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. (DOC)本数据集包含如下文件： 图版S1：atdfb-3突变体与AtDFB互补（COM，complemented）株系的鉴定。(A) AtDFB（At5g05980）基因结构图，方框代表外显子，线条代表内含子，标注了该突变体的T-DNA（转移DNA，Transfer DNA）插入位点，箭头指示反转录聚合酶链式反应（RT-PCR，reverse transcription polymerase chain reaction）所用引物（F和R）的位置。(B) ProAtDFB:AtDFB-HWG互补构建体的示意图，LB与RB分别代表左边界与右边界，Hyg代表潮霉素抗性基因（hygromycin resistance gene）。(C) 野生型（WT，wild-type）、atdfb-3突变体与一株代表性COM株系中的AtDFB转录本水平。总RNA提取自光照培养14天的幼苗，以ACTIN2转录本作为上样对照。 图版S2：6日龄黄化野生型与atdfb-3突变体在不同浓度NH4+（铵离子）(A、B)或有机氮(C)处理下的下胚轴表型。 图版S3：在9.4 N或0.3 N培养条件下，2日龄与3日龄光照培养的AtDFB:GUS转基因幼苗的GUS染色结果。 图版S4：氮限制条件下经5-F-THF（5-氟四氢叶酸）或5-M-THF（5-甲基四氢叶酸）处理后，野生型与atdfb-3突变体的下胚轴长度。(A) 氮限制条件下施加不同浓度5-F-THF后，7日龄黄化野生型与atdfb-3幼苗的下胚轴长度。(B) 氮限制条件下施加不同浓度5-M-THF后，6日龄黄化野生型与atdfb-3幼苗的下胚轴长度。(C) 将6日龄野生型与atdfb-3黄化幼苗先在含50 µM 5-F-THF的0.3 N培养基上培养，随后转移至不含5-F-THF的0.3 N培养基中完成剩余培养天数，统计其下胚轴长度。 图版S5：6日龄黄化野生型与atdfb-3突变体在0 N或添加NH4+并经5-F-THF处理条件下的下胚轴表型。(A) 0 N或3 mM NH4+条件下施加5-F-THF后，6日龄野生型与atdfb-3突变体的下胚轴表型图像。(B) 在0 N（上半部分）或含50 µM 5-F-THF的3 mM NH4+条件下（下半部分）培养的6日龄黄化野生型与atdfb-3幼苗的下胚轴长度。 图版S6：在9.4 N或0.3 N培养条件下，2日龄野生型与atdfb-3萌发种子中的叶酸谱。 图版S7：参与叶酸生物合成与代谢的基因的转录水平。 图版S8：黑暗条件下培养6日龄的野生型与atdfb-3幼苗的铵离子含量。 表S1：黑暗条件下在9.4 N或0.3 N培养基中培养的2日龄野生型与atdfb-3萌发种子的总叶酸及各类叶酸组分谱。 表S2：在9.4 N或0.3 N培养基中培养的6日龄野生型、atdfb-3及AtDFB互补（COM）株系黄化幼苗的总叶酸及各类叶酸组分谱。 (DOC)