Abstract
Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. 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 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. 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 in response to a cold environment. 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.
Highlights
Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses
A Universal Sample Preparation Workflow for Plant Phosphoproteomics—An optimized sample preparation protocol for plant samples is critical to enable the identification of low abundant phosphoproteins in plant cells and to deepen the coverage of the plant phosphoproteome
Various groups have reported the use of Tris-HCl with salts [24], Tris-HCl with 30% sucrose [38], or SDS coupled with Filter Aided Sample Prep (FASP) [39] for extraction of proteins from plant tissues
Summary
As the tomato is one of the most important horticultural crops in the world, we utilized our proteomic approach to investigate the underlying mechanisms of their cold tolerance. Pimpinellifolium), displays a coldsensitive phenotype under cold conditions These tomatoes are ideal materials to study the important cold tolerance signaling pathways in tomato. Significant analytical difficulty is encountered in plant phosphoproteomics because of the high dynamic range of the plant proteome, the rigidity of plant cell walls, and the interference from chlorophyll and secondary metabolites (20 –22) These challenges hamper the sensitivity and efficiency of detecting low abundance phosphorylation events in plants through MS. This in-depth phosphoproteomic resource reveals the phosphorylation sites implicated in kinase activation and cold-responsive gene expression. Upon coupling this data with in vitro kinase screening, we discovered a connection between SnRK2s activation and cold tolerance through phosphorylation of their downstream kinases, which sheds light upon which tomato phosphoproteins are critical for conferring cold tolerance
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