The Arabidopsis WRKY transcription factors WRKY12 and WRKY13

oppositely regulate flowering under short-day conditions

Wei Li, Houping Wang, and DiqiuYu

Supplemental Figure 1. Characterization of wrky mutants. (A) T-DNA insertion

positions in wrky12-1 (Salk_127750), wrky12-2 (Salk_080995)), wrky13-1
(Salk_064346C), and wrky13-2 (Salk_032912) mutants. The promoters used for GUS
staining and promoter-swap assays are shown in the figure. The untranslated regions,
exons, and introns are represented by white boxes, black boxes and black lines,
respectively. (B) and (C) WRKY expression levels in mutants. 25-day-old soil-grown
plants were harvested. Total RNA was isolated and analyzed by quantitative reverse
transcribed (qRT) PCR analyses. Error bars show SD from three independent RNA
extractions. The symbol ** indicates significant differences compared with WT based
on Student’s t test (P < 0.001).
Supplemental Figure 2. Restoration of flowering phenotype of wrky mutants. Bar
graph of flowering time of T2 transgenic lines under SD conditions. Error bars
indicate SD from 25 independent plants. WT, wild type.
Supplemental Figure 3. Flowering phenotype of WRKY12 and WRKY13
overexpression (OE) lines. (A) Flowering phenotypes of WRKY12OE#4 and
WRKY13OE#8 under SD conditions. Photograph was taken on 65 days after sowing.
WT, wild type. (B) Histogram of flowering time of WRKY12OE and WRKY13OE
under SD conditions. Error bars represent SD from 20 independent plants. The symbol
** indicates significant differences compared with WT based on Student’s t test (P <
0.001). (C) and (D) Expression levels of WRKY genes in OE lines. Error bars
represent SD from three independent RNA extractions. The symbol ** indicates
significant differences compared with WT based on Student’s t test (P < 0.001).
Supplemental Figure 4. Expression profiles in promoter-swap transgenic plants. (A)
and (B) RNA extracted from 20 lines of T2 transgenic lines were used to perform
assays. Error bars represent SD from three independent RNA extractions.
Supplemental Figure 5. ChIP results on SOC1 and LFY promoters. (A) Schematic of
SOC1 and LFY promoters. Bars indicate W-boxes (TGAC). Lines beneath bars
indicate the sequences detected by ChIP assays. (B) to (G) qPCR of ChIP. Methods
and plant materials are the same as for Fig. 5. WT, wild type. Error bars represent SD
from three independent ChIP assays.
Supplemental Figure 6. Expression levels of FUL genes in OE lines. (A) Analysis of
FUL expression levels in WT and FULOE transgenic plants. (B) Analysis of FUL
expression levels in WT and wrky12-2FUL transgenic plants. Error bars represent SD
from three independent RNA extractions.
Supplemental Figure 7. WRKY regions required to interact with DELLAs. (A) and
(C) Immunoblot analysis of proteins expressed in yeast. Total proteins were extracted
from the yeast cells and analyzed by immunoblot using anti-MYC or anti-FLAG
antibody (Sigma-Aldrich). (B) Left: Representation of natural and amino acids mutant
proteins. The empty pGADT7 prey vector was used as a negative control.
Supplemental Figure 8. GA level profiles and GA effects on WRKY gene expression
levels. (A) The GA level profile in the wild type. Error bars represent SD (n = 3). (B)
GA effects on WRKY genes. We sprayed 18-day-old wild type seedlings with 50 µm
of GA3 or pure ethanol 1%, Tween 0.1% (MOCK). Error bars represent SD (n = 3).
Supplemental Figure 9. Expression patterns of FUL. qRT-PCR analysis of FUL
expression under SD conditions. Shoot apices and leaves were collected at different
time points and used for expression analyses by quantitative real-time PCR. Error bars
indicate SD from three independent RNA extractions.
Supplemental Figure 10. Alignment of WKRY12 and WKRY13 amino acid
sequences. Identical residues are indicated by black boxes. Conserved residues
(WRKY and C2H2) are indicated by triangles.
Supplemental Figure 11. A model for age-dependent equilibrium in flowering time
regulation in Arabidopsis thaliana. See text for details.
Supplemental Table 1. Primer sequences used in this study.
Use genes Primer forward Primer reverse

