This research also found that when NRT1.1 was phosphorylated at a low nitrate concentration, it was involved in maintaining the low level primary response; when it was dephosphorylated under a high nitrate concentration, it led to a high level primary response. More recent work has shown that NRT1.1 mediated regulation is quite complex in that it activates distinct signaling mechanisms. Furthermore, a rice homolog of AtNRT1.1has been identified, and variations in this gene in the rice sub species indica have been identified as boosting the absorption of nitrate and the transport of nitrate from roots to shoots, and potentially enhance NUE in rice.Another important nitrate regulator is the transcription factor NLP7, which belongs to the NIN like protein family in Arabidopsis. The NIN protein family was originally found to function in the initiation of nodule development in legume species and these family members are conserved in higher plants and algae. The NIT2 protein is a homologue of the NIN family in Chlamydomonas and can bind to the promoter of the nitrate reductase gene. In Arabidopsis, NLP7 has been demonstrated to be an important positive regulator of primary nitrate response as the induction of the nitrate responsive genes NIA1, NIA2, NRT2.1, and NRT2.2 is inhibited and nitrate assimilation is also impaired in nlp7 mutants. The function of NLP7 in nitrate signaling was further confirmed by the identification of the nlp7 mutant in an effort to explore novel nitrate regulators by using a forward genetics approach. ChIP chip analysis revealed that NLP7 could bind 851 genes including genes involved in N metabolism and nitrate signaling, such as NRT1.1, CIPK8, LBD37/38, and NRT2.1.

A recent study found that NLP7 could regulate the expression of NRT1.1 in the presence of ammonium and bind directly to the promoter of NRT1.1. These findings illustrate that NLP7 works upstream of NRT1.1 directly when ammonium is present. NLP7 can also activate or repress nitrate responsive genes. The Arabidopsis thaliana genome encodes nine NLPs,vertical planters for vegetables all of which contain the conserved RWP RK domain and the PB1 domain. Members of this family can be divided into four subgroups: NLP1 and 2, NLP4 and 5, NLP6 and 7, and NLP8 and 9. Yeast one hybrid screening using four copies of the nitrate response cis element illustrated that all NLPs could bind to the NRE element. In response to nitrate, the transcript levels of NLP genes are not regulated, but examination of an NLP7 green fluorescent protein fusion revealed that localization of NLP7 was modulated by nitrate via a nuclear retention mechanism. Recently, this localization of NLP7 was identified to occur when Ser205 in NLP7 was phosphorylated in vivo in the presence of nitrate. Suppression of the NLP6 function resulted in the down regulation of nitrate responsive genes, indicating that NLP6 is also a master nitrate regulatory gene involved in primary response. Further characterization has shown that the N terminal region of NLP6 is necessary for its activation in response to nitrate signaling. Furthermore, using over expression lines, NLP7 was revealed to significantly improve plant growth under nitrogen poor and rich conditions. Moreover, ZmNLP4 and ZmNLP8, maize homologs of AtNLP7, play essential roles in nitrate signaling and assimilation and promote plant growth and yield under low nitrate conditions, implying that they may be potential candidates for improving the NUE of maize. In addition to NLPs, reverse genetics has identified LBD37/38/39 to be important nitrate regulators. LBD37/38/39 belong to a gene family encoding zinc finger DNA binding transcription factors and are strongly induced by nitrate.

