Vegetables are important dietary sources of folate for human nutrition. The influence of different nitrogen doses and forms on changes in primary nitrogen metabolism, such as amino acid and protein synthesis, in plants is well established. However, the impacts of the nitrate-N (NO 3 − )-to-ammonium-N (NH 4 + ) ratio on folate synthesis and accumulation in vegetables are unclear. This study used a hydroponic experiment with six different NO 3 − /NH 4 + ratio treatments to investigate the effects of the integrated application of NO 3 − and NH 4 + on the folate constituents and contents of pakchoi (Brassica rapa subsp. chinensis). The results indicated that an appropriate NO 3 − /NH 4 + ratio in nutrient solution could promote pakchoi growth and increase folate contents by increasing polyglutamylated 5-formyl-tetrahydrofolate (5-CHO-THF) and polyglutamylated 5-methyl-THF (5-CH3-THF). The activities of enzymes related to folate biosynthesis (except folylpolyglutamate synthase (FPGS)) were lower with an NH 4 + -N supply at the same nitrogen concentration. The statistical results revealed a significant negative correlation between folate contents and 14 detected metabolites (including fructose, sucrose, glutamine (Gln), shikimate, citrate, succinate, malate, α-oxoglutarate, p-aminobenzoate (pABA), and 6-hydroxymethyldihydropterin pyrophosphate (HMDH-P2) in the folate biosynthesis pathway), implying that the enhancement of folates biosynthesis with NH 4 + -N supply increased the consumption of the folate precursors and intermediate metabolites. Additionally, NH 4 + -N supply could improve folate stability by increasing polyglutamylated folates and reducing γ-glutamyl hydrolase (GGH) activity; the latter could weaken folate deglutamylation. As the best growth and highest total folate content were obtained at the appropriate NO 3 − /NH 4 + ratio, strategic selection of the NO 3 − /NH 4 + ratio should be considered for the hydroponic cultivation of foliar vegetable crops.
