Supplemental Figures Legends
Figure S1. Identification of wrky41 mutants and complemented lines.
(a) Diagram of the structure of the WRKY41 gene (At4g11070). The positions of the
T-DNA insertion are shown (about -220bp upstream of ATG for SAIL_545_D09 and
1048bp downstream of ATG for SALK_028449). Exon (black boxes) and intron (lines)
are indicated. A pair of primers (black arrow) is used to check the expression of
WRKY41 in the mutant by RT-PCR in (b). The red line indicates the specific sequence
detected by qRT-PCR in (c).
(b-c) Expression of WRKY41 in the wild-type Col-0, wrky41-1, wrky41-2, and
wrky41-1 complemented lines (line 1-4) by RT-PCR (b) and qRT-PCR (c). Total RNA
extracted from 6-day-old seedlings was used for cDNA synthesis. UBQ10 was used as
internal control.
Figure S2. Phenotypic analysis of wrky41 mutants.
(a) Comparison and quantitative evaluation of seed germination on MS, or MS
supplemented with different concentrations of ABA, between wrky41-1, wrky41-2,
Col-0, abi3-17 and wrky41-1 complemented lines. Three independent experiments
were done with similar results. Data show one experiment, and about 100 seeds were
counted for each repeat. Error bars indicate SD (n=4).
(b) Water loss from detached leaves. The water loss of 0.5 g detached leaves from
Col-0, wrky41-1 and abi3 mutant, was measured at different times with triple
replicates. Five independent experiments were performed with similar results. Data
show one experiment. Error bars indicate SD (n=3).
Figure S3. ABA content assay in imbibed seeds under different temperatures.
After-ripened seeds were used for assay. Three independent experiments were done
with similar results. Error bars indicate SD (n=3).
Figure S4. Comparison of transcript accumulation in developing siliques of wild-type
Col-0, wrky41-1, wrky41-1 complemented line 2 and abi3-17 mutant. RNAs were
extracted from different developmental siliques. Black, Col-0; white, wrky41-1; gray,
complemented line2; brow, abi3-17. DPA indicates days after pollination. PP2A was
used as internal reference gene. Three independent experiments were done with
similar results. Error bars indicate SD (n=3).
Figure S5. Identification of WRKY41 overexpressing lines.
(a) Expression of WRKY41 in Col-0 and WRKY41-overexpressing lines by RT-qPCR.
Total RNA extracted from 6-day-old seedlings was used for cDNA synthesis. UBQ10
was used as internal control. Error bars indicate SD (n=3).
(b) Protein level of WRKY41-GFP in Col-0 and WRKY41-overexpressing lines by
western-blot assay. Total protein extracted from 6-day-old seedlings was used for
assay. Anti-GFP was used to detect the abundance of WRKY41 protein. Ponceau S
indicates Ponceau staining of the loaded proteins.
(c) Comparison of seed germination on MS, or MS supplemented with 0.6 μM ABA
between Col-0, wrky41-1 mutant and WRKY41-overexpressing lines. Three
independent experiments were done with similar results. Data show one experiment,
and about 100 seeds were counted for each repeat. Error bars indicate SD (n=4).
Figure S6. Identification of transgenic lines overexpressing ABI3 in wrky41-1 mutant.
(a) Expression of ABI3 in Col-0, wrky41-1 and wrky41-1 transformed with 35S-ABI3.
Total RNA extracted from 6-day-old seedlings was used for cDNA synthesis. UBQ10
was used as internal control. Error bars indicate SD (n=3).
(b) Comparison of seed germination on MS medium under normal condition. Freshly
harvested seeds with prechilling at 4℃ for 3d were used for assay. Three independent
experiments were done with similar results. Data show one experiment, and about 100
seeds were counted for each repeat. Error bars indicate SD (n=4).
Figure S7. WRKY41 does not act downstream of ABA signal.
(a) Expression of WRKY41 in Col-0 seedlings on MS medium without or with 0.2µM
ABA for 5 d after the end of stratification. UBQ10 was used as internal reference gene.
Three independent experiments were done with similar results. Error bars indicate SD
(n=3).
