Figure S1. Expression profiles of SKB1 and FIT in wild-type (Col-0), SKB1-lesion mutants
(skb1-1 and skb1-2), Complementary plant (Com), and SKB1-overexpression lines (OX12-2 and
OX2-5).
(a) Quantitative RT-PCR analysis of SKB1 in Col-0, skb1-1, skb1-2, Com, OX12-2, and OX2-5
plants. (b) Quantitative RT-PCR analysis of FIT in the roots of Col-0, skb1-1, skb1-2, Com,
OX12-2, and OX2-5 plants. Template RNAs were extracted from 14-day-old seedlings grown on
MS medium with 100 μM Fe-EDTA (a), and from roots of the plants grown on MS medium
with 100 μM Fe-EDTA or without iron supply for four days after grown on MS medium for ten
days (b). Expression of AtACTIN8 was used to normalize mRNA levels (n = 3, values are ± SD)
* and ** indicate significant differences in values in comparison with Col-0 by Student’s t-test
at levels of P < 0.05 and P < 0.01, respectively.
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Figure S2. Phenotype of Col-0, skb1-1 and OX12-2.
(a) The plants of Col-0, skb1-1 and OX12-2 on soil; (b) the seedling of Col-0, skb1-1 and
OX12-2 were cultivated on MS agar plates for 16 d.
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Figure S3. Analysis of the growth curve and relative growth rate of main root.
(a) The main root growth curve of Col-0 and skb1-1 on MS agar plates with 0 μM, 100 μM and
300 μM Fe-EDTA. The seedlings were cultured on MS plate with 100 μM Fe-EDTA for five
days, and then transferred onto the MS plate with different iron concentration as labeled,
respectively. The length of main roots was measured at the same time every day, and the record
was performed for five days (n = 20, values are ± SD). (b) The relative growth rate of main root
of Col-0 and skb1-1 treated with different iron concentration was calculated, respectively.
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Figure S4. Western blot analysis of IRT1 accumulation.
Total proteins were extracted from roots of wild type, irt1 mutant, fit-2 mutant and
FIT/AtbHLH38 over-expressing plants (ox29/38) cultured on MS plates with 100 μM Fe-EDTA
(+) and without iron supply (-) for four days after grown on MS plates for ten days. Tubulin
served as a loading control.
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Figure S5. ChIP-PCR analysis of FRO2 and IRT1.
The gene structures of FRO2 (a) and IRT1 (b). Bars labeled with lowercase letters indicate the
regions examined by ChIP-PCR. Black boxes represent exons. ChIP-PCR assay for FRO2 (c)
and IRT1 (d) using gene-specific primers (see Table S1) was performed on samples
immunoprecipitated with anti-SKB1 and anti-H4R3sme2 antibodies. Chromatin was extracted
from Col-0 and skb1-1 seedlings grown on MS medium for two weeks. Input: chromatin before
immunoprecipitation; mock: control sample without addition of the antibody during
immunoprecipitation; anti-SKB1 and anti-H4R3sme2: chromatin samples immunoprecipitated
with the respective antibodies.
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Figure S6. Phenotype and gene expression profiles of FRO2 and IRT1 in Col-0, fit-2,
skb1-1fit-2 and skb1-1 roots.
(a) Phenotype of Col-0, fit-2, skb1-1fit-2 and skb1-1 on MS agar medium after growth on MS
with 100 μM Fe-EDTA for ten days followed by four days without iron supply. (b) Multiplex
RT-PCR analysis of FRO2 and IRT1 expression in roots of Col-0, fit-2, skb1-1fit-2, and skb1-1
plants. RNAs were extracted from roots of seedlings grown on MS plates without iron supply
for four days. (n = 3, values are ± SD). * and ** indicate significant differences in values in
comparison with Col-0 by Student’s t-test at levels of P < 0.05 and P < 0.01, respectively.
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Figure S7. ChIP-PCR analysis of AtbHLH38, AtbHLH39, AtbHLH100, AtbHLH101 and FIT.
Gene structure diagrams of AtbHLH38 (a), AtbHLH39 (c), AtbHLH100 (e), AtbHLH101 (g) and
FIT (i). Black boxes represent exons. Black bars labeled with lowercase letters indicate the
regions examined by ChIP-PCR. PCR-assay results appeared in b for AtbHLH38, d for
AtbHLH39, f for AtbHLH100, h for AtbHLH101, and j for FIT. Analysis was performed using
gene-specific primers (see Table S1) on DNA samples immunoprecipitated with anti-SKB1 and
anti-H4R3sme2 antibodies. Chromatin was extracted from Col-0 and skb1-1 mutant seedlings
grown on MS medium for two weeks. Immunoprecipitated DNA was detected by PCR. Input:
chromatin before immunoprecipitation; mock; control sample which was not incubated with the
antibodies during immunoprecipitation; anti-SKB1 and anti-H4R3sme2: chromatin samples
immunoprecipitated with the corresponding antibodies.
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Figure S8. Expression profiles of AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101 genes
in Col-0 seedlings in response to iron deficiency and in the FIT-null mutants fit-2 and ox29/38
(a line double-overexpressing FIT and AtbHLH38) under iron-sufficient condition.
(a) Multiplex RT-PCR analysis of AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101
expression in Col-0 in response to iron deficiency. RNAs were extracted from the seedlings
grown on MS medium without iron supply for 6 h and 12 h after culturing on MS medium for
14 days. “+” indicates the untreated control. Expression of AtACTIN8 was used to normalize
mRNA levels (n = 3, values are ± SD). ** indicates significant differences in values in
comparison with the control by Student’s t-test at the level of P < 0.01. (b) Multiplex RT-PCR
analysis of AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101 expression in fit-2, Col-0 and
ox29/38. RNAs were extracted from seedlings grown on MS medium with 100 μM Fe-EDTA
for two weeks. The UTR region of AtbHLH38 was selected to design the primer (designated as
M-endo-38 in Table S1) and to analyze expression driven by the endogenous
AtbHLH38-negative promoter rather than the 35S promoter in ox29/38. Expression of
AtACTIN8 was used to normalize mRNA levels (n = 3, values are ± SD). ** indicates
significant differences in values in comparison with the control by Student’s t-test at the level of
P < 0.01.
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Figure S9. ChIP-seq analysis of SKB1 association with chromatin containing FIT, FRO2 and
IRT1 genes.
Gene structure diagrams of FIT (a), FRO2 (d) and IRT1 (f). Black boxes represent exons.
ChIP-seq assays were performed using the HiSeq sequencing platform. Libraries were
constructed from DNA samples immunoprecipitated with anti-SKB1 antibody. Chromatin was
extracted from the roots of Col-0 seedlings grown on MS medium for seven days. The images
of enriched binding sites in the chromatin of FIT (b), FRO2 (e) and IRT1 (g) genes were
generated using IGV software. TIS: translation initiation site; purple arrows: regions upstream
from TIS; blue boxes behind arrows indicate exons; -500 and -1000: sites 500 and 1000 bp
upstream of the TIS. Signals of peak means the enrichment of sequencing reads mapped to the
binding sites associated with SKB1. (c) Multiplex RT-PCR analysis of FIT expression in roots
of Col-0, skb1-1 and OX12-2 seedlings. RNAs were extracted from the roots of seedlings grown
on MS medium for seven days. Expression of AtACTIN8 was used to normalize mRNA levels
(n = 3, values are ± SD).
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