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Updated: Aug 6, 2025
Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis
Published on: June 25, 2020
Fast Identification of In Vivo Protein Phosphorylation Events Using Transient Expression in Leaf Mesophyll
Ellen Broucke1,2, Filip Rolland3,4, Nathalie Crepin5,6
1Plant Metabolic Signaling Lab, Biology Department, KU Leuven, Heverlee, Leuven, Belgium. ellen.broucke@kuleuven.be.
Abstract:
Phosphorylation/dephosphorylation is a key posttranslational mechanism for signal transduction and amplification. Several techniques exist for assessing protein phosphorylation status, but each has its own drawbacks. The fast, straightforward, and low-tech approach described here uses transient overexpression of peptide-tagged proteins in Arabidopsis leaf mesophyll protoplasts and immunoblotting with Phos-tag™ SDS-PAGE and commercial anti-tag antibodies. We illustrate this with two relevant examples related to the SnRK1 protein kinase, which mediates metabolic stress signaling: Arabidopsis thaliana SnRK1 activation by T-loop (auto-)phosphorylation and SnRK1 phosphorylation of the Arabidopsis RAV1 transcription factor, which is involved in seed germination and early seedling development.
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