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 three-week-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).

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, 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 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)

Supplemental Table S1. List of primers used in this study

Name / Sequence / Experiments
miR399f Antisense probe / CCGGGCAAATCTCCTTTGGCA / microRNA northern blot assay
miR399f promoter-F1 / ATTTGTCAAATAGCTAATGATCAGTTACA / EMSA assay
miR399f promoter-R1 / GTAGCACCTTTGGAGATTTTATGATT
miR399f promoter-F2 / GGTGCTACAAGAATAGGATATATACAAGC
miR399f promoter-R2 / AAATCAGTTATACAATTAAGTCAACTTTACCA
miR399f promoter-F3 / GTACAAAAAAGCAGGCTCACTAACGTTATGGGGTTATT / GUS expression assay
miR399f promoter-R3 / GTACAAGAAAGCTGGGTATCACCTTCTCTATGTATTTTTCATC
AtMYB2 promoter-F / GTACAAAAAAGCAGGCTCACTAGCTTATTTGGGTTGTCGTC
AtMYB2 promoter-R / GTACAAGAAAGCTGGGTAGTTTGAGTTTATTCGCTCGTA
AtMYB2-F(XbaI) / GCTCTAGAATGGAAGATTACGAGCGAA / transient expression assay
AtMYB2-R(BamHI) / CGGGATCCATTATACGAATACGATGTC
AtMYB2-F(gateway) / GTACAAAAAAGCAGGCTCAATGGAAGATTACGAGCG / plant transformation
AtMYB2-R(gateway) / GTACAAGAAAGCTGGGTAATTATACGAATACGATGTCGTATC
AtMYB2-F(BamHI) / CGGGATCCATGGAAGATTACGAGCGAAT / recombinant protein
AtMYB2-R(SalI) / ACGCGTCGACATTATACGAATACGATGTCG
AtMYB2-F-RT / ATGGAAGATTACGAGCGAA / RT-PCR
AtMYB2-R-RT / ATTATACGAATACGATGTC
AtMYB2-F / TTTGGATGCCGAGATTAGTGG / quantitative real-time PCR
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 / mutant genotyping
SAIL_47_E01(UBC24)-RP / CCGCTTCTTAAGGTACGTTCC
Salk_045455(AtMYB2)-LP / ATCCACAAAACCATTCACACC
Salk_045455(AtMYB2)-RP / AAACGTGACGCAATTGAATTC

Supplemental Table S2. MBS elements in Pi-responsive microRNA promoters

Gene No. / microRNA / Position a / Strand b
At2g25095 / miR156a / -129 / +
At4g30972 / miR156b / No / No
At4g31877 / miR156c / No / No
At5g10945 / miR156d / No / No
At5g11977 / miR156e / -1355 / +
At5g26147 / miR156f / No / No
At2g19425 / miR156g / No / No
At5g55835 / miR156h / No / No
At1g29265 / miR399a / No / No
At1g63005 / miR399b / -1496 / -
At5g62162 / miR399c / -654 / -
-872 / -
At2g34202 / miR399d / No / No
At2g34204 / miR399e / No / No
At2g34208 / miR399f / -574 / +
-833 / -
At2g41616 / miR778a / No / No
At3g59884 / miR827a / No / No
At3g09285 / miR2111a / -561 / -
At5g02035 / miR2111b / 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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