Further characterization revealed that over expression of LBD37/38/39 can repress the expression of nitrate responsive genes including NRT2.1, NRT2.2, NIA1, and NIA2, indicating that the three LBD members function as negative regulators in nitrate signaling. Recently, following advances in bio informatics and global sequencing analysis, systems biology approaches have been developed and successfully applied to plant nitrogen research. The transcription factors SPL9, TGA1, and TGA4 have been sequentially identified by systems approaches. SPL9 was predicted to be a potential regulatory hub and may target sentinel primary nitrate responsive genes. Research has demonstrated that miR156 can target SPL9 and a mutation in the miR156 caused over expression of SPL9. Accordingly, miR156 resistant SPL9 transgenic plants were investigated and it was revealed that the transcript levels of NRT1.1, NIA2, and NIR significantly increased in response to nitrate, demonstrating that SPL9 plays a negative role in the primary nitrate response. TGA1 and TGA4 belong to the bZIP transcription factor family and are induced by nitrate in roots. Interestingly, induction of TGA1 and TGA4 is inhibited in chl15 and chl19 mutants after nitrate treatment, implying that the regulation of TGA1 and TGA4 by nitrate is affected by nitrate transport, but not the signaling function of NRT1.1. Transcriptome analysis of the roots of tga1 tga4 double mutant plants revealed that most of the genes differentially expressed in the double mutant were regulated by nitrate. Among these target genes of TGA1 and TGA4, induction of NRT2.1 and NRT2.2 was substantially reduced in the double mutants. Further analysis demonstrated that TGA1 could bind to NRT2.1 and NRT2.2 promoters to positively regulate their expression . These results all serve to suggest that TGA1 and TGA4 play important roles in the primary nitrate response.Recently, Shuichi’s lab found that nitrate inducible GARP type transcriptional repressor1 proteins act as central regulators in nitrate signaling.

Co transfection assays revealed that NIGT1 clade genes including NIGT1.1/HHO3, NIGT1.2/HHO2, NIGT1.3/HHO1, and NIGT1.4/HRS1 were all induced by nitrate and were redundant in repressing the nitrate dependent activation of NRT2.1. EMSA and chromatin immunoprecipitation–quantitative PCR analysis further showed that NIGT1.1 could directly bind to the promoter of NRT2.1. Transcriptome and co transfection analysis also illustrated that the expression of NIGT1s was auto regulated and controlled by NLPs. In addition, NIGT1.1 can suppress the activation of NRT2.1 by NLP7. Further investigation suggested that the regulation of NRT2.1 by NIGT1.1 and NLP7 is independent due to their distinct binding sites. A genome wide survey discovered the potential target genes that might be regulated by both NLP mediated activation and NLP NIGT1 transcriptional cascade mediated repression or the NLP NIGT1 cascade alone. Furthermore, phosphate starvation response 1 , the master regulator of P starvation response, also directly enhanced the expression of NIGT1 clade genes,vertical farming technology serving to reduce nitrate uptake. CIPK8 and CIPK23 are calcineurin B like interacting protein kinases. CIPK8 is induced rapidly by nitrate and down regulated in the chl15 mutant. Analysis of two independent T DNA insertion lines showed that induction of NRT1.1, NRT2.1, NIA1, NIA2, and NiR by nitrate was reduced in cipk8 mutants indicating that CIPK8 works as a positive regulator in the primary nitrate response. Further investigation revealed that CIPK8 regulated the nitrate induced expression of NRT1.1 and NRT2.1 under higher but not lower nitrate conditions , suggesting that CIPK8 functions as a positive regulator when nitrate is replete. CIPK23 can be induced by nitrate and downregulated in the chl15 mutant like CIPK8. Expression of the nitrate responsive gene NRT2.1 was upregulated in the cipk23 mutants after nitrate treatment, indicating that CIPK23 plays a negative role in primary nitrate response . This gene is essential for the affinity switch of NRT1.1: it interacts with NRT1.1 and phosphorylates NRT1.1 at T101 under low nitrate concentrations to enable NRT1.1 to operate as a high affinity nitrate transporter, while it dephosphorylates NRT1.1 when nitrate is replete so that NRT1.1 functions as a low affinity nitrate transporter. CPK10, CPK30, and CPK32 are subgroup III Ca2+ sensor protein kinases .They have all been identified as master regulators that orchestrate primary nitrate responses. Analysis of the single cpk10, cpk30, and cpk32 mutants has shown that they only trivially affect nitrate responsive genes. However, in the double mutants cpk10 cpk30, cpk30 cpk32, and cpk10 cpk32 and the triple mutant cpk10 cpk30 cpk32, nitrate responsive marker genes were reduced. Transcriptomic analysis showed that CPK10, CPK30, and CPK32 modulated various key cellular and metabolic functions immediately activated by nitrate. Furthermore, CPK10, CPK30, and CPK32 can phosphorylate NLP7 at Ser205 in vivo in the presence of nitrate, and trigger the nitrate CPK NLP signaling network. Recently, three other nitrate regulatory genes NRG2, CPSF30L, and FIP1 were identified using a forward genetics method. Two independent NRG2 T DNA insertion lines showed reduced induction for nitrate responsive sentinel genes , indicating that NRG2 plays an essential role in nitrate signaling.

