ORF
cDNA
M
Genomic
A
1Kb
750bp
B
5’atggctgacgcgaacgcccccttcgctctcgtcagccgcctctccccggccgcgcgcctg
M A D A N A P F A L V S R L S P A A R L
cccatccgtgcctggagggccgcgaggccagcgccgctctcgaccggcgggagaacccgc
P I R A W R A A R P A P L S T G G R T R
ccgctctccgtggcctccgcggcgcaggagaacagggacaactccgtcgacgtccaagtc
P L S V A S A A Q E N R D N S V D V Q V
agccaggcccagaacgccggaaaccagcagggcaacgcagtccagcgccgccctcgtcgc
S Q A Q N A G N Q Q G N A V Q R R P R R
gctggatttgacatctccccgttcgggctagtggacccgatgtcgccgatgaggacgatg
A G F D I S P F G L V D P M S P M R T M
cggcagatgcttgacacgatggaccggctgttcgacgacgccgtggggttccccacggcg
R Q M L D T M D R L F D D A V G F P T A
cggcgctcgctggcggcggcgagcgagatgccgcggatgccgtgggacatcatggaggac
R R S L A A A S E M P R M P W D I M E D
gacaaggaggtgaagatgcggttcgacatgcccgggctgtcgcgggaggaggtgaaggtg
D K E V K M R F D M P G L S R E E V K V
atggtggagggcgacgcgctcgtcatccgcggcgagcacaagaaggaggccggcgaaggc
M V E G D A L V I R G E H K K E A G E G
cagggcgaagcagcggaaggcggcgacgggtggtggaaggagcgcagcgtgagctcctac
Q G E A A E G G D G W W K E R S V S S Y
gacatgcgcctggctctgccggacgagtgcgacaagagtcaggtgcgcgccgagctcaag
D M R L A L P D E C D K S Q V R A E L K
aacggcgtgctgctcgtgtccgtgcccaagagggagaccgagcgcaaggtcatcgacgtg
N G V L L V S V P K R E T E R K V I D V
Aaggtccagtga 3’
K V Q -
Supplementary Fig. 1. (A) PCR amplification of wheat TaHSP26 genomic, cDNA and ORF
clones.
(B) ORF and protein sequence of TaHSP26. An arrowhead indicates the putative
processing site. Nucleotide sequence in dark blue box represents chloroplast transit
peptide (AA 1-44) and conserved α-crystallin domain (ACD) (136-230), respectively,
and sequence in red box show conserved amphipathic α-helix.
Td
Tm
Ta-g
810
Ta-e
Ta-mTdu
Ta-i 965
175
456
Hv
384 124
Cr
As sb
Zm
Os
Ps
622
1000
1000
Rc
Gm 996
999
Pt
993
998
625
441
1000
340
308
At
Ga
801
Nt
Ph
Sm
817
998
1000
Le
1000
Vv
Fh
Pp
0.1
Supplementary Fig. 2. Phylogenetic analysis of chloroplastic HSP26. The tree was constructed using
CLUSTALX by Neighborhood joining method, with the respective bootstrap values.
Ta-g (Triticum aestivum g allele, AAC 96315.1)
Ta-e (Triticum aestivum e allele, AAC 96314.1)
Ta-m (Triticum aestivum m allele, AAC 96317.1)
Ta-i (Triticum aestivum i allele, AAC 96316.1)
Tm (Triticum monococcum, CAI 96517.1)
Td (Triticum dicoccum, CAI 96514.1)
Tdu (Triticum durum, CAI 96515.1)
Hv (Hordeum vulgare, AAB 28590.1)
As (Agrostis stolonifera, AAN 74538.1)
Sb (Sorghum bicolor, XP_002468181.1)
Vv (Vitis vinifera, XP_002272418.1)
Gm (Glycine max, CAA30168.0)
At (Arabidopsis thaliana, NP_194497.1)
Rc (Ricinus communis, XP_002532054.1)
Ps (Pisum sativum, CAA30167.1)
Nt (Nicotiana tabacum, BAA29065.1)
Ga (Gossypium arboreum, ABI97958.1)
Pp (Physcometrilla patens, XP_001767809.1)
Fh (Fumaria hygrometrica, AAF19021.1)
Sm (Selaginella moellendorffii, XP_002963639.1)
Zm (Zea mays, NP_001105583.1)
Os (Oryza sativa, NP_001049541.1)
Pt (Populus trichocarpa, XP_002318460.1)
Cr (Chlamydomonas reinhardtii, XP_001701015.1)
A
B
Supplementary Fig. 3. Location of chloroplastic TaHSP26 on wheat homologous
chromosomes.
BLAST against DNA/protein collections mapped in, or associated with, plant
genomes (PLANTBLAST) NCBI/ MapViewer
WT
3.1 3.2
5.1
5.3
6.2
6.3
6.4
7.1
7.3
A
B
C
Supplementary Fig. 4. Analysis of different T3 homozygous Arabidopsis transgenic
lines over-expressing chloroplastic TaHSP26. RT-PCR of NPTII gene (A),
andTaHSP26 gene (B) and Western analysis with anti TaHSP26 antibodies
(C).
A
WT-C WT-HS
AS2
AS6
AS10 AS11 AS12
AS13
AS14
AS15 AS16 AS18
At HSP26
At actin 1
B
At HSP26
WT-HS
AS2
AS6
AS10
AS11
AS12
AS13
AS14
AS16
AS18
Supplementary Fig. 5. Induction of AtHSP26 gene in various antisense lines. (A) Semiquantitative RT-PCR and (B) Western analysis after 2h heat stress at 39oC.
A
M
L1
L2
L3
L4
L5
B
M
L1
L2
L3
L4
L5
3000bp
3000bp
1000bp
1000bp
C
Wheat Actin gene
Wheat HSP26
cDNA
D
3000bp
1000bp
Supplementary Fig. 6. Cloning of putative promoter of Ta HSP26 through directional genome walking.
L1-L5 represents different adapter ligated genomic libraries. (A) Result of first walking, a
~1200 bp fragment was cloned and sequenced.(B) Result of second walking, a ~1500 bp
fragment was cloned and sequenced. (C) Nucleotide BLAST result of combined sequence of
first and second genome walking (2718 bp). (D) Final putative promoter of TaHSP26-G
of1514 bp length.
Promoter activity after heat stress
Relative fold change
A
100
80
60
40
20
0
Heat stress
B
C
10’
20’
30’
40’
50’
60’
2h
24h
C
10’
20’
50’
24 h
Supplementary Fig.7. Induction of GUS gene governed by TaHSP26, P26 promoter in
transgenic Arabidopsis plants at different time points of heat stress. (A & B)
quantitative RT-PCR and semi-quantitative RT-PCR for GUS transcript, and GUS
histochemical assay (C).
A
D
B
C
E
F
K
L
G
H
I
J
Supplementary Fig. 8. Analysis of P26 promoter of TaHSP26 activity in different
tissues of transgenic Arabidopsis plants after a 2 h heat stress
treatment at 37oC. (A) Control inflorescence, (B) GUS accumulation in
inflorescence at various stages, (C-D) GUS in a mature flower and
petals and (E-F) in pollen. (G) wild type silique, (H&I) mature silique
from transgenic plants, (J) developing seeds and mature seeds from
transgenic plants (K & L).