Identification of cell wall proteins in roots of phosphate-deprived white clover plants

White clover (Trifolium repens L.) plants were grown in liquid media with the sole source of phosphate (KH2PO4) removed. After 21 days, the roots were harvested, extracted first with 25 mM 2-mercaptoethanol, the cell wall fraction washed thoroughly with water before incubation with 1.0 M NaCl to extract ionically-bound cell wall proteins. The cell wall proteins were separated using hydrophobic column chromatography, then SDS-PAGE, and selected proteins subjected to N-terminal sequencing. Two of these proteins have been identified using data-base searching. The first is a c. 22 kDa protein with identity to an α-fucosidase purified from Pisum sativum, while the second is a c. 35 kDa protein with identity to a class III endochitinase purified from Nicotiana tobaccum. Specific hydrophobic column fractions were purified further using ion-exchange column chromatography and a further protein identified as a c. 41 kDa protein with highest identity to polygalacturonase inhibitors purified from several plant species.