Diel Regulation in Spinach Synchronizes Nitrogen Assimilation via Transcriptional Gating

Plants synchronize metabolism with the day-night cycle, yet the interplay between circadian rhythms and nitrogen metabolism in leafy vegetables remains unexplored. We profiled spinach ( Spinacia oleracea L.) grown under high-nitrogen and low-nitrogen during four diel phases (end of dark, middle of light, end of light, middle of dark), integrating data from RNA-sequencing with measurements of gas exchange, chlorophyll fluorescence, nitrogen forms, amino acids, and enzyme activities. Nitrogen sufficiency enhanced biomass, CO 2 assimilation, chlorophyll content, and photosynthetic efficiency, with maxima in the middle of light phase. Transcriptom e ice analyses revealed that the greatest gene reprogramming occurred at the dark-to-light transition, where the expression of the morning oscillator component LHY expression peaked and coincid ed ing accumulation of dawn-biased nitrate-assimilation transcripts ( NIA and NiR ) and transporters. Peaks enzyme activities lagged transcript peaks by nearly one phase, suggesting transcriptional priming followed by daytime nitrogen assimilation, aligned with photosynthetic energy and carbon skeleton synthesis. Amino acid profiles reflected this coordination, with glutamine elevated during the day and glutamate enriched at night. Nitrogen deficiency suppressed growth, caused accumulation of reactive oxygen species, and activated circadian-regulated DNA repair genes (e.g., MSH2 and RPA2A , indicating genotoxic stress. Together, these findings support a model in which a diel/clock program, amplified by nitrogen sufficiency, gates nitrogen transport and reduction at dawn to couple daytime assimilation with photosynthesis while adjusting oxidative and DNA repair responses. This mechanistic framework clarifies how temporal regulation and nutrient status interact to shape nitrogen use efficiency and stress resilience in leafy vegetables. • Diel RNA-seq reveals circadian regulation of nitrogen assimilation in spinach. • High nitrogen enhances LHY-driven transcription of NIA, NiR, and nitrate transporters. • Nitrogen modulates diel activities of NR–NiR–GS–GOGAT and amino acid rhythms. • Low nitrogen triggers ROS buildup, triggering circadian-controlled DNA repair. • Circadian–nutrient integration model explains synchronized N and C metabolic fluxes.