Characterization of two phosphate transporters from barley; evidence for diverse function and kinetic properties among members of the Pht1 family

Putative phosphate transporters have been identified in a barley ( Hordeum vulgare L.) genomic library by their homology to known phosphate transporters from dicot species. The genes designated HORvu; Pht1; 1 and HORvu; Pht1; 6 encode proteins of 521 and 535 amino acids respectively with 12 predicted membrane-spanning domains and other motifs common to the Pht1 family of phosphate transporters. HORvu; Pht1; 1 is expressed exclusively in roots and is strongly induced by phosphate deprivation. HORvu; Pht1; 6 is expressed in the aerial parts of the plant with strongest expression in old leaves and flag leaves. In situ hybridization showed that HORvu; Pht1; 6 is expressed in the phloem of vascular bundles in leaves and ears. In order to study the biochemical properties of HORvu; Pht1; 1 and HORvu; Pht1; 6, the genes were expressed in transgenic rice ( Oryza sativa L.) plants under the control of the rice actin promoter and suspension cell cultures were generated. Cells derived from transgenic plants were able to take up phosphate at a much higher rate than control cells, demonstrating that both genes encode functional phosphate transporters. The estimated Km for phosphate for cells expressing HORvu; Pht1; 1 was 9.06 +/- 0.82 muM, which is characteristic of a high-affinity transporter. The rate of phosphate uptake decreased with increasing pH, suggesting that HORvu; Pht1; 1 operates as a H+/H2PO4- symporter. In contrast, the estimated Km for phosphate for cells expressing HORvu; Pht1; 6 was 385 +/- 61 muM, which is characteristic of a low-affinity transporter. Taken together, the results suggest that HORvu; Pht1; 1 functions in uptake of phosphate at the root surface, while HORvu; Pht1; 6 probably functions in remobilization of stored phosphate from leaves.