Journal: Science Advances

Article Title: The TaCEP15 peptide signaling cascade modulates primary root length and drought tolerance in wheat

doi: 10.1126/sciadv.ady1949

Figure Lengend Snippet: ( A ) TaCEPRL-mediated phosphorylation analysis of TaSnRK1α. The phosphorylation status of TaSnRK1α was examined in a phos-tag gel assay [anti–maltose-binding protein (MBP)]. Red asterisks and red arrow indicate the shifted phosphorylation bands of TaSnRK1α, and the black arrow indicates the nonphosphorylated TaSnRK1α proteins. WB, Western blot. ( n = 3 biologically independent experiments). ( B ) Examination of TaSnRK1α stability in response to TaCEPRL in N. benthamiana leaves. The relative abundance of TaSnRK1α was quantified using ImageJ software. The relative protein levels of TaSnRK1α without TaCEPRL-KD were defined as 1.00. ( n = 3 biologically independent experiments). ( C ) Examination of TaSnRK1α stability in response to TaCEPRL in a cell-free degradation assay. The relative protein abundance of TaSnRK1α was quantified using ImageJ software. The relative protein level of TaSnRK1α at 0 min was defined as 1.00. ( n = 3 biologically independent experiments). ( D ) TaCEP15 peptide enhances TaCEPRL-mediated phosphorylation of TaSnRK1α. The phosphorylation status of TaSnRK1α was examined in a phos-tag gel assay [anti–green fluorescent protein (GFP)]. Red asterisks and red arrow indicate the shifted phosphorylation bands of TaSnRK1α protein, and the black arrow indicates the nonphosphorylated TaSnRK1α proteins. WB, Western blot. ( n = 3 biologically independent experiments). ( E ) TaCEP15 accelerates TaCEPRL-mediated TaSnRK1α degradation in N. benthamiana leaves. The relative abundance of TaSnRK1α was quantified using ImageJ software. The relative protein levels of TaSnRK1α without TaCEPRL and TaCEP15 were defined as 1.00. Groups labeled with different alphabets were considered significantly different [ P < 0.05; one-way ANOVA and Tukey’s post hoc test ( n = 3 biologically independent experiments)]. ( F ) TaCEP15 peptide enhances TaCEPRL-mediated TaSnRK1α degradation in a cell-free degradation assay. The relative protein abundance of TaSnRK1α was quantified using ImageJ software. The relative protein level of TaSnRK1α at 0 min was defined as 1.00. ( n = 3 biologically independent experiments).

Article Snippet: The constructs p35S :TaSnRK1α-GFP and p35S :TaCEPRL-KD-Flag and p35S :TaSnRK1α-GFP, p35S :TaCEPRL-Flag, and TaCEP15 peptide were transformed into A. tumefaciens (strain GV3101) and infiltrated into N. benthamiana leaves in the presence or absence of 50 μM MG132 (133407-82-6, MCE, USA).

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Software, Degradation Assay, Quantitative Proteomics, Labeling