Functional Analysis Of Two Phosphate Transporters OsPT2 And OsPT6 From Rice In Heterologous Expression System

Phosphorus (P) is one of key macronutrients required for plant growth and metabolism. It plays an important role in energy transfer through the formation of energy-rich phosphate ester and is also an essential component of macromolecular such as nucleotides, phospholipids, and sugar phosphates. The concentration gradient from the soil solution P to plant cell exceeds about 2000-fold with an average inorganic phosphate concentration of 1μM in soil solution. Hence, in most soils, mobilization and acquisition of phosphate often limit plant growth and the crop yield.Phosphate uptake systems in plant roots operate over a broad range of phosphate concentrations and contain high-affinity and low-affinity transport systems. By analogy with other transporters in the Pht1 family, it is likely that there are 13 Pht1 family members in rice which functions as absorbing and transporting phosphate from soils. Further dissection of the expression pattern in rice showed OsPT2 and OSPT6 are strongly expressed by phosphate-starvation. For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake, two phosphate transporter cDNAs (OsPT2 and OsPT6) of rice are expressed in heterologous expression system.1. Semi-RT-PCR of Pht1 family members were done to dissection of the expression pattern in rice. The plants were treated under high and low concentration of phosphate for 21 days and reverse transcription PCR was done by comparing expression of the housekeeping gene, Actin. The transcripts of both OsPT2 and OsPT6 were predominately expressed in response to Pi deprivation in the roots, but expression in the leaves also increased upon Pi starvation, especially for OsPT6.2. Functional analysis of OsPT2 and OsPT6 in yeast mutant MB192. The yeast mutant complementation analysis suggests that the transporter OsPT6, but not OsPT2 was able to complement a yeast Pi uptake mutant in the high affinity concentration range. When expressed in a Pi-uptake deficient yeast mutant, the phosphate transporter (OsPT6) protein exhibits an apparent Km of 95.97μM and is putative a high-affinity phosphate transporter.3. The pH experiment suggests that the activity of OsPT6 is dependent on the electrochemical membrane potential mediates by the yeast P-type H+-ATPase.4. When expressed in HEK293 cells by either OsPT6: YFP or OsPT6: CFP proteins, both OsPT2- and OsPT2-EYFP fusion proteins were targeted to the plasma membrane in HEK 293 cells. This work demonstrates that OsPT6 has a similar function as PHO84, which is a phosphate transporter in yeast Saccharomyces Cerevisiae and OsPT6 plays an important role in Pi acquisition, while OsPT2 is a low-affinity Pi transporter and functions in translocation of the stored Pi in the plant.In conclusion, the phosphate transporter OsPT6 can complement the yeast Pi uptake mutant MB192 and is a high affinity transporter co-transporting with proton. OsPT2 failed to complement a yeast Pi uptake mutant in the high affinity concentration range. However, OsPT2 and OsPT6 were expressed in HEK293 and targeted to the plasma membrane.