Figure 1: Experimental setup and knocking down the SnRK1α2.

(a) Overview of the experimental setup for the analysis of the phosphoproteome of SnRK1 mutants. A dense time course (0, 20, 40, 60, 80, 100, 120, 180, and 360 min of extended night plus sample in the middle of the day) was sampled of the snrk1α1/α2 mutant and the wild type during extended night. Metabolites were extracted with methanol:chloroform:water extraction solution, derivatized and measured with GC-MS. Proteins were isolated, digested and desalted by a combined C18/graphite carbon method. Subsequently, phosphopeptides were enriched by TiO₂ MOAC and analysed by LC-MS/MS. (b) The expression of SnRK1α1 and SnRK1α2 in Col-0 and snrk1α1/α2 in the time series experiment for 3 time points (Light, 0 min and 360 min of extended night) is shown with pAMPK Thr¹⁷² antibody, which recognizes both SnRK1α1 (upper band 61.2 kDa) and SnRK1α2 (lower band 58.7 kDa). (c) The abundance of T-loop phosphorylation is also shown by the level of DGHFLK[T^175/176]SCGSPNYAAPEVISGK peptide in Col-0 and snrk1α1/α2. (d) Phenotype of Col-0 and SnRK1 mutant lines in 12 h light/12 h dark conditions. Knocking down of SnRK1α2 was started 22 days after germination (DAG) by spraying plants daily with 10 μM β-estradiol or mock solution without β-estradiol. Because SnRK1α2 protein is relatively stable its knocking down takes 5–6 days (Pedrotti et al. in preparation). This picture was taken 11 days (32 days after germination (DAG)) after the start of induction (phenotype development time course in Supplementary Fig. S1).