Updated FTP location. Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. In order to address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure to facilitate profiling plant phosphoproteomes. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5,500 phosphoproteins, as the largest tomato phosphoproteomic resource. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species under cold environment. In particular, the activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms.