Figure 1: SDCpro-GFP expression and DNA demethylation caused by R175Q mutation in MTHFD1.

(a) GFP fluorescence micrographs of WT, #162 M2, MTHFD1/mthfd1-2 F1 and #162/mthfd1-2 F1 seedlings. F1 are progeny of #162 M2 x MTHFD1/mthfd1-2. Dashed boxes indicate magnified areas shown in lower panels. Inlets show bright-field images. (b) Gene structure, positions of mutations and conserved domains of MTHFD1. The EMS mutation in #162 lead to a R175Q substitution of a conserved residue required for NADP binding²⁸. (c) PCR-based genotype analysis of 13 F1 seedlings and two control samples. Arrowheads mark bands corresponding to WT/mthfd1-1 (upper) and mthfd1-2 (lower). The mthfd1-2 allele co-segregates with GFP fluorescence in F1 (+: present, −: absent). L, ladder. (d) Habit of different genotype plants 20 days after germination. Scale bar, 10 mm. (e) DNA blot analysis of non-CG methylation at the MEA-ISR locus. Genomic DNA was digested with methylation-sensitive MspI; upper and lower bands correspond to methylated (m) and unmethylated (u) fragments, respectively. Ratios of band intensities for each lane are shown under the gel image. (f) Levels of non-CG methylation at the AtSN1 locus by quantitative chop PCR analysis of genomic DNA after digestion with methylation-sensitive HaeIII relative to undigested DNA. Mean values±s.d. (n=3). Different letters above bars indicate significant differences between pairwise comparisons by Student’s t-test (P<0.05).