As AtNLP7 has been reported to be regulated by nitrate via a nuclear retention mechanism , we next tested the subcellular localization of ZmNLP6 and ZmNLP8 proteins under nitrate starvation and nitrate re-addition conditions. Both ZmNLP6 and ZmNLP8 were found to be localized in cytosol after nitrate starvation while in the nucleus when nitrate was resupplied . Thus, ZmNLP6 and ZmNLP8 are mainly localized in the nucleus and slightly in cytosol in the presence of nitrate while both proteins are localized in cytosol when nitrate is absent. Previous studies have shown that nitrate assimilation is impaired and the nitrate content is increased in nlp7 mutants . To test if ZmNLP6 and ZmNLP8 affect nitrate assimilation, we measured nitrate content in transgenic lines and found that the increased nitrate content in nlp7-4 was recovered to WT levels . To investigate if this recovery is associated with nitrate reduction, we examined the NR activity and found that this activity was restored in the transgenic lines . Furthermore, the deficiency of amino acid in nlp7-4 mutant was completely rescued when ZmNLP6 and ZmNLP8 were over expressed in nlp7-4 mutant . Taken together, these data indicate that ZmNLP6 and ZmNLP8 are involved in regulating nitrate assimilation when over expressed in Arabidopsis. In order to further explore the underling mechanism whereby ZmNLP6 and ZmNLP8 affect nitrate content, we examined nitrate accumulation and the expression of several genes involved in nitrate assimilation. Plants were grown on 2.5 mM ammonium succinate for 7 days and then treated with 5 mM KNO3 for 0, 0.25, 0.5, 1, 2, hydroponic nft system and 4 h in the presence of 2.5 mM ammonium succinate. The nitrate content in whole seedlings was investigated and the results showed that no difference was found in nitrate accumulation among the WT, nlp7-4, and transgenic lines . However, the expression of Gln1.1, Gln1.3, NIA2, and NiR in transgenic lines was recovered to the levels even higher than in WT .

These findings suggest that ZmNLP6 and ZmNLP8 can regulate the nitrate assimilation more strongly than nitrate accumulation. It has been found that Arabidopsis NLP proteins can bind nitrate regulatory elements to regulate the nitrate responsive genes . To investigate whether ZmNLP6 and ZmNLP8 can bind NREs in maize, Y1H assay was performed. Firstly, we searched for NREs with a module from the 50 and 30 franking sequences of nitrate uptake and assimilation genes. Nine putative NREs were obtained as shown in Supplementary Table S2. Then, the candidate NREs were used for testing the binding activity of ZmNLP6 and ZmNLP8. The results showed that ZmNLP6 and ZmNLP8 could bind the NRE-like motifs of ZmNRT1.2 and ZmNiR2 , respectively . But no binding activity was found between ZmNLP6/ZmNLP8 and other NREs . These data suggest that the ZmNLP6 and ZmNLP8 proteins can bind putative NREs similar to what was reported for Arabidopsis. Low NUE in agriculture system is a global problem and therefore we assessed the potential of both genes to affect root architecture and NUE of in Arabidopsis. We first examined the primary root length and lateral root number in plants grown vertically on the media with different nitrate concentrations . The results showed that the length of primary roots and number of lateral roots were higher in ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines than in WT and nlp7-4 mutant under these three nitrate conditions . To determine whether ZmNLP6 and ZmNLP8 can enhance NUE in plants, we investigated the biomass of WT, nlp7-4, and ZmNLP6/nlp7- 4 and ZmNLP8/nlp7-4 transgenic lines under different nitrate concentrations. The results showed that the transgenic seedlings grew bigger than WT and nlp7-4 and the biomass of the whole seedlings increased by 15, 35, and 40% more than WT under 0.2, 2.5, and 5 mM KNO3 conditions , indicating that the ZmNLP6 and ZmNLP8 can rescue the defificient growth phenotype of nlp7-4 mutant and promote plant growth under both low and high nitrate conditions. Seed yield is an important trait for agricultural production and also for assessing the NUE of plants. Thus, we investigated the seed yield of ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines grown under both high and low nitrate conditions.