At the physiological level, NRG2 affects accumulation of nitrate in plants. Further investigation revealed that it regulates nitrate uptake by roots and the translocation of nitrate within plants. These effects might be achieved through modulating NRT1.1 and NRT1.8 as the expression of both genes was altered in the mutants. Genetic and molecular data suggest that NRG2 can regulate the expression and work upstream of NRT1.1, but function independently, with NLP7 in regulating nitrate signaling. In addition, transcriptomic analysis showed that four clusters in the differentially expressed genes in nrg2 mutant were involved in the regulation of nitrate transport and response, confirming that NRG2 plays essential roles in nitrate regulation. Interestingly, NRG2 can directly interact with NLP7 in vitro and in vivo, as revealed by yeast two hybrid and BiFC experiments. All these results demonstrate that NRG2 is an important nitrate regulator. In addition, the Arabidopsis genome harbors 15 members that are homologous with the NRG2 protein. All members of the NRG2 family contain two unknown conserved domains: DUF630 and DUF632. Whether and which other members of the NRG2 family are involved in nitrate signaling and what functions the two domains play are interesting and pertinent directions for future research. The CPSF30 gene encodes 28 kD and 65 kD proteins. The 28 kD protein was identified as a cleavage and polyadenylation specificity factor; the protein contains three characteristic CCCH zinc finger motifs and functions as both an endonuclease and an RNA binding protein. An additional YTH domain, along with the three zinc finger motifs, are contained in the 65 kD protein. A mutant allele of CPSF30, cpsf302 with a G to A mutation in the first exon of gene CPSF30, was identified by genetic screening and used to explore the functions of CPSF30. The expression of nitrate responsive genes can be down regulated in response to nitrate in cpsf302 compared to wild type and restored to wild type levels in a complemented CPSF30L/cpsf302 line, indicating that CPSF30L is involved in nitrate signaling. CPSF30L can regulate nitrate accumulation and assimilation at the physiological level. Transcriptomic analysis showed that genes involved in six nitrogen related clusters, including nitrate transport and assimilation, were differentially expressed in the cpsf302 mutant. Further study revealed that CPSF30 could work upstream of NRT1.1 and independently of NLP7. CPSF30 can also affect NRT1.1 mRNA 30 UTR alternative polyadenylation. All these results demonstrate that CPSF30 plays an important role in the primary nitrate response. FIP1, a factor interacting with poly polymerase 1, was identified as a positive nitrate regulatory gene using the fifip1 mutant and a FIP1/fifip1 line. Nitrate induced expression of NIA1, NiR, and NRT2.1 is repressed in the fifip1 mutant and can be restored to the wild type in the FIP1/fifip1 line. Furthermore, FIP1 can affect nitrate accumulation through regulating the expression of NRT1.8 and nitrate assimilation genes. Further research found that FIP1 could interact with CPSF30 and both genes can regulate the expression of CIPK8 and CIPK23. In addition, FIP1 can affect the 3 0 UTR polyadenylation of NRT1.1, a similar function to CPSF30. CPSF30, FIP1, and some other components such as CPSF100 can form a complex involved in poly processing. Together, these findings suggest that the complex composed by CPSF30 and FIP1 may play important roles in nitrate signaling. In the extant literature, key molecular components involved in primary nitrate responses, covering nitrate sensors, transcription factors, protein kinases, and polyadenylation specificity factors, have been identified. Methodologically, this has been achieved by using forward and reverse genetics as well as systems biology approaches . In summary, in the presence of both ammonium and nitrate , NRT1.1 functions as a sensor. NLP7, NRG2, and CPSF30 have been revealed to work upstream of NRT1.