Integration of Non-Invasive Micro-Test Technology and 15N Tracing Reveals the Impact of Nitrogen Forms at Different Concentrations on Respiratory and Primary Metabolism in Glycyrrhiza uralensis

<i>Glycyrrhiza uralensis</i> is a highly valued medicinal species worldwide. However, a paradox arises in its cultivation in that high nitrogen fertilization boosts yield at the expense of root quality, a problem linked to nitrogen's regulation of tricarboxylic acid (TCA) cycle-driven respiration. It remains unclear how different nitrogen forms coordinate respiratory and primary metabolism. We examined the regulatory mechanisms of nitrate (NO₃^-) versus ammonium (NH₄⁺) on these processes in cultivated <i>G. uralensis</i> by supplying seedlings with varying concentrations of K¹⁵NO₃ or (¹⁵NH₄)₂SO₄ in a modified Hoagland solution (HNS). Using non-invasive micro-test technology (NMT) and ¹⁵N tracing, we found that <i>G. uralensis</i> employs distinct nitrogen acquisition strategies: sustaining uptake at optimal NH₄⁺ and low-to-moderate NO₃^-, while declining uptake under high NO₃^-. These strategies drove form-specific differences in the activity of key nitrogen assimilation enzymes, nitrate reductase and nitrite reductase (NR/NiR), as well as glutamine synthetase and glutamate synthase (GS/GOGAT), and subsequent glutamate and glutamine accumulation. Ammonium nutrition enhanced primary ammonia assimilation and gamma-aminobutyric acid (GABA) metabolism, leading to greater glutamate and endogenous GABA levels. In contrast, nitrate nutrition preferentially stimulated the TCA cycle, resulting in higher accumulation of <i>α</i>-ketoglutarate (KGA) and succinate. The concomitant increase in GABA catabolism supported this nitrogen-responsive respiratory metabolism, acting as a compensatory mechanism to maintain KGA homeostasis. Our findings inform nitrogen form strategies for <i>G. uralensis</i> cultivation.