In Vivo and In Vitro SUMOylation Assays in Arabidopsis.

Small ubiquitin-like modification (SUMOylation) is a crucial post-translational modification that modulates protein stability, localization, and interaction dynamics. Despite the identification of thousands of putative small ubiquitin-like modifier (SUMO) substrates, functional validation remains challenging due to the low abundance and highly dynamic nature of SUMOylated proteins. Here, we present a protocol for detecting protein SUMOylation, integrating bioinformatic site prediction, and rapid substrate screening via in vivo tobacco transient expression and in vitro E. coli assay, followed by precise validation using transgenic Arabidopsis lines. However, detection of low-abundance SUMOylated proteins may require coupling with mass spectrometry, and the in vitro system does not fully recapitulate the complex regulatory network in vivo. This workflow provides a useful tool for studying SUMOylation in plants. Key features • Integrates bioinformatic prediction, in vitro validation, and in vivo confirmation for SUMOylation analysis. • E. coli co-expression system enables rapid SUMOylation detection without protein purification. • Arabidopsis transgenic line system confirms SUMOylation under physiological conditions. • The protocol is applicable to most Arabidopsis proteins.