Identification And Characterization Of Nitrate Regulatory Genes T5120 And NSIG In Arabidopsis

Nitrogen is one of the most important macronutrient for plant growth and development.The application of nitrogen fertilizer is the key to maintain high and stable yields of crops.A large amount of nitrogen fertilizer is applied to increase the yields of crops in agriculture.However,due to the low utilization rate of nitrogen fertilizer,the nitrogen that is not absorbed by plants is lost to the environment,causing serious non-point source pollution.Therefore,it is necessary to strengthen the researches on the nitrate uptake laws and regulatory networks of plants,to improve the nitrogen utilization rate of crops and promote the sustainable development of agriculture.The mechanisms of nitrogen uptake and utilization are delicate and complex,resulting in the researches on nitrogen regulation are very limited.In this study,we used the RNA-seq technology and forward genetics method respectively,to clone two nitrate regulatory genes named T5120 and NSIG,and identified their functions in nitrate signal and metabolism.Long non-coding RNAs(lncRNAs)have been reported as crucial regulators in many biological processes in plants.However,it remains unknown whether lncRNAs can respond to nitrate or function in nitrate regulation.Here,we identified six lncRNAs responding to nitrate using the RNA-seq technology and qPCR validation,and one of them named T5120 had the highest nitrate induction.Further investigations showed that the nitrate induction of T5120 was higher than that in WT,suggesting that T5120 is directly induced by nitrate but not its reduction products.The qPCR assay showed that the nitrate induction of several nitrate responsive genes in the T5120 overexpressing lines was significantly increased,indicating that T5120 can regulate the nitrate primary response.Physiological detection found that the nitrate content of the T5120 overexpression line was significantly reduced.Further investigation showed that the nitrate-reductase(NR)activity and amino acid content of T5120 overexpression lines were significantly increased,while the nitrate uptake was unchanged.The detection of molecular level showed that the expression of some nitrate assimilatory genes in T5120 overexpression lines was significantly increased.Our results indicate that T5120 can promote the nitrate response and nitrate assimilation.Moreover,overexpression of T5120 improved the plant growth under both nitrate-limiting and-sufficient conditions.To better understand the function mechanisms of T5120,we analyzed the promoter sequence of T5120 and found a nitrate-responsive-like element(NRE-like).Y1 H and ChIP-qPCR assays demonstrated that NLP7 bound to the promoter of T5120.Moreover,the dual luciferase reporter system analysis showed that NLP7 could activate the transcription of T5120.Further studies found that NLP7 could regulate the expression and the nitrate reduction of T5120,indicating that NLP7 can regulate the expression of T5120 through directly binding.In addition,the roots fluorescence of nlp7-4 lines expressing T5120 was significantly stronger than that of the nlp7-4 mutant.The NR activity and the expression of several nitrate assimilatory genes of T5120/nlp7-4 were higher than that of nlp7-4 mutant,suggesting that T5120 could partially restore the defects of nlp7-4 mutant in nitrate signal and assimilation.These results reveal that T5120 works downstream of NLP7 to regulate the nitrate signaling.Forward genetics is an effective way to find new genes.Here,we also used the nitrate regulatory genes screening system to clone a new gene NSIG.In the absence of nitrate,the roots fluorescence of WT seedlings containing nitrate-responsive promoter(NRP-YFP)was not observed,and we screened a mutant E7 with strong fluorescence in the roots.The map-based cloning and genome-wide sequencing verified the gene mutated in E7 and named NSIG.Inaddition,NSIG protein was localized in the nucleus.The transcriptome analysis showed that in the absence of nitrate,the expression of many nitrate responsive genes in E7 was incresed,demonstrating that NSIG negatively regulates nitrate signaling.Further investigations displayed that NSIG still negatively regulated nitrate signaling in the presence of nitrate,indicating that the negative regulation of nitrate signaling by NSIG is independent of nitrate.To characterize the function of NSIG in nitrate metabolism,we examined the nitrate content in E7 mutant,and found that the nitrate content in E7 was reduced.Subsequent assays exhibited that the nitrate uptake in the E7 mutant was decreased,while the NR activity and amino acid content was increased.Molecular level analysis found that the expression of the transport gene NRT1.1 in the E7 mutant was down regulated,while the expression of several nitrate assimilatory genes were upregulated,suggesting that NSIG regulates the nitrate uptake and assimilation.To explore the relationship between NSIG and NRT1.1 in nitrate regulation,we performed genetics analysis.Our result displayed that the expression of NRT1.1 in the E7 mutant was depressed,while the expression of NSIG in the nrt1.1 mutant was unchanged.Then we obtained the double E7 chl1-13 mutant and observed its fluorescence,ultimately found that NSIG and NRT1.1 regulate nitrate signaling in different pathways.Taken together,NSIG can regulate the transport function of NRT1.1 but not affect its signaling function.Furthermore,the similar study on the relationship between NSIG and NLP7 found that NSIG and NLP7 are also independent in the nitrate signaling pathway.In this study,we cloned two new nitrate regulatory genes T5120 and NSIG respectively,identified their functions on nitrate regulation,and elucidated their function mechanisms.Our findings will lay a foundation for deciphering nitrate regulatory mechanisms and their networks of plants and will provide theoretical basis and direction for improving nitrogen utilization of crops.