Epigenetic effects of biofortification with folate and microelements in food plants

The increasing interest in nutrients and microelements linked with epigenetic modifications, brought to focus on folate (vitamin B9), the transporter of methyl groups for DNA methylation the most studied epigenetic feature of DNA. The principal approaches to overcome folate and microelements deficiencies have been either mandatory fortification or voluntary supplementation by pills of synthetic B9 vitamin. These strategies, however, have potential drawbacks due to the fact that folic acid is an unnatural compound with potential adverse effects. Some research, therefore, lately focused on developing genetically engineered plants that contains more folates than natural plants, but this approach also raises a considerable concern for the potential noxious effects of metabolic products from genetically modified plants. Since folate metabolism needs microelements as enzyme cofactors, as selenium (Se) and zinc (Zn), whose content is often inadequate in natural soil, there is a growing interest in finding novel strategies to increase both natural folate and microelement contents in food plants. This agronomic biofortification approach required the development of adequately enriched fertilizer formulas. The study is aimed at developing an agronomic biofortification model of lettuce through the development of specific foliar fertilizers containing Zn, Se and para-aminobenzoic acid, a natural precursor of folate, in a setting of growth chamber, hydroponic and open field culture. Microelements analyses were performed after setting up a novel ICP-MS method by using NIST 1570A, Trace Elements in Spinach Leaves, as certified reference material (CRM). The improvement of the levels of microelements in plant leaves was demonstrated and a clear enrichment of folate content in lettuce plants was confirmed by UPL/MC qTOF analysis. Cell culture model experiments were used to test the effects of lettuce extracts on HepG2 transcriptome through RNA-Seq sequencing that highlighted either a u- and down-regulation of an ample number of genes in several pathways including those linked to Se and Zn transport and folate metabolism. The epigenetic regulation at promoter region by DNA methylation was tested on selected genes involved in folate and microelements metabolism such as MT2A and FOLR1 were investigated showing a possible role of lettuce extract exposure on gene expression modulation through the epigenetic feature i.e. DNA methylation. For a deeper focus on novel mechanism of cell-to-cell communication, attention was given also to exosomes, microvesicles released by all fluids and cells. Exosomes mRNA cargo was analysed after the treatment of HepG2 and also on Ea.hy926, endothelial cell model, with extracts of lettuce control and biofortified and gene expression compared between the two model showing that cells exposed to biofortified lettuce have higher number of exosome production by the cells potentially affecting significantly the cell-to-cell communication of mRNA of genes differentially modified by lettuce extracts exposure. Biofortification using a novel Se, Zn and p-ABA-enriched formula is effective to ameliorate the content of such microelements and 5methylTHF in lettuce and lead to gene expression and gene specific DNA methylation modulation in HepG2 and Ea.hy926 cell culture models. Future perspectives include specific investigation of biofortified lettuce nutritional and microelements bioavailability and specific epigenetic modulation in humans for possible correction of widespread microelements and vitamin deficiencies with the purpose of reducing the risk of several major chronic diseases related to nutritional deficiencies.