File S1 - Arabidopsis Plastidial Folylpolyglutamate Synthetase Is Required for Seed Reserve Accumulation and Seedling Establishment in Darkness

Contains the following files: Figure S1. Identification of atdfb-3 and AtDFB complemented (COM) line. (A) Gene map of AtDFB (At5g05980). Boxes indicate exons and lines indicate introns. T-DNA insertion site for the mutant is indicated. Arrows indicate the positions of the primers (F and R) used for RT-PCR. (B) Schematic diagram of the ProAtDFB: AtDFB-HWG complemented construct. LB and RB indicate the left and right borders, respectively, and Hyg indicates the hygromycin resistance gene. (C) AtDFB transcripts in wild-type (WT), atdfb-3, and one representative COM plant. Total RNA was prepared from 14-day-old seedlings grown in light. ACTIN2 transcripts were used as a loading control. Figure S2. Hypocotyl phenotypes of 6-day-old etiolated WT and atdfb-3 at various concentrations of NH4+ (A, B) or organic nitrogen (C). Figure S3. GUS staining of 2- and 3-day-old light-grown AtDFB: GUS seedlings under 9.4 N or 0.3 N conditions. Figure S4. Hypocotyl length of WT and atdfb-3 under N-limited conditions with 5-F-THF or 5-M-THF treatment. (A) Hypocotyl length of 7-day-old etiolated WT and atdfb-3 seedlings after application of various concentrations of 5-F-THF under N-limited conditions. (B) Hypocotyl length of 6-day-old etiolated WT and atdfb-3 seedlings after application of various concentrations of 5-M-THF under N-limited conditions. (C) Hypocotyl length of 6-day-old WT and atdfb-3 etiolated seedlings grown on 0.3 N medium with 50 µM 5-F-THF and then transferred to 0.3 N medium without 5-F-THF for the remaining days. Figure S5. Hypocotyl phenotype of 6-day-old etiolated WT and atdfb-3 under 0 N or NH4+ with 5-F-THF treatment. (A) Image of hypocotyl phenotype of 6-day-old WT and atdfb-3 under 0 N or 3 mM NH4+ with 5-F-THF. (B) Hypocotyl length of 6-day-old WT and atdfb-3 etiolated seedlings grown on 0 N (upper panel) or 3 mM NH4+ with 50 µM 5-F-THF (lower panel). Figure S6. Folate profiles in 2-day-old WT and atdfb-3 germinating seeds under 9.4 N or 0.3 N. Figure S7. Transcript levels of genes involved in folate biosynthesis and metabolism. Figure S8. Ammonium content in 6-day-old WT and atdfb-3 seedlings in the dark. Table S1. Profiles of total folates and various folate species in 2-day-old WT and atdfb-3 germinating seeds grown on 9.4 N or 0.3 N medium in the dark. Table S2. Profiles of total folates and various folate species in 6-day-old WT, atdfb-3, and AtDFB complemented (COM) etiolated seedlings grown on 9.4 N or 0.3 N medium.
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