amplicon in both native FT and transgenic FT:GFP transcripts, shows that native FT expression is downregulated in ftip1-1 versus wild-type, whereas total FT expression (including minor native FT and major transgenic FT:GFP expression) remains unchanged in SUC2:FT:GFP ftip1-1 versus SUC2:FT:GFP. Although the former indicates that FTIP1 affects the steady-state levels of native FT expression, the latter implies that FTIP1 does not directly affect FT mRNA stability. Amplification of fragment c, which only detects the amplicon in transgenic FT:GFP transcripts, further supports that FTIP1 does not directly affect FT mRNA stability as transgenic FT:GFP expression is not changed in ftip1-1. Amplification of fragment b, which only detects the amplicon in native FT transcripts, shows that native FT expression is also downregulated in SUC2:FT:GFP transgenic plants. 9-d-old seedlings grown under LDs were harvested for expression analysis by quantitative real-time PCR. Results were normalized against the expression of TUB2. Asterisks indicate that the expression of fragment c was undetectable in wild-type and ftip1-1 seedling. Error bars indicate SD. (C) Western blot analysis using anti-GFP antibody shows the comparable abundance of FT:GFP protein in wild-type and ftip1-1 plants. Ponceau S staining of the membrane is used as a loading control. (D) GUS staining of rosette leaves of 9-d-old FT:GUS and FT:GUS SUC2:FT seedling.