(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) (B) (C) (D) (E) (F) Mannitol (mM) Polyethylene glycol (%) Day 0 0 100 200 300 400 pBIN-GFP Hsp31_#1 Day 0 Day 4 Mannitol (mM) 0 10 20 30 40 Polyethylene glycol (%) (A) (B) (C) (D) (E) (F) Hsp31_#1 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) (B) (C) Day 0 Day 4 Control 10 mM H2O2 10 mM MG 300 mM NaCl pBIN-GFP Hsp31_ #2 Hsp31_ #3 Hsp31_ #4 Hsp31-GFP_ #1 Hsp31-GFP_ #2 (A) (B) (C) * 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.

27 35 71 51 42 kDa 26 52 Marker pBIN-GFP Hsp31_ #2 Hsp31_ #3 Hsp31-GFP_ #1 Hsp31-GFP_ #2 Hsp31-m1 Hsp31-m2 Hsp31-m3 WB α Hsp31 CBB staining 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.

pBIN-GFP Hsp31 Hsp31-GFP Hsp31-m1 #1 #2 #3 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).

H2O2 Supplementary Fig. 6 Methylglyoxal levels in the transgenic Hsp31, Hsp31-m1 and pBIN-GFP vector control plants after 4 days in response to 10 mM H2O2, 10 mM Methyglyoxal and 300 mM NaCl. 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.

Media Extract pBIN-GFP Hsp31_#1 Hsp31-m1 Blank Control H2O2 MG NaCl (A) (B) Supplementary Fig. 7 Malondialdehyde (MDA) levels in the transgenic Hsp31, Hsp31- m1 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 pBIN-GFP vector control transgenic plants exposed to 300 mM NaCl.

NtCAT1 NtPER12 NtAPX6 NtMSD1 NtGNS NtGST21 NtPR1 NtCOX1 NtCSD1 NtGAPDH Water 10 mM H2O2 300 mM NaCl 10 mM MG pBIN-GFP Hsp31_#1 Hsp31-m1 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) (B) (C) 0 mM H2O2 5 mM H2O2 GFP Overlay nptII Hsp31 NOS terminator 35S promoter RB LB pBIN-Hsp31-eGFP eGFP (A) (B) pBIN-GFP pBIN-Hsp31- eGFP (C) 0 mM H2O2 5 mM H2O2 GFP Overlay 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