Functional Study Of Methylenetetrahydrofolate Reductase Gene In Switchgrass

Switchgrass(Panicum virgatum)has been developed into a model crop for bioethnal production.Genetic improvement on reducing lignin is important for developing new switchgrass cultivars.Downregulation of caffeic acid O-methyltransferase(COMT),a key enzyme in lignin biosynthesis,led to altered lignification and increased biofuel yield in switchgrass.Methylenetetrahydrofolate reductase(MTHFR)mediated metabolic process provides methyl units consumed by COMT,further affects lignin biosynthesis,but other functions of MTHFR gene remains unclear.Here,we study the function of MTHFR in switchgrass,the synergetic effect between MTHFR and COMT,and also the effect of inhibition of these two genes on lignin biosynthesis.These results are summarized as follow:(1)Two 1785bp full-length homologous alleles of MTHFR encoding 594 amino acids were isolated from switchgrass by one-step PCR.The function of MTHFR protein was conserved,it contained FAD and CH2-THF binding site,SAM regulatory site and ATP synthetase site that typically existed in plants.MTHFR was constitutively expressed in switchgrass.The expression levels of MTHFR gene in different tissues from high to low were shown as follow:intemode,node,root,inflorescence,leaf sheath,and leaf blade.It also showed higher expressions in younger stems and leaves among all the stems and leaves in different developmental stages.Subcellular localization suggested that MTHFR protein was located in cytoplasm.(2)MTHFR singe gene knockdown lines were generated by RNAi.MTHFR/COMT double gene knockdown lines were generated by re-transformation using COMT-RNAi as a background plant.? The most knockdown level of MTHFR in PvMTHFR-RNAi lines was 56%,while in double gene knockdown lines was 92%,suggested it was unable to generate green shoot with heavy knockdown level of MTHFR single gene during tissure culture.Double transformants were classified into three groups based on gene knockdown levels:COMT was strong downregulated while MTHFR remained unchanged,both two genes were moderately downregulated,and both two genes were heavily downregulated.?Suppressing MTHFR didn't affect switchgrass growth but delayed its flowering,however,strongly co-suppressing MTHFR and COMT heavily affected the growth and development of switchgrass.?Suppressing MTHFR didn't affect lignin biosynthesis.Lignin content in double tranformants was similar with that in COMT-RNAi line(both decreased 8%-9%),but G-lignin was significantly decreased in double tranformants(9%-13%),suggested that MTHFR was mainly involved in G-lignin biosynthesis.?It was unexpected that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic cell death phenotype.Molecular analyses revealed that the phenotype was caused by the the induction of genes related with redox,defense,signaling and hormone due to co-silencing of MTHFR and COMT genes,suggesting a synergetic effect between them.The increased accumulation of amino acids and stress-related phenolic acids were affected by the formation of the lesions.(3)To decipher the reason why there was no green shoot generated due to the heavily downregulation of MTHFR single gene in switchgrass,we generated the Arabidopsis double mutants with partially or completely loss of function of MTHFR gene.Phenoytyping and genotyping revealed 50%of residue activity of MTHFR was able to survive the plant while completely knock out MTHFR failed to acquire homozygous double mutant,which was consistent with the conclusion in switchgrass that completely loss of function of MTHFR was lethal.Heterozygous double mutants were used for the following genetic analysis.After self-pollination,the seed development,later the seed germination and the growth of seedlings were normal.The segregation ratio of progeny and reciprocal cross results demonstrated that it was male gametophytic lethal.Further,some small and shriveled pollen grains were seen,and pollen viability decreased by 50%,suggesting completely loss of function of MTHFR led to male sterile in Arabidopsis.We first propose the novel roles of MTHFR in switchgrass and Arabidopsis,which have important biological significance.Besides,we also provide the theoretical base for further studying the interaction network between Cl and secondary metabolisms.