The double mutant chl1-13 nlp7-4 exhibited significant lower nitrate content than that in WT and the nlp7 plants, and similar to that in the chl1 mutants . The nitrate reductase activity of the two single mutants was much lower than that of WT, and the double mutant chl1-13 nlp7-4 showed similar nitrate reductase activity to the chl1-13 mutant . In addition, we analyzed the nitrate content in whole seedlings after treatments with different nitrate concentrations for 2h and with 5mM KNO3 for different times under the ammonium succinate condition. The results showed that the nlp7 mutants displayed the same nitrate content as WT, but lower in the chl1-13 mutant than in WT under both conditions . The double mutant chl1-13 nlp7-4 showed the same nitrate content as the chl1-13 mutant . Because NRT1.1 is epistatic to NLP7 in these experiments, these results indicate that NLP7 and NRT1.1 may be involved in the same pathway and NLP7 may regulate nitrate signaling upstream of NRT1.1.In order to provide more evidence to support the conclusion that NLP7 regulates nitrate signaling upstream of NRT1.1, we over expressed NRT1.1 in the nlp7-4 mutant . The transgenic lines NRT1.1/nlp7-4 exhibited higher expression of NRT1.1 than that in the WT and nlp7 plants . The YFP signal in the NRT1.1/ nlp7-4 plants grown under the ammonium nitrate condition was much stronger than that of the nlp7-4 mutant, plastic grow bag and weaker than that of WT . Quantifying the fuorescence intensity of the NRT1.1/nlp7-4 plants showed that the YFP levels in the NRT1.1/nlp7-4 plants were 70% higher than that in the nlp7-4 mutant and reached 71% of that in WT .

Then we tested the nitrate content and nitrate reductase activity in the NRT1.1/nlp7-4 plants. As shown in Fig. 3c, the nitrate content in the NRT1.1/nlp7-4 plants was lower than that in the nlp7-4 mutant, and similar to that in WT . The nitrate reductase activity in the NRT1.1/nlp7-4 plants was higher than that in the nlp7-4 mutant, and reached the levels of WT . In addition, we examined the transcript levels of NIA1, NiR, NRT2.1, HHO1, and HRS1 which are responsive to nitrate in a short time. We found that the transcript levels of the nitrate inducible genes were significantly higher in the NRT1.1/nlp7-4 plants than that in the nlp7-4 mutant and completely or partially restored to the WT levels , and there was no significant difference between WT and NRT1.1/nlp7-4 plants before nitrate treatment . Taken together, the phenotypes of NRT1.1/nlp7-4 plants were completely or partially recovered to that of WT, further indicating that NLP7 functions upstream of NRT1.1. Since NRT1.1 functions in nitrate signaling pathway dependently on NH4 + while NLP7 is NH4 + independent, NLP7 may also function in another nitrate signaling pathway independent on NRT1.1 in the absence of NH4 +.Firstly, we compared the expression of nitrate inducible genes in the WT and the chl1-13 mutant roots after nitrate treatment. The result showed that 152 genes could respond to nitrate in both WT and the chl1-13 mutant . In contrast, 438 genes lost their nitrate response in the chl1-13 mutant. 47 genes showed a gain of nitrate response in the chl1-13 mutant compared with WT. In summary, 485 genes were differentially expressed showed altered nitrate responses in the chl1-13 mutant compared to WT.We also compared the gene expression between WT and the nlp7-4 mutant in response to nitrate. The results showed that there were 253 genes could respond to nitrate in both WT and the nlp7-4 mutant . The expression of 337 genes was responsive to nitrate in WT, but not in the nlp7-4 mutant. 191 genes were responsive to nitrate exclusively in the nlp7-4 mutant, but not in WT. In total, 528 genes were differentially expressed in the nlp7-4 mutant.

