Root hairs are single tubular cells formed from the differentiation of epidermal cells on roots. They are involved in water and nutrient uptake and represent the infection site on leguminous roots by rhizobia, soil bacteria that establish a nitrogen-fixing symbiosis. Root hairs develop by polar cell expansion or tip growth, a unique mode of plant growth shared only with pollen tubes. A more complete characterization of root hair cell biology will lead to a better understanding of tip growth, the rhizobial infection process, and also lead to improvements in plant water and nutrient uptake. We analyzed the proteome of isolated soybean (Glycine max) root hair cells using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and shotgun proteomics (1D-PAGE-liquid chromatography and multidimensional protein identification technology) approaches. Soybean was selected for this study due to its agronomic importance and its root size. The resulting soybean root hair proteome reference map identified 1,492 different proteins. 2D-PAGE followed by mass spectrometry identified 527 proteins from total cell contents. A complementary shotgun analysis identified 1,134 total proteins, including 443 proteins that were specific to the microsomal fraction. Only 169 proteins were identified by the 2D-PAGE and shotgun methods, which highlights the advantage of using both methods. The proteins identified are involved not only in basic cell metabolism but also in functions more specific to the single root hair cell, including water and nutrient uptake, vesicle trafficking, and hormone and secondary metabolism. The data presented provide useful insight into the metabolic activities of a single, differentiated plant cell type.

2-D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non-abundant proteins within seeds is difficult when 60-80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) to precipitate soybean (Glycine max) seed storage globulins, glycinin and beta-conglycinin. This method removes 87+/-4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of >or=50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5-fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-D electrophoretic separation and an in-gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.