Supple Figs for Plant Cell-R3.ppt · 9/4/2013 · The expression of glutaredoxin, HSFA2, CRF2,...
8
Supplemental Figure 1. Inhibition of SAA to HL or HS by catalase. Related to Figure 1. (A) Experimental design showing the application of stress to local leaves (arrow), the placement of water or catalase between the local and systemic tissue (circle), and the subsequent application of stress to systemic leaves (jagged arrow). (B) Images showing the protection of systemic leaves from HL in acclimated plants and its inhibition by catalase (two independent experiments). (C) Same as (B), but for HS (two independent experiments). HL Exp 1 Exp 2 Exp 1 Exp 2 HS Control -Catalase Acclimation -Catalase Control +Catalase Acclimation +Catalase HL or HS treatment Control, HL or HS acclimation Control/Acclimation under +/-Catalase Water or Catalase Control -Catalase Acclimation -Catalase Control +Catalase Acclimation +Catalase A B C Bar=5 mm Bar=5 mm Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Transcript of Supple Figs for Plant Cell-R3.ppt · 9/4/2013 · The expression of glutaredoxin, HSFA2, CRF2,...
Supplemental Figure 1. Inhibition of SAA to HL or HS by catalase. Related to Figure 1.
(A) Experimental design showing the application of stress to local leaves (arrow), the
placement of water or catalase between the local and systemic tissue (circle), and the
subsequent application of stress to systemic leaves (jagged arrow).
(B) Images showing the protection of systemic leaves from HL in acclimated plants and its
inhibition by catalase (two independent experiments).
(C) Same as (B), but for HS (two independent experiments).
HL
Exp 1
Exp 2
Exp 1
Exp 2
HS
Control-Catalase
Acclimation-Catalase
Control+Catalase
Acclimation+Catalase
HL or HS treatment
Control, HL or HS acclimation
Control/Acclimation under +/-Catalase
Water or Catalase
Control-Catalase
Acclimation-Catalase
Control+Catalase
Acclimation+Catalase
A B
C
Bar=5 mm
Bar=5 mm
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Supplemental Figure 2. Detection of H2O2 in local (cotyledons) and systemic (root tip) tissues in WT
and rbohD seedlings in response to local application of 1 mM H2O2. Related to Figure 2.
(A) Experimental design showing the application of H2O2 to local cotyledon tissue (top dashed box) and
the detection of H2O2 in systemic root tissue with Amplex® Red (bottom dashed box).
(B) Images showing the detection of H2O2 in local and systemic tissues of WT and rbohD seedlings (two
independent experiments). H2O2 was imaged with 2 M Amplex® Red.
WT
rbohD
Bright field
Amplex Red
Bright field
Amplex Red
Control H2O2
Bright field
Amplex Red
Bright field
Amplex Red
H2O2Control
Local
Systemic WT
rbohD
A B
Local
Systemic
Bar=1 mm
Exp 1 Exp 2 Exp 1 Exp 2
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Supplemental Figure 3. Expression of stress response transcripts in systemic tissues. Related to
Figure 4.
The expression of glutaredoxin, HSFA2, CRF2, GPT2, JAS1, JAZ6, AOC3 and PBP1 was
determined using qRT-PCR analysis in systemic tissues of plants sampled at 15 or 45 min
following heat stress (HS), high light stress (HL) or wounding (Wound) (see, “Stress Treatments for
Microarray and Metabolome Analyses” in “Methods”). Transcript level relative to control (Ctrl)
corresponding to each stress is shown (n=3). Error bars indicate SD. **, Student’s t-test significant
at P<0.01.
Glutaredoxin (At4g15680) HSFA2 (At2g26150)
JAS1 (At5g13220) JAZ6 (At1g72450)
CRF2 (At4g23750) GPT2 (At1g61800)
AOC3 (At3g25780) PBP1 (At5g54490)
** **
**
****
** **
**** **
**
**** **
45 min 15 min 45 min
15 min 45 min 15 min 45 min
15 min 45 min 15 min 45 min
15 min 45 min 15 min 45 min
Rel
ativ
e tr
ansc
ript
leve
l
Rel
ativ
e tr
ansc
ript
leve
l
Rel
ativ
e tr
ansc
ript
leve
l
Rel
ativ
e tr
ansc
ript
leve
l
Rel
ativ
e tr
ansc
ript
leve
l
Rel
ativ
e tr
ansc
ript
leve
lR
elat
ive
tran
scrip
t le
vel
12
14
10
8
6
4
2
0Rel
ativ
e tr
ansc
ript
leve
l 3500
3000
2500
2000
1500
1000
500
0
2.5
2.0
1.5
1.0
0.5
0.0
16
14
12
108
6
4
2
0
12
10
8
6
4
2
0 0.0
1.0
2.0
3.0
4.0
5.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.00.51.01.52.0
2.53.03.54.04.5
15 min
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Supplemental Figure 4. Categorization of expression patterns of the transcripts elevated by heat stress
(HS), high light stress (HL) or wounding (Wound) in systemic tissues. Related to Figure 4.
