Identification of an OPR3-independent pathway for jasmonate biosynthesis. Department of Plant Molecular Genetics, National Centre for Biotechnology,

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1 Supplementry Informtion Identifition of n OPR3-independent pthwy for jsmonte iosynthesis Andre Chini 1, Isel Monte 1, Angel M. Zmrreño 2, Mts Hmerg 3, Steve Lssueur 4, Philippe Reymond 4, Slly Weiss 5, Annik Stintzi 5, Andres Shller 5, Andre Porzel 6, José M. Grí-Min 2 nd Roerto Solno 1 * 1 Deprtment of Plnt Moleulr Genetis, Ntionl Centre for Biotehnology, Consejo Superior de Investigiones Científis (CNB-CSIC), 2849 Mdrid, Spin 2 Environmentl Biology Deprtment, University of Nvrr, Nvrre, Spin 3 Division of Physiologil Chemistry II, Deprtment of Medil Biohemistry nd Biophysis, Krolinsk Institutte, Stokholm, Sweden 4 Deprtment of Plnt Moleulr Biology, University of Lusnne, CH-115 Lusnne, Switzerlnd 5 Institute of Plnt Physiology nd Biotehnology, University of Hohenheim, 7593 Stuttgrt, Germny 6 Deprtment of Bioorgni Chemistry, Leiniz Institute of Plnt Biohemistry, Hlle, Germny. * Corresponding uthor: rsolno@n.si.es Nture Chemil Biology: doi:1.138/nhemio.254

2 Supplementry Results plstidil memrne lipids LIPASES 18:3 16:3 13-LOX AOS AOC OPDA dnopda hloroplsts OPDA dnopda OPR3 OPC-8 OPC-6 OPC-4 peroxisomes JA JAR1 JA JA-Ile ytoplsm Supplementry Fig. 1. Biosynthesis nd intrellulr flux of jsmontes in Aridopsis Sheme of the iosyntheti pthwy of JA-Ile generted from plstidil memrne lipids. Pthwy intermedites (desried in the text) re 18:3 (!-linoleni id), 16:3 (hexdetrienoi id), OPDA (12- oxo-phytodienoi id), dnopda(dinor-oxo-phytodienoi id), OPC-8 (8-(3-oxo-2-(pent-2-enyl) ylopentyl)otnoi id), OPC-6 (6 (3-oxo-2-(pent-2-enyl)ylopentyl)hexnoi id), OPC-4 (4-(3- oxo-2-(pent-2-enyl)ylopentyl)utnoi id), JA (jsmoni id) nd JA-Ile (jsmonoyl-isoleuine). Blk rrows define the otdenoid pthwy, lue rrows indite the prllel hexdenoid pthwy. Biosyntheti enzymes (desried in the text) re shown in green: 13-LOX (13-lipoxygense), AOS (llene oxide synthse), AOC (llene oxide ylse), OPR3 (OPDA redutse 3) nd JAR1 (jsmoni id-mido synthetse). Nture Chemil Biology: doi:1.138/nhemio.254

3 genomi DNA T-DNA T-DNA Reltive expression OPR OPR3 C # B C $%&'()# B C # A C $%&'()# A Supplementry Fig. 2. opr3 mutnts in Aridopsis () Sheme of the OPR3/AT2G65 lous, inluding the T-DNA insertion tht defines the llele 18,33 in ession nd (SK24765) llele in Col ession. () Expression of OPR3 fter fungl infetion nd wounding of Col- nd mutnt plnts (n = 1) mesured y rel-time PCR in untreted plnts (ontrol, C) nd plnts hllenged for 3 dys with B. inere (B) or 9 dys with A. rssiiol (A). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. Experiments were repeted twie with similr results. Sttistilly signifint expression ompred to untreted plnts is highlighted (Student s t test; ** p <.1; p <.1). Nture Chemil Biology: doi:1.138/nhemio.254

