(A)
(B)
Supplementary Fig. 1 Sequence alignment and Phylogenetic analysis of DJ-1 homologs. (A).
Multiple sequence alignment of DJ1 homologs from A. thaliana, PARK7 and HSP31. (B).
Phylogenetic relationship between the sequences presented in (A). Sequence alignments were
performed in MultAlin (http://multalin.toulouse.inra.fr/multalin/).
(A)
(D)
Mannitol (mM)
0
100
200
300
Polyethylene glycol (%)
400
0
10
20
30
40
Day 0
pBIN-GFP
Hsp31_#1
Day 4
pBIN-GFP
Hsp31_#1
(B)
(E)
70
pBIN-GFP
Hsp31_#1
50
Fresh weight (mg)
Fresh weight (mg)
60
40
30
20
10
0
60
*
50
pBIN-GFP
Hsp31_#1
40
30
20
10
0
0
100
200
300
Mannitol (mM)
400
10
20
30
Polyethylene glycol (%)
40
(F)
pBIN-GFP
Hsp31_#1
800
700
*
600
500
400
300
200
100
0
0
100 200 300
Mannitol (mM)
400
pBIN-GFP
Hsp31_#1
800
Total chlorophyll content
(μg/g tissue)
Total chlorophyll content
(μg/g tissue)
(C)
0
700
600
500
400
300
200
100
0
0
10
20
30
40
Polyethylene glycol (%)
Supplementary Fig. 2 Osmotic stress tolerance in Hsp31 overexpression tobacco plants: (A) Leaf
disc senescence under Mannitol stress. 8 week old tobacco plant leaf discs were kept for 4 days at
different concentrations of Mannitol (0, 100, 200, 300 and 400 mM). (B) Fresh weight of the tobacco
plant leaf discs exposed to Mannitol stress. (C) Total chlorophyll content of the tobacco plant leaf
discs exposed to Mannitol stress. (D) Leaf disc senescence under Polyethylene glycol stress. 8 week
old tobacco plant leaf discs were kept for 4 days at different concentrations of Polyethylene glycol (0,
10, 20, 30 and 40%). (E) Fresh weight of the tobacco plant leaf discs exposed to Polyethylene glycol.
(F) Total chlorophyll content of the tobacco plant leaf discs exposed to Polyethylene glycol stress. The
values are mean ± SD from three independent experiments. The data are means of three independent
experiments. Bars indicate ±SE. Means designated with the asterisks (*) are significantly different
according to DMRT at P<0.05.
(A)
Control
Day 0
10 mM H2O2
10 mM MG
300 mM NaCl
Control
Day 4
10 mM H2O2
10 mM MG
300 mM NaCl
(C)
Fresh weight (mg)
60
50
40
30
20
10
0
*
*****
** ***
* ***
700
Total chlorophyll content
(μg/g tissue)
(B)
600
500
400
*****
**
** *
pBIN-GFP
* * ***
Hsp31_#2
300
Hsp31_#3
200
Hsp31_#4
Hsp31-GFP_#1
100
Hsp31-GFP_#2
0
Supplementary Fig. 3 Hsp31 and GFP fused Hsp31 overexpressing tobacco plants offer abiotic
stress tolerance. (A) Leaf disc senescence assay for Hsp31 and Hsp31-GFP overexpressing plants
under abiotic stress. Stress conditions used are H2O2 (10 mM), MG (10 mM) and NaCl (300 mM). (B)
Fresh weight of the tobacco plant leaf discs exposed to H2O2, MG and NaCl stress. (C) Total
chlorophyll content of the tobacco plant leaf discs exposed to H2O2, MG and NaCl stress. The values
are mean ± SD from three independent experiments. The data are means of three independent
experiments. Bars indicate ±SE. Means designated with the asterisks (*) are significantly different
according to DMRT at P<0.05.
kDa
52
WB
α Hsp31
26
71
51
42
35
CBB
staining
27
Supplementary Fig. 4 Immuno-blot analysis of Hsp31 protein accumulation in tobacco
plants over expressing Hsp31, Hsp31-GFP and Hsp31 mutants (m1, m2 and m3). About 40
μg of total protein from each sample were separated in a 12% SDS-PAGE gel. Blot was
probed with polyclonal anti-Hsp31 antibody (1:2000 dilution) and detected as described in
methods.
#2
#1
#3
Hsp31
#2
Hsp31-m1
pBIN-GFP
#2
#1
#1
#2
Hsp31-GFP
Supplementary Fig. 5 Hsp31 and Hsp31-GFP overexpressing tobacco plants offer
biotic stress tolerance. Symptoms of the brown spot disease in transgenic and control
plants after 7 days post infection with A. alternata, the characteristic necrosis symptom
was visible on the infected tobacco leaves (indicated with arrowheads).
