Extended Data Fig. 4: Metabolic characterization in rosette leaves and functional complementation of RE1 and RER1 in the arg11 yeast mutant.
From: RETICULATA1 is a plastid-localized basic amino acid transporter
Relative metabolite levels per mg fresh weight (FW) of (a) Arg, (b) His, (c) Lys, (d) Citr, (e) Orn, and (f) Aminoadipate from 4-week-old rosette leaves of Col-0, re-6, rer1-1, rer1-2, rer1-3, re-6 OEX1 and re-6 OEX2. Samples were harvested in the middle of the light period. Data are shown as mean ± SD of four biological replicates. Different letters indicate statistically significant differences between means (P < 0.05; one-way ANOVA with Tukey’s test). Relative response of (g) Asp, (h) Gln, (i) Glu, (j) His, and (k) Lys in isolated chloroplasts from 3-week-old Col-0 and re-6 plants. Relative response was normalized for chlorophyll content in μg/mL. Data are presented as box and whiskers (min to max) of six biological replicates. Different letters indicate statistically significant differences between means (P < 0.05; one-way ANOVA with Tukey’s test). The boxplots (g–k) show the median (horizontal line), interquartile range (box), and whiskers extending to 1.5× the IQR. Exact P values (a–k) are shown in source data with 95% confidence intervals. (l) Growth behavior of the yeast strain arg11 transformed with pDR195 expressing the mitochondrial targeting peptide from the S. cerevisiae cytochrome c oxidase 4 (ScCOXIV), the S. cerevisiae mitochondrial Orn carrier (ScORT1p), the mitochondrial basic amino acid carrier from Arabidopsis (AtmBAC1) fused with an N-terminal His-tag, and the mature forms of RE1 and RER1 fused to the mitochondrial targeting peptide of ScCOXIV with and without a N-terminal His-tag. Cells from positive transformants were inoculated to an initial OD600 of 1, diluted tenfold four times, and grown on control and selective plates for 2 d at 30 °C. (m) Verification of protein expression. Mitochondria were isolated from the yeast strain arg11 transformed with pDR195 and pDR195 expressing the mature form of RE1 fused to the mitochondrial targeting peptide of ScCOXIV. 25 µg mitochondrial protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. RE1 protein was visualized by immunodetection with an antibody raised against the RE1 protein.