4 Reltive expression ChiB Reltive expression %# %! $# $! # * * Thio Reltive expression # / + % $ +" %" $" JAZ1 Reltive expression /! +! %! $! JAZ5 &'() '*.+",'-$" '*.+3,'-$" C B C B C B C B C &'() A C A '*+" C,'-$" A '*.+,'-$" C A C Sl C Sl C Sl C Sl &'() '*.+)+",'-$)+,'-$)+)"'*.+)+" C W C W 1" '*.+" C,'-$" W C '*.2,'-" W 45 Col- oi1-3 oi1-3 B. inere Col- oi1-3 oi1-3 Col- oi1-3 oi1-3 Col- oi1-3 oi1-3 4 spores/m Col Col- Smple 1 Smple 2 Smple 3 Smple 4 oi1-3 oi1-3 d spores/m A. rssiiol e Col- f Reltive expression (x 1 3 ) Smple 1 Smple 2 Smple 3 Smple 4 PDF1.2 C 4" B C '*.+)$" B Col- PDF1.2 C 4" A C '*.+)$" A " '*.+)$" C W C W Reltive expression oi1-3 JAZ7 Reltive expression (x 1 3 ) Reltive expression AOS ** 4" '*.+)$" C W C W oi1-3 Supplementry Fig. 3. Responses of opr3 mutnts to fungl infetion Nture Chemil Biology: doi:1.138/nhemio.254

5 () Expression of JA-regulted genes fter fungl infetion, inset hllenge nd wounding of Col- nd mutnt plnts (n = 1). ChiB, Thio2.1, JAZ1 nd JAZ5 were mesured y rel-time PCR in untreted plnts (ontrol, C) nd plnts hllenged for 3 dys with B. inere (B) or 9 dys with A. rssiiol (A), or 48 h with S. littorlis lrve (Sl) or 3 min fter wounding (W) (n = 1). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. Experiments were repeted twie with similr results. Sttistilly signifint wound-indued expression ompred to untreted plnts re highlighted (Student s t test; * p <.5; ** p <.1; p <.1). ( nd ) Infetion of Aridopsis plnts (n = 15) with B. inere. Quntifition of the spores of B. inere grown on different mutnt lines ws lulted 72 h post-inoultion. Dt shown s ox-plots of men of 5 iologil replites of 5 leves eh. Letters ove olumns indite signifint differenes (one-wy ANOVA/post-ho Tukey HSD Test, p <.5). The experiment ws repeted three times with similr results. Representtive leves of plnts infeted with B. inere re shown in (). Sle rs, 1 m. (d nd e) Infetion of Aridopsis plnts with A. rssiiol. Fungl spores, grown on different mutnt lines were ounted 9 dys post-inoultion, re shown s ox-plots of men of 5 iologil replites of 5 leves eh. Letters ove olumns indite signifint differenes (one-wy ANOVA/post-ho Tukey HSD Test, p <.5). The experiment ws repeted three times with similr results. Representtive leves of plnts infeted with A. rssiiol re shown in (e). Sle rs, 1 m. (f) Expression of JA-regulted genes fter fungl infetion nd wounding of Col- nd mutnt plnts (n = 1). PDF1.2, JAZ7 nd AOS were mesured y rel-time PCR in untreted plnts (ontrol, C) nd plnts hllenged for 3 dys with B. inere (B) or 9 dys with A. rssiiol (A) or 3 min fter wounding (W) (n = 1). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. Experiments were repeted twie with similr results. Sttistilly signifint wound-indued expression ompred to untreted plnts is highlighted (Student s t test; ** p <.1; p <.1). Nture Chemil Biology: doi:1.138/nhemio.254

