Response Of The Plasma Membrane H~+-ATPase Of Rice Seedling To Ammonium And Nitrate Nutrition

Rice (Oryza sativa L.) is one of the most important crops in China. The growth and development of the crops are largely depending on nitrogen (N). Ammonium (NH₄⁺-N) and nitrate (NO₃^--N) are the predominant inorganic N forms in soil which are utilized by crops. Since nitrification is strongly inhibited in paddy soil, ammonium becomes the major N form. Rice roots can aerate the surrounding soil (rhizosphere) by excreting oxygen (O₂) and this activity promotes the process of nitrification and the conversion of NH₄⁺ to NO₃^- which can be absorbed. The transport of nutrient by plant cells must pass through the plasma membrane. Both high-affinity and low-affinity transport systems for ammonium and nitrate are found in plant plasma membrane. Ammonium is transported by uniporters into plant cell which controled by the plasma membrane electrochemical gradient. Nitrate is cotransported with more than two H⁺ by symporters against a concentration and an electrical gradient, which needs to be energized by the plasma membrane H⁺-ATPase. Both ammonium and nitrate transport are closed related to the plasma membrane H⁺-ATPase. In this study, rice plants (Oryza sativa L. japonica ssp. cv. Nanguang) were fed with ammonium (NH₄⁺-N) or nitrate (NO₃^--N) as sole nitrogen in hydroponics culture experiment and at the seedling stage the plasma membrane vesicles of roots or leaves were isolated by two-phase system. The plasma membrane H⁺-ATPase hydrolytic activity, the Kinetic characteristics and the immuno-detection of plasma membrane H⁺-ATPase were analyzed respectively to expound the effect of the ammonium or nitrate nutrition on the plasma membrane H⁺-ATPase from rice roots and leaves. The obtained results showed that:The purity of the rice seedling roots plasma membrane was above 95%. The plasma membrane H⁺-ATPase hydrolytic activity and ATPase Mechaelis constant K_m and maximum initial velocity V_max from NH₄⁺-N fed rice root were markedly higher than those from NO₃^--N fed rice root. The pH optimum of the plasma membrane H⁺-ATPase of NH₄⁺-N fed rice root was 6.0, but was about 6.2 for that of NO₃^--N fed rice root. The immuno-detection of plasma membrane H⁺-ATPase showed that the enzyme concentration of plasma membrane H⁺-ATPase from NH₄⁺-N fed rice root was higher than that from NO₃^--N fed rice root. The results indicated that the higher activity of H⁺-ATPase from NH₄⁺-N fed rice root resulted from the increased number of H⁺-ATPase units per membrane area. Furthermore, the different H⁺-ATPase isoforms may exist in the NH₄⁺-N fed rice root in comparison with the NO₃^--N fed rice root. The higher activity of plasma membrane H⁺-ATPase of rice root may be an adaptation mechanism to the NH₄⁺-N nutrition.The purity of rice seedling leaves plasma membrane was above 80%, and both plasma membrane H⁺-ATPase hydrolytic activity V_max and K_m obtained from NO₃^--N fed rice leaves were markedly higher than those with NH₄⁺-N. The pH optimum was 6.2 for the plasma membrane H⁺-ATPase from rice leaves of both NH₄⁺-N and NO₃^--N fed plants. 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 with NH₄⁺-N. The results indicated 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 was higher than that of NH₄⁺-N. As the NO₃^--transport across the plasma membrane is an active transport process in relation to the plasma membrane H⁺-ATPase, it is may 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.