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BONCAT enables time-resolved analysis of protein synthesis in native plant tissue

Glenn, Weslee S. and Stone, Shannon E. and Ho, Samuel H. and Sweredoski, Michael J. and Moradian, Annie and Hess, Sonja and Bailey-Serres, Julia and Tirrell, David A. (2017) BONCAT enables time-resolved analysis of protein synthesis in native plant tissue. Plant Physiology, 173 (3). pp. 1543-1553. ISSN 0032-0889. doi:10.1104/pp.16.01762.

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Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal non-canonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis thaliana seedlings. BONCAT relies on the translational incorporation of a non-canonical amino acid (ncAA) probe into cellular proteins. In this study, the probe is the methionine surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse, and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry (LC-MS) to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and post-translational regulation in plants.

Item Type:Article
Related URLs:
URLURL TypeDescription Data
Glenn, Weslee S.0000-0001-7733-7291
Stone, Shannon E.0000-0002-6617-3874
Ho, Samuel H.0000-0001-7647-0752
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Bailey-Serres, Julia0000-0002-8568-7125
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2017 American Society of Plant Biologists. Free via OPEN. Received November 17, 2016; Accepted January 14, 2017; First Published on January 19, 2017. We would like to thank the laboratory of Professor Elliot Meyerowitz, especially Arnavaz Garda and Dr. Paul Tarr, for seeds, space in growth chambers and helpful discussions. We would also like to thank Roxana Eggleston-Rangel for helpful discussions on sample preparation for mass spectrometry. This work was supported by the Gordon and Betty Moore Foundation through grant GBMF2809. W.S.G. was supported by a National Research Council Ford Foundation Post-Doctoral Fellowship and a United Negro College Fund/Merck Foundation Post-Doctoral Fellowship. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation, through Grant GBMF775, the Beckman Institute and the NIH through Grant 1S10RR029594-01A1. Author Contributions: W.S.G. conceived of and implemented the project, and performed most of the experiments with contributions from all other authors. S.H.H., J.B-S and W.S.G. designed and conducted gel imaging and immunoblotting experiments. A.M., S.H. and W.S.G. designed and ran the mass spectrometry experiments. S.E.S., M.J.S., S.H., J. B-S and W.S.G. conducted all bioinformatic analyses of the mass spectrometry data. S.E.S., M.J.S. and W.S.G. constructed figures with input from all other authors. W.S.G, J.B-S and D.A.T. wrote the article with valuable contributions from all authors.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
Ford FoundationUNSPECIFIED
United Negro College FundUNSPECIFIED
Merck FoundationUNSPECIFIED
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:3
Record Number:CaltechAUTHORS:20170123-111643957
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73620
Deposited By: Tony Diaz
Deposited On:26 Jan 2017 00:56
Last Modified:11 Nov 2021 05:19

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