Abstract: 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 (NO3-)-to-ammonium-N (NH4+) ratio on folate synthesis and accumulation in vegetables are unclear. This study used a hydroponic experiment with six different NO3-/NH4+ ratio treatments to investigate the effects of the integrated application of NO3- and NH4+ on the folate constituents and contents of pakchoi (Brassica rapa subsp. chinensis). The results indicated that an appropriate NO3-/NH4+ 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 NH4+-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 NH4+-N supply increased the consumption of the folate precursors and intermediate metabolites. Additionally, NH4+-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 NO3-/NH4+ ratio, strategic selection of the NO3-/NH4+ ratio should be considered for the hydroponic cultivation of foliar vegetable crops.

水培条件下营养液中硝铵比对小白菜叶酸成分及含量变化的影响

[1]AkhtarTA, OrsomandoG, MehrshahiP, et al., 2010. A central role for gamma-glutamyl hydrolases in plant folate homeostasis. Plant J, 64(2):256-266.

[2]BaileyLB, 1998. Dietary reference intakes for folate: the debut of dietary folate equivalents. Nutr Rev, 56(10):294-299.

[3]BaileyLB, StoverPJ, McNultyH, et al., 2015. Biomarkers of nutrition for development—folate review. J Nutr, 145(7):1636S-1680S.

[4]BlancquaertD, de SteurH, GellynckX, et al., 2014. Present and future of folate biofortification of crop plants. J Exp Bot, 65(4):895-906.

[5]BlancquaertD, van DaeleJ, StrobbeS, et al., 2015. Improving folate (vitamin B₉) stability in biofortified rice through metabolic engineering. Nat Biotechnol, 33(10):1076-1078.

[6]BoschieroBN, MarianoE, AzevedoRA, et al., 2019. Influence of nitrate-ammonium ratio on the growth, nutrition, and metabolism of sugarcane. Plant Physiol Biochem, 139:246-255.

[7]DelchierN, HerbigAL, RychlikM, et al., 2016. Folates in fruits and vegetables: contents, processing, and stability. Compr Rev Food Sci Food Saf, 15(3):506-528.

[8]Díaz de la Garza RI, Ramos-ParraPA, Vidal-LimonHR, 2019. Biofortification of crops with folates: from plant metabolism to table. In: Jaiwal PK, Chhillar AK, Chaudhary D, et al. (Eds.), Nutritional Quality Improvement in Plants. Springer, Cham, p.137-175.

[9]EstebanR, ArizI, CruzC, et al., 2016. Review: mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci, 248:92-101.

[10]US Preventive Services Task Force, 2017. Folic acid supplementation for the prevention of neural tube defects: US Preventive Services Task Force recommendation statement. JAMA, 317(2):183-189.

[11]GaoHD, YueXZ, LiZT, et al., 2023. The molecular regulation of folate accumulation in pakchoi by low intensity white light-emitting diode (LED) illumination. Sci Hortic, 314:111937.

[12]GorelovaV, AmbachL, RébeilléF, et al., 2017. Folates in plants: research advances and progress in crop biofortification. Front Chem, 5:21.

[13]GräwertT, FischerM, BacherA, 2013. Structures and reaction mechanisms of GTP cyclohydrolases. IUBMB Life, 65(4):310-322.

[14]HachiyaT, SakakibaraH, 2017. Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants. J Exp Bot, 68(10):2501-2512.

[15]HansonAD, GregoryIII JF, 2011. Folate biosynthesis, turnover, and transport in plants. Annu Rev Plant Biol, 62:105-125.

[16]HeYN, SongSH, LiC, et al., 2022. Effect of germination on the main chemical compounds and 5-methyltetrahydrofolate metabolism of different quinoa varieties. Food Res Int, 159:111601.

[17]KołtonA, Długosz-GrochowskaO, WojciechowskaR, et al., 2022. Biosynthesis regulation of folates and phenols in plants. Sci Hortic, 291:110561.

[18]KrappA, 2015. Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces. Curr Opin Plant Biol, 25:115-122.

[19]LeeKT, LiaoHS, HsiehMH, 2023. Glutamine metabolism, sensing and signaling in plants. Plant Cell Physiol, 64(12):1466-1481.

[20]LiWC, LiangQJ, MishraRC, et al., 2021. The 5-formyl-tetrahydrofolate proteome links folates with C/N metabolism and reveals feedback regulation of folate biosynthesis. Plant Cell, 33(10):3367-3385.

[21]LianT, WangXX, LiS, et al., 2022. Comparative transcriptome analysis reveals mechanisms of folate accumulation in maize grains. Int J Mol Sci, 23(3):1708.

[22]LiuY, von WirénN, 2017. Ammonium as a signal for physiological and morphological responses in plants. J Exp Bot, 68(10):2581-2592.

[23]NakamichiY, KobayashiJ, ToyodaK, et al., 2023. Structural basis for the allosteric pathway of 4-amino-4-deoxychorismate synthase. Acta Crystallogr D Struct Biol, 79:895-908.

[24]PatrickJW, BothaFC, BirchRG, 2013. Metabolic engineering of sugars and simple sugar derivatives in plants. Plant Biotechnol J, 11(2):142-156.

[25]Ramos-ParraPA, Urrea-LópezR, Díaz de la Garza RI, 2013. Folate analysis in complex food matrices: use of a recombinant Arabidopsis γ-glutamyl hydrolase for folate deglutamylation. Food Res Int, 54:177-185.

[26]SainiRK, NileSH, KeumYS, 2016. Folates: chemistry, analysis, occurrence, biofortification and bioavailability. Food Res Int, 89:1-13.

[27]ScaglioneF, PanzavoltaG, 2014. Folate, folic acid and 5-methyltetrahydrofolate are not the same thing. Xenobiotica, 44(5):480-488.

[28]TyagiK, SunkumA, GuptaP, et al., 2023. Reduced γ-glutamyl hydrolase activity likely contributes to high folate levels in Periyakulam-1 tomato. Hortic Res, 10:284-296.

[29]WanX, HanLD, YangM, et al., 2019. Simultaneous extraction and determination of mono-/polyglutamyl folates using high-performance liquid chromatography-tandem mass spectrometry and its applications in starchy crops. Anal Bioanal Chem, 411(13):2891-2904.

[30]WangM, ShenQR, XuGH, et al., 2014. New insight into the strategy for nitrogen metabolism in plant cells. Int Rev Cell Mol Biol, 310:1-37.

[31]WangY, WangJS, DongEW, et al., 2023. Foxtail millet [Setaria italica (L.) P. Beauv.] grown under nitrogen deficiency exhibits a lower folate contents. Front Nutr, 10:1035739.

[32]WatanabeS, OhtaniY, TatsukamiY, et al., 2017. Folate biofortification in hydroponically cultivated spinach by the addition of phenylalanine. J Agric Food Chem, 65(23):4605-4610.

[33]XuGH, FanXR, MillerAJ, 2012. Plant nitrogen assimilation and use efficiency. Annu Rev Plant Biol, 63:153-182.

[34]YokoyamaR, KlevenB, GuptaA, et al., 2022. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase as the gatekeeper of plant aromatic natural product biosynthesis. Curr Opin Plant Biol, 67:102219.