The Functional Study On The Mitochondrial Folylpolyglutamate Synthetase DFC Under Low Nitrogen Stress In Arabidopsis

Folates are essential co-factors involved in the plant one-carbon transfer reaction.DFC(DHFS-FPGS Homolog C)encodes folylpolyglutamate synthetase 2(FPGS2),which is reportedly the mitochondrial isoform,responsible for catalysing the polyglutamylation reaction of various folate derivatives by sequential conjugation of additional glutamate residues through ?-peptide linkage.In previous work,we isolated an Arabidopsis T-DNA insertion mutant that the T-DNA inserted in the 15th exon of DFC,therefore called dfc.The dfc mutant did not show any obvious phenotype in normal growth conditions.But when cultivated on a low nitrogen medium which contained 0.3 mM N03-,the primary root length of the dfc mutant was about half of that of the wild type,and an accumulation of anthocyanin in leaves was observed.Besides,the contents of total folates,5-methyltetrahydrofolate and 5-formyltetrahydrofolate in the mutant were obviously less than those in the wild type.Based on the previous study,the effects of overexpression of DFC on folate content and the responses to low nitrogen stress had been further investigated.Moreover,elevated CO2 was found to recover the low nitrogen phenotype,the contents of photorespiratory amino acids and folates contents in the dfc mutant.The main results are listed as follows:1.The DFC cDNA overexpression vector drived by CaMV 35S promoter was transformed in the Columbia wild type Arabidopsis.Through resistant screen and PCR analysis,fifteen T1 transgenic lines were obtained,among which,seven lines were homozygous.2.Semi-quantitative RT-PCR and Western blot analysis were performed for the seven homozygous transgenic lines.The DFC transcriptional expressions of the seven lines were all higher than that in the wild type,and the transcript in DFC OE5 was the highest.But for the translational analysis,only five lines were higher than the wild type,and DFC OE5 contained the most amount of DFC protein.3.Folates contents assay by HPLC/MS/MS was carried out in the wild type plants and seven overexpressed transgenic lines.The contents of total folates,5-methyltetrahydrofolate and 5-formyltetrahydrofolate in the seven lines were all much higher than those in the wild type,and displayed a significant difference or greatly significant difference compared with the wild type,among which,folates contents in DFC OE5 were the highest.But none of them showed a better growth under low nitrogen stress compared with the wild type.4.The phenotype of the dfc mutant on low nitrogen medium was analysed under elevated CO2 condition.The mutant had a similar primary root length with the wild type and the leaves were green instead of purple.5.Contents of amino acids in the dfc mutant under ambiant air and elevated CO2 condition was assayed.An accumulation of glycine in the dfc mutant under ambiant air condition was observed,and the content of glycine decreased dramatically to the level of the wild type when cultivated under elevated CO2 condition.6.Content of folates in the dfc mutant under ambiant air and elevated CO2 condition was assayed.Folates content in the dfc mutant recovered to the wild type level when cultivated under elevated CO2 condition.All of these results suggest that DFC plays an important role in the folate biosynthesis metabolism,and it has an direct effect on the metabolism integrity and the folates composition.But overexpression of DFC is not sufficient to enhance the low nitrogen tolerance of the Arabidopsis.Elevated CO2,which inhibit photorespiration,results in the recovery of low-nitrogen phenotype,glycine/serine ratio and folates content of the dfc mutant,implying that DFC is probably involved in the responses to low nitrogen stress by affecting photorespiration metabolism.