35S P.tAPX or GUS intronNOS T. G P.XVET3A Lex A P.ccdB G P.XVET3A Lex A P. tAPX or GUS intron Xba I pGWB80/tAPX (or GUS) pMDC7 A B C pMDC7/tAPX (or GUS) RNAi Xba I Spe I Maruta et al., Supplemental Fig. S1. Plasmid construction for estrogen inducible RNAi. To construct the plasmid for estrogen-inducible silencing of tAPX, DNA fragment containing the 3’-untranslational region of tAPX was cloned into the donor vector, pDONR201, and then recloned into the destination vector, pGWB80. An open reading frame of the beta-Glucuronidase (GUS) gene was also cloned into pGWB80 as a control. PCR and in vitro BP and LR recombination reactions were carried out according to the manufacturer’s instructions (Invitrogen). The specific primers with attB1 and attB2 sequences were as follows: attB1-tAPX3’ (5’-AAAAAGCAGGCTGAGGACAGTCATGGACAGTG-3’), attB2-tAPX3’ (5’-AGAAAGCTGGGTTCACCACGTAATTATGTATGTAGGT-3’), attB1-GUS (5’- AAAAAGCAGGCTATGTTACGTCCTGTAGAAAC-3’), and attB2-GUS (5’- AGAAAGCTGGGTTCATTGTTTGCCTCCCTGCT-3’). DNA sequencing was performed using the dideoxy chain terminator method with an automatic DNA sequencer (ABI PRISMTM 310, Applied Biosystems, The plasmids obtained, pGWB80/tAPX and pGWB80/GUS (A), were digested with a restriction enzyme Xba I, and then blunted by a Mung Bean Nuclease (Takara). The estrogen-inducible expression vector, pMDC7 (B), was also digested with restriction enzymes, Xba I and Spe I, and blunted. The regions of an RNAi construct having an inverted repeat of tAPX and GUS were ligated to the blunted pMDC7.

RNAi- trigger XVE tAPX Actin8 RNAi- trigger tAPX Actin8 wild type IS-GUS-2 IS-tAPX-2 IS-tAPX-19 wild type IS-GUS-2 IS-tAPX-2 IS-tAPX-19 AB C (h) tAPX Actin8 Maruta et al., Supplemental Fig. S2. Association between expression of tAPX and dsRNA in the IS-tAPX-19 plants during treatment with estrogen. (A) IS-GUS-2, IS-tAPX-2, and IS-tAPX-19 plants (T 2 generation) were grown under normal light for 17 days. The transcript levels of tAPX and XVE were determined by sem-quantitative RT-PCR. (B) Seventeen-day-old IS-GUS-2, IS-tAPX-2, and IS-tAPX-19 plants were sprayed with a 100 µM estrogen. At 24 h after the estrogen treatment, the expression of tAPX and RNAi-triggers was determined by semi-quantitative RT-PCR. The expression of RNAi-triggers was indicated using a primer set for the intron region of RNAi-triggers (Supplemental Fig. S1C). Primer sequences were as follows; intron-F (5’-GGTGAGATCTTACTTCTCCT- 3’), intron-R (5’-CGAAACTATTTCGCCGAATC-3’). (C) Seventeen-day-old IS-tAPX-19 plants were sprayed with a 100 µM estrogen. A time-course analysis of the expression of tAPX and RNAi-triggers was performed.

IS-tAPX IS-GUS-2-17 KO-tAPX wild type AtbZIP65hydrolase ACS7PDIL1-1 UP F-box ANAC Relative mRNA level * * * * * * * * * * * * * * Maruta et al., Supplemental Fig. S3. The transcript levels of RTS genes in the estrogen-treated IS-tAPX or the KO-tAPX plants. (A) Seventeen-day-old IS-GUS-2-17 and IS-tAPX plants were sprayed with a 100 µM estrogen, and kept under normal light. At 48 h after the estrogen treatment, the transcript levels of RTS genes (AtbZIP65, ACS7, UP, F-box, hydrolase, PDIL1-1, and ANAC074) were measured by q-PCR. Error bars indicate SD (n = 3). Significant differences: *P < 0.05 vs. the value for IS-GUS-2-17 plants. (B) The transcript levels of RTS genes in 17-day-old wild-type and KO-tAPX plants were measured by q-PCR. Error bars indicate SD (n = 3). Significant differences: *P < 0.05 vs. the value for wild-type plants.

