Table S1. ESTs of putative bag genes from various plants (obtained from the EST

database search using default parameters of NCBI BLAST).

Plant

name

Library description EST

Accession number

Amino acid

similarity to BD of

AtBag4 (%)

ABIOTIC barley

(Hordeum

vulgare)

sorghum

(Sorghum

bicolor)

soybean

(Glycine max)

sugar beet (Beta

vulgaris)

grape (Vitis

vinifera)

BIOTIC barley

(Hordeum

vulgare)

grape (Vitis

vinifera)

legume

(Medicago

truncatula)

TISSUE - hot pepper

(Capsicum

annuum)

legume

(Medicago

truncatula)

sorghum

(Sorghum

bicolor)

grape (Vitis

vinifera)

STRESS seedling shoot (2 days of cold

stress)

mix of 5-week old plants (7 and

8 days of water stress)

3 days drought stressed leaf

tissue of one month old plants

4-day germinated seedlings

under stress (salt/NaCl,

dehydration/Mannitol and

anaerobic stress) mixed 8, 9, 11, 13, 15, 16 weeks

abiotic stressed berries

STRESS seedling green leaf (Blumeria

challenged)

mid-season development leaves

infected with the bacterial

pathogen Xylella fastidiosa

elicited root tissues derived cell

culture (induced with yeast cell

wall extracts)

SPECIFIC EXPRESSION flower bud

developing flower

ovary

developing preanthesis pannicles

berries (post-veraison stage)

fruit (green stage)

BF622329

BF622327

BE592352

BE593047

BG043600

BI073238

CB912188

BE216889

CB342561

BF646049

BM066508

BI272171

BG356714

BI140027

CB980485

CB980084

CB980018

BQ799356 BQ799253

60

62

67

67

59

43

77

62

54

59

73

62

62

48

79

79

79

66

65

cotton bolls

(Gossypium

arboreum)

potato (Solanum

tuberosum)

wheat (Triticum

aestivum)

soybean

(Glycine max)

apple (Malus x

domestica)

pine (Pinus

pinaster)

berries (veraison stage)

fibers isolated from bolls

mature tuber

roots

roots of five day old etiolated

seedlings

seedling shoot

immature seed coats

cultured shoots

differentiating xylem

BQ798817

BQ798556

BQ798294

CB347936

BQ403711

BF268630

BF460358

BM108950

BE405084

BE426477

AI960691

AU223481

BX249429

65

65

54

65

44

42

56

58

64

62

59

63

61

Methods

In Vitro Protein Binding Assays

The plasmid pGEX2T-701, a GST fusion of the Arabidopsis hsc70-1 gene (accession

number – X74604), was kindly provided by C. Guy, University of Florida. GST-Hsc70

was expressed in E. coli BL21 cells as described (Takayama et al., 1995) and purified

using Glutathione Sepharose 4B in accordance with the Amersham Health Inc. protocol.

A GST-Bcl-2 fusion protein was obtained from T. Ostersdolf (Idun Pharmaceuticals,

Inc.).

To determine whether or not the BAG domain of AtBAG4 is responsible for binding to

Hsc70, site-directed mutagenesis of this domain was performed. Two binding residues

were substituted using following primer pairs: 5’-

CCGCTCGAGCGGCATTGTCGAAAAACGATGATGC-3’ + 5’-

TCATAAGAAGCCCTGCAGCCATG-3’ and 5’-CATGGCTGCAGGGCTTCTTATGA-

3’ + 5’-GCTCTAGAGCATGTCAGTCAAATTTCTCCCAATC-3’ (AtBAG4m1:

Glu179 (Fig. 2B) (corresponding residue of hBAG1 E212, Fig. 2A) ! Gly; A XhoI site

was introduced at the 5’ end and a XbaI site at 3’ end ). AtBAG4m1 was cloned into

TOPO vector and amplified in E coli (Invitrogen). Isolated plasmids were used as

template for introduction of the second point mutation. The second point mutation was

introduced with following primer pairs: 5’-

GGTGAATTCATGATGCATAATTCAACCGAA-3’ + 5’-

ATTCCGGACAATTTGAGCAGCTGCCTCATA-3’ and 5’-

TTGTCCGGAATTGAGGCTGAAGGGGACG-3’ + 5’-

CCGCTCGAGTCAGTCAAATTTCTCCCAATC-3’ (AtBAG4m1m2: Asp189 (Fig. 2B)

(corresponding residue in hBAG1 D222, Fig. 2A) ! Ser by introducing a BspE1 site;

