Functional Characterization Of The Polyglutamyl Synthetase AtDFC Under Nitrogen Limitation In Arabidopsis

In plants, folate (or vitamin B9) and its derivatives are involved in many metabolic pathways as an important cofactor for one-carbon transfer reactions. In the folate biosynthesis pathway, the polyglutamyl synthetase (FPGS) catalyzes addition of polyglutamyl residues to theγsite of the first glutamate of tetrahydrofolate. AtDFB, AtDFC and AtDFD are folylpolyglutamate synthase (FPGS) localized in plastid, mitochondria and cytosol respectively in Arabidopsis. In this study, the function of mitochondria-targeted AtDFC was investigated using T-DNA insertion mutant. The mutant could not adapt to nitrogen limitation of 0.3 mM NO3-, mainly characterized by shorter primary root than the wild type. So we intended to uncover the molecular and physiological mechanisms underlying the inadaptability of the loss-of-function mutant Atdfc under nitrogen limitation. First, the content of the monoglutamate folate (5-CH3-THF and 5-CHO-THF) were analyzed and we found no significant difference between the wild type and the Atdfc. Then we measured the free amino acids, and found that the content of many amino acids are much higher in Atdfc than in wild type. This may indicate that the endogeneous nitrogen is not sufficient to satisfy Atdfc growth, thus the mutant needs to promote protein lysis into amino acids to maintain its development. It was found by real-time RT-PCR that the expression patterns of the genes involved in nitrogen metabolism, amino metabolism and photorespiration changed significantly in the mutant as compared to wild type when subjected to nitrogen limitation, thus AtDFC could be the connection point between the nitrogen metabolism and the photorespiration. At last, the transgenic complementation lines were obtained and the phenotype was recovered to wild type under nitrogen limitation, proving the phenotype of the mutant Atdfc was caused by the T-DNA insertion in the locus of AtDFC.