At3g08970At2g29490At2g29470At2g29460At1g17170At2g43510At1g10585At3g53230 At4g37370At3g49620At4g37990At2g41380At4g22530At3g54150At1g26420At1g26380 At3g26830At3g28210At1g57630At4g01870At3g11340At2g43820At1g22400At1g19020 At1g05340At2g21640At3g09350At1g13340At4g39670At1g62300At5g13080At2g32190 Relative mRNA level Relative mRNA level Relative mRNA level Relative mRNA level IS-tAPX-19-23IS-GUS-2-17 Maruta et al., Supplemental Fig. S4. The transcript levels of general oxidative stress response markers in the estrogen-treated IS-tAPX plants. Seventeen-day-old IS-GUS-2-17 and IS- tAPX plants were sprayed with a 100 µM estrogen, and kept under normal light. At 48 h after the estrogen treatment, the transcript levels of “general oxidative stress response markers” were measured by q-PCR. Error bars indicate SD (n = 3).

Maruta et al., Supplemental Fig. S5. Effect of lack of tAPX on cold acclimation under LL. Seventeen-day-old IS-GUS and IS-tAPX plants were sprayed with a 100 µM estrogen solution or water (mock), and transferred to cold stress conditions (10 µmol photons m -2 s -1, 4°C) for 2 weeks. The treatment with estrogen was performed every 3 days in order to maintain the tAPX silencing. At 14 days after cold stress, the IS-GUS-2-17 and IS- tAPX plants were photographed. The same results were obtained in three independent experiments. Estrogen Treatment Mock IS-tAPX-19-23IS-GUS-2-17

KO-tAPXwild type B A COR15BCOR6.6 COR414- TM1 COR414- TM2 Relative mRNA level KO-tAPXwild type * * F v /F m BC Maruta et al., Supplemental Fig. S6. Effect of lack of tAPX on cold acclimation. (A) The wild-type and KO-tAPX plants were grown under light for 17 days. The transcript levels of RTS genes (CBF1/DREB1B, CBF2/DREB1C, COR6.6, COR15B, COR414-TM1, and COR414- TM2) were measured by q-PCR. Error bars indicate SD (n = 3). Significant differences: *P < 0.05 vs. the value for wild-type plants. (B) Seventeen-day-old wild- type and KO-tAPX plants were transferred to cold stress conditions (100 µmol photons m -2 s -1, 4°C). At 2 weeks after cold stress, the wild-type and KO-tAPX plants were photographed. (C) F v /F m values in the leaves of wild-type and KO-tAPX at 2 weeks after cold stress were measured using a Closed FluorCam 800MF. Error bars indicate SD (n = 3). CBF1/ DREB1B CBF2 DREB1C

ICS2RLP7RLP23RLP34 Relative mRNA level KO-tAPXwild type RLP39RLP41AtNUDX6NIMIN-3LCR68LCR70 Relative mRNA level * * * * * * * * * * TIR domain protein TolB-related protein Maruta et al., Supplemental Fig. S7. Effect of lack of tAPX on the transcript levels of RTS genes involved in disease resistance. The wild-type and KO-tAPX plants were grown under normal light for 17 days. The transcript levels of RTS genes (ICS2, TolB, TIR, RLP7, RLP23, RLP34, RLP39, RLP41, NIMIN3, NUDX6, LCR68, and LCR70) were measured by q-PCR. Error bars indicate SD (n = 3). Significant differences: *P < 0.05 vs. the value for wild- type plants.

KO-tAPX wild type Free SA (µg g -1 FW) Total SA (µg g -1 FW) Maruta et al., Supplemental Fig. S8. Effect of lack of tAPX on the levels of SA. The wild-type and KO-tAPX plants were grown under normal light for 17 days. The levels of free and total SA in the wild-type and KO-tAPX plants were measured as described under ‘‘Experimental Procedures.’’

Maruta et al., Supplemental Fig. S9. Effect of tAPX silencing on HL sensitivity. Seventeen-day-old IS-GUS-2-17 and IS- tAPX plants were sprayed with a 100 µM estrogen solution or water (mock), and kept under NL. At 48 h after the treatments, IS-GUS-2-17 and IS-tAPX plants were exposed to HL (1000 µmol photons m -2 s -1 ). (A) At 72 h after HL, the IS- GUS-2-17 and IS-tAPX plants were photographed. The same results were obtained in three independent experiments. (B) F v /F m values in the leaves of IS-GUS and IS-tAPX were measured using a Closed FluorCam 800MF. Error bars indicate SD (n = 5). Estrogen Treatment Mock IS-tAPX-19-23IS-GUS-2-17 A (h) F v /F m IS-tAPX IS-GUS-2-17