WRKY12 ATAGGATCCATGGAAGGAGGAGGGAGAA ATATCTAGATTAAAAGGAAGAGAGACAATCATG

GA G
Overexpres
WRKY13 ATAGGATCCATGGGTGCGATAAACCAAGG ATATCTAGATCACCAGAAGAAGTTGGAGAGAG
sion
A G

WRKY12 ATAGAATTCATGGAAGGAGGAGGGAGAA ATAGGATCCTTAAAAGGAAGAGAGACAATCATG

GA GTC

WKRY13 ATAGAATTCATGGGTGCGATAAACCAAGG ATAGGATCCTCACCAGAAGAAGTTGGAGAGAG

A G

RGA ATAGGATCCATGAAGAGAGATCATCACCA ATAGAGCTCTCAGTACGCCGCCGTCGAGAGT

ATTC

GAI ATAGAATTCATGAAGAGAGATCATCATCA ATAGGATCCAGCCACCATCTAATTGGTGGA

TCATCA

Yeast Two RGL1 ATACATATGATGAAGAGAGAGCACAACC ATACCCGGGTTATTCCACACGATTGATTCGCCAC

Hybrid ACCGT GC

RGL2 ATACATATGATGAAGAGAGGATACGGAGA ATACCCGGGTCAGGCGAGTTTCCACGCCGAG

AAC

RGL3 ATACCCGGGATGAAACGAAGCCATCAAG ATAGAGCTCCTACCGCCGCAACTCCGCCGCT

AAACG

GAI ATATCTAGAATGAAGAGAGATCATCATCA ATAGGATCCATTGGTGGAGAGTTTCCAAGCC

TCATCATC

RGL1 ATATCTAGAATGAAGAGAGAGCACAACC ATAGGATCCTTCCACACGATTGATTCGCCAC

ACCGTG
BiFC
WRKY12 ATAGGATCCATGGAAGGAGGAGGGAGAA ATATCTAGAAAAGGAAGAGAGACAATCATGGTC

GA A

WKRY13 ATAGGATCCATGGGTGCGATAAACCAAGG ATATCTAGACCAGAAGAAGTTGGAGAGAGGAG

A G

WRKY12 TATGGATCCTTGATAACTTACGTATTTACG TATACTAGTGTTTTTAATCTCAATCTCTCCTTGTT

AACCCAA CC
GUS
WKRY13 TATGGATCCTCATTCTCACGGACCGATGT TATGAGCTCCTCGCAAAAGCTTGACGAAG

FUL ATAAAGCTTTAACCGTAACAGTAGTTGGG ATAGGATCCATCTCTCTCTCTTCAAAATCTCAAG

AGAACT AAC

WRKY12 AGCCTTCACCCCAGGAGTTAT CAATCTTCCGATAGTCGCTCCA

WKRY13 CACCCAACATCCCAGGAGC TGGGTCATCTGCTAATCTCTCC

FUL AGAGAAGGTCTGGTTTGCTCAAGA TGGAAGAGAAGACGATGAGAGCA

SOC1 ATGGTGAGGGGCAAAACTCAG CCCAATGAACAATTGCGTCTC

LFY TTGGTGGTTTAGAGGGACTATTCG GCCGTAAAACCTAACTCCGCTA

ACT2 ATTCAGATGCCCAGAAGTCTTGT GAAACATTTTCTGTGAACGATTCC

qRT-PCR
GA3ox-1 CATCACCTCAACTACTGCGA GCTGACCCCAAGTGAATTTAG

GA2ox-1 TTGAGTACTTGTTGATGAACGC TTCCAATGCGTTTCTGAAAGTT

GA2ox-2 TGAGTATCTCCTCCTCAATGC CCACCGACTCACGGAAAAT

 GA2ox-6 CTTTCAAGTTCAGCTCGGC CTTCGCTAGACTTCTGTCCC

GA20ox-1 CCGTTCAAGATTACTTCTGCG TACTCTTGATACACCTTCCCAAA

GA20ox-2 AATCCATTCGTCCCAATCCC CCGTTCGATAGAGCCATGA

pFUL-1 ACAAGAGAGAGAAACGACAACCC CCCAGAGAGACATAAGAAAGAAAGAG

pFUL-2 TGGGTTTCGCGTGAGTCTACA TCATCGTCTATGTATTCACGTCACA

pFUL-3 TGCACGGAGAAATTTTAATTTTATT GCACAATGTTTCTCCCTGTTTAA

pFUL-4 AGTACACGAAAGAATTTGACCCAA CCGTGTTATTTTTGTGTTTGCAA

