Abstract Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass-spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling. Significance Statement Chemical genetics, which investigates the biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecule. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3 interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylate the auxin carrier PIN-FORMED1 (PIN1) and regulated its polarity that is required for the vascular patterning in the leaf.

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling