The nuclear retention is controlled by the phosphorylation of the conserved serine205 by AtCPK10/30/32 in the N-terminal region of AtNLP7 . In addition, other members of the AtNLP family can bind the NRE and modulate nitrate responsive genes . Maize is an essential food and cash crop in many parts of the world. Much of the world’s nitrogen fertilizer is used to support the growth and high yield of maize. However, little is known about the regulation of nitrate assimilation in maize at the molecular and genetic levels. It has been reported that the expression levels of maize nitrate transporters ZmNRT2.1 and ZmNRT2.2 exhibit two distinct peaks in different growth stages under low nitrate conditions . On the other hand, the expression of ZmNRT2.1 and ZmNRT2.2 in the roots of seedlings and adult plants are inhibited by local low nitrate while enhanced by local high nitrate . Additionally, most ZmNRT2 family members are responsive to nitrogen treatments . GS gene ZmGln1-3 is localized in mesophyll cells and plays an important role in grain production . However, there have been no nitrate regulatory genes characterized in maize. In this study, we identified all nine ZmNLP genes in maize and analyzed their expression profiles. Genetic and molecular analyses suggest that ZmNLP6 and ZmNLP8 play essential roles in nitrate signaling and assimilation. Both genes are localized in the nucleus. Y1H assay revealed that ZmNLP6 and ZmNLP8 could bind NRE of ZmNRT1.2 and ZmNiR2 directly in vitro. Furthermore, constitutively over expressing ZmNLP6 and ZmNLP8 in Arabidopsis can promote growth and yield under low nitrate conditions,nft growing system implicating their potential in improving maize NUE. To character the NLP members, the protein sequences of NLP family in Arabidopsis and rice were used as query sequences to perform BLASTp in 34 species in plant genomics resource1 .

The BLASTp were used with an e-value cutoff set to 1e−003. The isolated protein sequences were examined using the domain analysis program Hidden Markov Model of SMART2 and Pfam3 with the default cutoff parameters . We obtained the sequences with the Pfam number PF02042.10 and PF00564.19, which contained atypical RWP-RK and PB1 domains, from the maize genome sequences using a Perl-based script. The full length NLP protein sequence of Arabidopsis, maize, sorghum, and rice were used for constructing phylogenetic tree with the neighbor-joining method of the MEGA7 program4 using the p-distance and complete default option parameters.The images of the phylogenetic trees were drawn using MEGA7. The genomic and CDS of NLP members were downloaded from the plantGDB database5 . The gene structures of NLP genes were generated using the GSDS6 . The position of GAF-Like, RWP-RK and PB1 domain was indicated from the Pfam results.The amino acid sequences of ZmNLPs were aligned using the ClustalX program with BLOSUM30 as the protein-weight matrix. The chromosomal location was retrieved from the maize genome data7 , and the genes were mapped to the chromosomes using MapDraw program. The isoelectric points and molecular weights of these proteins were obtained with the help of the proteomics and sequence analysis tools on the ExPASy Proteomics Server8 . The conserved motif was discovered by Motif discovery in MEME9 using 248 NLP proteins in 34 species. The logos were created and downloaded by Multiple Em for Motif Elicitation. Maize B73 inbred seeds were grown in the fields every year and a part of 0–20 cm underground roots and ear leaves were collected at the stage vegetative 3 , vegetative 5 , vegetative 9 , vegetative 13 , reproductive 1 , and vegetative 18 . These are important stages for the growth and development of maize.

A part of 0–20 cm underground roots, ear leaf , ear stem , ear sheath , tassels , and grains in reproductive 1 stage were collected for testing the gene expression every year. Maize plants used for testing the induction of nitrate responsive genes were grown on matrix watered with B5 solution to 3-leaf stage, and then the endosperm were removed from the seedlings. The uniform seedlings were transferred to hydroponics system with 2.5 mM ammonium succinate for 2 days, followed by the treatments with 0.2 mM or 10 mM KNO3/KCl for 2 h.Arabidopsis plants used for testing the YFP fluorescence intensity were grown on plates with nitrate medium for 4 days and observed under a fluorescence microscope . The fluorescence of roots was quantified using Image J. For Arabidopsis plants used for qPCR analysis of nitrate responsive genes, seedlings were grown in aseptic hydroponics as previously described for 7 days and then treated with 10 mM KNO3 or KCl for 2 h. RNA was extracted from the roots for testing the expression of nitrate responsive genes.For nitrate content detection, seedlings of WT, nlp7-4, and transgenic lines were grown in 1/2 MS medium for 7 days and then collected 0.05– 0.1 g of the whole seedlings. The samples were milled into powder using a RETCH MM400 and then added into l mL ddH2O followed by boiling at 100 ◦C for 30 min. The cooled sample was centrifuged at 13000 rpm for 10 min and 400 µL supernatant was collected to the flow cell. The nitrate content was detected by hydrazine reduction method using AutoAnalyzer 3 continuous flow analytical system . Nitrate reductase activity was detected by sulfanilamide colorimetric method as described previously . Amino acid content in tissue was determined using ninhydrin colorimetric analysis as described previously .To analyze the phylogenetic relationship of NLP proteins, the unrooted phylogenetic tree was generated based on the full length amino acid sequences of NLP proteins from Arabidopsis , maize , sorghum , and rice using MEGA7.0. As shown in Figure 1A, the NLPs were divided into 3 groups . In each group, the NLP proteins from maize, sorghum, and rice were closely clustered. Exon/intron structures of these NLPs, shown in Figure 1B, revealed an obvious difference among groups: a module with four exons and three introns was found in most group I members with the smallest size in the second exon.

The group II members mainly contain five exons and four introns, and two smaller exons separate three bigger ones. A module of five exons and four introns also found in group III members, but the third exon is the smallest one. Thus, the length and position of introns make great contributions to the diversity of gene structure in each group. For the ZmNLP proteins,nft hydroponic system the RWP-RK and PB1 domains were found to be well conserved in all tested members, and GAF-like domain were also found in the N-termini of ZmNLPs . In addition, the results revealed two novel motifs, GAF motif and GSL motif, in the NLP family by using MEME analysis . The GAF motif , which is located in the N-termini of NLPs, is the main part of the GAF-Like domain and critical for nitrate signaling transduction in Arabidopsis . GSL motif exists just in front of the RWP-PK domain and with conserved GSL residues as a core. The GSL motif was well conserved in 236 NLP proteins in 34 species, but not found in the RKD family proteins, so that it can be regarded as a marker for distinguishing the NLP proteins from RKD proteins. To better understand the function of each gene in the ZmNLP family, we examined their temporal and spatial expressions in maize B73 inbred lines by using qPCR. The results showed that different ZmNLP genes exhibited various expression patterns . Among these genes, ZmNLP6 and ZmNLP8 showed significant higher expression levels in almost all tested stages and tissues, while the expression levels of ZmNLP1 and ZmNLP7 were much lower. ZmNLP4 and ZmNL9 exhibited the lowest expression levels in diverse organs and stages. Remarkably, the expression of ZmNLP6 and ZmNLP8 was the highest in the vegetative 13 stage in leaves and reproductive 1 stage in roots. V13 is the stage of tasseling and R1 is grouting stage, both of which are essential for maize production, suggesting that ZmNLP6 and ZmNLP8 may play outsized roles in these stages critical to maximizing maize yield. The expression of ZmNLP6 and ZmNLP8 was higher in roots, tassels, and grains, while ZmNLP2 and ZmNLP3 was higher in leaves than in other organs . The expression of ZmNLP1 was relatively uniform, while ZmNLP7 was more highly expressed in both roots and tassels. The expression of ZmNLP5 was relatively low, mainly expressed in leaves, tassels, and grains. We also used the second housekeeping gene ZmUBCE for testing the expression of these genes and similar results were found . These results suggest that ZmNLP6 and ZmNLP8 may play more important roles in different developmental stages and tissues compared with the other members of ZmNLPs in maize. To test if ZmNLPs are responsive to different nitrate conditions, we investigated the expression of all ZmNLP genes in roots. Seedlings were grown on 1/2MS medium for 2 weeks, and then transferred into 2.5 mM ammonium succinate for 2 days followed by treatments with 0.2 or 10 mM KNO3 for 2 h. As shown in the Figure 2C, ZmNLP3 and ZmNLP4 were induced by 0.2 mM KNO3 in roots. After 10 mM KNO3 treatment, the expression of most ZmNLPs was not induced except ZmNLP2 . Thus, most ZmNLP genes were not induced by nitrate. To test the expression patterns of ZmNLP genes under various nitrate conditions, 2-week-old seedlings were grown under 0, 0.2, 2.5, 5, and 10 mM KNO3 conditions and RNAs were extracted from the shoots and roots for qPCR analysis.

