Functional Analysis Of The Nodule Inception-like Proteins AtNLP7 And OsNLP4 In Nitrate Assimilation And Signaling

Nitrogen is one of the most critical macronutrients for plant growth,development,and production.In agriculture,nitrogen fertilizer has been widely used to maintain the high yield of crops.Because of low nitrogen use efficiency(NUE),nitrate cannot be completely absorbed by plants and therefore a large part is lost to the environment,resulting in serious environmental pollution and increased production cost.To deal with this problem,tremendous efforts have been invested worldwide to increase the NUE of crops.However,only few regulators of nitrate assimilation have been identified in higher plants to date.Legume-specific symbiotic nitrogen fixation is under the control of the putative transcription factor,NIN,in Lotus japonicus.Here we reported the Arabidopsis thaliana homologue AtNLP7(NIN-LIKE PROTEIN 7)was a potential candidate to improve NUE,which one can regulate nitrate assimilation effectively.Overexpression of AtNLP7 in Arabidopsis significantly promoted the growth and development of plants,increased plant biomass under both nitrogen-poor and rich conditions with better-developed root system,reduced shoot/root ratio and larger seeds.AtNLP7-overexpressing plants showed a significant increase in nitrogen absorption,key nitrogen metabolites,total nitrogen content,and expression levels of genes involved in nitrogen assimilation and signaling.Overexpression of AtNLP7 also enhanced photosynthesis rate and carbon assimilation.Moreover,AtNLP7 improved plant growth and N use in transgenic tobacco(Nicotiana tabacum)as in Arabidopsis.Our results demonstrated that AtNLP7 significantly improved plant growth under both N-poor and rich conditions by coordinately enhancing nitrogen and carbon assimilation and shed new light on crop improvement.Rice is one of the most important food crops in the world,provides 35%-60%calories for 3 billion people.Nitrate is a key nutrient that affects rice growth and yield,and much has yet to be learned about nitrate regulatory genes and mechanisms in rice.Here,we analyzed the functions of OsNLP4 in nitrate assimilation.We found that OsNLP4 and AtNLP7 have shown similar functions in nitrate metabolism,when OsNLP4 was overexpressed in the Arabidopsis mutant nlp7-1,nitrate assimilation and induction of nitrate-responsive genes in the transgenic plants were recovered to WT levels.Knockout of OsNLP4 showed dwarf growth and low nitrogen use efficiency.OsNLP4 expressed in leaves and roots primarily.Interestingly,we observed that nitrate might serve as a signal to regulate positively the translation of OsNLP4 mRNA.Furthermore,we found that OsNLP4 could physically interact with OsNLP2 and OsNLP3 to form heterodimers,which may serve as a regulatory mechanism.Overexpression of OsNLP4 in rice improved growth and development,increased plant biomass under different nitrogen concentrations in hydroponic conditions,promoted the absorption and assimilation of nitrate,and regulated the expression of the genes in nitrate metabolism.Field experiment results showed that OsNLP4 mainly affected the tiller numbers of rice to increase the yield.These data suggest that OsNLP4 could significantly improve the rice NUE and yield.