The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress. Legumes (Fabaceae) are one of the most economically important crop families in the world and are characterized by their unique ability to fix atmospheric nitrogen through a symbiotic relationship with soil bacteria. These bacteria, collectively termed Rhizobia and which include genera such as Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium, and Azorhizobium, form specialized organs within the plant, and nitrogen fixation occurs via the conversion of N 2 into NH 3 by bacterial nitrogenases. The mutualistic interactions provide a plentiful supply of nitrogen to the plants that in turn results in very high protein levels in legumes. Therefore, legumes have been assimilated as a major dietary source of protein for both humans and animals. Legumes also provide nitrogen to the soil, thus reducing the need for exogenous fertilizers. Legumes are also a unique source of natural products such as isoflavonoids, alkaloids, and saponins, many of which have documented antimicrobial and pharmacological properties (1-3). Based on the above traits, legumes have significant economic and ecological value. For example, soybeans provide more than one-third of human protein intake and approximately half of the world's supply of oilseed. Over 73 million acres of soybeans (Glycine max) and 76 million acres of alfalfa (Medicago sativa) were cultivated in the United States in 2003 and have estimated values of $17.5 billion and $7.5 billion, respectively (4).The mutualistic interactions between legu...

The temporal proteome response of Medicago truncatula suspension cell cultures to yeast elicitation (which mimics fungal infection) was investigated using two-dimensional polyacrylamide gel electrophoresis (2-DE) and nanoliquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Reproducibility of 2-DE was assessed using the number of the visualized protein spots and spot volume. Average coefficient of variation was determined to be less than 6% for the number of spots and around 50% for spot volume. About 4% of the total visualized proteins, that is, 34 out of 861, were differentially accumulated in the suspension cells 24 h after yeast elicitation, including isoflavononid biosynthetic enzymes and a putative laccase. The induction of the putative laccase was highly correlated with the polymerization of phenolics such as 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, and ferulic acid into cell walls. In contrast, lignin was only induced at the later stages of the temporal study, indicating that this specific laccase is primarily involved in cell wall modifications and/or fortifications rather than in lignification in response to yeast elicitation.