The results showed that the expression levels of the ZmNLP4, ZmNLP6, and ZmNP8 were generally increased with the higher concentrations of nitrate in shoots, while the highest peak was observed at 2.5 mM nitrate concentration for ZmNLP6 and ZmNLP8 in the roots . However, the expression of ZmNLP3 and ZmNLP7 generally decreased in both shoots and roots with the increase of nitrate concentrations. For ZmNLP1, the expression was stable in shoots while decreasing with increased nitrate concentrations . These data indicate that different ZmNLPs exhibit various expression profiles. The expression of ZmNLP6 and ZmNLP8 in shoots was the highest and increased with increasing nitrate concentrations, indicating that both genes are the strongest candidates for playing important roles in regulating nitrate metabolism in maize. The second housekeeping gene ZmUBCE were used for testing the expression of these genes and the results were similar to those obtained by ZmActin1 . Therefore, we focused on ZmNLP6 and ZmNLP8 in the following studies.AtNLP7 has been reported as a key regulator in nitrate signaling . The Arabidopsis mutant nlp7-4 with a mutation at the position 62, converting Gln to a stop codon, was identified by a forward genetic screen . The nlp7-4 plants contain NRP::YFP construct and are defective in nitrate regulation of nitrate responsive genes. To investigate if ZmNLP6 and ZmNLP8 are involved in nitrate signaling, we cloned them from the cDNAs of B73 inbred line and over expressed them in the mutant nlp7-4, respectively, using a 35S promoter. Transgenic plants over expressing the ZmNLP6 and ZmNLP8 were generated and two independent homozygous lines were selected for following investigation . Nitrate-responsive expression was monitored using a construct containing the YFP fused to a nitrate-responsive synthetic promoter . The transgenic lines were grown on nitrate medium and YFP fluorescence of these lines was found to be partially or completely restored to WT levels , indicating that the ZmNLP6 and ZmNLP8 may function in nitrate signaling. Furthermore, we investigated the expression of endogenous nitrate-responsive genes including NIA1, NiR, and NRT2.1 and found that the induction levels of all three genes were recovered in ZmNLP6 and ZmNLP8 transgenic lines after nitrate treatments . The expressions of these genes were also normalized to the percentage of second housekeeping gene AtActin8 and similar results were found . These results suggest that ZmNLP6 and ZmNLP8 can complement the Atnlp7 mutant and thus play important roles in nitrate signaling. To investigate the sub-cellular localization of ZmNLP6 and ZmNLP8 proteins, the full length CDS of the two genes driven by 35S promoter were fused with GFP in N-terminal. The 35S::GFPZmNLP6 and 35S::GFP-ZmNLP8 constructs were transiently transformed into Nicotiana benthamiana epidermal cells and Arabidopsis mesophyll protoplasts, the results showed that both ZmNLP6 and ZmNLP8 were mainly localized in the nucleus and slightly in cytosol .