The Biological Function And Mechanism Of OsNAR2.1 Involved In Nitrogen Use In Rice

Rice(Oryza sativa L.)is a major staple food crop for a large part of the world population.Nitrogen(N)nutrition affects all levels of plants functions from metabolism to resource allocation,growth and development.Conventional cultivation of rice is mostly in flooded environment where nitrification is inhibited;therefore inorganic nitrogen in the rhizosphere soil mostly is in the form of ammonium(NH4+).However,the root system of rice can transport and secrete the oxygen from shoots into the rhizosphere through its developed ventilated tissue,so that the nitrifying bacteria in the rhizosphere will oxidize part of the ammonium into nitrate.Therefore,in the actual growth environment of rice,the roots are in the mixed nutrition of ammonium and nitrate.Studies have shown that,even if it has been grown in the flooded soil,5%-40%of the absorption of total nitrogen in rice is in the form of nitrate.Plants absorb nitrate from the soil mainly by the nitrate-nitrogen transport system.The nitrate-nitrogen system can be divided into high affinity transport system(HATS)and low affinity transport system(LATS),so that plants can cope with both high and low NO3-concentrations in soil.Since the nitrate in the roots is in the process of being absorbed and produced continuously,NO3-in the rhizosphere of rice is always at a low concentration,which causing the uptake of NO3-is mainly due to the high affinity transport system.And the high affinity transport system of NO3-is mainly composed of OsNRT2 family.There is a two-component NRT2-NAR2 system in the NO3-transport process of rice.Some OsNRT2 family members need to be combined with OsNAR2.1 not OsNAR2.2 to exercise the function of absorbing and transporting nitrates.Studies have shown that inhibition of OsNAR2.1 expression by RNA interference(RNAi)can reduced the expression of OsNRT2.1,OsNRT2.2 and OsNRT2.3a in the mutant roots,which affected the uptake of NO3-in rice.We hoped to obtain rice lines with improved grain yield and nitrogen use efficiency by coordinate expression of OsNAR2.1 and OsNRT2.1 or OsNAR2.1 and OsNRT2.3a in rice.In order to verify additional biological functions of OsNAR2.1,we also obtained a new protein OsNramp3,which is an interaction protein of OsNAR2.1,by pull down experiment,and achieved new function by studying the interaction of OsNramp3 and OsArAR2.1.The main results were as follows:1.We enhanced the expression of the OsNRT2.1 gene,which encodes a high-affinity NO3-transporter,using a ubiquitin(Ubi)promoter and the NO3--inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants.Transgenic lines expressing p Ubi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited increased total biomass including yields of approximately 21%and 38%compared with wild-type(WT)plants.The agricultural NUE(ANUE)of the pUbi:OsNRT2.1 lines decreased to 83%of that of WT plants,while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128%of that of WT plants.The dry matter transfer(DMT)into grain decreased by 68%in the pUbi:OsNRT2.1 lines and increased by 46%in the pOsNAR2.1:OsNRT2.1 lines relative to the WT.The expression of OsNRT2.1 in shoot showed that Ubi enhanced OsNRT2.1 expression by 7.5-fold averagely and OsNAR2.1 promoters increased by about 80%higher than the WT.Interestingly we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines,however for pOsNAR2.1:OsNRT2.1 lines OsNAR2.1 expression was only increased in leaf sheaths and inter nodes.The ratio of OsNRT2.1 to OsNAR2.1 expression was altered in the transgenic lines with ratios of approximately 11.3:1 and 4.7:1 in thep Ubi:OsNRT2.1 and pOsNAR2.1:OsNRT2.1 lines,compared with a ratio of 7.2:1 in WT plants.We show that increased expression of OsNRT2.1,especially in combination with a relative lower expression ratio with its partner gene OsNAR2.1,can improve yield and NUE in rice.2.We transfered another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants.The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level,compared with wild type(WT),and 15N influx in roots increased 21-32%in response