Effects of ammonium- and nitrate-nutrition on the plasma membrane H⁺-ATPase and proton pump of rice leaves

Rice plants (Oryza sativa L. japonica ssp. cv. Nipponbare) were cultivated with ammonium (NH4+-N) or nitrate (NO3--N) as sole nitrogen in a hydroponics experiment, and the plasma membrane vesicles of leaves were isolated by two-phase system at the seedling stage. The plasma membrane H+-ATPase hydrolytic activity and proton pump activity were analyzed for elucidating the responses of the plasma membrane H+-ATPase of rice leaves to ammonium or nitrate nutrition. The purity of plasma membrane is above 80%. The optimum pH for the plasma membrane H+-ATPase is 6.2 under both NH4+-N and NO3--N cultivated plants. The plasma membrane H+-ATPase hydrolytic activity, Vmax and Km obtained from the NO3--N fed rice leaves are significantly higher than those from the NH4+-N fed rice leaves. In addition, the Western Blot showed that the enzyme concentration of plasma membrane H+-ATPase from rice leaves of NO3-- N fed plants was higher than that of NH4+-N fed plants. These results indicate that the higher activity of H+-ATPase from rice leaves of NO3--N fed plants is resulted from the increased number of H+-ATPase units per membrane area and up-regulated expression of H+-ATPase. The H+- pump activity of the plasma membrane from NO3--N fed rice leaves is larger than that of NH4+-N fed rice leaves. As the NO3- -transport across the plasma membrane is an active transport process in relation to the plasma membrane H+-ATPase, it is might be true that higher activity of plasma membrane H+-ATPase of rice leaves with NO3--N nutrition is induced by uptake of large amount NO3--N in to the leaf cells.