We compared the 528 differentially expressed genes in the nlp7-4 mutant and 485 differentially expressed genes in the chl1-13 mutant and found 342 genes were regulated by both NLP7 and NRT1.1 while 143 and 186 genes were regulated only by NRT1.1 and NLP7, respectively . Gene Ontology analysis of 342 shared genes showed four clusters related to nitrogen, including cellular amino acid bio-synthetic process, cellular amino acid metabolic process, response to nitrogen compound, and nitrogen compound transport . In these four clusters, genes involved in amino acid synthesis and nitrate transport were enriched . These data indicate extensive overlap in the target genes modulated by NLP7 and NRT1.1 and provide additional support that both NLP7 and NRT1.1 work in the same pathway in nitrate signaling. The 186 and 143 genes exclusively regulated by NLP7 and NRT1.1, respectively were further investigated by GO analysis. Among the 186 genes regulated by NLP7, there are two nitrogen-related clusters , including genes involved in amino acid transport and nitrate transport . Among the 143 genes modulated by NRT1.1, three nitrogen-related clusters were found . In the three clusters, gene involved in nitrate transport was enriched . These data imply that NLP7 may function in nitrate signaling pathway independent of NRT1.1.NRT1.1 have been characterized to be a nitrate transceptor in previous reports and can regulate the expression of nitrate regulatory genes CIPK8 and CIPK23. Moreover, NRT1.1 can be phosphorylated by CIPK23 at amino acid Tr-101 under low nitrate concentration. NLP7 is another key nitrate regulator. As a transcription factor, it can bind to the nitrate response cis-elements. ChIP-chip and ChIP-qPCR assays have illustrated that NLP7 binds to many genes functioning in the nitrate regulation and assimilation, for instance NRT2.1, NRT2.2, and LBD37. It has been reported that NLP7 could bind NRT1.1, but it is not known if NLP7 binds to the promoter of and regulates NRT1.1.

In this paper, our results revealed that the transcript levels of NRT1.1 were down-regulated in the nlp7 plants when NH4 + is present. The nitrate induction of NRT1.1 was reduced as well in the mutants . These data indicate that the expression and induction of NRT1.1 can be modulated by NLP7 in the presence of NH4 +. Our genetic analysis showed that the nitrate-responsive YFP signal, nitrate content, nitrate reductase activity, and nitrate uptake in the double mutant chl1-13 nlp7-4 were strongly reduced than those in WT and close to those in chl1-13 mutant . These results indicate that NLP7 participates in nitrate signaling in the same pathway as NRT1.1 and NLP7 may work upstream of NRT1.1. We also over expressed the NRT1.1 in the nlp7-4 mutant and found that the YFP signal, nitrate content, nitrate reductase activity, and the induction levels of the endogenous genes after nitrate treatment in the NRT1.1/nlp7-4 plants were completely or partially recovered to WT levels . Even though the levels of NRT1.1 mRNA were higher in the transgenic plants, one was similar to WT levels and both lines gave similar results. These results demonstrate that NLP7 regulates nitrate signaling upstream of NRT1.1. We found that the nitrate content was significant higher in the nlp7 mutants than in WT ,pe grow bag consistent with previous studies. The reason for the higher nitrate content in the mutants may come from higher nitrate uptake and/or lower nitrate reduction. We tested the nitrate accumulation and found no significant difference between the nlp7 mutants and WT , similar to the report that nitrate uptake activity of the nlp7-1 mutant at 5mM 15NO3 − external concentration was the same as that of WT. We also tested the total nitrogen content and similar levels were found in the nlp7 mutants and WT, indicating that the nitrate import was not changed in the nlp7 plants . However, the nitrate reductase activity in the nlp7 plants was much lower than that in WT . These findings indicate that the higher nitrate content in the nlp7 mutants is caused by the reduced nitrate assimilation, rather than altered nitrate uptake. In addition, it has been reported that the nitrate content is lower and the nitrate uptake ability is reduced in chl1 mutants. Interestingly, our data revealed that the nitrate reductase activity