The Molecular Mechanisms Of NRG2 And NLP7 Regulating NRT1.1 In Arabidopsis

Nitrogen is one of the most important macronutrient for plant growth and development and most terrestrial plants absorb nitrate as the main nitrogen source.In agriculture system,nitrate supply directly affects plant growth and crop productivity.In many developped and developping countries,excessive nitrogen fertilizers have been applied in agriculture while the nitrogen use efficiency of the crops is low and the fertilizers could not be completely taken up by the plants.So a large part of nitrogen is lost into the environment,resulting in serious environmental pollutions like eutrophication,soil acidification,etc.These problems must be solved and improving the nitrogen use efficiency is the key to address this issue.Elucidating the mechanisms of nitrate regulation and deciphering the network of nitrate regulated genes will definitely provide the theoretical base and guidance for the improvement of nitrogen use efficiency.In the last few years,several nitrate regulatory genes functioning in nitrate signaling have been characterized.However,our understanding of the regulatory mechanisms and genes involved in nitrate signaling in plants is incomplete.NRG2(Nitrate Regulatory Gene 2)is a new nitrate regulatory gene identified using forward genetics.In this research,we studied further NRG2 gene in nitrate regulation.To test if NRG2 was regulated by different nitrogen conditions,we investigated the expression levels of the gene after nitrate,ammonium,and nitrogen starvation treatments.The results did not show significant changes,indicating that the expression of NRG2 was not modulated by nitrate,ammonium and nitrogen starvation.NRG2 was found to be localized in the nucleus in both present of nitrate or nitrogen starvation.To investigate the mechanisms for the lower nitrate accumulation in nrg2 mutants,we tested the time-course of nitrate accumulation treated with KNO3,nitrate content treated with various concentrations of nitrate,NR activity,the expression of nitrate transport genes and nitrate assimilation genes.We found that the nitrate accumulation and the expression of NRT1.1(Nitrate Transporter 1.1)were lower,but NR activity and the expression of nitrate assimilation genes did not show significant changes in nrg2 mutants.The results indicated that the lower nitrate content in nrg2 mutants may be caused by nitrate uptake affected in nrg2 mutants.To understand the relationship of NRG2 and NRT1.1,a double mutant nrg2-2 chl1-13 was obtained.We inspected the nitrate content and the expression of nitrate responsive genes in the double mutants.The results showed that the nitrate content and the expression of nitrate responsive genes in the double mutants were similar to those in single mutant chl1-13.Additionally,when the cDNA of NRT1.1 was transformed into the nrg2-2 mutant,the nitrate content and the expression of nitrate responsive genes were recovered to WT levels.These results suggest that NRG2 and NRT1.1 may work in the same nitrate signaling pathway and NRG2 functions upstream of NRT1.1.Transcriptome analysis showed that 278 genes were regulated by both NRT1.1 and NRG2.These genes were further investigated by GO analysis,and the result showed that a nitrogen compound transport cluster was involved.These data further confirmed that NRG2 worked in the same nitrate signaling pathway as NRT1.1.NRG2 contains two uncharacterized functional domains and it was predicted that NRG2 could bind DNA.In order to verify the hypothesis,we designed ChIP assay.The results indicated that NRG2 could bind the promoter of NRT1.1 in vivo.To investigate the relationship between NRT1.1 and NLP7(Nodule Inception-like Protein 7)in the process of regulating nitrate response,we measured NRT1.1 transcript levels in nlp7 mutants and NLP7 transcript levels in nrt1.1 mutants.The expression of NRT1.1 in the nlp7 mutants was significantly decreased in ammonium nitrate media and no change was found for the expression of NLP7 in the nrt1.1 mutants.This indicates that the expression of NRT1.1 is regulated by NLP 7.We tested the YFP fluorescence levels,nitrate content and NR activity in the chl1-13 nlp7-4 double mutants.The results showed that the YFP fluorescence levels,nitrate content and NR activity in the double mutants were similar to those in single mutant chl1-13,indicating that NLP7 may work in the same nitrate signaling pathway as NRT1.1.Additionally,when the cDNA of NRT1.1 was transformed into the nlp7-4 mutant,the YFP fluorescence levels,NR activity and the expression of nitrate responsive genes of nlp7-4 mutant were recovered to WT levels.These results suggested that NLP7 and NRT1.1 were in the same nitrate signaling pathway and NLP7 functioned upstream of NRT1.1 Transcriptome analysis showed that 342 genes were regulated by both NRT1.1 and NLP7.These genes were further investigated by GO(Gene Ontology)analysis,and the result showed that two nitrogen compound metabolic clusters were involved.These data further confirmed that NLP7 worked in the same nitrate signaling pathway as NRT1.1.Futhermore,GO analysis the 186 genes exclusively regulated by NLP7 showed that there were two nitrogen-related clusters,including genes involved in amino acid transport and nitrate transport,implicating that in addition to NRT1.1-dependent way,NLP7 could also be involved in nitrate signaling independent of NRT1.1.Previous study has shown that NLP7 may bind NRT1.1 by ChIP-chip test.Our ChIP and EMS A assays showed that NLP7 could bind the promoter of NRT1.1 directly.In this study,we characterized the function of NRG2 in nitrate regulation.We also illustrated the relationship between NRG2 and NRT1.1 and the relationship between NLP7 and NRT1.1 These findings offer insights into the mechanism of nitrate regulation in plants,and expand our understanding of nitrate signaling networks.