6 '! &! OPC-4 Col- $!!! #&!! JA pmoles/fw (g) pmoles/fw (g) %! $! #! '! &! %! $! #! 3 m 1 h 3 h ( %!)" #*" %*" OPC-4 pmoles/fw (g) d pmoles/fw (g),!! $!! #!! '! %! $! #!," +" &" $" 3 %)" m 1 #*" h %*" 3 h JA-Ile 3 m 1 h 3 h ( %!)" #*" %*" 3 % m 1 #" h 3 %" h Supplementry Fig. 4. Aumultion of JA derivtives in wild-type plnts nd opr3 mutnts Aumultion of OPC-4 (pmoles/fresh weight (g)) (-), JA (pmoles/fresh weight (g)) () nd JA-Ile (pmoles/fresh weight (g)) (d) in wounded wild-type (grey rs) nd opr3 mutnts (lk rs) plnts. Four-week-old plnts (n = 1) were wounded nd dmged leves olleted t the time indited. Dt for ompred to Col- re shown in (), wheres ompred to is shown in () () nd (d). Dt shown s men ± SD of four iologil replites. The experiment ws repeted three times with similr results. Nture Chemil Biology: doi:1.138/nhemio.254

7 (+)-4,5-ddh-JA (9) (-)-4,5-ddh-JA (1) (+)-4,5-ddh-JA-(S)-Ile (11) (-)-4,5-ddh-JA-(S)-Ile (12) pure (+)-4,5-ddh-JA-Ile WT Supplementry Fig. 5. Strutures of 4,5-ddh-JA nd of 4,5-ddh-JA-Ile () Chemil strutures of 4,5-ddh-JA, used s n enntiomeri mixture of the (-)- nd (+)-forms, (-)-4,5-ddh-JA-Ile nd (+)-4,5-ddh-JA-Ile. () Chromtogrm of pure (+)-4,5-ddh-JA-Ile (top pnel) nd of WT nd plnts. Nture Chemil Biology: doi:1.138/nhemio.254

8 )! (! 4,5-ddh-JA Col- %! 4,5-ddh-JA pmoles/fw (g) '! &! %! $! pmoles/fw (g) $! #! #! 3 m 1 h 3 h %*" #+" %+" 3 m 1 h 3 h %*" #+" %+" Supplementry Fig. 6. Aumultion of 4,5-ddh-JA in wild-type plnts nd opr3 plnts ( nd ) Aumultion of 4,5-ddh-JA (pmoles/fresh weight (g)) in wounded wild-type (grey rs) nd opr3 mutnts (lk rs) plnts. Four-week-old plnts (n = 1) were wounded nd dmged leves olleted t the time indited. Dt for ompred to Col- re shown in (), wheres ompred to re shown in (). Dt shown s men ± SD of four iologil replites. The experiment ws repeted three times with similr results. Nture Chemil Biology: doi:1.138/nhemio.254

9 &!! JA pmoles/fw (g) %$ $! #$ Col- '()* (+,-" 4,5-ddh-JA Supplementry Fig. 7. JA umultion fter exogenous 4,5-ddh-JA tretment in Col- nd plnts. Dt shown s men ± SD of four iologil replites fter sutrtion of sl JA levels. JA umultion ws not sttistilly different (Student s t-test p <.1) in Col- ompred to plnts (n = 1). The experiment ws repeted twie with similr results. Nture Chemil Biology: doi:1.138/nhemio.254

reltive expression 5 4 3 2 1 Col- C W C W C W C W C W C W Col- Col-

T-DNA opr2-1 T-DNA Col- opr1-1 opr2-1 d WT opr1-1 WT opr2-1 OPR1 e

JA-Ile 3 min 6 min 18 min Col- opr1-1 opr2-1 opr1-1 opr2-1 A.

3 4 5 Smple opr2-1 6 Reltive expression Reltive expression i j 8 6 4

opr2-1 6 opr2-1 4 2 8 6 4 2 opr1-1 Col- Col- PDF1.

