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-à-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na+ (10–100 mM). 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 >10 mM, 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-à-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 xylem sap desalinization, and differences in xylem sap Na+ concentrations in both species are attributed to differences in Na+ transport affinity/conductance between the transporters.