Foliar folic acid application enhances nitrogen assimilation and improves quality-related metabolites accumulation in Dendrobium officinale

Folates are essential cofactors in one-carbon metabolism, yet their roles in coordinating nitrogen assimilation and the accumulation of quality-related metabolites in medicinal orchids remain poorly understood. Here, we profiled folate derivatives across tissues and stem developmental stages of Dendrobium officinale and evaluated how foliar nutrient treatments affect folate accumulation and nitrogen metabolism. Folate profiling revealed distinct tissue-specific patterns: leaves were the major sink for total and reduced folates, whereas flowers preferentially accumulated formylated folates. Foliar application of balanced nutrient solutions, particularly Huaduoduo No. 1, markedly increased total folate levels. In vitro enzyme assays showed that folates, especially 5-formyltetrahydrofolate (5-F-THF), enhanced nitrate reductase (NR) and nitrite reductase (NiR) activities, with a stronger effect on NiR. Consistent with these findings, foliar application of folic acid (FA; 15 mg L -1 ) increased NR and NiR activities in planta , accompanied by higher nitrate and lower nitrite levels. Untargeted metabolomic analysis further showed that FA treatment reprogrammed amino acid and alkaloid metabolism, increasing the abundance of tryptophan-derived metabolites and several bioactive alkaloids. Molecular docking further predicted favorable interactions between folates and the catalytic pockets of nitrogen metabolism-related enzymes, supporting a potential direct role of folates in regulating nitrogen assimilation. Collectively, these findings indicate that folate-associated regulation of nitrogen assimilation is linked to metabolic reprogramming in D. officinale . This study provides a practical folate-based strategy for enhancing the nutritional and medicinal quality of this economically important orchid.