FIGURE 1.

GCN2 kinase activation by cold is light dependent. (A) Top: Schematic of the light regimen. Wild-type Landsberg [Wt (Ler)] seedlings were grown for 14 days at 22°C in a 16 h light/8 h dark cycle and shifted to 4°C starting 2 h after lights-on [8 a.m., zeitgeber time (ZT)2]. The red arrow at ZT2 indicates the start of sampling right before the beginning of cold treatment. Bottom: Immunoblot showing the time course of eIF2α phosphorylation in 14-day-old Wt(Ler) and gcn2-1 mutant (gcn2-1) seedlings subjected to cold stress as described in (A). Upper panel: Probed with phospho-specific antibody against eIF2α-P (38 kDa). Middle panel: Rubisco large subunit (∼55 kDa) as a loading control after Ponceau S staining of the blot. Lower panel: Probed with antibody against eIF2α (38 kDa). (+), arbitrary amount of total protein extract from glyphosate treated Wt seedlings indicating unphosphorylated (eIF2α) or phosphorylated (eIF2α-P) protein; (10, 30, 120) sampling time in minutes; (M) Molecular weight marker. Also shown on the right is the variation in eIF2α-P levels (percent intensity) across the tested time periods in Wt seedlings. Error bars represent standard. deviation from five biological replicates. (B) Time course of eIF2α phosphorylation as in (A) but with Wt seedlings maintained at 22°C as a control. A cropped band at the top of the blot indicates non-specific binding of the antibody. (C) eIF2α phosphorylation in Wt seedlings under 4°C in the dark. Seedlings were grown in a 16 h light/8 h dark cycle, dark-acclimated for 24 h and shifted to 4°C in the dark. Time = 0 indicates the start of sampling in dark right before the cold treatment.