Nitrogen nutrition remains a major challenge for sustainable crop production, particularly under the current need to reduce fertilizer inputs. This study evaluated whether inoculation with arbuscular mycorrhizal fungi (AMF) improves the physiological, nutritional, and metabolic performance of escarole (Cichorium endivia L.) plants under nitrogen-limited conditions. Escarole plants were grown under two nitrogen regimes, full nitrogen supply and 25% of the optimal nitrogen concentration, either inoculated or non-inoculated with Rhizophagus irregularis. Plant growth, mineral composition, soluble sugars, proteins, pigments, and non-targeted LC-MS metabolomic profiles were analyzed. The results showed that mycorrhizal symbiosis mitigated the negative effects of nitrogen deficiency, maintaining shoot development and overall productivity, while reducing root growth, likely due to improved nutrient acquisition via fungal symbionts. Although total nitrogen content decreased under limiting conditions, AMF-inoculated plants exhibited higher levels of glucose, soluble proteins, and chlorophylls, alongside sustained or improved uptake of other essential minerals. Metabolomic profiling revealed AMF-dependent changes in the chemical profile and in multiple metabolic pathways, with 7165 metabolic features altered under nitrogen limitation and 58% of them increased in mycorrhizal plants. Principal component analysis further separated mycorrhizal and non-mycorrhizal plants, with PC1 explaining 49% of the chemical variability under nitrogen limitation. This metabolic response led to the accumulation of bioactive compounds such as riboflavin, L-citrulline, sarcosine, cyclic pyranopterin monophosphate, and 5-hydroxy-L-tryptophan. Overall, these findings demonstrate that AMF enhance plant performance under nitrogen limitation not by increasing nitrogen content per se, but by optimizing nitrogen utilization and reshaping metabolic networks associated with C/N balance.
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