Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/11/2011
Publication Date: 6/3/2011
Citation: Lewandowska-Gnatowsk, E., Johnston, M.L., Antoine, W., Szczegielniak, J., Muszynska, G., Miernyk, J.A. 2011. Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding. Phytochemistry. 72:1285-1292. Interpretive Summary: A specialized and very sensitive probe was utilized for analysis of chemical reactions in corn leaves. Changes in the extent of the chemical reaction was measured after using mechanical wounding of corn leaves in order to simulate attack by an insect pest. By subjecting the results to a thorough statistical analysis, it was possible to arrange a time-line of events after the simulated insect feeding. By detailed analysis of the time line, it was possible to demonstrate that one type of signaling molecule was not involved in the response of corn leaves to insect feeding. A large group of corn leaf chemicals was separated into five groups, each of which shares common properties. Knowing the distribution of these chemical groups will help understand the results from future studies of insect feeding. This information will be useful to scientists in their efforts to improve agricultural crop resistance to pests through both classical breeding and application of biotechnology strategies.
Technical Abstract: Mechanical wounding of 2-week old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. Multiplex-staining of high resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) allowed us to quantify changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were grouped into five clusters. Forty one spots were excised from the gels, digested with trypsin, and the resultant tryptic peptides analyzed by mass spectrometry. A reliable identity was assigned to 21 of the selected proteins, all but two of which are annotated as being involved in either signaling or central metabolism. The results provide a platform for future analyses of maize leaf responses to mechanical wounding.