Fig. 7. Flow diagram showing the phosphoproteomic approach used in this study to identify the targets of SnRK2.8 kinase. Root protein was isolated from wild-type Arabidopsis, an SnRK2.8 overexpression line, and a knockout line. Protein was separated on a 2-DE gel and stained with Pro-Q Diamond. Image analysis was conducted, and protein spots that were present or more abundant in the overexpression lines compared with the knockout were picked, and proteins were identified with tandem MS. Eleven candidate proteins were chosen for in vitro phosphorylation assays. Protein was obtained from nine candidates by expression in E. coli. Each purified protein was incubated with the SnRK2.8 kinase and [γ^-32P] dATP. The gel at the lower left is a representation of a coomassie stained gel used to determine protein loading. At the lower right is a representation of an autoradiograph of the gel containing candidate proteins incubated with [γ^-32P] dATP and the SnRK2.8 kinase. The autoradiograph shows the autophosphorylated kinase in lane 1, no signal in lane 2 (containing the target protein alone), and the autophosphorylated kinase and a phosphorylated kinase in lane 3.