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) ( ), respectively, and sequence in red box show conserved amphipathic α-helix. 5’ 3’ A B cDNAGenomic M ORF 1Kb 750bp

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 )At (Arabidopsis thaliana, NP_ ) Ta-e (Triticum aestivum e allele, AAC )Rc (Ricinus communis, XP_ ) Ta-m (Triticum aestivum m allele, AAC )Ps (Pisum sativum, CAA ) Ta-i (Triticum aestivum i allele, AAC )Nt (Nicotiana tabacum, BAA ) Tm (Triticum monococcum, CAI )Ga (Gossypium arboreum, ABI ) Td (Triticum dicoccum, CAI )Pp (Physcometrilla patens, XP_ ) Tdu (Triticum durum, CAI )Fh (Fumaria hygrometrica, AAF ) Hv (Hordeum vulgare, AAB )Sm (Selaginella moellendorffii, XP_ ) As (Agrostis stolonifera, AAN )Zm (Zea mays, NP_ ) Sb (Sorghum bicolor, XP_ )Os (Oryza sativa, NP_ ) Vv (Vitis vinifera, XP_ )Pt (Populus trichocarpa, XP_ ) Gm (Glycine max, CAA )Cr (Chlamydomonas reinhardtii, XP_ )

BLAST against DNA/protein collections mapped in, or associated with, plant genomes (PLANTBLAST) NCBI/ MapViewerNCBIMapViewer Supplementary Fig. 3. Location of chloroplastic TaHSP26 on wheat homologous chromosomes. A B

Supplementary Fig. 4. Analysis of different T 3 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). WT A B C

Supplementary Fig. 5. Induction of AtHSP26 gene in various antisense lines. (A) Semi- quantitative RT-PCR and (B) Western analysis after 2h heat stress at 39 o C. WT-C WT-HS AS2 AS6 AS10 AS11 AS12 AS13 AS14 AS15 AS16 AS18 At HSP26 At actin 1 WT-HS AS2 AS6 AS10 AS11 AS12 AS13 AS14 AS16 AS18 A B At HSP26

M L1 L 2 L3 L4 L5 3000bp 1000bp A 3000bp M L1 L 2 L3 L4 L5 B 3000bp 1000bp D 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. C Wheat HSP26 cDNA Wheat Actin gene

10’ 20’ 50’ 24 h C 10’ 20’ 30’ 40’ 50’ 60’ 2h 24h Promoter activity after heat stress Relative fold change Heat stress A B 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). C

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 37 o C. (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). A B C D E F G H I J K L