Transcripts upregulated by each stress were divided into three groups; (i) transcripts responsive to H2O2
(H2O2 responsive), (ii) transcripts responsive to O2- and/or 1O2, but not to H2O2 (Other ROS responsive) and
(iii) transcripts not responsive to ROS (No ROS responsive). Transcripts that belong to each group were, then
categorized into three different expression patterns that are indicated as red, blue or green lines in graphs.
Blue: transcripts that showed maximum expression level at 15 min following stress treatment. Red: transcripts
that were continuously elevated by 15 and 45 min stress treatment. Green: transcripts that spiked at 45 min
following stress treatment. The number of transcripts that belong to each expression pattern was counted
and the percentage relative to the total number of transcripts in each group is shown in the different graphs.
60.9
11.8
Other ROS responsive No ROS responsive
27.2
65.6
13.4
21.0
75.7
5.4
18.9
26.6
30.9
42.5
60.9
13.0
26.1
80.0
0
20.0
H2O2 responsive
34.7
40.4
24.9
35.7
30.4
33.9
33.3
16.7
50.0
Exp
ress
ion
patte
rn(H
S)
Exp
ress
ion
patte
rn(H
L)E
xpre
ssio
n pa
ttern
(Wou
nd)
Time (min) Time (min) Time (min)
Time (min) Time (min) Time (min)
Time (min) Time (min) Time (min)
0 15 45
0 15 45
0 15 45 0 15 45
0 15 45
0 15 45
0 15 45
0 15 45
0 15 45
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Supplemental Figure 5. Suppression of metabolites in systemic tissues of plants subjected to a local
wound stimulus. Related to Figure 4 and 10.
The level of metabolites was determined using LC-MS analysis in systemic tissues sampled at 15 and
45 min following injury (see “Methods”). b-Aminoisobutyric acid, ethanolamine, homoserine and
sarcosine were suppressed in systemic tissues in response to local wounding (n=5). Error bars
indicate SE. **, Student’s t-test significant at P<0.01.
**
b-A
min
oiso
buty
ric a
cid
(% o
f Con
trol
)
Time (min)
ControlWound
Eth
anol
amin
e (%
of C
ontr
ol)
Time (min)
**
ControlWound
Hom
oser
ine
(% o
f Con
trol
)
Time (min)
**
ControlWound
Time (min)
Sar
cosi
ne (
% o
f Con
trol
)
**
ControlWound
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
MBF1c-GFP DAPIDIC Merge0.5
0.4
0.3
0.2
0.1
0.0
No.
of n
ucle
i (G
FP
)N
o. o
f nuc
lei (
DA
PI)
Bar=25 m
Supplemental Figure 6. Nuclear localization of MBF1c in systemic tissues during SAA to HS is
attenuated by catalase. Related to Figure 8.
(A) Images of root tips showing inhibition of nuclear localization of the MBF1c-GFP protein by
catalase during SAA to HS. Experimental design is similar to Figure 6A.
(B) Quantification of MBF1c-GFP nuclear localization in control and systemic tissues of seedlings
subjected to a local HS stimulus (n=100) with or without catalase application. Error bars indicate SD.
* or **, Student’s t-test significant at P<0.05 or P<0.01, respectively.
**
*
A B
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Time (min)
WT rbohD
Time (min)
Time (min)
1
-1
0
1 2 3 4 50
4
-4
0
5 10 15 200
1 2 3 4 50
1
-1
0
A B
-45 10 15 200
4
0
Vol
ts
Vol
tsV
olts
Vol
ts
Time (min)
HL
R
A
Supplemental Figure 7. Association between the ROS wave and systemic potential variations during
the SAA response of plants to HL stress. Related to Figure 9.
(A) Experimental design used to monitor changes in systemic potential variations using phloem-
feeding green peach aphids (Myzus persicae) placed on a systemic leaf of wild-type (WT) and rbohD
plants subjected to a local HL stimulus.
(B) Representative phloem systemic potential variation plots from WT and rbohD plants subjected to a
local HL stimulus. The period of HL stress is indicated in yellow and the systemic HL signal is
highlighted in gray (n=30).
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
Target gene Forward primer Reverse primer
EF1-a GAGCCCAAGTTTTTGAAGA TAAACTGTTCTTCCAAGCTCCAglutaredoxin TGATCTCCGAGAAGTCGGTAGTG ATCGTTGGATTCACGCCAAAHSFA2 GCAGGACAAAAGCATCTCTTGA CCCGTCGAAACCGTATTGCCRF2 GGAAGAGCCATCCATGACAAC AACCACTGAGCTCCGGAAATCGPT2 CCATTTTCTATTGCCGTGGAA GTGCCACTACCCACCAGACAJAS1 CCGGTCCACGCGTCTCT TTCACCAGCTTTGTTACGAGACAJAZ6 CCAAGCCAGAGATGGTTGCT TCCATCGGCATATGGTTATGGAOC3 GGTGCGTACGGACAGGTTAAG CGTAACCGCCGTTCCAGTAAPBP1 TGGAGAAGGTCTGATCGAAGAGA CAAGAACCGTCGATGCGTTT
Supplemental Table 1. Primer pairs used for qRT-PCR. Related to Figure 4A and Supplemental Figure 3 online.
Supplemental Data. Suzuki et al. (2013). Plant Cell 10.1105/tpc.113.114595
