File S1 - Characterizing the Crucial Components of Iron Homeostasis in the Maize Mutants ys1 and ys3

Supporting Information. Figure S1. Biological replicates for quantitative real-time PCR for the expression changes in maize of genes homologous to those involved in Fe homeostasis in rice. The expression changes in maize [wild type (YS3WT), which was the same cultivar and had the same genetic background as the ys3 mutant], of genes homologous to those involved in Fe homeostasis in rice. Quantitative real-time PCR of the genes homologous to those involved in the methionine cycle (ZmMTN, GRMZM2G171111; ZmAPT, GRMZM2G093347; ZmMTK, GRMZM2G464137; ZmIDI2, GRMZM2G139533; ZmFDH, GRMZM2G049811; ZmIDI4, GRMZM2G067265; ZmRPI, GRMZM2G035599; ZmPRPPs, GRMZM2G065030), transcription (ZmIRO2, GRMZM2G057413; ZmIRO3, GRMZM2G350312), MAs biosynthesis (ZmNAS1, GRMZM2G034956; ZmNAS3, GRMZM2G478568; ZmDMAS1, GRMZM2G060952), and transport (ZmYS1, GRMZM2G156599; ZmTOM1, GRMAM2G063306; ZmTOM2, GRMZM5G877788; ZmTOM3, GRMZM2G141081; ZmIRT1, GRMZM2G118821; ZmNRAMP1, GRMZM2G178190; ZmMATE2/ZmPEZ1, GRMZM2G170128) was performed with appropriate primers (Table S1 in File S1). The data are shown as ratios relative to the expression in Fe-sufficient shoots. The ubiquitin gene (UBQ) was used to normalize data. S.D. was calculated from the biological replicates (n = 5). Column bars followed by different letters are significantly different from each other according to the Tukey-Kramer HSD test (n = 5, PFigure S2. Biological replicates for quantitative real-time PCR to determine the differences in expression levels of Fe deficiency-inducible genes among YS1, YS3 [wild-type (YS3WT), which is the same cultivar and has the same genetic background as the ys3 mutant] and ys1 or ys3 mutants. Quantitative real-time PCR of genes involved in the methionine cycle (ZmIDI2, GRMZM2G139533; ZmFDH, GRMZM2G049811; ZmIDI4, GRMZM2G067265; ZmRPI, GRMZM2G035599), transcription (ZmIRO2, GRMZM2G057413; ZmIRO3, GRMZM2G350312), and MAs biosynthesis (ZmNAS1, GRMZM2G034956; ZmNAS3, GRMZM2G478568) was performed with appropriate primers (Table S1 in File S1). The data are shown as ratios relative to the expression in Fe-sufficient WT shoots. The ubiquitin gene (UBQ) was used to normalize data. S.D. was calculated from the biological replicates (n = 3–6). Column bars followed by different letters are significantly different from each other according to the Tukey-Kramer HSD test (n = 3–6, PFigure S3. Biological replicates for quantitative real-time PCR to determine differences in the expression levels of Fe deficiency-inducible genes among YS1,YS3 [wild-type (YS3WT), which is the same cultivar and has the same genetic background as the ys3 mutant] and ys1 or ys3 mutants. Quantitative real-time PCR of the genes involved in MAs biosynthesis (ZmDMAS1, GRMZM2G060952) and transport (ZmYS1, GRMZM2G156599; ZmTOM1, GRMAM2G063306; ZmTOM2, GRMZM5G877788; ZmTOM3, GRMZM2G141081; ZmIRT1, GRMZM2G118821; ZmNRAMP1, GRMZM2G178190; ZmMATE2/ZmPEZ1, GRMZM2G170128) was performed with appropriate primers (Table S1 in File S1). The data are shown as ratios relative to the expression in Fe-sufficient YS3WT shoots. The ubiquitin gene (UBQ) was used to normalize data. S.D. was calculated from the biological replicates (n = 3–6). Column bars followed by different letters are significantly different from each other according to the Tukey-Kramer HSD test (n = 3–6, PFigure S4. Unspliced introns of ZmTOM1 in ys3. GRMZM2G063306_T02orf, ZmTOM1 cDNA sequence predicted by GRAMENE; ZmTOM1pcr, partial sequence of ZmTOM1 used for quantitative real-time PCR; ZmTOM1orf, sequenced ZmTOM1 from the WT; Pattern_1, intron insertion version of ZmTOM1 cDNA in ys3; Pattern_2, intron insertion version of ZmTOM1 cDNA in ys3; Pattern_3, intron insertion version of ZmTOM1 cDNA in ys3. Figure S5. Genomic sequence of GRMZM2G063306. Colored and bold font indicates the assembled plant exon in this region. Pink font shows the region where sequencing was not perfect. Figure S6. Full-length cDNA of ZmTOM1. GRMZM2G063306_T02orf, ZmTOM1 cDNA sequence predicted by GRAMENE; ZmTOM1pcr, partial sequence of ZmTOM1 used for quantitative real-time PCR; ZmTOM1orf, sequenced ZmTOM1 from the WT. Figure S7. Phylogenetic tree of TOM1 and ENA1 homologs in maize. GRAMENE was searched for homologous genes of ENA1 and TOM1. Scale bar, 0.3 substitutions/site. Table S1. (PDF)