Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivalling several other major systems, such as transcription regulation and epigenetics, I describe the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarize the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I propose a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I present new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of ubiquitin E3 ligase genes.
Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity
Abstract Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.
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- Award ID(s):
- 1951094
- NSF-PAR ID:
- 10224819
- Date Published:
- Journal Name:
- Plant Physiology
- Volume:
- 185
- Issue:
- 4
- ISSN:
- 0032-0889
- Page Range / eLocation ID:
- 1943 to 1965
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/plphys/kiab011
