The Function Of Plasma Membrane H~+ ATPase OsA1 In The Improvement Of Carbon And Nitrogen Utilization By Rice

Rice(Oryza sativa L.)is one of the important staple foods worldwide,therefore,increasing the yield of rice crop is of great significance to ensure food security.Carbon and nitrogen are two essential nutrients for plants,and the main components of plant organism.However,carbon is obtained in the form of CO2 via stomatal opening of leaves,while nitrogen is absorbed from the soil by plant roots.Since paddy rice grows in flooded soils,ammonium is the main nitrogen form available for rice roots.Enhancing further the uptake of C and N would have a practical significance for the high productivity in agriculture.Proton pump(plasma membrane H+-ATPase)is a master enzyme in plants,and has the important physiological functions.The PM H+-ATPase can control expansion and contraction of guard cells to regulate stomatal opening,which is a key factor influencing the carbon dioxide entering the leaves.In roots,PM H+-ATPase can pump H+ outside the cells to maintain a membrane potential for the NH4+ uptake;additionally,the H+ produced by ammonium assimilation could also be exported outside cells to keep H+homeostasis in the cytoplasm.Ten plasma membrane proton pump genes OsAs were identified in rice.OsAl has very high homology with other isogenes of OsAs.In this research,we constructed OsAl over expression transgenic rice with 35S promoter and screened Tos-17 insertion osal mutant and obtained homozygote lines.We then analyzed stomatal conductance,net photosynthetic rate,and ammonium uptake of these rice lines.In addition,we checked the rice yield by conducting field trials.The results were as follows:1.The homology of OsA1 and other rice plasma membrane H+-ATPase genes of the family is up to 79%,and OsAl gene contains 8 transmembrane domains.According to the micro array results,the OsAl gene is widely expressed in roots and leaves,etc.Relative expression levels of OsAl gene in leaves and roots of over-expression OsAl transgenic rice are about 6 to 10 times higher than wild-type.Via field experiment,we obtained three lines of stably OsAl over-expression transgenic rice plant.The protein content of proton pump in leaves and roots of OsA1over-expression transgenic rice increased more than 1.5 times than the wild-type and Tos-17 insertion mutants.2.Compared to the wild-type,stomatal opening rate,stomatal conductance and net photosynthetic rate was higher in OsAl over-expression transgenic rice.Potassium influx channel gene OsKAT1 and OsAKT1 in guard cells showed no difference between the transgenic rice,wild-type and mutants,which indicates that OsAl overexpression influenced the stomatal opening rather than potassium channel genes.3.The biomass and nitrogen content of OsAl over-expression transgenic rice were significantly higher than that of the wild-type under ammonium nutrition ranging from 1 mM to 4 mM.It shows that over expression of OsAl can significantly enhance the absorption of both ammonium nitrogen and carbon.4.Wild-type rice showed depressed growth at high ammonium concentration(4 mM),while OsA1over-expression transgenic rice showed improvement in the growth.It was found that the concentration of H+ in the rhizosphere of transgenic rice was 1.3 to 1.5 times higher than that of wild-type,moreover,phosphorylation level and total protein of proton pump increased in the leaf and root with increased PM H+ATPase activity.The results here indicated that OsA1over expression can contribute to the improved uptake of C and N.In addition,over expression of OsAl has no influence on the expression of other plasma membrane proton pump genes in leaves and roots.Further,over expression of OsAl induced higher expression of OsGS1;2 gene,which is important for the assimilation of ammonium in roots with enough capacity.5.Through the field trials with different levels of nitrogen fertilizer(0,100,200,300kg N/ha),we found that the stomatal conductance and CO2 assimilation rate of OsAl over-expression transgenic rice were significantly higher than that of wild-type plants under every nitrogen level.The OsAl over expression transgenic plants can also enhance nitrogen absorption.The effective tillers and panicle grain number of OsA1 over expression transgenic plants were significantly higher than that of wild-type plants under different nitrogen levels.With the increased amount of nitrogen fertilizer,the tillers and panicle grain number of OsAl over expression transgenic plants was increases from 6%to 15%,which enhanced the grain weight from 10.6%to 18.7%.In summary,plasma membrane proton pump gene OsA1 played an important role in the regulation of stomatal opening for CO2 uptake and photosynthesis,and also in the ammonium uptake and assimilation in roots,which in turn significantly enhanced the rice yield.