EcoR1 at 5’ and XhoI at 3’ end). The deletion of the BAG domain was made using

following primer pairs: 5’-GGTGAATTCATGATGCATAATTCAACCGAA-3’ + 5’-

TCCCCCCCGGGCACCGCAACTTGCGTCCCTCCA-3’ and 5’-

TCCCCCCCGGGAACTTGCAGGAGGCTGTGGATA-3’ + 5’-

CCGCTCGAGTCAGTCAAATTTCTCCCAATC-3’ (BAG-del: all conserved amino

acid residues within the BAG domain removed (Fig. 2A) + XmaI site introduced; An

EcoR1 site introduced at 5’ end and a XhoI at 3’ end). AtBAG4m1m2 (BAG-mut) and

BAG-del PCR products were digested, ligated into pCDNA3(+) and cloned into E. coli.

In vitro translated, [35S] labeled AtBAG4, BAG-mut and BAG-del were synthesized from

pAtBAG7.1 (wt), BAG-mut in pCDNA3(+) and BAG-del in pCDNA3(+) using TnT

Quick Transcription/Translation System (Promega). 2 !g of pAtBAG7.1 plasmid DNA

was incubated with 40 µl of TnT Quick Master Mix containing 20 µCi of [35S]methionine

at 30°C for 90 min. The purified GST-proteins were immobilized with 10 µl of

Glutathione-Sepharose beads and incubated with 5 !l in vitro translated AtBAG4 in 275

µl of binding buffer (50 mM Tris-HCl, ph 7.6, 150 mM NaCl, 0.1% Triton X-100) at 4°C

for 3 h. The beads were washed three times with 1 ml of binding buffer, resuspended in 5

!l of SDS-loading buffer, boiled for 3 min., and subjected to SDS-PAGE, followed by

exposure to x-ray film. The use of equivalent amounts of GST-fusion proteins and in

vitro translated AtBAG4 was confirmed by SDS-PAGE analysis, followed by Coomassie

staining, or autoradiographically, respectively.

Plasmid Constructions and Plant Transformation

The following PCR primers: 5’–TGTCGAAAAATCATGATGCATAATTCAACCG –

3’ and 5’–CCAAGGCAACGGAGCTCTACTAAAATGTC–3’, were designed such that

a BspH1 site introduced at the 5’-end of the AtBAG4 cDNA and Sac-I site at the 3’-end.

PCR was performed with pAtBAG7 plasmid DNA. The PCR product was digested with

BspH1 and Sac-I and subcloned into the plant expression cassette pRTL2. The expression

cassette containing atbag4 under the control of the cauliflower mosaic virus 35S

promoter and the Agrobacterium nopaline synthase terminator was subcloned into the

binary vector pPZP211 as HindIII fragment. The resulting construct is referred to as

pZPRTL2BAG. The binary vector pZPRTL2BAG was mobilized into Agrobacterium

tumefaciens strain C58C1 by triparental mating for further tobacco transformation.

Agrobacterium transconjugants were selected on YEP medium supplemented with

rifampicin (50 mg/liter), gentamycin (50 mg/liter), spectinomycin (100 mg/liter), and

streptomycin (100 mg/liter). Tobacco leaf tissue (cv. Xanthii, genotype NN) was

transformed and plants were regenerated as described (Horsch et al., 1985).

Analysis of gene expression

Total RNA from different tissues (leaf, stem, flower, and root), leaves at different ages,

and at different time points after heat treatment or after cold stress, was extracted using

TRIzol reagent (Invitrogen). For first-strand cDNA synthesis, RT-PCR was performed

using the following components: reverse transcription buffer (50 mM Tris-HCl ph 8.3, 50

mM KCl, 10 mM MgCl2, 10 mM DTT, and 0.5 mM spermidine), 400 µM of each dNTP,

40 µM random primer, 2 µg total RNA, and DEPC-treated water added up to 25 µl final

reaction volume. AMV Reverse Transcriptase (10 units, FisherBioReagents) was added

and the reaction incubated at 55°C for 30 min. The resulting RT-PCR fragments were

amplified using specific primers designed from the coding region and spanned the

intron/exon junctions of atbag4 (5` - TTCTACGTACCTCAGC-CTTACGC - 3` and 5` -

ACGCCACTTTCGGGGATGTAAAG - 3) or atbag6 (5` -

TGCTGTCGAGGGATTGCATCC - 3` and universal primer 5` -

TCACTAGCATTCTCCGGGTCC - 3) to eliminate possible DNA contamination.

