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

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 (Kub) 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 Kub 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.赖氨酸泛素化（lysine ubiquitination）是一种被广泛研究的翻译后修饰（posttranslational modification），在真核细胞的各类生物学过程中发挥至关重要的作用。尽管已有多项研究对植物泛素化组（ubiquitylome）展开了分析，但作为分子生物学模式植物的烟草（Nicotiana tabacum）却尚未有相关研究报道。本研究采用液相色谱-串联质谱（LC–MS/MS）与高灵敏度免疫亲和纯化联用技术，对烟草中的赖氨酸泛素化修饰进行了全面分析，最终在572个蛋白质中鉴定出964个赖氨酸泛素化（Kub）位点。深入的生物信息学分析显示，这些蛋白质分布于细胞质、叶绿体、细胞核、质膜等多种细胞亚区。值得关注的是，25%的泛素化蛋白质定位于叶绿体，其中21种酶类参与了光合作用与碳固定等重要通路。蛋白质免疫印迹（Western blot）分析结果表明，烟草花叶病毒（TMV）侵染可引发泛素化水平发生改变。本研究为首次针对烟草开展的赖氨酸泛素化全面蛋白质组学分析，阐明了泛素化在多种生理生化过程中的关键作用，同时为现有赖氨酸泛素化研究图谱提供了极具价值的补充。