Supplemental data
Regulation of miR399f transcription by AtMYB2 affects phosphate-starvation responses
in Arabidopsis
Supplemental Figure S1. Chlorosis phenotype in AtMYB2 OE plants. A, Leaves of threeweek-old AtMYB2 OE plants display chlorosis phenotype. Seeds were grown on MS medium
for three weeks under constant light condition. B, Total chlorophyll contents were measured
in three-weeks-old wild type (WT) and AtMYB2 OE seedling shown in (A). Bars represent
means ± standard error of three replicates with 9 seedlings per replicate. Asterisks represent
significant differences from the WT (p-value ≤ 0.05 from a Student’s t-test).
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Supplemental Figure S2. Pi starvation responses of atmyb2-3. A, Schematic representation
AtMYB2 showing the T-DNA insertion site in the atmyb2-3 mutant. Lines indicate introns,
black boxes indicate exons and the triangle marks the site of insertion of T-DNA. B, RT-PCR
analysis of AtMYB2 mRNA abundance in total RNA extracted from 10 day-old seedlings of
wild type (Col-0) and atmyb2-3. TUBULIN2 mRNA is shown for normalization. C, Seedlings
were grown on MS medium for 5 days, transferred to high Pi, low Pi or Pi deficiency media,
and then incubated for 7 d before photography. Shown are representative seedlings from a
total of 16 from each line. D, Northern blot analysis of miR399f expression was performed on
total RNA extracted from the above seedlings. 5S rRNA is shown as loading control. E,
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Inorganic Pi contents were measured in root and shoots of 10 day-old MS-grown seedlings.
Bars indicate mean ± standard deviation for two biological replicates.
Supplemental Figure S3. AtMYB2 is localized in the nucleus. Protoplasts prepared from
leaves of wild type seedlings were transiently transformed with a CaMV35S:AtMYB2-sGFP
or CaMV35S:sGFP vector construct and examined under a fluorescent microscope 12 h later.
DAPI stain was used to visualize nuclei. Shown are the white light (light), GFP (green signal),
DAPI (blue signal) and merged GFP and DAPI images (Merge) of a transformed protoplast.
Bar indicates 20 µm.
Supplemental Figure S4. Expression patterns of miR399b and miR399c in AtMYB2 OE
plants. WT and three independent lines of AtMYB2 OE plants were grown in MS medium for
10 d. Expression levels of miR399b and miR399c were analyzed by qRT-PCR. TUBULIN2
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was used for normalization. Bars represent mean ± standard deviation for two biological
replicates with two technical replicates each.
Supplemental methods
Measurements of Chlorophyll Content
Chlorophyll was extracted by 10-20 mg of the homogenized tissue in 1 ml of 80%
acetone. To remove chlorophyll-free sediment, the homogenate was centrifuged at 1,500 g for
1 min. Chlorophyll was analyzed by using UV spectrophotometer at 645 nm (chlorophyll B)
and 663 nm (chlorophyll A). Total chlorophyll was calculated using fallowing formula:
Chlorophyll (ug/mg F.W.) = [ ( OD645 x
20.2 ) + ( OD663 x 8 ) ] / Fresh weight (mg)
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Supplemental Table S1. List of primers used in this study
Name
Sequence
miR399f Antisense probe
CCGGGCAAATCTCCTTTGGCA
miR399f promoter-F1
ATTTGTCAAATAGCTAATGATCAGTTACA
miR399f promoter-R1
GTAGCACCTTTGGAGATTTTATGATT
miR399f promoter-F2
GGTGCTACAAGAATAGGATATATACAAGC
miR399f promoter-R2
AAATCAGTTATACAATTAAGTCAACTTTACCA
miR399f promoter-F3
GTACAAAAAAGCAGGCTCACTAACGTTATGGGGTTATT
miR399f promoter-R3
GTACAAGAAAGCTGGGTATCACCTTCTCTATGTATTTTTCATC
AtMYB2 promoter-F
GTACAAAAAAGCAGGCTCACTAGCTTATTTGGGTTGTCGTC
AtMYB2 promoter-R
GTACAAGAAAGCTGGGTAGTTTGAGTTTATTCGCTCGTA
AtMYB2-F(XbaI)
GCTCTAGAATGGAAGATTACGAGCGAA
AtMYB2-R(BamHI)
CGGGATCCATTATACGAATACGATGTC
AtMYB2-F(gateway)
GTACAAAAAAGCAGGCTCAATGGAAGATTACGAGCG
AtMYB2-R(gateway)
GTACAAGAAAGCTGGGTAATTATACGAATACGATGTCGTATC
AtMYB2-F(BamHI)
CGGGATCCATGGAAGATTACGAGCGAAT
AtMYB2-R(SalI)
ACGCGTCGACATTATACGAATACGATGTCG
AtMYB2-F-RT
ATGGAAGATTACGAGCGAA
AtMYB2-R-RT
ATTATACGAATACGATGTC
AtMYB2-F
TTTGGATGCCGAGATTAGTGG
AtMYB2-R
GCTGAATTGAACGAAACCCG
UBC24-F
TGAGATGCTTGTGAAGGACC
UBC24-R
CGGTGGAATTTGTGGTTGAG
AtPT1-F
CTCTCAACGCCTCCTCAAGTTGAC
AtPT1-R
TGTAACGGGCAGTTTCAGGC
AtPT2-F
ACCCAATGCTACAACCTTCG
AtPT2-R
CTGGGTTCTGAGCCAAGTAC
AtPS2-F
CACGACTTCACTAAATCCCCTC
AtPS2-R
GCTCCATCTCCTAGATAGATCATTTT
AtPS3-F
CTCTACTGGCTCCCATTTTACG
AtPS3-R
CTCTTGCACCAATTCGGTCT
AtIPS1-F
CTGATTCAGACTGCGAGTTTTG
AtIPS1-R
GGAGTGGGTACAACCCAAACA
AtRNS1-F
TGATGCCTCTAAACCATTCGAT
AtRNS1-R
TACCATGCTTCTCCCATTCG
SAIL_47_E01(UBC24)-LP
TCTGACCTTGATGAGAAACGG
SAIL_47_E01(UBC24)-RP
CCGCTTCTTAAGGTACGTTCC
Salk_045455(AtMYB2)-LP
ATCCACAAAACCATTCACACC
Salk_045455(AtMYB2)-RP
AAACGTGACGCAATTGAATTC
Experiments
microRNA northern blot assay
EMSA assay
GUS expression assay
transient expression assay
plant transformation
recombinant protein
RT-PCR
quantitative real-time PCR
mutant genotyping
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Supplemental Table S2. MBS elements in Pi-responsive microRNA promoters
Gene No.
microRNA
Position a
Strand b
At2g25095
At4g30972
At4g31877
At5g10945
At5g11977
At5g26147
At2g19425
At5g55835
At1g29265
At1g63005
At5g62162
miR156a
miR156b
miR156c
miR156d
miR156e
miR156f
miR156g
miR156h
miR399a
miR399b
miR399c
At2g34202
At2g34204
At2g34208
miR399d
miR399e
miR399f
At2g41616
At3g59884
At3g09285
At5g02035
miR778a
miR827a
miR2111a
miR2111b
-129
No
No
No
-1355
No
No
No
No
-1496
-654
-872
No
No
-574
-833
No
No
-561
No
+
No
No
No
+
No
No
No
No
No
No
+
No
No
No
a
Positions were assigned relative to the transcription start site of the microRNA precursors. No indicates no
predicted MBS in the region up to 1500 bp upstream.
b
(+) and (-) indicate sense or antisense DNA strands.
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