Circadian rhythm, a vital adaptive mechanism in green organisms, synchronizes plants' physiological processes with daily and seasonal environmental changes. Circadian clock oscillator genes significantly regulate transcriptional and post-transcriptional changes, emphasizing their role in mediating plant responses to environmental stresses. Understanding these regulatory mechanisms is essential for optimizing plant growth, enhancing productivity, and improving resilience to ecological changes, contributing to sustainable agriculture and food security. Nitrogen is an essential plant nutrient; both depleted and excessive nitrogen fertilization can negatively impact plant growth, development, and yield. Overapplication of nitrogen fertilizers can disrupt soil properties, limiting nutrient availability and altering soil composition (including soil acidification, salinization, and disruption of beneficial microbial communities). Additionally, excessive nitrogen usage contributes to harmful gas emissions from the soil into the atmosphere, which can affect human health, climate, and overall ecosystems. Effective nitrogen management is crucial for promoting healthy plant growth and minimizing environmental damage, making a balanced approach essential for sustainable agriculture. This study evaluated the daily regulation of transcriptional changes in nitrogen metabolism under nitrogen depletion (Low N: 50 ppm) and spinach leaf repletion (High N: 200 ppm) conditions. The RNA-Seq analysis reveals that high nitrogen (HN) conditions induce more significant transcriptomic changes than low nitrogen (LN), particularly in nitrogen assimilation, transport, and amino acid metabolism genes. Expression patterns of these genes vary across time points, with distinct regulation during light and dark cycles. Validation through qPCR and RNA-Seq confirms that nitrogen assimilation peaks at the end of the dark cycle. In contrast, nitrogen transport (NRT1) and amino acid synthesis are more pronounced during the light cycle under HN conditions. The circadian clock gene Late Elongated Hypocotyl (LHY) regulates the timing of nitrogen assimilation. LHY expression increases at the end of the dark cycle, correlating with higher expressions of nitrogen assimilation genes, including Nitrate Reductase (NIA) and Nitrite Reductase (NiR). These results underscore the significance of circadian rhythms, mainly through LHY, in optimizing nitrogen acquisition and utilization.
