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Nitrate is a key nutrient that affects maize growth and yield, and much has yet to be learned about nitrate regulatory genes and mechanisms in maize. Here, we identified nine ZmNLP genes in maize and analyzed the functions of two ZmNLP members in nitrate signaling. qPCR results revealed a broad pattern of expression for ZmNLP genes in different stages and organs with the highest levels of transcript expression of ZmNLP6 and ZmNLP8. When ZmNLP6 and ZmNLP8 were overexpressed in the Arabidopsis nitrate regulatory gene mutant nlp7-4, nitrate assimilation and induction of nitrate-responsive genes in the transgenic plants were recovered to WT levels, indicating that ZmNLP6 and ZmNLP8 can replace the essential roles of the master nitrate regulatory gene AtNLP7 in nitrate signaling and metabolism. ZmNLP6 and ZmNLP8 are localized in the nucleus and can bind candidate nitrate-responsive cis-elements in vitro. The biomass and yield of transgenic Arabidopsis lines overexpressing ZmNLP6 and ZmNLP8 showed significant increase compared with WT and nlp7-4 mutant line in low nitrate conditions. Thus, ZmNLP6 and ZmNLP8 regulate nitrate signaling in transgenic Arabidopsis plants and may be potential candidates for improving nitrogen use efficiency of maize.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

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Nitrate is not only an important nutrient but also a signaling molecule for plants. A few of key molecular components involved in primary nitrate responses have been identified mainly by forward and reverse genetics as well as systems biology, however, many underlining mechanisms of nitrate regulation remain unclear. In this study, we show that the expression of NRT1.1, which encodes a nitrate sensor and transporter (also known as CHL1 and NPF6.3), is modulated by NIN-like protein 7 (NLP7). Genetic and molecular analyses indicate that NLP7 works upstream of NRT1.1 in nitrate regulation when NH4+ is present, while in absence of NH4+, it functions in nitrate signaling independently of NRT1.1. Ectopic expression of NRT1.1 in nlp7 resulted in partial or complete restoration of nitrate signaling (expression from nitrate-regulated promoter NRP), nitrate content and nitrate reductase activity in the transgenic lines. Transcriptome analysis revealed that four nitrogen-related clusters including amino acid synthesis-related genes and members of NRT1/PTR family were modulated by both NLP7 and NRT1.1. In addition, ChIP and EMSA assays results indicated that NLP7 may bind to specific regions of the NRT1.1 promoter. Thus, NLP7 acts as an important factor in nitrate signaling via regulating NRT1.1 under NH4+ conditions.