Quantitative proteomics reveals extensive lysine ubiquitination and transcription factor stability states in Arabidopsis

• DOI: 10.1093/plcell/koae310
• Publication Date: 2024-11-21T18:32:09Z
• Authors: Gaoyuan Song; Christian Montes; Damilola Olatunji; Shikha Malik; Chonghui Ji; Natalie M Clark; Yunting Pu; Dior R Kelley; Justin W Walley
• Keywords: Protein Stability; Ubiquitin-Protein Ligases

Abstract Protein activity, abundance, and stability can be regulated by post-translational modification including ubiquitination. Ubiquitination is conserved among eukaryotes plays a central role in modulating cellular function; yet, we lack comprehensive catalogs of proteins that are modified ubiquitin plants. In this study, describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling enable quantification up 18-multiplexed samples. This identified 17,940 lysine sites arising from 6,453 Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, rosette leaves. Gene ontology analysis indicated associated numerous biological processes hormone signaling, plant defense, protein homeostasis, metabolism. We determined residues directly regulate the three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using vivo degradation assays. Furthermore, codon mutation create K166R conversion prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led early flowering phenotype increased expression FLOWERING LOCUS T (FT). These site-level ubiquitinome profiles provide wealth data for future functional studies related modulation mediated