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Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stress-related events, defense gene expression, and the hypersensitive response

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SUPPLEMENTAL MATERIAL

SUPPLEMENTAL FIGURES

0 10 20 30 40 50

Total catalase activity (U.mg-1proteins) 0

20 40 60

H2O2half-life (s)

0 2 4 6 8

Total peroxidaseactivity (U.mg-1proteins) Col-0 pad2-1

A B C

Col-0 pad2-1 Col-0 pad2-1

Supplemental Figure S1. Measurement of H2O2 half-life, total peroxidase and catalase activity in Col-0 and pad2-1. A, Half-life of H2O2 was determined by measuring the absorbance of H2O2 at 240 nm. B, Total peroxidase activity determined by measuring the absorbance of oxidized guaiacol substrate at 470 nm. C, Total catalase activity determined by measuring the absorbance of H2O2 at 240 nm. Results are the mean of three independent experiments. No significant difference was observed between Col-0 and pad2-1 for assays of H2O2 half-life, peroxidase and catalase activity.

0 2 000 4 000 6 000 8 000 10 000 12 000 14 000 16 000 18 000

water OG cPTIO + OG

NO production (relative fluorescence) Col0

pad2

*

water OG OG + cPTIO

Col-0 pad2-1

Supplemental Figure S2. Intracellular NO production at 8 h after treatment with OG in Col-0 and pad2-1 plants. Leaf discs were vacuum-infiltrated with 2.5 g L-1 OG and 20 µM of NO- specific fluorescent probe DAF-2DA ± 500 µM NO scavenger cPTIO. NO production was monitored by measuring DAF-2T fluorescence. Values are means ± SE of eight parallels in a representative experiment out of three independent ones. Asterisk indicates statistically significant difference between OG-elicited Col-0 and pad2-1 plants, using Student’s t test (p

< 0.05).

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-100 000 -50 000 0 50 000 100 000 150 000 200 000

0 10 20 30 40 50 60

Plasma membrane potential changes (relative fluorescence)

Time (min) Col-0 + OG Col-0 + OG + Nif

0 20 40 60 80 100 120

0 10 20 30 40 50 60 70 80 90

H2O2production (%)

Time (min) Col-0 + OG Col-0 + OG + Nif control

A

B

Supplemental Figure S3. Effect of the anionic channel inhibitor niflumic acid (Nif) on OG- induced changes of plasma membrane potential and H2O2 production in Col-0 cell suspensions. A, Cells were incubated with the voltage-sensitive fluorescent probe DiBAC4(3) (10 µm) in the dark and were treated with 200 µm niflumic acid 10 min before 0.5 g L-1 OG treatment. Plasma membrane depolarization was monitored by following the variation of DiBAC4(3) fluorescence. Values are means of three measurements of a representative experiment out of three independent ones. B, Effect of niflumic acid on OG-dependent H2O2 production in Col-0 cell suspensions. Cells were treated with 200 µM niflumic acid 10 min before OG treatment (0.5 g L-1). H2O2 production was measured using chemiluminescence of luminol. Data are expressed in percentage of maximal H2O2 production in OG-treated cells and are means of three measurements ± SE. Results are representative of two independent experiments.

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0 10 20 30

40 CYP79B2

0 100 200 300

400 CYP81F2

0 500 1.000 1.500 2.000

PAD3

Relative transcript accumulation

0 2 4 6 8 10 12

14 CYP79B3

*

mock P. brassicae

Col-0 pad2-1 Col-0 pad2-1

#

#

#

#

#

#

Supplemental Figure S4. Expression of genes involved in camalexin and glucosinolate biosynthesis in response to P. brassicae at 24 hpi. Plant leaves were infected by agar plug inoculation of 8-day-old mycelium. Mycelium-free plugs were used for mock treatment. The transcript accumulation of CYP79B2, CYP79B3, CYP81F2 and PAD3 (CYP71B15) was analyzed by real-time quantitative PCR. After normalization with UBQ10, results are expressed as the fold changes in transcript level compared to the t=0 sample (mean ± SE;

n=3) from three biological independent experiments. Hash marks (#) indicate statistically significant difference between mock and infected samples (p < 0.05) and asterisks (*) indicate statistically significant difference between infected Col-0 and infected pad2-1 samples (p <

0.05), using unpaired heteroscedastic Student's t-test.

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SUPPLEMENTAL TABLE

Gene Primer sequences 5' - 3'

Forward Reverse

CYP79B2 At4g39950 CTCGCGAGACTTCTTCAAGG CCATAACCAACGGTTTAGCC CYP79B3 At2g22330 CCAGCCTTTGCTTACCGCTGAT ATTCCACGGCGTTTGATGGGTT CYP81F2 At5g57220 TGGCTATGCGTAAACTCGTG CCGGTAAACTTCAAAATGGTG GR1 At3g24170 TTGGGATACATCCATCTTCTG CAAATCCTTTATTGGGACACGAG GSTF6 At1g02930 CCAGCCTTTGAAGATGGAGA CTTGCCAGTTGAGAGAAGGTTG ICS1 At1g74710 GGGATAAGGGGTTCTCAC AACAATCATAACAGCTAGGC NPR1 At1g64280 ACCGTCTCACTGGTACGAAGAGAA AGCCAGTTGAGTCAAGTCCTCACA PAD3 At3g26830 GGGTACCATACTTGTTGAGATGG TTGATGATCTCTTTGGCTTCC PR-1 At2g14610 ACTACAACTACGCTGCGAACAC TGGCTTCTCGTTCACATAATTCCC RBOHD At5g47910 TCCACAAGGTTATTGCAAGCG CTGCTCCGTGCTTTCAGATCAA UBQ10 At4g05320 GGCCTTGTATAATCCCTGATG GAGATAACAGGAACGGAAACATAG Supplemental Table S1. Primers used for qPCR.

SUPPLEMENTAL PROTOCOL

Supplemental Protocol S1. Measurement of H2O2 half-life, total peroxidase activity and total catalase activity. Total proteins from Col-0 and pad2 cell suspension were extracted with 100 mM potassium phosphate buffer supplemented with 1% polyvinylpolypyrrolidone and 1 mM phenylmethylsulfonyl fluoride, pH 7. H2O2 half-life and catalase activity were determined by measuring the absorbance of H2O2 at 240 nm with a spectrophotometer. H2O2

solution was added in 95 µL of water + 5 µL of total proteins to measure an absorbance of 0.500. The absorbance was measured for 10 min. The decrease in absorbance was expressed in specific catalase activity (U mg-1 proteins). For peroxidase activity measurement, 50 mM of peroxidase substrate gaïacol and 2 mM H2O2 were added to total proteins and the absorbance of oxidized gaïacol was measured at 470 nm and was expressed in specific peroxidase activity (U mg-1 proteins).

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