Dose-dependent effects of nitrate on growth dynamics and metabolic profiling in Medicago sativa L.

Nitrate (NO3−) is a major inorganic nitrogen source regulating plant growth, yet its dose-dependent effects on forage legumes are poorly defined. This study evaluated how increasing nitrate concentrations influence growth, physiological performance, and secondary metabolism in Medicago sativa L. var. Gabès. Plants were grown under controlled environmental conditions and supplied with 0, 1, 2, 4, or 8 mM NO3−. The 1 mM NO3− treatment promoted the highest biomass accumulation and chlorophyll content, whereas exposure to 8 mM NO3− led to a 45.6% reduction in total dry matter and a 30% decline in chlorophyll, indicating growth inhibition under nitrate excess. Elevated nitrate caused a marked rise in tissue nitrate storage (4–5 times higher than the control) and triggered strong metabolic reprogramming, including substantial increases in total polyphenols (92%), flavonoids (200%), and the predominance of phenolic acids such as protocatechuic and chlorogenic acid. These responses illustrate a coordinated shift between primary metabolism and defensive secondary pathways in relation to nitrate availability. Since the experiment was conducted under controlled growth-chamber conditions, future field validation is required to determine the extent to which these responses occur under variable environmental and agronomic settings. These findings indicate that moderate nitrate supply (∼1 mM) promoted optimal growth under controlled growth-chamber conditions. However, field studies are required before translating these results into agronomic fertilizer recommendations.