Fig. 5: Genome editing-based knockout and overexpression in tomato confirm GAME8’s role in SGA biosynthesis.
From: Enzymatic twists evolved stereo-divergent alkaloids in the Solanaceae family
a LC-MS analysis of SGA (α-tomatine) in either tomato hairy roots with knocked out SlGAME8 (SlGAME8-ko), or expressing the eggplant SmGAME8 in the tomato SlGAME8-ko background. Hairy roots expressing GFP only were used as a control. b LC-MS analysis of aglycones (tomatidine and soladulcidine) in tomato hairy roots with knocked out SlGAME8 (SlGAME8-ko), or expressing SmGAME8 in SlGAME8-ko background. c, d LC-MS analysis of intermediates cholesterol-GlcA (c) and 22S-hydroxycholesterol-GlcA (d) in the same hairy root lines. Identity of metabolites was assigned based on accurate mass and our previous studies24. e Percentage of 25 R α-tomatine in control and SmGAME8 expressing tomato hairy roots (n = 14 of independent replicates for each genotype, P = 2.32756E-45). f Extracted ion chromatograms (EICs) showing accumulation of the steroidal saponin uttroside B and 25-epi-uttroside B in WT and SlGAME8 expressing Solanum nigrum leaves. g Percentage of 25-epi-uttroside B in WT and SlGAME8 expressing S. nigrum (n = 5 of independent replicates for each genotype, P = 1.11608E-07). Asterisks indicate statistically significant differences as compared to control or WT determined by two-sided Student’s t-test. Box plots show all data points, center line represents median value, bounds correspond to upper and lower quartile, and whiskers represent min and max values.
