Transcription factor ZmNLP8 modulates nitrate utilization by transactivating ZmNiR1.2 in maize

The excessive use of nitrogen fertilizer boosts agricultural production, but poses serious threats to the environment. It therefore highlights an urgent need to achieve the required yield increases while reducing nitrogen fertilizer use. Improving crop nitrogen use efficiency (NUE) is regarded as a promising approach to solve this dilemma. Here, we show that loss-of-function mutations in maize NIN-like protein 8 (ZmNLP8) cause earlier senescence and result in over 70% grain yield losses per plant under normal nitrogen nutrition in field trials, while the transgenic line that overexpressed ZmNLP8 exhibits remarkable growth vigor in fluctuating nitrate conditions, indicating that ZmNLP8 is indispensable in regulating nitrogen utilization in maize. ZmNLP8, acting as a transcription factor, binds to the nitrate-responsive cis-element and transactivates the expression of ZmNiR1.2. Moreover, a pull-down assay, combined with luciferase complementation assay and mass spectrometry analysis, revealed that ZmNLP8 physically interacted with ZmTCP8. Knockout of ZmTCP8 impairs vegetative growth under different nitrate conditions, indicating that ZmTCP8 functions in a common pathway as ZmNLP8. The dual-luciferase transient expression system reveals that ZmNLP8 synergistically associates with ZmTCP8 to regulate the expression of ZmNiR1.2. Analysis of a panel of 1210 maize germplasms reveals that 92.6% of modern improved maize lines carry abundant variations and have larger nucleotide difference per site (Dxy) in the ZmNLP8 promoter, which results in a reduction in the transcriptional activity of ZmNLP8. Our results demonstrate that ZmNLP8 has probably been selected through breeding and plays a pivotal role in nitrogen utilization, at least in part, by regulating the expression of ZmNiR1.2.