In addition, the scale of the application required to have an impact on the atmospheric C level is unclear at this point. More long-term and standardized studies, under different environmental conditions, of below ground carbon fluxes, integrating models and measurements are needed. C sequestration through plant-microbe interaction is still in its exploratory phase. As more worldwide attention is drawn towards mitigating elevated atmospheric C level, hopefully more global collaborative interdisciplinary research efforts will be directed towards assessing the conditions required for successful application of plant-microbe C sequestration. Nitrogen is an essential macro-nutrient for plant growth and development and most terrestrial plants absorb nitrate as their main nitrogen source. In agricultural systems, nitrate supply directly affects plant growth and crop productivity. In many developed and developing countries, excessive nitrogen fertilizer is applied in agriculture,commercial greenhouse supplies while the nitrogen use efficiency of crops is low. Therefore, a large fraction of the applied nitrogen cannot be taken up by plants and is lost into the environment, resulting in serious problems such as eutrophication and nitrate pollution of underground water. These problems must be addressed. One approach is to improve the NUE of crops, which could reduce the load of nitrogen fertilizers on farm land and natural ecosystems. Elucidating the mechanisms and the underlying network of nitrate regulation would provide a theoretical basis and guiding framework for improving NUE.A part of the nitrate imported into cells is reduced and assimilated into amino acid through a series of enzymes including nitrate reductase , nitrite reductase , glutamine synthase , and glutamate synthase .
Nitrate acts as a nutrient and as an important signal to regulate gene expression, plant growth, and development.The short-term effect is referred to as the primary nitrate response, in view of the fact that many genes can be regulated after a short period of exposure to nitrate inputs. Indeed, some genes involved in nitrate transport and reduction are induced in a matter of minutes. The long-term effects include the impact of nitrate on plant growth and development after a longer period of time, including effects on the morphogenesis of roots, plant flowering, seed dormancy, stomatal closure independent of abscisic acid, the circadian rhythm, and the transport of auxin. Among these aspects, the effects of nitrate on root development are well studied and several essential genes involved in this process have been identified. Here we review the genes involved in the primary nitrate response and describe their functions in nitrate signaling . Then we summarize the relationship between nitrate availability and root system architecture and the roles of the characterized genes that control root growth and development in response to local and systemic nitrate signals .In the late 1990s, molecular components involved in nitrate signaling were identified in bacteria and fungi. In Escherichia coli, both NARX and NARQ containing a P-box domain were found to be responsible for nitrate binding and could activate the nitrate-regulating proteins NarL and NarP, which are essential for nitrate sensing. Therefore, these two genes are nitrate regulators in E. coli. In fungi, two transcription factors NirA and Nit4 have been identified as important nitrate regulators. NirA is needed for the expression of nitrate reductase and Nit4 may interact with nitrate reductase directly. Both proteins were demonstrated to activate their target genes that can respond to nitrate. In plants, some genes encoding proteins required for nitrate assimilation, transport, and energy and carbon metabolism are rapidly induced after nitrate treatment.
These are regarded as primary nitrate-responsive genes. Scientists have characterized a few of the regulators playing important roles in primary nitrate responses, mainly by employing methodologies in forward and reverse genetics as well as systems biology. NRT1.1, also called CHL1 and NPF6.3, belongs to the NRT1/PTR family. Previously, NRT1.1 was identified as a dual-affinity nitrate transporter working in both low and high nitrate concentrations. Subsequently, it was shown that NRT1.1 controlled root architecture by acting as a potential nitrate sensor. Then in 2009 it was found that NRT1.1 is involved in the primary nitrate response. Using a forward genetic screen, the Crawford lab identified a mutant with a defective response to nitrate, and the mutation was localized to NRT1.1. Characterization of the mutant revealed that expression of the primary nitrate-responsive genes NIA1, NiR, and NRT2.1 was significantly inhibited when plants were grown in the presence of ammonium. Interestingly, the regulatory role of NRT1.1 was lost when ammonium was absent because the expression of these nitrate-responsive genes was restored in the mutant without ammonium, indicating that other nitrate sensor were present and dominated in the absence of ammonium. The Tsay lab also showed that a null mutant of NRT1.1, chl1-5, lost both nitrate uptake and primary nitrate response functions. They then described an allele of NRT1.1that was defective in nitrate uptake but not nitrate regulation. These results indicate that the primary nitrate response was defective in the mutant chl1-5 but not in chl1-9, and the function of NRT1.1 in nitrate signaling is independent of its uptake activity, thereby identifying NRT1.1 as a nitrate sensor. 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,vertical grow 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, 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.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 chl1-5 and chl1-9 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, 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 chl1-5 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 down regulated in the chl1-5 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.