CPK10, CPK30, and CPK32 are subgroup III Ca2+-sensor protein kinases . The activity of CPKs can be enhanced in response to nitrate within 10 min. 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, CPSF30-L, 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,indoor growers 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.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, cpsf30-2 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 cpsf30-2 compared to wild-type and restored to wild-type levels in a complemented CPSF30-L/cpsf30-2 line, indicating that CPSF30-L is involved in nitrate signaling. CPSF30-L 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 cpsf30-2 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 NRT1.1. NRG2 can interact with NLP7 whilst NLP7 can interact with, and be phosphorylated by, CPK10. In addition, NLP7 binds to the promoter of NRT1.1 as revealed by ChIP and EMSA assays. NRT1.1 works upstream of, and regulates, TGA1/TGA4. Furthermore, CIPK23 interacts with and phosphorylates NRT1.1. CPSF30 can interact with FIP1 and regulate the expression of both CIPK8 and CIPK23. NIGT1.1 can suppress NLP7-activated NRT2.1. In the presence of nitrate but absence of ammonium , NRT1.1 works only as a nitrate transporter, but not as a nitrate regulator. The other nitrate regulatory genes, including NRG2, NLP7, CPSF30, FIP1, LBD37/38/39, SPL9, NIGT1s, CIPK8, and CIPK23, still play an important role in the nitrate signaling.
Serving as an important molecular signal, nitrate also regulates plant growth and development and has been particularly well studied in the context of root system architecture. Root system architecture controls the absorption and utilization of nutrients and affects the growth and biomass of plants. Lateral root growth is dually regulated by nitrate availability, including local induction by NO3− and systemic repression by high NO3. Several key genes and miRNAs functioning in nitrate-regulated root architecture have been characterized. The ANR1 gene, encoding a member of the MADS-box family of transcription factors,danish trolley was the first gene to be identified as an essential component in nitrate-regulated root growth. Nitrate can inhibit the growth of lateral roots when seedlings are grown on media with higher nitrate concentrations compared to lower nitrate concentrations . However, ANR1 down regulated lines obtained by antisense or co-suppression exhibited reduced lateral root length when grown on media with various nitrate concentrations, indicating the enhanced sensitivity of lateral root growth to nitrate inhibition in those lines. Over expression of ANR1 in roots resulted in increased lateral root growth and this phenotype was strongly dependent on the presence of nitrate, suggesting post translational control of ANR1 activity by nitrate. Interestingly, the expression of ANR1 in nrt1.1 mutants was dramatically diminished and these mutants exhibited reduced root elongation in nitrate-rich patches, similar to what was observed with the ANR1-repressed lines. This suggests that NRT1.1 works upstream of ANR1 in terms of local nitrate-induced lateral root growth. Recently, the auxin transport role of NRT1.1 was characterized in lateral root primordia when seedlings were grown on media without nitrate or with low nitrate concentrations; under these conditions, NRT1.1 represses the growth of pre-emerged LR primordia and young LRs by inhibiting the accumulation of auxin. Subsequently, Gojon’s lab revealed that the NRT1.1-mediated regulation of LR growth was dependent on the phosphorylation of NRT1.1 and the non-phosphorylated form of NRT1.1 could transport auxin in the absence of nitrate or in low nitrate concentrations. Further investigation indicated that in the presence of nitrate, the promoter activity of NRT1.1 was stimulated and mRNA stability was increased, while protein accumulation and auxin transport activity were repressed in LRPs, resulting in accelerated lateral root growth. Altogether, NRT1.1 offers a link between nitrate and auxin signaling during lateral root development. However, the mechanisms by which nitrate induces the expression of NRT1.1 while repressing NRT1.1 protein accumulation and auxin transport activity in LRPs remain unclear. Previous reports have also documented that several genes involved in hormone biosynthesis or response regulate the root system architecture response to changes in nitrate availability. NRT2.1, a high-affinity nitrate transport gene, is induced by nitrate and sugar. Wild-type seedlings grown on media with high carbon/nitrogen ratios exhibited significantly repressed lateral root initiation compared to a standard growth medium.
