Functional characterisation of SiFPGS2 gene of foxtail millet in folates accumulation and root development

Abstract Folates content in foxtail millet (Setaria italica) are higher than that in major crops. Polyglutamylation of folates could enhance their compartmentalization, reducing their transport, increase their affinities with enzymes and increase the stability of the folates pool in Arabidopsis. The polyglutamylation reaction are catalyzed by folylpolyglutamate synthetase (FPGS). But FPGS function in foxtail millet is still elusive. In this study we identified Setaria italica FPGS2 (SiFPGS2) genes in foxtail millet by blast analysis with AtFPGS1/2/3 protein sequences. The phylogenetic tree analysis, protein function domain analysis and docking analysis results showed that SiFPGS2 belong to FPGS subfamily, possessed the tetrahydrofolypolyglutamate synthase domain, could bind tetrahydrofolate (THF) as substrate. Subcellular localization predicted that SiFPGS2 localized in the chloroplast. Cis-acting elements prediction analysis indicated that there were meristem expression motif and root specific motif in SiFPGS2 promoter. Over-expressed SiFPGS2 in Arabidopsis not only increased the folates content, but also increased the root length, and further propidium iodide staining result suggested that the increased root length was owning to the promotion of cell division in root apical meristem zone. SiFPGS2 catalyzed the polyglutamylation of THF and lead to the increase of folates content and root length by promotion of root apical meristem zone cell division. SiFPGS2 can be used as a candidate gene for biofortification of folates in Gramineae crops via genetic engineering.