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Fig. S1
a
Ws
mutant
b
Fig. S1 Morphology of the low-iron-sensitive mutant of Arabidopsis.
(a) Wild type (Ws) and the low-iron-sensitive mutant (mutant) germinated on one-halfstrength MS agar medium for four days. Mutant exhibited a longer hypocotyl and a larger
and paler hypocotyldeons compared to wild type. (b) Wild type (left) and the low-ironsensitive mutant (right) grown on soil for two weeks. The leaves of the mutant showed
light green in comparison to wild type.
Fig. S2
NGA8
Chromosome 4
Recombinant
SSLP3624
SSLP1475
CIW5
a
c
5 1
0
3
med16-4
GT-AT
b
ATG
TAG
med16-2
med16-3
c
Ws
med16-4
AtMED16
AtACTIN8
Fig. S2 Map-based cloning of the mutation gene of the low-iron sensitive mutant (med16-4)
of Arabidopsis and the affection of the mutation on MED16 expression.
(a) A genetic and physical mapping of the low-iron sensitive mutant (med16-4) . A total of 326 F2
progenies homozygous for mutant were used to determine the approximate position of the
mutation gene. The mutation gene was linked to the markers CIW5 and NGA8 on chromosome
IV. Further analysis of 2876 F2 mutant plants delimited the mutation gene to a region of 3.1 Mbp
between markers SSLP1475 and NGA8. The number of recombination events linked to markers
is marked. (b) Structure of the MED16 gene (At4g04920), the MED16 mutation site, and the
insertion sites of the T-DNA in sfr6-2 (med16-2) and sfr6-3 (med16-3). The filled boxes represent
exons and the lines indicate introns. (c) RT-PCR analysis of MED16 expression in med16-4
mutant. ACTIN8 was used as loading control. RNAs were extracted from seedlings grown on onehalf-strength MS agar medium with iron for 10 days.
Fig. S3
a
root
b
leaf
Fig. S3 Subcellular localization of Arabidopsis MED16.
The plasmid 35S:MED16-GFP was constructed and transferred to wild
type of Arabidopsis. Transformed plants with stable expression of
MED16-GFP were selected, and the green GFP signal were observed in
root tips of one-week-old seedlings (a) and in leaf (b) of one-month-old
plants under a Carl Zess confocol microscope.
Fig. S4
WT
sin4
MED16
ACT1
Fig. S4 Characterization of the yeast MED16/SIN4-knocking out mutant.
RT-PCR analysis of knocking out of SIN4 (a MED16 homolog in yeast genome) in
sin4 yeast mutant, ACT1 was used as loading control.
Fig. S5
Bright Field
Epifluo
Col mesophyll protoplasts
a
med16-3 mesophyll protoplasts
b
bHLH38-CC/FIT-YN
Fig. S5 Observation of FIT/bHLH38 heterodimer formation in mesophyll cells of
the mutant med16-3 of Arabidopsis and its wild type.
The constructs BiFC-FIT-nYFP (FIT-YN) and BiFC-AtbHLH38-cCFP (bHLH38CC)were introduced into the mesophyll protoplasts of wild type (a) and med16-3 mutant
(b) by PEG transformation approach. After incubation for 16-20 h, the interaction signal
(green color) of FIT with bHLH38 were observed under a confocol microscope.
Fig. S6
a
WT
med16-3
med16-3ox29/38
MED16
ACTIN8
b
ATG
TAG
TGG-TAG
c
WT
med16-3
med16-5ox29/38
MED16
ACTIN8
med16-5ox29/38
Fig. S6 The med16 mutants (med16-3ox29/38 and med16-5ox29/38) of Arabidopsis in
the genetic background of ox29/38.
(a) RT-PCR verification of lacking MED16 expression in med16-3ox29/38 mutant
generated by crossing med16-3 with ox29/38, ACTIN8 was used as loading control. (b)
Gene structure diagram of MED16 and the position of the point mutation from TGG
to TAG in the mutant line med16-5ox29/38 obtained by screening a EMS mutation
population of ox29/38. (c) RT-PCR analysis of MED16 expression in med16-5ox29/38,
ACTIN8 was used as loading control.