Homology modeling identifies crucial amino-acid residues that confer higher Na⁺transport capacity of OcHKT1;5 from Oryza coarctata Roxb

HKT1;5 loci/alleles are important determinants of crop salinity tolerance. HKT1;5s encode plasmalemma-localized Na + transporters, which move xylem Na + into xylem parenchyma cells, reducing shoot Na + accumulation. Allelic variation in rice OsHKT1;5 sequence in specific landraces (Nona Bokra OsHKT1;5-NB/Nipponbare OsHKT1;5-Ni) correlates with variation in salt tolerance. Oryza coarctata, a halophytic wild rice, grows in fluctuating salinity at the seawater-estuarine interface in Indian and Bangladeshi coastal regions. The distinct transport characteristics of the shoots and roots expressing the O. coarctata OcHKT1;5 transporter are reported vis-a-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na + (10- 100mM). Electrophysiological measurements in Xenopus oocytes expressing O. coarctata or rice HKT1;5 transporters indicate that OcHKT1;5, like OsHKT1;5-Ni, is a Na + -selective transporter, but displays 16-fold lower affinity for Na + and 3.5-fold higher maximal conductance than OsHKT1;5-Ni. For Na + concentrations >10mM, OcHKT1;5 conductance is higher than that of OsHKT1;5-Ni, indicating the potential of OcHKT1;5 for increasing domesticated rice salt tolerance. Homology modeling/simulation suggests that four key amino-acid changes in OcHKT1;5 (in loops on the extracellular side; E239K, G207R, G214R, L363V) account for its lower affinity and higher Na + conductance vis-a-vis OsHKT1;5-Ni. Of these, E239K in OcHKT1;5 confers lower affinity for Na + transport, as evidenced by Na + transport assays of reciprocal site-directed mutants for both transporters (OcHKT1;5- K239E, OsHKT1;5-Ni-E270K) in Xenopus oocytes. Both transporters have likely analogous roles in xylemsap desalinization, and differences in xylem sap Na + concentrations in both species are attributed to differences in Na + transport affinity/ conductance between the transporters.