A DUF630 and 632 Domains-containing Protein, ZmNRL1, Acts as a Positive Regulator of Nitrogen Stress Response in Maize

Nitrogen (N) is a key macronutrient whose availability often determines maize growth and productivity. Improving nitrogen use efficiency (NUE) is critical to increase maize yield while reducing N input, and more importantly, alleviating environmental pollution. However, only a few genes have been exploited for maize NUE improvement thus far. Here, we evaluate NUE-related traits in 296 maize inbred lines. A total of 87 genetic variants, resolved to a total of 44 candidate genes, were found to associated with NUE-related traits through a genome-wide association study. We further found that the natural variations in ZmNRL1, encoding a DUF630 and DUF632 domains-containing protein, strongly associated with chlorophyll content under N starvation. Loss of ZmNRL1 function causes accelerated leaf senescence and reduces biomass, nitrate content and nitrate reductase activity under N limitation, while overexpression of ZmNRL1 enhanced N stress tolerance. Comparative transcriptome analysis revealed that ZmNRL1 has a broad impact on the expression of many N utilization and signaling genes. Furthermore, heterologous expression of ZmNRL1 in Arabidopsis also improved the growth and yield under N-limiting conditions. Thus, ZmNRL1 plays a key role in regulating maize adaptation to N limitation and could be a potential target for breeding high-yield maize with enhanced NUE. Overall design: For maize RNA-seq analysis, 10-day-old maize seedlings were treated with modified Hoagland solutions supplemented with 15 mM NO3- or 0.15 mM NO3- for 48 hours and roots were harvested for total RNA isolated. A total of 18 cDNA libraries of six samples (WT+N, ZmNRL1-OE+N, zmnrl1-mu+N, WT–N, ZmNRL1-OE–N and zmnrl1-m–N) with three repeats were constructed and sequenced.