Comprehensive Proteomic Analysis of Lysine Ubiquitination in Seedling Leaves of Nicotiana tabacum

[Image: see text] Lysine ubiquitination, a widely studied posttranslational modification, plays vital roles in various biological processes in eukaryotic cells. Although several studies have examined the plant ubiquitylome, no such research has been performed in tobacco, a model plant for molecular biology. Here, we comprehensively analyzed lysine ubiquitination in tobacco (Nicotiana tabacum) using LC–MS/MS along with highly sensitive immune-affinity purification. In total, 964 lysine-ubiquitinated (K(ub)) sites were identified in 572 proteins. Extensive bioinformatics studies revealed the distribution of these proteins in various cellular locations, including the cytoplasm, chloroplast, nucleus, and plasma membrane. Notably, 25% of the K(ub) proteins were located in the chloroplast of which 21 were enzymatically involved in important pathways, that is, photosynthesis and carbon fixation. Western blot analysis indicated that TMV infection can cause changes in ubiquitination levels. This is the first comprehensive proteomic analysis of lysine ubiquitination in tobacco, illustrating the vital role of ubiquitination in various physiological and biochemical processes and representing a valuable addition to the existing landscape of lysine ubiquitination.