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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.
- Phosphorylation
- Mass Spectrometry
- Signal Transduction
- Phosphoproteome
- Stress response
- Cold Stress
- Tomato Phosphoproteome
Footnotes
Author contributions: C.-C.H., Y.Z., P.W., J.-K.Z., and W.A.T. designed research; C.-C.H., Y.Z., P.Z., and I.-H.C. performed research; C.-C.H., Y.Z., J.V.A., J.S.P., and W.A.T. analyzed data; C.-C.H., J.V.A., J.-K.Z., and W.A.T. wrote the paper.
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This article contains supplemental material. The authors have declared no conflict of interest.
- Received March 11, 2018.
- Revision received June 21, 2018.
- © 2018 Hsu et al.
Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.