was notably depressed in the chl1 mutants, indicating that the reduced nitrate content in the chl1 mutants may result from both decreased nitrate uptake and reduction.NRT1.1 is an essential component of nitrate uptake and nitrate signaling, respectively. But its function in nitrate signaling has been found to work mainly when NH4 + is present. In this paper, we found that the expression of NRT1.1 could be modulated by NLP7 when NH4 + is present and the nitrate uptake of nlp7 mutants was not affected . Combining with our results that NLP7 works upstream of NRT1.1, these findings indicate that NLP7 regulates the nitrate signaling function but does not affect the transport function of NRT1.1. NRG2, another important nitrate regulatory gene, has been reported to modulate the expression of NRT1.1 in both presence and absence of NH4 +, indicating that NRG2 can affect both nitrate transport and signaling functions of NRT1.1. These findings demonstrate that both NRG2 and NLP7 can regulate NRT1.1 but in different ways. NRT1.1′s functioning as a regulatory gene is NH4 +-dependent, while NLP7 is NH4 +-independent and NRT1.1 can restore the phenotype of the nlp7-4 mutant . Accordingly, these findings suggest that NLP7 functions in nitrate signaling independent of NRT1.1 in the absence of NH4 +. Our RNA-sequencing analysis showed that many genes were regulated by both NLP7 and NRT1.1 in response to nitrate . GO analysis of these genes revealed that there were four nitrogen-related clusters, including cellular amino acid bio-synthetic process, cellular amino acid metabolic process, response to nitrogen compound, and nitrogen compound transport, modulated by both NLP7 and NRT1.1 .

These data provide further evidences that NLP7 modulates nitrate regulation in the same pathway as NRT1.1 under these conditions. Since NLP7 is a transcription factor and ChIP-chip test has shown that it could bind NRT1.1, we performed ChIP-qPCR, ChIP-PCR, and EMSA to investigate the binding activity of NLP7 to the promoter of NRT1.1. These results indicate that NLP7 can bind to the promoter of NRT1.1 . ChIP assays showed that NLP7 could bind the Q4 and Q8 region of NRT1.1 promoter , while EMSA assay revealed that NLP7 could bind to Q4 region, but not Q8 region of NRT1.1 promoter , implicating that NLP7 may bind Q8 region indirectly. These results were obtained from the conditions with both ammonium and nitrate. Since ammonium is crucial for the regulation of NRT1.1 by NLP7, the presence of NH4 + might be also important for the binding of NLP7 to the promoter of NRT1.1. Without NH4 +, the NLP7 protein may not bind to the promoter of NRT1.1, resulting in the lost regulation function of NLP7 to NRT1.1. Taken together, in the primary nitrate response, NLP7 regulates nitrate signaling upstream of NRT1.1 in the presence of NH4 + and may modulate an NRT1.1-independent pathway in the absence of NH4 + in nitrate signaling. Moreover, NLP7 may regulate NRT1.1 via binding to the promoter of NRT1.1. Therefore we propose the working model as shown in Fig. 6. NLP7 plays a key role through regulating NRT1.1 in the presence of NH4 + while through an NRT1.1-independent pathway in nitrate signaling when NH4 + is absent. NLP7 can interact with NRG2 and NRG2 works upstream of NRT1.1 when NH4 + is present and/or absent. NRT1.1 regulates the transcript levels of CIPK8 and CIPK23 and can be phosphorylated by CIPK23 to switch its nitrate affinity. The genes regulated by NLP7 in the nitrate signaling pathway without NH4 + need to be investigated. The characterization of the regulation relationship of NLP7 and NRT1.1 has provided insights into the regulatory mechanisms of the genes functioning in nitrate regulation.The protein is composed of 121 amino acids and its monomer has a mass of approximately 13 kDa. Griffithsin forms a homodimer with six binding pockets with high affinity for mannose, a common sugar found at the terminal end of oligosaccharides on the surface of many enveloped viruses. The protein is thought to inhibit the entry of enveloped viruses into host cells as well as viral maturation and transmission events by binding to oligosaccharides on the viral envelope surface. Native Griffithsin and its analogs are the most potent HIV-1 entry inhibitors yet described, with EC50 values in the picomolar range .