(b) Expression of WRKY41 and ABI5 in 6-day-old Col-0 seedlings on MS medium
with 100µM ABA for different time. The expression data have been converted to fold
changes between ABA treatment and control set in each time points. UBQ10 was used
as internal reference gene. Error bars indicate SD (n=3).
(c) Expression of WRKY41 and ABI3 in after-ripened seeds (two months) imbibed in
30µM ABA for indicated hours. PP2A was used as internal reference gene. The
expression data have been converted to fold changes between ABA treatment and
control set in each time points. Error bars indicate SD (n=3).
(d) Expression of WRKY41, ABI3 and At2S3 in different developmental siliques.
PP2A was used as internal reference gene. Three independent experiments were done
with similar results. Error bars indicate SD (n=3).
(e) Protein level of WRKY41-GFP under normal or ABA treatment was detected by
western-blot assay. Total protein extracted from 6-day-old seedlings was used for
assay. Anti-GFP was used to detect the abundance of WRKY41 protein. Ponceau S
indicates loading control.
Figure S8. Expression of ABA metabolic genes and other ABA signaling genes in
wild-type and wrky41-1 mutant.
(a) Expression of ABA metabolic genes in developing siliques from wild-type (Col-0)
and wrky41-1 plants. PP2A was used as internal reference gene. Three independent
experiments were done. Error bars indicate SD (n=3).
(b) Expression of ABA signaling related genes in wild-type, wrky41-1 and abi3-17
after-ripened seeds (two months) imbibed in water with or without 30µM ABA for
24h. PP2A was used as internal reference gene. Three independent experiments were
done. Error bars indicate SD (n=3).
Figure S9. Relationship between WRKY41 and seed development controlling genes.
(a) Expression of FUS3, LEC2 and LEC1 in different developmental siliques of Col-0
and wrky41-1 mutant. PP2A was used as internal reference gene. Three independent
experiments were done with similar results. Error bars indicate SD (n=3).
(b) Expression of WRKY41 and ABI3 in different developmental siliques from Col-0,
fus3-8, lec2-1 and lec1-1. Three independent experiments were done with similar
results. Error bars indicate SD (n=3).
(c) Expression of PIL5 in imbibed seeds of Col-0 and wrky41-1 mutant. Three
independent experiments were done with similar results. Error bars indicate SD (n=3).
Figure S10. The binding of WRKY41 to ABI3 promoter is unaffected by ABA.
ChIP analysis showing the in vivo binding of WRKY41 to the ABI3 promoter.
5-day-old 35S:WRKY41-GFP seedlings with or without 100µM ABA treatment for 6
h were used for ChIP assay. The F1 fragment (indicated in Figure 8) was determined
in the ChIP assay. IgG was used as negative control. Three independent repeats were
done. Error bars indicate SD (n=3).
Figure S11. Expression of ABI3, ABI4 and ABI5 in wild-type, wrky41-1, abi3-17 and
abi4 mutants.
RNAs were extracted from seedlings of the different genotypes on MS medium with
0.2µM ABA 5 d after the end of stratification. UBQ10 was used as internal reference
gene. Three different experiments were repeated with similar results. Error bars
indicate SD (n=3).
Figure S12. A model for network between WRKY41 and ABA signaling.
ABI3 acts downstream of ABA to control seed maturation and germination. WRKY41,
under developmental signal, positively regulates primary seed dormancy and
thermoinhibition by directly regulating ABI3 expression independently of ABA signal.
Meanwhile, WRKY41 is possibly negatively regulated by high concentration of ABA.
Supplemental Tables
Table S1. Primers used in this study.
Additional Remarks
Additional remarks 1: WRKY41 regulates ABI3 expression via a mechanism
independent of ABA metabolism or ABA signaling.
Additional remarks 2: Regulation of ABI3 by WRKY41 is independent of known
regulators
Additional remarks 3: WRKY41 indirectly affects the expression of ABI4 and ABI5
Supporting Methods
Supporting Method 1: Methods used in details including Plant materials and growth
conditions, Germination and root growth assay, Water loss assay and drought
treatment, ABA content assay, GUS staining, Gene expression analysis and sequence
analysis, Protein extraction and Western blot assay, Trans-activation assay in N.
benthamiana leaves, Purification of recombinant protein and electrophoretic mobility
shift assay (EMSA), ChIP Assay