pFUL-5 GCCTATATTCCATAACTTATTCACTTGA TCTCTTCCCATAAATAGGCCACT

pFUL-6 CTGGTAACTGGTAAATTGGTAATGTACT ATTGACAGAAAAGGTTTTATTTTCGT

pFUL-7 TGTAGGATTAACACATCACACTAAGCT AAGTTGAACCTTCGGAAGAAATTA

pFUL-8 AACAACATTCATGTCGGTCCC TTCCACTTTTCAGACCAATATATGTTAT

pFUL-9 GGAGCTACCAATCTTCCAAACC CTTAATTGATCATAAACACATAAATTTTAGTG

pWRKY1 AAGCAAGAGAAGATAAACCGTCAA GTTTTTAATCTCAATCTCTCCTTGTTCCTTTTTGT

2-1 TG

pWRKY1 TCACATGAAGTTGACTGCCAAAG AAAGGGTAGGGCTTCGTGTGTAT

2-2

pWRKY1 TTGTAAACGTTATTGGTATGATTATTTTG GGTTATTCCACAACATTCTAATATCGT

2-3

ChIP qRT- pWRKY1 ATGGATGGAAGCTTTGATGTACC ATGAAGGACATTGTTCATTTCACA

PCR 2-4

pWRKY1 TCCACCTCAATCATGTTTTATGGT ACTTCCTCCACAATAGCAACACA

2-5

pWRKY1 CAAGAAGACCTTAGCTTTTGAAACTAC TTACCGAGTTACTGACACCAAGAGT

2-6

pWKRY1 GGATGGATTGGATTGTCTCAACT GCTTGACGAAGGTGAGATTGTGT

3-1

pWKRY1 AATGAATAATTTTCATGCATAGATCAA TTACAGTTTTGTGTTTTGGTAGCAGT

3-2

pWKRY1 AACGCCGTTTGAGGGACGTA GATAGAACAAAAAGCAGAAAACGC

3-3

pWKRY1 CGAAAAATGTCAAATAGGAGGCT GGAGGAGCAATGATGAAAAATCT

3-4

pSOC1-1 CTTTTTGGGTACTTAATCTTTCGTTG ATAACAAGATTAGTCACACACATAAAACA

pSOC1-2 AGCTTCCTCCTGGTTAGGTTTTTAT AAACCCATATAAGTTTATAGATCCATCA

pSOC1-3 TCATATTTGCGACACATATGACTGA TTCTTTTCTATTTTATTTCTTGGAGAAA

pSOC1-4 TTTTTCATATCAATTACAAGTGGGG CCCATAAATATCTAGTTGGATGGAA

pSOC1-5 CATTGTCTCTGTACTAGCCTCCAAA TATGACATAATCTTACATCTCCATGTGA

pLFY-1 ATGCTTTATTGCAAAAATAGCGA TGTAAAAATGACCACTCGAGCAA

pLFY-2 TGTCGATGAAAGAAAAAGTTTGACT TCGCTATTTTTGCAATAAAGCAT

pLFY-3 GAGAATCACCACAGTGAAAACCC TTTTCTTTCATCGACATTCTTTTTC

pLFY-4 AAAGACGTAGGTTGGCCATGTAT CATTTATGACAAAAATATTTGTTACGATAG

pLFY-5 CGTCCATGCCAATACAGTTAAAG GGAAAGTTGGCAATACTGGCTAT

 pLFY-6 AAATCAGGGAGATGACAACACGT AAATGCAGTGTGAAAACTCCAGA

pLFY-7 TTTCCCCACTTTAATTTGTAAGTTCT GAAAACCGAATCAATTTGATAAG

pLFY-8 TTTTATCGGCTCAATTCCAATGT ACGTAAACTTTGTGACTAAACAGTGAA

pLFY-9 AACTGAGGGAGTGAGACAGTCAAA ATGTTGGAAAGGTAGCCAATGAG

WRKY12 AAACAATGGAAGGAGGAGGGA GTAATACGACTCACTATAGGGCGAAAAAACGCA

antisense TTGAAAGTTTGAA

WRKY12 GTAATACGACTCACTATAGGGCGAAACAA AAAAAACGCATTGAAAGTTTGAA

In situ
sense TGGAAGGAGGAGGGA
probe
WRKY13 ATGGGTGCGATAAACCAAGG GTAATACGACTCACTATAGGGCGGCAAGGCAAG

antisense GTTGATTGATT

WRKY13 GTAATACGACTCACTATAGGGCGATGGGT GCAAGGCAAGGTTGATTGATT

sense GCGATAAACCAAGG