铵、硝营养对水稻叶细胞膜H^+-ATPase和质子泵活性的影响

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

用两相法分离铵态氮(NH4+-N)和硝态氮(NO3--N)培养的水稻苗期叶细胞膜,并测定了细胞膜H+-ATPase水解活性和质子泵活性,以期阐明铵、硝营养对水稻叶细胞膜H+-ATPase的影响。结果表明,叶细胞膜H+-ATPase活性最佳pH值均为6.2。NO3--N培养的水稻叶细胞膜H+-ATPase的水解活性、Vmax和Km均显著高于NH4+-N培养的水稻叶;Western Blot分析结果看出,NO3--N培养的水稻叶细胞膜H+-ATPase酶浓度也高于NH4+-N培养的水稻叶,说明NO3--N培养的水稻叶中单位细胞膜上的H+-ATPase酶分子数量大于NH4+-N培养的水稻叶,这与细胞膜上H+-ATPase蛋白的表达量升高有关。此外,NO3--N培养的水稻叶质子泵初速度和膜囊体内外H+浓度梯度均高于NH4+-N培养。由于NO3-的跨膜运输是与细胞膜上H+-ATPase紧密联系的主动运输过程,NO3--N培养的水稻叶片细胞膜H+-ATPase活性和质子泵活性高可能与水稻叶细胞吸收大量NO3-有关。

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 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.