RESEARCH ARTICLE Quantitative iTRAQ proteome and comparative transcriptome analysis of elicitor-induced Norway spruce (Picea abies) cells reveals elements of calcium signaling in the early conifer defense response Dustin N. Lippert 1 , Steven G. Ralph 1 * , Michael Phillips 2 ** , Rick White 3 , Derek Smith 4 , Darryl Hardie 4 , Jonathan Gershenzon 2 , Kermit Ritland 5 , Christoph H. Borchers 4, 6 and Jörg Bohlmann 1, 5 1 Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada 2 Max Planck Institute for Chemical Ecology, Jena, Germany 3 Department of Statistics, University of British Columbia, Vancouver, BC, Canada 4 University of Victoria – Genome BC Proteomics Centre, Victoria, BC, Canada 5 Department of Forest Sciences, University of British Columbia, Vancouver, BC, Canada 6 Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada Long-lived conifer trees depend on both constitutive and induced defenses for resistance against a myriad of potential pathogens and herbivores. In species of spruce (Picea spp.), several of the late events of pathogen-, insect-, or elicitor-induced defense responses have previously been characterized at the anatomical, biochemical, transcriptome, and proteome levels in stems and needles. However, accurately measuring the early events of induced cellular responses in a con- ifer is technically challenging due to limitations in the precise timing of induction and tissue sampling from intact trees following insect or fungal treatment. In the present study, we used the advantages of Norway spruce (Picea abies) cell suspensions combined with chitosan elicitation to investigate the early proteome response in a conifer. A combination of iTRAQ labeling and a new design of iterative sample analysis employing data-dependent exclusion lists were used for pro- teome analysis. This approach improved the coverage of the spruce proteome beyond that achieved in any prior study in a conifer system. Comparison of elicitor-induced proteome and transcriptome responses in Norway spruce cells consistently identified features associated with calcium-mediated signaling and response to oxidative stress that have not previously been observed in the response of intact trees to fungal attack. Received: 26 March, 2008 Revised: 14 July, 2008 Accepted: 6 August, 2008 Keywords: Blue stain fungus / Cell suspension culture / Ceratocystis polonica / Conifer pathogen interaction / Plant defense 350 Proteomics 2009, 9, 350–367 Correspondence: Dr. Jörg Bohlmann, Michael Smith Labora- tories, University of British Columbia, 321-2185 East Mall, Van- couver, BC, Canada V6T 1Z4 E-mail: bohlmann@msl.ubc.ca Fax: 11-604-822-2114 Abbreviations: CaM, calmodulin; MEP, methyl erythritol phos- phate; TAIR, The Arabidopsis Information Resource * Current address: Department of Biology, University of North Dakota, Grand Forks, ND, USA. ** Current address: Departament de Genètica Molecular Vegetal, Consorci CSIC-IRTA, Barcelona, Spain. DOI 10.1002/pmic.200800252  2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com