The results showed that the yield per plant was higher in ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines by 44 and 45%, respectively, than in WT under low nitrate conditions . However, no significant difference was found between the transgenic lines and WT when grown under high nitrate conditions . These data suggest that ZmNLP6 and ZmNLP8 may improve plant NUE under low nitrate conditions. It has been reported that Arabidopsis NLP genes are involved in nitrate regulation, but the functions of maize NLP genes remain unknown. As maize is one of the main crops of the world, identifying the genes associated with nitrate signaling and deciphering the corresponding gene networks are of great importance for improving NUE and reducing environmental pollution. In this study, we identified nine ZmNLP genes containing RWP-RK and PB1 domains by genome-wide analysis in maize. The RWP-RK super family includes NLP and RKD families, both of which contain RWP-RK domain . NLP family is conserved in the land plants we searched, especially in maize , sorghum , rice , and Arabidopsis . This family can be divided into three subfamilies and each subfamily shows different gene structure characteristics from each other . A previous study also reported a similar subfamily division in Arabidopsis, rice, and Lotus japonicus . We found two novel NLP motifs among these searched 34 species: GAF motif and GSL motif . The GAF motif exists in the N-terminus of NLP protein, a region involved in receiving the nitrate signal . The most conserved signature structure in GAF motif is the first serine, the fifth phenylalanine, and GLPGR.The GSL motif, located in the front of the RWP-RK domain, is conserved only in the NLP proteins but not in RDK proteins. The expression profiles of ZmNLP genes showed that ZmNLP6 and ZmNLP8 exhibited the highest expression levels among the whole gene family, especially in R1 in roots and V13 in leaves . As roots of R1 and the leaves of V13 are important for absorbing and remobilizing nitrate to pool organs and critical for yield of maize , ZmNLP6 and ZmNLP8 may be important for nutrient uptake and translocation. The nitrate induction was tested for all ZmNLP genes and the results showed that only the expression of ZmNLP3 and ZmNLP4 were induced by low nitrate, and induced poorly or not at all by high nitrate , implying that a post-transcriptional regulation response to nitrate may exist.

Under different nitrate conditions, ZmNLP1, ZmNLP2, ZmNLP3, and ZmNLP7 exhibited relatively higher expression in roots under nitrogen starvation and they mainly belongs to ZmNLP Group I, implying that the function of Group I may be involved in nitrogen starvation . Moreover, ZmNLP6 and ZmNLP8 showed the highest expression levels under higher concentrations of nitrate in shoots while the highest levels on 2.5 mM nitrate condition in roots and may participated in nitrate absorb and allocation. The expression patterns of ZmNLPs implicate that functional redundancy of ZmNLP family members in nitrate regulation may exist,nft channel and these NLP genes may play important roles in nitrate regulation in different stages and organs under various nitrate conditions. In Arabidopsis, several nitrate regulatory genes have been identified and these genes can modulate genes involved in nitrate transport, assimilation, and response. But so far, no nitrate regulators have been reported in maize. We investigated the function of maize NLP genes and found that over expression of ZmNLP6 and ZmNLP8 in Arabidopsis nlp7-4 mutant could recover the YFP fluorescence from the NRP-YFP transgene product and the induction of nitrate responsive genes to WT levels , indicating that ZmNLP6 and ZmNLP8 can restore the nitrate signaling in nlp7-4 mutant. Previous studies have reported that NRE works as an important nitrateresponsive cis-acting element and can be bound by NLPs in Arabidopsis . Our Y1H results showed that ZmNLP6 and ZmNLP8 proteins can bind potential NREs of ZmNRT1.2 and ZmNiR2 in vitro , suggesting a direct regulation of ZmNRT1.2 and ZmNiR2 by ZmNLP6 and ZmNLP8 may exist in maize. In Arabidopsis, NLP7 has a profound influence on nitrate responsive genes at the transcriptional level and some target genes of NLP7 are regulated by binding to their NREs . Thus, the ZmNLPs may bind NRE to regulate nitrate signaling in maize similar to that in Arabidopsis, and this regulation mechanism may be conserved in monocots and dicots. The subcellular localization of ZmNLP6 and ZmNLP8 is regulated by nitrate , similar to a mechanism controlling AtNLP7 localization in Arabidopsis. Our physiological and molecular analyses further revealed that the nitrate reduction process could be recovered in the ZmNLP6/nlp7-4 and ZmNLP8/nlp7-4 transgenic lines, indicating that both ZmNLP6 and ZmNLP8 can modulate nitrate assimilation when constitutively over expressed in Arabidopsis.

