Involvement Of Plasma Membrane H~+ATPase Of Rice Roots In The Ammonium-Nutrition

Plasma membrane H+-ATPase,also known as proton pump,is a kind of enzyme located on the plasma membrane.It’s main physiological function is to hydrolyze ATP and get energy to pump intracellular H+ out of the cytoplasm,which in turn establishing membrane potential and maintaining electrochemical gradient across the plasma membrane.Meanwhile,proton driving force can be used to assist transport of other ions across the plasma membrane.Rice is an important crop in China.It grows in the flooded soil.Because nitrification is inhibited under anaerobic conditions,a large amount of nitrogen exists in the soil in the form of ammonium.Since nitrogen is the most important mineral nutrient absorbed by plants,about 70%of cations absorbed by rice roots are NH4+.Previous research showed that proton pump activity needs to be up-regulated to adapt to the low rhizosphere pH under ammonium nutrition.Even rice is a crop species adapted to ammonium nutrition,our further investigation found that paddy rice would be inhibited under high concentration of ammonium nutrition.In this study,plasma membrane H+-ATPase OsA1 overexpression and osa1 mutant rice lines were cultivated under different concentrations of ammonium and PM H+ ATPase activity was analyzed at molecular and protein levels.The involvement of Plasma membrane H+-ATPase in relation to the rice yield was tested in field trials.The results are as follows:1.the expression level of the four PM H+-ATPase genes(OsA1,OsA2,OsA7,and OsA9)in the OsAl overexpression lines was higher than those in the wild-type rice,and PM H+ ATPase protein amount and phosphorylation level were significantly increased,also the activity.On the contrary,expression level of five proton pump genes(OsA1,OsA2,OsA8,OsA9,and OsA10)in the osa1 mutant was lower than that of the wild-type rice and PM H+ ATPase activity was significantly lower than that of the wild-type rice.PM H+-ATPase protein amount and phosphorylation level of the mutant were significantly reduced.2.OsA1 overexpression lines,osa1 mutants and wild-type rice were treated with different concentrations of ammonium nutrient for 2 weeks.The nitrogen content of OsA1 overexpression lines under different ammonium concentrations was higher than that of the wild-type rice.After 5min treatment of rice roots with NH4+ labeled with isotope 15N,it was found that the absorption rate of 15N in the roots of OsAl overexpression lines was significantly higher than that of the wild-type rice,and the osa1 mutants was lower than that of the wild-type rice.The results indicated that PM H+-ATPase regulated the absorption of ammonium in rice roots.3.When treated with different concentrations of ammonium for two weeks,it was found that the accumulation of potassium in OsAl overexpression lines was higher than that in the osa1 mutants.There was no significant difference in the accumulation of P among OsAl overexpression lines and osa1 mutants.However,with the increase of external ammonium concentration,the accumulation of P in rice also increased.4.Field plot experiments with different amount of nitrogen fertilizer showed that tiller,panicle number and grain number per panicle of OsAl overexpression lines increased significantly,with enhanced photosynthetic rate.In osa1 mutants,all the parameters mention above decreased significantly.Taken together,OsAl plays an important role in regulating the absorption of ammonium in rice roots,which may contribute to the photosynthesis and grain yield in rice crops.