Figure 2

Seed imbibition at supraoptimal temperature activates the FUS3 promoter and induces de novo FUS3 mRNA synthesis and FUS3 protein accumulation. (A) Relative FUS3 mRNA levels measured by qPCR in seeds imbibed at 21°C or 32°C for up to 72 h under constant light. The mean value of three replicates was normalized using ACTIN 7 as the internal control. Results are plotted as the ratio to the lowest detected level. Two independent experiments were conducted with similar results and one representative is shown. (B) Confocal images showing GFP fluorescence in the epidermis of FUS3:GFP embryos of seeds imbibed at 21°C or 32°C. HAI, hours after imbibition. (C, D) Confocal images (C) and immunoblots (D) showing FUS3-GFP fluorescence and protein accumulation in FUS3p:FUS3-GFP (FFG) seeds imbibed at 21°C and 32°C for up to 72 h under constant light. WT in D) is shown as the negative control. FUS3-GFP (~MW 61 kDa) was detected with anti-GFP antibody. Comparable confocal settings were used in all images shown in B) and C). Duplicate experiments were conducted and one representative is shown.