Quantitative Phosphoproteomics Study on cold signaling in Arabidopsis

Plants suffer external stresses in their whole life cycle and respond to that mainly through internal regulation. Growth and development of plants need proper temperature, and cold stress is a major environmental factor that adversely impacts plant growth and reduces crop yields. Plants have evolved a sophisticated regulatory network to respond to the challenge of low temperature, which involves plenty of proteins. Protein phosphorylation has been reported to be essential in cold signaling of plants, supported by the characterized roles of various protein kinases and phosphatases in cold response. Plant cold response is rapid, and the first wave of gene expression comes by 15 minutes of cold treatment. However, the changes in phosphorylation events that happen within a few minutes or even short term are still unclear. In this proposed research, we plan to employ the 15N stable isotope metabolic labeling-based quantitative PTM proteomics method to study the alterations in phosphoproteome of Arabidopsis thaliana in response to cold stress of 40 seconds. In a manner of high-throughput and high accuracy, the application of this approach is likely to discover the previous unknown components and facilitate the improvement of this signaling network. The phosphoproteins detected from the quantitative proteomics analysis will be first confirmed with biochemical assays. Polyclonal antibodies against the phosphosites will be generated, and immunoblotting will be performed to validate the results of phosphoproteomics and profile the time- (various durations) and dose-dependent (different temperatures) phosphorylation changes. After that, the functions of these phosphoproteins in cold response will be further explored with molecular genetics and physiological approaches. The mutants will be screened with the wild type at different low temperatures to study the role of each protein and select the chilling- and freezing-tolerant mutants. The transcriptome of various genotypes will also be compared to explore the downstream genes of these signaling proteins. Combined together, these results will provide information underlying the roles of the phosphorylation relays in cold stress response of plants and to delineate the plant signaling pathways. The outcome of this research will potentially contribute to the development of stress-resistant breeds to improve crop productivity to feed billions of people worldwide.