Supplemental Figure 1
CGTTGACAAATAAGTTTGATCGACATTGTGTGACAAATTTAAAACACATCATTAATAACAACGAGGACAAATACAGTTCAGATATCGTCT
W-BOX
-908
ACTATTAAAACACTTCTTCTATAGGAGCAAAGAAAAATTGTCGGCAACGAACTGGGGACCAATAATATTCCGAGTTTGAGTTCAAACTGA
DRE
-818
GTAATTTTATTTTGAGAAAATTTGCCAAGTTAACTTATAATTCTGGTTTACGTGTAGTAATTTATTAAGTTGTTATAGGAAAATGAGAAA
MYB ABRE
WUN
-728
ATACTAAGACACATCCAAGAAAGTTTCACACGAAATTTACTTACAAAAAGATTGTTTATTTAATACTTCCGTATATAGATATATAAATAT
WUN
-638
TTAACACATTAATTATAAAGTTCAAGATAATTGATTATCTATCTTTTTTTGTCATCTGAAATTATTATCGCTCAAACGAAGTAATTCTGA
MYC
-548
GGAAAGTTGTTTACAAACTAGTTATTTCATTATTGTCTACTTATATAATAGAATTAAAAAAAATTATTGCTTAATGCAATTTAGTTTTAG
-458
ATAAAATCATTAAACTTAATAGATTATATAAGTTAGATATCAATAATTGGGCTTGCTTAAAAACATAAATATAAAATATTATTGGGCCGT
-368
TACGTGCATACAAAACGAACCTTCTAACAAACAAGTGTGAACGTTACGACTTCAAAATTAAAAAAAAACACAACAACTATGTCCACACGT
ABRE
MYC
ABRE
-278
AATCTCATATGATTCAGATTCCAAGGAGAACAAAATTAAAAACAAATCTCGTAAACATACATACACTTCACATAAAACAAAAGGTACAGT
MYC
CAAT
-188
ATATACCATAAATCTCCGAGATTCTTTTGATGTATCTGTCCATTTCATTATTACACAAACTAGGAAACTGATATCTCTCTATTCACATTC
TATA
-98
CTCTGATTCTATTTCTCTTTATATATATTCACCCATTAACCATCTCAATCTTATAACCCTCAAAATCACAATCTTCTCTTACAAAAAACT
-8
TTGAAAGATG
Supplemental figure. Structure of the RNS1 promoter. One kb of genomic sequence upstream of the start codon
(box) of RNS1 was analyzed to identify the presence of motifs with significant similarity to previously
identified wounding- and ABA-related cis-acting elements (grey boxes). These include CAAT and TATA boxes,
wound-responsive elements (W-box, WUN), a dehydration-responsive element (DRE), ABA-responsive elements
Supplemental figure S1: Structure of the RNS1 promoter. One kb of genomic
(ABRE), and MYC binding sites.
sequence upstream of the start codon (box) of RNS1 was analyzed to identify the
presence of motifs with significant similarity to previously identified wounding- and
ABA-related cis-acting elements (grey boxes). These include CAAT and TATA
boxes, wound-responsive elements (W-box, WUN), a dehydration-responsive
element (DRE), ABA-responsive elements (ABRE), and MYB and MYC binding
sites.
Supplemental Figure 2
abi2
A
C
A
W
RNS1
EF1-a
B
ABA
C
12h
24h
Wnd
12h
RNS1
Supplemental figure S2: (A) Induction of RNS1 by ABA treatment is dependent of
abi2 signaling. Northern blot analysis of RNA extracted from abi2 seedlings 4
hours after wounding (W) or after treatment with 100 μM ABA for 4 h (A) or water
(C). (B) Increase in RNS1 activity in response to ABA. Plants were treated with
100 μM ABA or wounded for the indicated times. Protein extracts were prepared
from these plants and 90 μg of each sample were analyzed. RNase activity was
detected by an in gel activity assay. The band corresponding to RNS1 was
determined by comparison with an extract from plants wounded for 12 h.
Wounding samples are included as positive controls for the induction of RNS1
mRNA and activity.
Supplemental Figure 3
Col
C
W
abi4
C W
Ws
C
W
abi5
C W
RNS1
EF1-α
Supplemental figure S3: Induction of RNS1 by wounding is not regulated by ABI4
and ABI5. Northern blot analysis of RNA extracted from WT (Col or Ws) and abi4
and abi5 mutant seedlings 4 h after wounding. Individual bands were quantified
and normalized using EF-1α as loading control.
Supplemental Figure 4
control
ABA
control
wounding
Supplemental figure S4: Patterns of RNS1 promoter-driven luciferase expression
in response to wounding and ABA. Four-week-old plants were treated with 100 μM
ABA (a, c, right) or wounded (b, d, right). Control plants were mock-treated (a, c,
left), or not treated (b, d, left). Six hours after treatment, plants were imaged using
a CCD camera without illumination to register luciferase activity (c, d). White light
images are also shown (a, b). Arrows in (b) indicate wounding sites. The bar below
panels c-d indicates the arbitrary scale of luciferase activity measured in those
panels.