Expression from the Arabidopsis actin1 gene using specific primers also spanning

intron/exon junctions (5` - TCGTACTACCGGTATTGTGCTCGAC - 3` and 5` -

TTTGCGATCCACATCTGCTGGAAGG - 3`) were used to ensure equivalent RNA

loading.

Figure Legends

Figure S1. Gene structure of the Arabidopsis BAGs. Similar exon-intron organization

within the BAG were found in genes, the products of which are predicted to localize in

the same subcellular compartment (see text for details).

Figure S2. In vitro binding assay of AtBAG4 to Arabidopsis Hsc70. GST-Hsc70 was

immobilized on glutathione-Sepharose beads and tested for in vitro binding to in vitro

translated 35S-labeled AtBAG4. Samples were analyzed by autoradiography to detect

bound AtBAG4 to the immobilized Hsc70 protein (“pull-down”). Binding of AtBAG4 to

GST coupled to glutathione-Sepharose beads was used as an unspecific binding control.

In vitro translated 35S-labeled AtBAG4 was loaded in the gel to compare the amount of

input versus bound AtBAG4 (“supernatant”). BAG: AtBAG4, BAG-mut: AtBAG4 with

two mutated binding residues, BAG-del: AtBAG4 without BAG domain.

Figure S3. RT-PCR analysis of atbag4 and atbag6 expression patterns. A. The atbag4

and atbag6 transcript abundance in various tissues of flowering Arabidopsis plants.

Arabidopsis leaf tissue of prior to and during flowering plants was also included to

compare atbag4 and atbag6 expression in different developmental stages. B. atbag4 and

atbag6 transcripts levels are differentially regulated by cold treatment. Arabidopsis

flowering plants were cold treated at -20ºC for 10 min. and leaf tissue samples were

collected at 0 min., 20 min., and 90 min. after exposure. C. atbag4 and atbag6 transcripts

levels are differentially regulated by heat treatment. Arabidopsis plants were heat treated

and leaf tissue samples were collected at 0 min., 30 min., 60 min., and 90 min. of

exposure to 37ºC. Actin-1 expression was used as a control of total RNA equal loading to

RT-PCR reaction mixes.

Figure S4. Oxidative stress resistance in atbag4 low and intermediate level expressing

transgenic tobacco plants. Phenotypes of detached wild-type and selected transgenic (L-2

and I-5) tobacco leaves after two weeks of 5mM menadione treatment.

Figure S5. Phenotypes of UV-irradiated leaf discs from wild-type and transgenic

tobacco plants: L-2, I-5 and H-12 (atbag4 high level expressing line) one week after

treatment with UV-C light (32kj/m2).

Figure S6. Mean number of live leaf discs from wild-type and transgenic tobacco

plants L and I lines after 24 and 48 h of 10 !M paraquat treatment. Leaf discs were

scored as dead when their disc areas were stained more than 20 percent by Evans blue.

Figure S7. Freezing tolerance in low-level atbag4 expressing line. A. Chlorophyll a

and b contents in wild-type and transgenic leaves 24 h. after the cold stress at -20°" for

10 min. Chlorophyll was extracted with 80% aqueous acetone. The concentrations of

chlorophyll were determined spectrophotometrically at 652 nm. The content is expressed

as a percentage of the mock leaves. B. Mean number of live leaves after 40h cold stress

at -20°" for 6 min. Leaves were scored as dead when their leaf areas were stained more

than 20 percent by Evans blue.

Figure S8. DNA fragmentation in cold stressed wild-type and low level atbag4

expressing tobacco leaves 2h. after cold treatment at -20°" for 10 min. Ethidium

bromide-stained agarose gel of DNA isolated from the transgenic line L-17 (lane1) and

wild-type tobacco (lane 2 and 3) after cold stress, and L-17 (lane 4) and wild-type

tobacco (lane 5) without any treatment.

Figure S9. RT-PCR data of atbag4 and atbag6 expression patterns in response to (A) B.

cinerea stress, and (B) hormone treatment; in (C), atbag4, atbag5, and atbag6

expression pattern in response to divalent metal ions treatment are shown( lane5, the

plants were pretreated with EDTA 12 h. before Ca2+ treatment). Arabidopsis plants

without any treatment exposure were used as a reference control. Actin expression was

used as an equal loading control.

Supplementary fig. 1

Supplementary fig.2

Supplementary fig.3

Supplementary fig.4

Supplementary fig.5

Supplementary fig.6

Supplementary fig.7a

Supplementary fig.7b

Supplementary fig.8

Supplementary fig.9