Identifying SUMOylation target sites in plants poses a significant challenge due to their relatively low abundance and high dynamics. Through this lysine-null SUMO1 method and the optimized two-step purification procedure, we successfully identified over 2,000 SUMOylation sites from 1,300 putative SUMO acceptors, significantly deepening the current SUMOylome in plants. Identification of thousands of SUMOylation sites offers a unique opportunity to comprehend the biological function and site specificity of SUMOylation at the proteome level.