Identification and characterisation of two distinct acid phosphatases in cell walls of roots of white clover

Abstract

White clover ( Trifolium repens L.) plants were grown in liquid media and subjected to phosphate starvation by removal of the sole phosphate source. After 21 d, roots were harvested and ionically-bound (1 M salt-extractable) cell wall proteins isolated. Two distinct acid phosphatases (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2), designated APase I and II, were identified using hydrophobic column chromatography with each protein consisting of two isoforms resolved by ion-exchange column chromatography. For APase I, both isoforms (IA and IB) are glycosylated (as determined by binding to a monoclonal antibody, mAb 2.23, which is specific to xylose/fucose-containing complex-type N-linked glycans, and a Galanthus nivalis (GNA) lectin which recognises terminal mannose sugars), and exist as active monomers of 52 kDa as determined by SDS-PAGE and by gel filtration. APase IA and IB have pH optima for p-nitrophenyl phosphate of 5.8 and 6.2, and pIs of 7.3 and 6.5, respectively. For APase II, both isoforms exist as active monomers of 113 kDa by SDS-PAGE and 92 kDa by gel filtration, with pH optima of 5.8 and 6.8, and pIs of 4.4 and 5.2 to 5.3 for IIA and IIB, respectively. Isoform IIA was not recognised by the two glycan probes, while IIB was recognised by mAb 2.23. The activity of all four isoforms was severely inhibited by Cu 2+, Zn 2+ and Mo 2+, while each showed activity against a range of phosphate monoester substrates with highest substrate specificity ( V max/ K m) for ATP and PPi. Activity associated with APase I and II is detectable in roots isolated from plants maintained in P-containing media, but the activity of both is increased with the onset of P-deficiency. However, a temporal difference in response between APase I and II is observed over a 28-d time course of P-deprivation.