Measuring the turnover rates of Arabidopsis proteins using deuterium oxide: an auxin signaling case study.
Rapid environmental responses in plants rely on endogenous signaling mechanisms, which in many cases are mediated by changes in protein turnover rates. It is therefore necessary to develop methods for measuring protein dynamics that monitor large sets of plant proteins to begin to apply a systems biology approach to the study of plant behavior. The use of stable isotope labeling strategies that are adaptable to proteomic methods is particularly attractive for this purpose. Here, we explore one example of such methods that is particularly suitable for plants at the seedling stage, where measurement of amino acid and protein turnover rates is accomplished using a heavy water labeling strategy. The method is backed by microarray evaluation to define its feasibility for specific experimental approaches, and the CULLIN-ASSOCIATED AND NEDDYLATION DISSOCIATED 1 (CAND1) and TRANSPORT INHIBITOR RESPONSE 1 (TIR1) proteins are used to illustrate the potential utility in understanding hormonal signaling regulation. These studies provide insight not only into the potential utility of the method, but also address possible areas of concern regarding the use of heavy water labeling during plant growth. These considerations suggest a prescription for specific experimental designs that minimize interference resulting from the induction of treatment-specific gene expression in the results obtained.
Yang
Xiao-Yuan
XY
Department of Plant Biology, University of Minnesota, St. Paul, MN 55108, USA.
Chen
Wen-Ping
WP
Rendahl
Aaron K
AK
Hegeman
Adrian D
AD
Gray
William M
WM
Cohen
Jerry D
JD
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D017666:Deuterium Oxide / Q000378:metabolism* / Q000494:pharmacology; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D007210:Indoleacetic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
D000596:Amino Acids; D029681:Arabidopsis Proteins; C488785:CAND1 protein, Arabidopsis; D044783:F-Box Proteins; D007210:Indoleacetic Acids; D011956:Receptors, Cell Surface; C501405:TIR1 protein, Arabidopsis; D017666:Deuterium Oxide