MTX releases DNA methylation of LUCL. (A-J)Luciferase luminescence of LUCL seedlings treated with variouscompounds. (A) DMSO-treated LUCL seedlings. (B-D)D-MTX-treated LUCL seedlings. (E-G) LUCL treatedwith a mixture of D- and L-MTX. (H-J) L-MTX-treated LUCLseedlings. The concentrations of the chemicals are as indicated in(B-J). (K) Chemical structures of L-MTX (top) andD-MTX (bottom). The arrows indicate the position of chirality of the twoforms. (L) McrBC-PCR-based methylation assay of LUCLseedlings treated with D-MTX. DC: DMSO-treated Col-0 control, D:DMSO-treated LUCL. The gray triangle represents increasingconcentrations of MTX (2 μM for the left lane and8 μM for the right lane). (M) MTX inhibits SAMbiosynthesis to indirectly affect gene silencing via DNA methylation.MTX inhibits the conversion of DHF to THF. Under normal circumstances,the energy given off by the conversion of THF to 5-methyl THF promotesthe production of methionine from homocysteine and vitamin B12.(N) Expression of LUCL and six endogenous RdDM lociin DMSO (control)- and MTX-treated seedlings as determined by RT-PCR.(O) McrBC-PCR-based methylation assay of LUCLseedlings treated with DMSO (D) or MTX (M), and non-treatednrpe1-11 seedlings (n). Two biological replicates gavesimilar results and only one is shown here. +: McrBC digested; -:non-digested. The six loci in the bottom panel are known to undergoRdDM. LUCp1 to LUCp4 correspond to regions 1 to 4 ofthe LUCL transgene in Figure 2D.Chr2_1882324 is a region that harbors DNA methylation in wild type.At2g19920 is a gene that does not harbor any DNAmethylation and is used as an internal loading control. DHF:dihydrofolate; DMSO: dimethyl sulfoxide; McrBC-PCR: digestion of genomicDNA by McrBC followed by PCR; MTX: methotrexate; RT-PCR: reversetranscription-PCR; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; THF: tetrahydrofolate.