8 4 Col- Col- ChiB MYC2 opr1-1, C A C W opr2-1 opr1-1 opr2-1

10 reltive expression Col- C W C W C W C W C W C W Col- Col- Col- g Col- opr1-1 opr2-1 OPR1 OPR2 OPR3 h opr1-1 opr2-1 opr1-1 T-DNA opr2-1 T-DNA Col- opr1-1 opr2-1 d WT opr1-1 WT opr2-1 OPR1 e pmoles/fw (g) f spores/m 2 EF OPR2 EF1 JA-Ile 3 min 6 min 18 min Col- opr1-1 opr2-1 opr1-1 opr2-1 A. rssiiol Smple Col- Smple opr1-1 Smple opr2-1 Smple Smple opr Smple opr2-1 6 Reltive expression Reltive expression i j C 12 B Col- 12 AOS C W 1 Col- ChiB opr1-1, opr1-1 opr2-1 6 opr opr1-1 Col- Col- PDF1.2 JAZ1, opr1-1 opr2-1 opr1-1 opr2-1 opr2-1 C 6 6 A d C 12 W 8 4 Col- Col- ChiB MYC2 opr1-1, C A C W opr2-1 opr1-1 opr2-1 Supplementry Fig. 8. Anlysis of OPR expression nd hrteriztion of opr mutnts Nture Chemil Biology: doi:1.138/nhemio.254

11 () OPR1, OPR2 nd OPR3 expression in Col- nd mutnt plnts (n = 1) 3 min fter wounding (W, drk grey rs). Unwounded plnts were inluded s ontrols (C, light grey rs). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. The experiment ws repeted three times with similr results. ( nd ) Genomi sheme of the loss-of-funtion opr1-1 () (SALK_145353) nd opr2-1 () (SALK_116381) mutnt lines nd the exogenous T-DNA insertions. (d) Expression nlysis y RT-PCR of WT plnts nd T-DNA insertion lines of OPR1 nd OPR2 using EF1 s ontrol trnsripts. The experiment ws repeted three times with similr results. (e) Aumultion of JA-Ile (pmoles/fresh weight (g)) in wounded Col-, opr single nd doule mutnt plnts (n =1). Five-week-old plnts were wounded nd dmged leves olleted t time (white rs), 3 (light grey), 6 (drk grey) or 18 min (lk) fter wounding. Dt shown s men ± SD of four iologil replites. The experiment ws repeted three times with similr results. (f) Box-plots of fungl spore quntifition of Col- nd mutnt plnts (n = 15) infeted with A. rssiiol (9 di). Letters ove olumns indite signifint differenes (one-wy ANOVA/post-ho Tukey HSD Test, p <.1). (g-h) Representtive leves of Col- nd mutnt plnts infeted with B. inere nd A. rssiiol re shown in (g) nd (h) respetively. Sle rs, 1 m. (i) Expression of JA-regulted ChiB nd PDF1.2 (in thousnds) fter fungl infetion of Col- nd mutnt plnts (n = 1) mesured y rel-time PCR in untreted plnts (ontrol, C) nd plnts hllenged for 7 dys with B. inere (B) or 9 dys with A. rssiiol (A). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. Experiments were repeted twie with similr results. Letters ove olumns indite signifint differenes ompred to expression in plnts (Student s t-test, P <.5). (j) Expression of erly JA-regulted genes fter wounding in Col- nd mutnt plnts (n = 1). AOS, JAZ1 nd MYC2 expression were mesured y rel-time PCR in untreted plnts (ontrol, C, grey rs) nd in plnts 1 h fter wounding (W, drk grey rs). Vlues re the men ± SD of three tehnil replites expressed s reltive fold hnge normlized to ACT8. The experiment ws repeted t lest twie with similr results. Letters ove olumns indite signifint differenes ompred to expression in plnts (Student s t-test, P <.5). Nture Chemil Biology: doi:1.138/nhemio.254