It has been reported that over expression AtNLP7 in Arabidopsis can increase fresh weight and modify root architecture under low and high nitrate conditions. In our study, over expression of ZmNLP6 and ZmNLP8 in nlp7-4 mutant can also enhance the biomass and root development. However, the nitrate content, amino acid content, and NR activity were increased in AtNLP7 over expression lines while restored to WT levels in ZmNLP transgenic lines. In addition, we found an increase in seed yield in ZmNLP6 and ZmNLP8 transgenic Arabidopsis lines under low nitrate conditions. It remains to be further investigated if the seed yield can be increased in AtNLP7 over expression lines. Results shown in this study suggest the function of the group III NLPs in Arabidopsis and maize may be partially conserved in nitrate regulation. Improving NUE of crops is of great importance for sustainable agriculture. Several nitrate-related genes have been implicating in improving NUE. OsDEP1, encoding a highly cysteine – rich G protein γ subunit, has been reported to increase rice harvest index and grain yield under moderate levels of nitrogen fertilization . OsNRT1.1B-indica variation has been identified to enhance the ability of nitrate uptake and root to-shoot transport to improve NUE in rice . In addition, over expression of OsNRT2.3b can improve grain yield and NUE by increasing the capacity of pH-buffering and uptake of N, Fe, and P in rice . In Arabidopsis, over expression of AtNLP7 can improve plant growth under both nitrogen-limiting and -sufficient conditions . In this paper, our results showed that ZmNLP6 and ZmNLP8 could promote plant growth under both low and high nitrate conditions, and increase seed yield under low nitrate conditions . Therefore, both ZmNLP6 and ZmNLP8 genes may be of great potential in improving NUE of maize. It would be also interesting to assess the role of other NLP members in promoting NUE of maize in the near future.Plastids originate from a single endosymbiontic event involving a cyanobacterium-related organism. In the course of endosymbiosis a massive gene transfer occurred, during which most plastidic genes were transferred to the host cell nucleus. Consequently, today the majority of plastidic proteins must be post-translationally imported back into the organelle. So far, two protein translocation complexes have been characterized in the outer and inner envelope membrane: Toc and Tic. After passing the outer membrane via the Toc translocon, the Tic complex catalyses import across the IE membrane. So far, seven components have been unambiguously described as Tic subunits: Tic110, Tic62, Tic55, Tic40, Tic32, Tic22 and Tic20 . Tic110 is the largest, most abundant and best studied Tic component. It contains two hydrophobic transmembrane-helices at its N-terminus, anchoring the protein in the membrane, and four amphipathic a-helices in the large C-terminal domain that are responsible for channel formation. At the intermembrane space side, Tic110 contacts the Toc machinery and recognizes preproteins. Moreover, loops facing the stroma provide a transit peptide docking site and recruit chaperones such as Cpn60, Hsp93 and Hsp70. Tic110 is expressed in flowers, leaves, stems and root tissues, indicating a role in import in all types of plastids. It is essential for chloroplast bio-genesis and embryo development. Heterozygous knockout plants are clearly affected: they have a pale green phenotype, exhibit defects in plant growth, display strongly reduced amounts of thylakoid membranes and starch granules in chloroplasts, coupled with impaired protein translocation across the IE membrane.