The Biological Function Of Folate Folypolyglutamate Synthase

Tetrahydrofolate (THF) and its derivatives-commonly grouped under the name of folates-are vital cofactors for enzymes that mediate one-carbon transfer reactions. Folates are involved in a wide range of key metabolic functions. Bacteria and plants can synthesize folate de nove. Human and animals don't have this ability and depends entirely on dietary supply. THF is a tripartite molecule, comprising pteridine, p-aminobenzoate (pABA) and glutamate moieties. The plant folate synthesis pathway is essentially the same as in bacteria. There are two branches in the plant folate biosynthesis pathway: pABA and pteridine branch. The pABA branch is in the plastid, and the pteridine branch is in the cytosol. Then they are coupled together in the mitochondria, glutamylated and reduced to produce THF. Other enzymes can catalyse THF to form THF derivatives. In plants, the four enzymes AtDFA, AtDFB, AtDFC and AtDFD have the ablity of adding glutamate. AtDFA is dihydrofolate synthase (DHFS) localized in mitochondria, it catalyzes one glutamate added to the dihydropteroate and produces dihydrofolat. AtDFB, AtDFC and AtDFD are folylpolyglutamate synthase (FPGS) localized in plastid, mitochondria and cytosol respectively. A short chain ofγ-linked glutamates can be added by FPGS.Folate molecules exist in vivo mainly as polyglutamates and these are preferred by folate-dependent enzymes. For this reason, FPGS are very important especially the one localized in mitochondria which is a major site for folate synthesis. In this study, Atdfc mutant was used to investigate the biological function of AtDFC.AtDFC was expressed ubiquitously both in different development stage and in various tissues. There was nearly no difference in visible phenotypes between wild type plants and Atdfc mutant plants during vegetable growth. However, Atdfc mutant plants bolted about one week later than wild type. Typical late-flowering mutants had more leaves than wild type, but the Atdfc mutants plants contain the same leaf number as the wild type . Total folate content of the Atdfc was 35.6% less than that of the wild type measured by microbiological method. The content of different folate derivatives was analyzed by HPLC/MS, and the results showed that 5-methyl-THF in Atdfc mutant was62.6% less than that of the wild type; 5-formyl-THF in Atdfc mutant was 32.7% less than that of the wild type. Microarray results showed that there were 10.8% differentially expressed genes involved in metabolism pathway. Four genes were involved in nitrogen metabolism; three genes were involved in metabolism of xenobiotics by cytochrome P450; seven were involved in amino acid metabolism. Then we measured the free amino acid content. Most of these amino acids have a higher content in Atdfc than in wild type. Among these amino acids, Asn, Arg and Gln participate in the synthesis of ammonium and amino acid; Ser participates in one-carbon metabolism; chorismic acid can be used to synthesize Tyr and Phe. In the process of dark-induced senescence, Atdfc mutant leaves senescence rapidly than do wild type leaves when treated with ddH2O. Treatment of the Atdfc mutant and wild type leaves with methotrexate (MTX,inhibitor of the folat synthesis pathway) accelerated dark-induced senescence. The Atdfc mutant showed severe yellowing and cell death. We recorded the length of primary root when treated the Atdfc mutant, nia1/nia2 (nitrate reductase double mutant) and wild type seedling with (MTX) or nitrogen starvation. The length of primary roots almost had no difference between the Atdfc mutant and the wild type in the normal condition. The primary root of nia1/nia2 mutant is a little shorter than the wild type primary root. Treatment of the Atdfc mutant, nia1/nia2 and wild type with MTX or nitrogen starvation inhibited the primary root growth. In MTX treatment, the percentage of inhibition of wild type was 65%; the ones of Atdfc and nia1/nia2 mutant were 85% and 86.7%. In nitrogen starvation treatment, the percentage of inhibition of Atdfc was 85%; the ones of wild type plants and nia1/nia2 mutant were 72% and 67.7%.Taken together, AtDFC is very important for folat synthesis. The loss of mitochondiral FPGS decreases the level of folate and has an effect on the metabolism of nitrogen and amino acid. Blocking of the folate synthesis pathway accelerated leaf senescence and resulted in an increased sensitivity to nitrogen starvation .