Received December 10, 2005
Returned for revision January 30, 2006
Accepted February 25, 2006
Proteomic analysis of seed filling in Brassica napus: Developmental characterization of metabolic isozymes using high-resolution two-dimensional gel electrophoresis
Martin Hajduch , Jill E. Casteel , Katherine E. Hurrelmeyer , Zhao Song , Ganesh Kumar Agrawal , and Jay J. Thelen *
Department of Biochemistry, Life Sciences Center, University of Missouri, Columbia, Missouri 65211, USA
Computer Science Department, University of Missouri, Columbia, Missouri 65211, USA
* Corresponding author; email: thelenj{at}missouri.edu.
Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed twelve different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography-tandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 non-redundant proteins. These proteins were classified into fourteen functional categories based upon the Arabidopsis genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to acetyl-CoA is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a novel, systematic analysis of metabolic processes operating in developing B.napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) which is accessible to the public domain.
