Abstract
Capturing molecular machines in action is essential to understanding the architecture of protein complexes, cellular regulation, and gene function. Here, we present a robust cross-linking mass spectrometry (XL-MS) platform that uses the PhoX cross-linker to map proteome-wide interactions in Arabidopsis thaliana under semi-native conditions. Using whole-cell lysates, chloroplasts, and nuclei, we identified 47,119 unique cross-links, including 3,527 inter-protein cross-links. We then constructed a high-confidence protein-protein interaction (PPI) network containing 1,229 proteins and 1,446 PPIs. In silico validation using STRING confirmed 637 heteromeric interactions with a combined confidence score of at least 0.4, including 532 interactions with a score of at least 0.7. The remaining interactions are novel. Our dataset provides direct experimental evidence of physical interactions and defines residue-level binding interfaces. XL-MS resolved the spatial topology of structurally challenging protein complexes, such as the chloroplast 70S ribosome stalks, and transient intermediates involved in photosystem repair. XL-MS also revealed both conserved and distinct interaction patterns across photosystem complexes, ribosomes, and chromatin-associated proteins. Notably, the data suggest that the ribosome undergoes ubiquitination and is regulated by the 26S proteasome. This finding is further supported by in vivo diGly proteomic analysis. Among the novel interactions uncovered is a metabolite enzyme that interacts with histones, which could potentially contribute to histone modification. This work establishes XL-MS as a powerful tool for advancing the functional annotation of genomes and the understanding of the architecture of protein complexes and cellular regulation in plants, providing a valuable resource for the plant research community.