Methyglyoxal (μMol/g tissue)
6000
*
5000
*
4000
3000
*
2000
1000
*
*
*
Control
H2O2
H2O2
MG
Supplementary Fig. 6 Methylglyoxal levels in the transgenic
pBIN-GFP vector control plants after 4 days in response to
Methyglyoxal and 300 mM NaCl. The data are means
experiments. Bars indicate ±SE. Means designated with
significantly different according to DMRT at P<0.05.
Hsp31-m1
Hsp31_#1
pBIN-GFP
Hsp31-m1
Hsp31_#1
pBIN-GFP
Hsp31-m1
Hsp31_#1
pBIN-GFP
Hsp31-m1
Hsp31_#1
pBIN-GFP
0
NaCl
Hsp31, Hsp31-m1 and
10 mM H2O2, 10 mM
of three independent
the asterisks (*) are
(A)
Media
pBIN-GFP
Hsp31_#1
Hsp31-m1
Extract
pBIN-GFP
Hsp31_#1
Hsp31-m1
(B)
1800
*
*
1400
1200
1000
800
400
*
*
*
pBIN-GFP
600
Hsp31-m1
MDA ( nMol/g tissue)
1600
*
200
Control
H
H2O2
2O2
MG
Hsp31-m1
Hsp31_#1
Hsp31_#1
pBIN-GFP
Hsp31-m1
Hsp31_#1
pBIN-GFP
Hsp31-m1
Hsp31_#1
pBIN-GFP
0
NaCl
Supplementary Fig. 7 Malondialdehyde (MDA) levels in the transgenic Hsp31, Hsp31m1 and pBIN-GFP vector control plants after 4 days in response to 10 mM H2O2, 10
mM Methyglyoxal and 300 mM NaCl. (A) MDA content among samples. (B)
Calorimetric quantification of MDA content. Comparison of MDA content. The data are
means of three independent experiments. Bars indicate ±SE. Means designated with
the asterisks (*) are significantly different according to DMRT at P<0.05.
NaCl
0 mM
300 mM
pBIN-GFP
Hsp31_#1
Hsp31-m1
Supplementary Fig. 8 Accumulation of H2O2 in response to NaCl treatments. In situ
detection of H2O2 − by DAB staining in the leaves of Hsp31, Hsp31-m1 and pBINGFP vector control transgenic plants exposed to 300 mM NaCl.
pBIN-GFP
Hsp31_#1
Hsp31-m1
NtCAT1
NtPER12
NtAPX6
NtMSD1
NtCSD1
NtGST21
NtCOX1
NtPR1
NtGNS
NtGAPDH
Supplementary Fig. 9 Semi-quantitative PCR analysis of anti-oxidant genes. Expression
profile of ROS-responsive genes in Hsp31, Hsp31-m1 and pBIN-GFP vector control
transgenic plants in response to H2O2 (10 mM), Methyglyoxal (10 mM) and NaCl (300
mM). CAT1, catalase 1 gene; PER12, peroxidase 12 gene; APX6, ascorbate peroxidase 6
gene; MSD1, manganese superoxide dismutase 1 gene; GST21, glutathione transferase
21 gene; GNS1, beta-1,3-glucanase 1 gene; PR1, pathogenesis-related protein 1 gene;
COX1, cytochrome c oxidase 1 gene; CSD1, copper/zinc superoxide dismutase 1 gene;
GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (internal control).
(A)
LB
NOS
promoter
NOS
35S
terminator promoter
nptII
NOS
terminator
Hsp31
RB
eGFP
pBIN-Hsp31-eGFP
(B)
pBIN-GFP
pBIN-Hsp31- eGFP
0 mM H2O2
5 mM H2O2
Overlay
GFP
(C)
Supplementary Fig. 10 Schematic diagram of the 35S: Hsp31-GFP fusion construct and
Transient expression of 35S:Hsp31-GFP in N. benthamiana leaves. (A) Schematic diagram of
the 35S:Hsp31-GFP fusion construct. LB, left border; RB, right border; 35S Pro, CaMV
35S promoter; Pnos, Nos promoter; Tnos, Nos terminator; nptII, Kanamycin resistance
gene; Hsp31-GFP, S. cerevisiae heat shock protein 31 gene fused with GFP in the C
terminal. (B) Transient expression of 35S-Hsp31-GFP in N. benthamiana leaves. (C)
Subcellular localization of Hsp31. Fluorescence images of the subcellular localization of
Hsp31 by GFP tagging. The sections were made from the transgenic Hsp31 plants which
were pre-exposed to with and without 5 mM H2O2 stress for 24 h. DAPI and Mitotracker
red were used for the labeling of nucleus and mitochondria respectively. Scale : 50 μm