Analysis of IGMT4. a) Quantitative effects of IGMT4 on leaf IGs in DE155 and DE096. Data pooled from 4 independent experiments show estimated marginal means of metabolite concentration in nmol per gram fresh weight (± SEM) of 4OHI3M (red) and 4MOI3M (blue) with IGMT4 intact (left) or defective (right) in DE155 (circles and solid lines) and DE096 (diamonds and dashed lines). Each genotype had n = 35–61 samples. The interaction of IGMT4 by background is highly significant for 4MOI3M (F_1,164 = 18.7; P < 0.0001) but not for 4OHI3M (F_1,164 = 0.0; P = 0.994). There were no significant genotype or genotype-by-background effects for I3M, 1MOI3M, and total IG. b) Sliding window analyses of the open Reading frame for nucleotide polymorphisms (gray) and Tajima's D (black), with a window size of 100 nucleotides and a step size of 25 nucleotides. Tajima's D is significantly elevated (D > 2.07; P < 0.05) from ca. 650 to ca. 850 bp (shaded gray). c) Functionally important amino acids. S-adenosyl-L-methionine binding sites are shown in blue, and the active site in red. Two polymorphic amino acids are highlighted in yellow.