However, the repression of lateral root initiation was diminished in nrt2.1 mutants under high C/N ratios where this phenotype was not dependent on nitrate uptake. These results demonstrate that NRT2.1 plays an important role in lateral root initiation under high C/N ratios. In addition, nrt2.1 mutants exhibited significantly reduced shoot-to-root ratios compared to wild-type and nrt2.2 mutant seedlings when grown in common hydroponic conditions . The reductions in shoot-to-root ratios were even greater for nrt2.1 nrt2.2, suggesting that both genes are involved in regulating plant growth with NRT2.1 playing a more important role. Moreover, nrt2.1 mutants exhibit reduced LR growth on media with limited nitrogen and this reduction was more severe in nrt2.1 nrt2.2 double mutant plants, indicating that both genes are important regulators involved in lateral root growth. Recently, Gutierrez’s lab determined that induction of NRT2.1 and NRT2.2 was directly regulated by TGA1/TGA4 in response to nitrate treatment. Further investigation showed that tga1 tga4 plants and nrt2.1 nrt2.2 plants exhibited similarly decreased LR initiation compared with wild-type plants, indicating that NRT2.1 and NRT2.2 work downstream of TGA1/TGA4 to modulate LR initiation in response to nitrate. Lateral root emergence was also affected in tga1 tga4 and nrt2.1 nrt2.2 mutants, and tga1 tga4 mutants displayed larger reductions in LR emergence than nrt2.1 nrt2.2 mutants, revealing that additional pathways are required for LR emergence controlled by TGA1/TGA4 besides NRT2.1 and NRT2.2. Moreover, primary roots in tga1 tga4 mutants were shorter than in wild-type and nrt2.1 nrt2.2 plants, suggesting that the modulation of primary root growth by TGA1/TGA4 is independent of NRT2.1 and NRT2.2. The protein kinase CIPK8 is not only involved in primary nitrate response, but also in long-term nitrate regulation on root growth. In the presence of nitrate, cipk8 mutants exhibited longer primary root length compared to the wild type, indicating that CIPK8 modulates primary root growth in a nitrate-dependent pathway.
Furthermore, the key nitrate regulator NLP7 has also been found to control root growth under both N-limited and N-rich conditions besides its essential roles in the primary nitrate response . nlp7 mutants developed longer primary roots and higher LR density on N-rich media. Interestingly, transgenic lines with over expression of NLP7 also exhibited increased primary root length and lateral root density under 1, 3, and 10 mM nitrate conditions.The underlying inter-phenotype mechanisms regulating root growth in the mutant and over expression lines are still unknown. These findings indicate that NLP7 plays an important role in nitrate-regulated root development. Recently, it has been shown that the Ca2+-sensor protein kinases CPK10, CPK30, and CPK32 are also involved in nitrate-specific control of root development. In response to nitrate, icpk mutants had reduced lateral root primordia density and reduced lateral root elongation compared to the wild type. In the last few years, microRNAs have emerged as important regulators involved in nitrate-regulated root growth. It has been reported that miR167 targets and controls expression of the auxin response factor ARF8, and both miR167 and ARF8 are expressed in the pericycle and lateral root cap. Levels of miR167 were repressed under nitrogen treatment, leading to accumulation of ARF8 in the pericycle. In contrast to wild-type plants, which displayed increased ratios of initiating vs. emerging lateral roots in response to nitrogen treatment, the miR167a over expression lines and arf8 mutants were insensitive to nitrogen in terms of lateral root emergence. These results indicate that the auxin response factor-miRNA regulatory module miR167/ARF8 plays an important role in controlling lateral root growth in response to nitrogen . In addition, miR393 was induced by nitrate treatment, specifically cleaved the auxin receptor AFB3 transcript, and modulated the accumulation of AFB3 mRNA in roots under nitrate treatment . The primary root of the wild type was shorter when treated with KNO3 compared to KCL, however the primary root of the miR393-overexpression line andafb3 mutant were insensitive to nitrate treatments. miR393/AFB3 also controlled lateral root growth as well as primary root growth. The miR393 over expression line and afb3 mutant showed diminished densities of initiating and emerging lateral roots compared to the wild type, which exhibited increased growth of lateral roots in response to nitrate treatments.