Phosphoproteomics
Previous tissue-specific transcriptomic experiments performed in the lab already revealed that several transporters and kinases were differentially regulated during auxin-mediated LR formation between Col-0 and CASP1pro::shy2-2. Since it was recently shown that auxin treatment evokes very rapid protein phosphorylation changes, we used phosphoproteomics to characterize differential protein phosphorylation between Col-0 and CASP1pro::shy2-2 after short auxin treatment (~5 minutes). These experiments were performed in collaboration with the laboratory of Prof. Dolf Weijers, which have developed an efficient workflow to detect rapid changes in protein phosphorylation after auxin treatment {Kuhn, Roosjen et al, 2024, PMID: 38128538.
We performed two types of experiments. In the first, both genotypes were treated with 0.1µM IAA for 5 minutes. In a second experiments, we used Col-0 and CASP1pro::shy2-2 plants that were expressing ccvTIR1 in the XPP (GATA23pro::CITRINE:ccvTIR1) and plants were treated with 0.1µM cvxIAA for 5 minutes. The ccvTIR1/cvxIAA pair allows for specific activation of TIR1 signalling via a synthetic receptor/ligand complex (Uchida, et al., 2018, PMID: 29355850). This should allow us to separate generic auxin-mediated changes in protein phosphorylation from TIR1-mediated changes in protein phosphorylation in the XPP. Although, the cvxIAA/ccvTIR1-mediated changes in phosphopeptides showed some overlap with the IAA-mediated changes, several candidates were specific for the cvxIAA/ccvTIR1 experiment. We are currently functionally characterizing several promising candidates.
(Unpublished data)
Early auxin-mediated changes in protein phosphorylation in wild type and spatial accommodation mutant. (A) Histogram showing the distribution of significantly hypo- or hyperphosphorylated peptides after short auxin treatment (5 min, 0.1µM IAA). Top panel shows the results or Col-0, lower panel for CASP1pro::shy2-2. (B) Histogram showing the distribution of significantly hypo- or hyperphosphorylated peptides after short convex auxin treatment (5 min, 0.1µM cvxIAA). Top panel shows the results for Col-0 + GATA23pro::ccvTIR1:CITRINE, lower panel for CASP1pro::shy2-2 + GATA23pro::ccvTIR1:CITRINE. Hypophosphorylated peptides in blue, hyperphosphorylated peptides in magenta. (C) Venn diagram showing comparison of identified phosphopeptides between the different genotypes after IAA and cvxIAA treatment.