to 0.2 mM,2.5 mM 15NO3-and 1.25 mM 15NH4 15NO3.Under these three N conditions,the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143,129,and 51%,and total N content increased 161,242,and 69%,respectively,compared to WT.Furthermore in field experiments we fould the grain yield,agricultural nitrogen use efficiency(ANUE),and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21,22,and 21%,compared to WT.We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field,found that post-anthesis N uptake differed significantly between them,and in comparison with the WT.Post-anthesis N uptake(PANU)increased approximately 39%and 85%,in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines,respectively,possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage.These results show that rice NO3-uptake,yield,and NUE were improved by increased OsNAR2.1 expression via its native promoter.3.We used the constitutive strong promoter 35S to express both OsNAR2.1 and OsNRT2.3a,and obtained the transgenic lines with co-overexpression of OsNAR2.1 and OsNRT2.3a.Field experiments showed that,compared with Wuyunjing7,the dry matter at anthesis(DMA),the dry matter at maturity(DMM),the total N accumulation at anthesis(TNAA)and the total N accumulation at maturity(TNAM)were increased 23.3%,18.6%,28.3%and 19.5%,respectively,in co-overexpression lines.And the dry matter transport efficiency(DMTE)and the nitrogen transport efficiency(NTE)were increased by 28.9%and 9.8%.And ultimately causing the grain yield(GY),the agronomic nitrogen use efficiency(ANUE)and the nitrogen recovery efficiency(NRE)increased 22.6%,28.6%and 21.2%,respectively.The results showed that,co-expression of OsNAR2.1 and OsNRT2.3a can increase the yield and agronomic nitrogen-use efficiency of rice.4.We obtained OsNramp3,an interaction protein of OsNAR2.1,by doing pull-down experiment,and used two-hybrid yeast system and bimolecular fluorescence complementation(BiFC)to verification.By analyzing the expression pattern,we found OsNAR2.1 and OsNramp3 had co-expression at the Node I site during grouting stage and at the junction of leaf sheath and rhizome during the seedling stage.Both OsNAR2.1 and OsNramp3 were induced by 0.2 mM N03-.Compared to WT,the absorption rate of 0.2mM 15NO3-reduced significantly in the knockout mutants of OsNramp3.After 24h grown in the 0.2mM 15NO3-solution,the transport of 15NO3-from shoot to root was inhibited in the knockout mutants of OsNramp3.And after 3 weeks in the 0.2mM 15NO3-solution,the biomass and total nitrogen content were significantly decreased in the knockout mutants.By crossing back yeast mutant strain,we found the co-expression of OsNAR2.1 and OsNramp3 had transport activity over Fe.And we also found,in 0.2mM NO3-,the absorption and distribution of Fe was affected after knock out OsNramp3.Analysis of OsNAR2.1 overexpression lines and OsNramp3 knockout mutants showed that overexpression of OsNAR2.1 could not cover up the growth phenotype of NG3081,which is a complete knockout mutant strain of OsNramp3,but it could cover up partial growth phenotype of NG4081,which is a partial knockout mutant of OsNramp3.And the results validated that OsNAR2.1 required the participation of OsNramp3 to improve rice grain yield and nitrogen use efficiency.5.We used RNAi and overexpression OsNAR2.1 lines to investigate the drought susceptibility of rice and found that OsNAR2.1 was involved in the drought tolerance.Compared with Wuyunjing 7,the drought resistance of pOsNAR2.1:OsNAR2.1 transgenic lines(Ox1,Ox2,Ox3)significantly improved.The photosynthetic rate and water use efficiency of pOsNAR2.1:OsNAR2.1 transgenic lines did not change under the condition of flooding,but increased by 39.2%and 11.8%under controlled water conditions.Compared with Wuyunjing 7,the grain yield of pOsNAR2.1:OsNAR2.1 transgenic lines increased 28.2%under controlled water and 70.7%under the condition of flooding.Physiological nitrogen use efficiency(PNUE)and nitrogen recovery index(NHI)of pOsNAR2.1:OsNAR2.1 transgenic lines didn’t change under the condition of flooding,but increased 14.6%and 13.2%,respectively,under controlled water.