Na+/H+ antiporter in tonoplast vesicles from rice roots

Abstract

The Na+/H+ antiporter in vacuolar membranes transports Na+ from the cytoplasm to vacuoles using a pH gradient generated by proton pumps; it is considered to be related to salinity tolerance. Rice (Oryza sativa L.) is a salt-sensitive crop whose vacuolar antiporter is unknown. The vacuolar pH of rice roots, determined by 31P-nuclear magnetic resonance (NMR), increased from 5.34 to 5.58 in response to 0.1 M NaCl treatment. Transport of protons into the tonoplast vesicles from rice roots was fluorometrically measured. Efflux of protons was accelerated by the addition of Na+. Furthermore, the influx of 22Na+ into the tonoplast vesicles was accelerated by a pH gradient generated by proton-translocating adenosine 5'-triphosphatase (H+-ATPase) and proton-translocating inorganic pyrophosphatase (H+-PPase). We concluded that this Na+/H+ antiporter functioned as a Na+ transporter in the vacuolar membranes. The antiporter had a K(m) of 10 mM for Na+ and was competitively inhibited by amiloride and its analogues. The K(i) values for 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and 5-(N,N-hexamethylene)-amiloride (HMA) were 2.2, 5.9, and 2.9 μM, respectively. Unlike barley, a salt-tolerant crop, NaCl treatment did not activate the antiporter in rice roots. The amount of antiporter in the vacuolar membranes may be one of the most important factors determining salt tolerance.