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Title:'Reac+ve'short1chain'leaf'vola+les'act'as'powerful .... S2 (Yamauchi) ) Vaporized...

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Title: Reac+ve short1chain leaf vola+les act as powerful inducers of abio+c stress1related gene expression Authors: Yasuo Yamauchi,* Mikiko Kunishima, Masaharu Mizutani, Yukihiro Sugimoto Affilia+ons: Graduate School of Agricultural Science, Kobe University, Rokkodai 1B1, Nada, Hyogo 657B8501, Japan *Correspondence to: [email protected] (Y.Y.)
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Title:'Reac+ve'short1chain'leaf'vola+les'act'as'powerful'inducers'of'abio+c'stress1related'gene'expression''Authors:'Yasuo&Yamauchi,*&Mikiko&Kunishima,&Masaharu&Mizutani,&Yukihiro&Sugimoto&

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Affilia+ons:'Graduate&School&of&Agricultural&Science,&Kobe&University,&Rokkodai&1B1,&Nada,&Hyogo&657B8501,&Japan&

*Correspondence&to:&[email protected]&(Y.Y.)�

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Fig. S1 (Yamauchi)�Transient increase in (E)-2-hexenal in Arabidopsis treated at 10 nmol cm–3. a, Treatment of plants with SLVs. b, Changes in (E)-2-hexenal content. Data are means ± SE (n = 3). c, Typical HPLC chromatogram. Arrows indicate peaks of DNP-(E)-2-hexenal. The retention times of authentic DNP-RSLVs and the internal standard (IS) are shown above the chromatogram.�

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Arabidopsis plants were exposed to various concentration of (E)-2-hexenal for 30 min to determine dose-dependence of HSFA2 expression. Expression of the HSFA2 gene was determined using qRT-PCR. Relative transcript levels were normalized to ACTIN2 mRNA. Data are means ± SE (n = 3). �

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To determine toxicity of the RSLVs, Arabidopsis plants were exposed to various concentrations of 2-propenal (open square), (E)-2-butenal (open diamond), (E)-2-pentenal (open circle), (E)-2-hexenal (open triangle), or (E)-3-hepten-2-one. After 90 min, residual PSII activity (Fv/Fm) was measured. Data are means ± SE (n = 3).�

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Fig. S4. (Yamauchi)�Pathways for production of RSLVs in plants. (E)-2-Hexenal and (E)-2-butenal are mainly produced via enzymatical (a) and non-enzymatical (b, referred from 34) pathways, respectively.�

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Fig. S5. (Yamauchi)�Determination of (E)-2-butenal as endogenous RSLV produced under oxidative stress. Arabidopsis leaflets were treated with 10 µM MV in the presence of light (80 µmol m–2 s–1) for an hour. a, Typical chromatogram of HPLC analysis of DNP-carbonyls of MV-untreated (top) and MV-treated Arabidopsis (bottom). Arrow indicates peak of DNP-(E)-2-butenal. The retention times of authentic DNP-RSLVs and the internal standard (IS) are shown above the chromatogram. b, Typical chromatogram (selected reaction monitoring) of 2-butenal-DNP by LC-MS/MS analysis. Other RSLVs did not increase significantly after MV treatment. c, Full-scan spectra of fragment ions of endogenous (top) and authentic 2-butenal (bottom).�

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Estimation of (E)-2-butenal contents and HSFA2 expression in MV-treated (a, b) or UVB-treated (c, d) Arabidopsis. a and c, (E)-2-Butenal contents at 0 time (-) and after stress treatment (+). Data are means ± SE (n = 4). Values followed by the same letter are not significantly different according to Tukey-Kramer (P <0.05). b and e, Expression of HSFA2 was enhanced in aor mutant under oxidative stress. HSFA2 mRNA expression in Col-0 and aor was determined by qRT-PCR. Relative transcript levels were normalized to ACTIN2 mRNA. The expression level of the 0 time sample was set to 1. Data are means ± SE (n = 3). (*, P < 0.05 vs Col-0, Student’s t-test). Absolute HSFA2 transcripts were approximately 5 copies ng-1 RNA in both Col-0 and aor. e, Alkenal/one oxidoreductase (AOR) catalyzes saturation of α,β-unsaturated carbonyl bonds by using NADPH (shown in dotted box). RSLVs are good substrates for AOR19.��

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Expression of related transcription factors by (E)-2-Hexenal treatment in Arabidopsis (a), tomato (b) and rice (c). Tested plants were treated with (E)-2-hexenal (10 nmol cm–3) for 30 min, and then expression of each transcription factor gene was determined by qRT-PCR. Relative transcript levels were normalized to ACTIN mRNA. For each gene examined, the expression level in the MeCN-treated control sample was set to 1. Data are means ± SE (n = 3).��

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Induction of HSFA2 expression and HSP17.6 producition by RSLVs in Arabidopsis seedlings. a, Arabidopsis seedlings were exposed to (E)-2-hexenal (10 nmol cm–3) for 30 min, and then expression of HSFA2 was determined using qRT-PCR. Relative transcript levels were normalized to ACTIN2 mRNA. The expression level of the 0 h exposure sample was set to 1. Data are means ± SE (n = 3). b, After the indicated time of RSLV treatment, HSP17.6 proteins was detected by western blot analysis. Quantified value of each band by densitometric analysis is indicated in parenthesis. Maximal intensity is set to 100. Rubisco was stained using Coomassie Brilliant Blue R-250 as a loading control. �

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Effect of RSLV treatment on thermotolerance. a, The 5-day-old dark-grown QK seedlings (Before) were pretreated at 38°C for 90 min to acquire thermotolerance (AT) or 10 µM (E)-2-hexenal for 2 h and then heat-stressed at 45°C for 2 h. Seedling were returned to 23°C in the dark and length was measured after 3 days. Length of seedlings before treatment was set to 100%, and elongation of each treatment was calculated. Schemes of treatment are shown in Fig. 3b. b and c, Effect of RSLVs on protection of PSII from heat (b) or UV-B treatment (c). Two-week-old Arabidopsis (Col-0) were pretreated with 10 nmol cm-3 RGLV for an hour, and then treated with heat (45°C for 2h) or UV-B (1 mW cm–2 for 30 min). Data are means ± SE (n = 4 or 5). Values followed by the same letter are not significantly different according to Tukey-Kramer (P <0.05). �

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Table S1 Descending order of Top100 up-regulated gene expression ratio [(E)-2-hexenal versus control] calculated from results of microarray.

The "Ratio" values are the mean of 3 independent experiments,

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Table S2

Primer sequences used in this study

Target gene Forward Reverse

Arabidopsis

Actin2 ACC AGC TCT TCC ATC GAG AA� GAA CCA CCG ATC CAG ACA CT

HSFA1a GAC GGG TTC TCA TCT CCA AA TCA TCA ATC TCG GGG TCT TC

HSFA1b GAG GTG GGG AAG TTT GGA AT TTG TGC TGC TTC GTT TAT CG

HSFA1d TCA GAA GCA ACC GAG AAC TG CCA TCC ATT TTG TTC CTG CT

HSFA1e ATC GAT GAA CGA TGC AAC AA CTG TCT CGC ATC CAA CAA GA

HSFA2 GCA AGG AAC GTC ATC ATC TG ATC AGC AAG GAT CTG GGA TG

HSFB1 TTG GTT CGC CTT CTG AGT CT CTT TCA ACC ACA CCC CAA AC

ZAT12 GGC GAA TTG TTT GAT GCT TT CAA GCC ACT CTC TTC CCA CT

ZAT10 GCT TCT CCG ATT CCT CCT TT GAC CAC CGA GAG CTT GGT AA

MBF1c GAG CAG ATA CCC AGG AGC AG TGA TCT GTT TCG CCA AAT CC

DREB2A GTG GAG TGG AGC CGA TGT AT ATC GTC GCC ATT TAG GTC AC

Tomato

Actin AGC AAT ACC AGG GAA CAT GG GGA TCT TGC TGG TCG TGA TT

HSFA1 AGG AGG TCC CAC CAA CTT CT TCC CAC TTT TCC CTC AAC TG

HSFA2 GAT CTG GTG CTT GCA TTG AA TGG GGG TCA TCG TTA GTC TC

HSFB1 CAA AGG ATT TGC TTC CCA AA CCG TGA ACT GGG ACA ACT TT

Rice

Actin1 GGA TCC ATC TTG GCA TCT C GTC AGA CTC GTC GTA CTC A

HSFA2c GAG CAG TCA GAG TTG GAT GGC A AAT CAA CTC TAT TTT GGA CTA A

HSFA2d CAA GAG ATG ATG CTG GGA TTC C CTA TTG CTT AGA TAA CCC AGC T

HSFA2e GCT CGA TGA GGA TAC CAG GAA C GCG ATT TGG TGC TGC TTG AGT T

DREB2A TAA GTG GGT GGC TGA GAT CC ATG AAG GTG CTG ATG TGC AG

Reference!!34. !Frankel, E. N. Lipid oxidation. Prog. Lipid Res. 19, 1-22 (1980).�!


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