Cloning And Functional Characterization Of Maize 5-Formyltetrahydrofolate Cycloligase In Folate Metabolism

Folates act as key cofactors mediating one-carbon transfer reactions and involved in many important reactions. 5-formyltetrahydrofolate cycloligase(5FCL) was reported to catalize the storage form 5-CHO-THF into active folate forms, here the function of maize 5FCL was studied through enzyme activity assay in vitro and overexpression in Arabidopsis. The express pattern of Zm5 FCL gene and subcellular localization of Zm5FCL::GFP fusion protein were analyzed. In addition, the distribution of folate in embryo and endosperm as well as transcription levels of folate synthesis and metabolism gene in maize were also investigated. The obtained results are as follows:1、Based on sequence similarity with At5 FCL, a homolog(GI: GRMZM5G807835) from maize was cloned and named as Zm5 FCL. Zm5 FCL contains a conserved 5-formyltetrahydrofolate cyclo-ligase superfamily domain and share 53.07% and 52.38% identity with the Arabidopsis and tomato 5FCL, 22%-28% similarity with human, yeast, and bacterial 5FCLs.2、By in vito enzymatic assay Zm5 FCL was found could catalyze 5-CHO-THF to 5,10-CH=THF. And after overexpression in Arabidopsis 5fcl mutant, the 5-CHO-THF level in leaves restored to wild type. It suggested that Zm5 FCL could complement the function of At5 FCL. And 5-CHO-THF levels decreased significantly after Zm5 FCL overexpression in wild type.3、By transcient experiment in maize protoplasts, Zm5FCL::GFP fusion protein localized in cytoplasm. And the transcripts analysis showed that during maize kernel development the gene expression level of Zm5 FCL are significantly higher in embryo than endosperm. The folate concentration analysis showed that 5-CH3-THF level was significantly higher in embryo than endosperm, and 5-CHO-THF level had no difference in embryo and endosperm.4、Furtherly, the transcription levels of genes involved in folate synthesis and folate metabolism were analyzed, among them ZmGTPCHI-2, ZmGDCH-1, ZmGDCT, ZmDHC5, Zm10-FDF, Zm5 FCL accumulate higher transcripts in embryo than endosperm, and ZmDHNA1, ZmHPPK, ZmADCL2, ZmDHFR-1, ZmDHFR-2, ZmDHFR-4, ZmGDCH-4, ZmGDCP, ZmDHC1, ZmDHC4, ZmMTHFR1, ZmMTHFR2 accumulate higher transcripts in endosperm embryo than embryo. It’s presumed that there existed different mechanism regulates folate biosynthesis and distribution in maize embryo and endosperm.