Adaptation Of Plasma Membrane H~+-ATPase To The Uptake Of Ammonium By Rice And The Accumulation Of Nitrate In Pakchoi

Rice plants prefer ammonium to nitrate in paddy soils.The mechanisms responsible for adaptation of rice roots to the NH₄⁺ nutrition-associated strong rhizosphere acidification were not fully elucidated.In the present study the adaptation of plasma membrane H⁺-ATPase activity in rice roots to ammonium versus nitrate nutrition was investigated at pH 6.5 and 3.0.Nutrient solution was gradually acidified to pH 3.0 to allow adaptation of rice roots to low pH.After 4 d cultivation at final pH levels plasma membrane was isolated from rice(Oryza sativa L."japonica") roots by two-phase method system.The results indicated that:1.In vitro,hydrolytic H⁺-ATPase activity,maximum initial velocity(V_max),Mechaelis constant(Kin),H⁺-pumping activity,H⁺ permeability of plasma membrane and pH gradient across the plasma membrane of membrane vesicles,were significantly higher for rice roots grown at pH 3.0 than pH 6.5,irrespective of nitrogen forms supplied to rice plants.2.Vanadate sensitivity of the plasma membrane H⁺-ATPase remained unchanged.3.Immunoassays using polyclonal antibodies against H⁺-ATPase showed significant higher enzyme concentration of the plasma membrane H⁺-ATPase in the membrane fraction obtained from rice roots grown at low pH than at high pH,irrespective of nitrogen forms supplied.4.On the transcriptional level,the expression of OSA1,OSA3,OSA7,OSAS,and OSA9 from roots at low pH was much higher than those at high pH.The transcription rate of OSA2 remained unchanged at low pH.However,for OSA4,OSA5,OSA6 and OSA10 no PCR products could be found.5.In conclusion,various isoforms of plasma membrane H⁺-ATPase of rice roots were highly regulated after adaptation to low nutrient solution pH,resulting higher hydrolytic activity and H⁺-pumping activity of plasma membrane H⁺-ATPase.This adaptation was independent of nitrogen forms supplied.Pakchoi is a typical vegetable with high nitrate conten.In order to investigate the adaptation of the plasma membrane H⁺-ATPase to the significant difference of the accumulation of nitrate in different tissues of Pakchoi(Brassica chinensis L.cv. 'Shanghaiqing'),the plasma membrane of petiole and leaf was isolated by two-phase method system.The hydrolytic activity of plasma membrane H⁺-ATPase in relation to the NO₃^transport was studied.The results were below:1.The purity of plasma membrane was above 90%.2.The H⁺-ATPase hydrolytic activity of petiole was significantly higher than that of leaf,in vitro.3.The pH optimum of the H⁺-ATPase activity for petiole was pH 6.6,but for leaf was between 6.4 and 6.6.In addition,the K_m for the petiole plasma membrane H⁺-ATPase was higher than that for the leaf.4.V_max at two temperatures(20℃and 30℃) was used to calculate the active energy by Arrhenius equation and results showed that the active energy for the H⁺-ATPase of petiole was higher than that of leaf.5.The westem blot showed that the enzyme concentration of plasma membrane H⁺-ATPase from petiole was higher than that from leaf.The results indicated that nitrate content was high in petiole and this was related to the high hydrolytic activity of its plasma membrane H⁺-ATPase,K_m value,active energy and the high enzyme concentration of H⁺-ATPase.It is may be true that the higher activity of plasma membrane H⁺-ATPase in petiole caused more uptake of NO₃^than in leaf.