12 Supplementry Tle 1 List of primers used Nme OPR1 qpcr-f OPR1 qpcr-r OPR2 qpcr-f OPR2 qpcr-r OPR3-3 qpcr-f OPR3-3 qpcr-r OPR3-3 qpcr-f2 OPR3-3 qpcr-r2 PDF1.2 qpcr-f PDF1.2 qpcr-r CYP79B3 qpcr-f CYP79B3 qpcr-r AOS qpcr-f AOS qpcr-r LOX3 qpcr-f LOX3 qpcr-r JAZ1 qpcr-f JAZ1 qpcr-r JAZ5 qpcr-f JAZ5 qpcr-r JAZ7 qpcr-f JAZ7 qpcr-r MYC2 qpcr-f MYC2 qpcr-r ACT8 qpcr-f ACT8 qpcr-r Nme LB_GW1 SLB1.3 OPR1_F1 OPR2_F1 OPR3_F1 Sequene 5'-ATCCAGGAGCATTAGGGCT-3' 5'-CGCTTTCCTCATCGGCAT-3' 5'-TCCAGAAGCATTAGGGCTGT-3' 5'-TGATGTTGAAAGCACATATAAAAGC-3' 5 -GCATGGAAGCAAGTTGTGGAAGCA-3 5'-CATGCGCCCCGTGGATCTCAAT-3 5'-ATCTCTCTCATCGAGTGGTT-3' 5'-CCTCCATTAGGTTGATACACTG-3' 5 -CACCCTTATCTTCGCTGCTC-3 5 -GTTGCATGATCCATGTTTGG-3 5'-CTTTGCTTACCGCTGATGAA-3' 5'-GCGTTTGA TGGGTTGTCTG-3' 5'-GCGACGAGAGATCCGAAGA-3' 5'-CTCGCCACCAAAACAACAAA -3' 5'-CACTGCAATTCACAAGCAACC-3' 5'-CAAAGGAGGAATCGGAGAAGC-3' 5'-CACGTCTGTGAGAAGCTAGGC-3' 5'-TTCTGAGTTCGTCGGTAGCC-3' 5'-AAAGATGTTGCTGACCTCAGTG-3' 5'-CCCTCCGAAGAATATGGTCA-3' 5'-TTCGGATCCTCCAACAATCCCA-3' 5'-TCAAGACAATTGGATTATTATGTTACAGT-3' 5'-GTGCGGGATTAGCTGGTAAA-3' 5'-ATGCATCCCAAACACTCCTC-3' 5'-CCAGTGGTCGTACAACCGGTA-3' 5'-TAGTTCTTTTCGATGGAGGAGCTG-3' Sequene 5'-GCTTTCGCCTATAAATACGACGGATCGT-3' 5'-ATTTTGCCGATTTCGGAAC-3' 5'-AACACACTACATTACATTATTGATAACA-3' 5'-GAAACACATTACATTACTGATAACACGA-3' 5'-GCATGGAAGCAAGTTGTGGAAGCA-3' Nture Chemil Biology: doi:1.138/nhemio.254

13 Supplementry Tle 2 Ioniztion soure working prmeters Instrumentl prmeters Vlue Sheth gs flow rte 44 u Auxiliry gs flow rte 11 u Sweep gs flow rte 1 u Spry voltge 3.5 kv Cpillry temperture 34 ºC S-lens RF level 5 Auxiliry gs heter temperture 3 ºC Nture Chemil Biology: doi:1.138/nhemio.254

14 Supplementry Tle 3 Msses of phytohormones nd internl stndrd nd their prinipl frgments Anlyte [M-H] -1 Phytohormone JA J-Ile OPDA dn-opda ,5-ddh-JA ,5-ddh-JA-Ile OPC OPC H 5 -JA H 5 -JA-Ile H 5 -OPDA H 5 -dnopda H 5 -tnopda H 5-4,5-ddh-JA H 5 -ddh-ja-ile H 5 -OPC H 5 -OPC H 5 -JA H 5 -JA-Ile H 5 -OPDA H 5 -dnopda Anlyte [M-H] -1 Frgment JA J-Ile OPDA dn-opda ,5-ddh-JA ,5-ddh-JA-Ile OPC OPC H 5 -JA H 5 -OPDA H 5 -dnopda H 5 -JA H 5 -JA-Ile H 5 -OPDA H 5 -dnopda Nture Chemil Biology: doi:1.138/nhemio.254

b a wt ccd8 dad2 Secondary branches 15 e cd normal low b 2

b a wt ccd8 dad2 Secondary branches 15 e cd normal low b 2 A 3 1 Shoot mss (g) B Root mss (g) C 8 Primry rnhes D 8 Seonry rnhes 1 e E 8 Shoot mss/rnhes norml low 1 F 8 8 Shoot mss/root mss 8 8 Supplementl Figure 1 Aitionl phenotypi t reore from the plnts esrie