Abstract
Tomato is a major crop plant and an important constituent of the human diet. Exclusive features such as bearing fleshy fruits and undergoing a phase transition from partially photosynthetic to fully heterotrophic metabolism make tomato fruit a model system for fruit development studies. Although the tomato genome has been completely sequenced, functional proteomics studies are still at their starting stage. Proteomics technologies, especially the combination of multiple approaches, provide a very powerful tool to accurately identify functional proteins and investigate certain sets of proteins in more detail. The direct binding of plant 14-3-3 proteins to their multiple target proteins modulates the functions of the latter, suggesting that these 14-3-3 proteins are directly involved in various physiological pathways. This chapter outline methods for the identification of 14-3-3 protein complexes in tomato fruit tissues. These methods include detailed protocols for protein extraction, coimmunoprecipitation, SDS-PAGE, SYPRO Ruby staining, in-gel trypsin digestion, and LC-MS/MS analysis for 14-3-3 interactomics.
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Acknowledgments
This work was supported by a Grant in-Aid for Scientific Research to T.S. [Nos. 15K18819 and 17K08190], by Grants in Aid for Scientific Research to J.Y. [Nos. 15H0116705, 262921888, and 18H02162] from the Japan Society for the Promotion of Science (JSPS), and by a grant from The NOASTEC foundation, Hokkaido University Young Scientist Support Program to T.S. Lu was supported by a JSPS research fellowships (2016-2018) and JSPS Postdoctoral fellowships for Research in Japan (2018-2020). Luo was supported by a Support Grant for Self-Supported International Graduate Student (Hokkaido University Faculty of Science: 2017-2018). This work was also supported by a Cooperative Research Grant of the Plant Transgenic Design Initiative, Gene Research Center, University of Tsukuba.
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Luo, Y., Lu, Y., Yamaguchi, J., Sato, T. (2020). Proteome Analysis of 14-3-3 Targets in Tomato Fruit Tissues. In: Jorrin-Novo, J., Valledor, L., Castillejo, M., Rey, MD. (eds) Plant Proteomics. Methods in Molecular Biology, vol 2139. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0528-8_21
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DOI: https://doi.org/10.1007/978-1-0716-0528-8_21
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