Valentin Marquis

Ekaterina Smirnova

Laure Poirier

Julie Zumsteg

Fabian Schweizer

Philippe Reymond

Thierry Heitz
email: thierry.heitz@ibmp-cnrs.unistra.fr

Stress-and pathway-specific impacts of impaired jasmonoyl-isoleucine (JA-Ile) catabolism on defense signaling and biotic stress resistance

Keywords: Arabidopsis, Botrytis, catabolism, defense, feedback, jasmonate, resistance, signaling, wounding

Jasmonate synthesis and signaling are essential for plant defense upregulation upon herbivore or microbial attacks. Stress-induced accumulation of jasmonoyl-isoleucine (JA-Ile), the bioactive hormonal form triggering transcriptional changes, is dynamic and transient, due to the existence of potent removal mechanisms. Two JA-Ile turnover pathways operate in Arabidopsis, consisting in cytochrome P450 (CYP94)-mediated oxidation and deconjugation by the amidohydrolases (AH) IAR3/ILL6. Understanding their impacts was previously blurred by gene redundancy and compensation mechanisms. Here we address the consequences of blocking these pathways on jasmonate homeostasis and defenses in double-2ah, triple-3cyp mutants, and a quintuple-5ko line deficient in all known JA-Ile-degrading activities. These lines reacted differently to either mechanical wounding/insect attack or fungal infection. Both pathways contributed additively to JA-Ile removal upon wounding, but their impairement had opposite impacts on insect larvae feeding. In contrast, only the ah pathway was essential for JA-Ile turnover upon infection by Botrytis, yet only 3cyp was more fungus-resistant. Despite of building-up extreme JA-Ile levels, 5ko displayed near-wild-type resistance in both bioassays. Molecular analysis indicated that restrained JA-Ile catabolism resulted in enhanced defense/resistance only when genes encoding negative regulators were This article is protected by

 (Chini, Gimenez-Ibanez, Goossens, & Solano, 2016). Distinct JA-Ile-triggered networks regulate leaf defenses against various aggressors and are integrated by separate sets of TFs. MYC2, a bHLH type TF, integrates (together with MYC3 and MYC4) simultaneous JA/abscisic acid (ABA) stimuli and defines a wound/insect-specific branch including the induction of typical responses such as vegetative storage proteins (VSPs) or glucosinolates (Fernandez-Calvo et al., 2011;[START_REF] Schweizer | Arabidopsis basic helix-loop-helix transcription factors MYC2, MYC3, and MYC4 regulate glucosinolate biosynthesis, insect performance, and feeding behavior[END_REF]; ERF1/ORA59 TFs integrate concomitant JA/ethylene (ET) signals into a microbe-specific branch controling expression of plant defensins, typically PDF1.2 (Pieterse, Van der Does, Zamioudis, Leon-Reyes, & Van Wees, 2012;[START_REF] Wasternack | Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in[END_REF].

JA signaling, like any hormonal pathway, needs to be tightly controlled in time and space, particularly because defense upregulation is costly and connected to overall growth inhibition (Guo, Major, & Howe, 2018). This antagonism was long thought to be merely imposed by limited resources that need to be re-allocated from developmental to defensive sinks (Havko et al., 2016), but recent advances have evidenced hardwire connections in the control of growth-defense tradeoffs (Campos et al., 2014;Guo et al., 2018). For appropriate control of JA responses, plants rely on several negative feedback mechanisms. In addition to JAZ repressor proteins, other negative regulators were identified that repress JA responses at the level of gene promoters, including JAV1 (Yan et al., 2018), and the JAM subclass of bHLH proteins that compete with MYC2/3/4 TFs [START_REF] Sasaki-Sekimoto | Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate responses in Arabidopsis[END_REF]. Another way to repress or terminate jasmonate action is at the metabolic level, either by diverting the flux to hydroxy-JA rather than to JA-Ile [START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF], or by modifying JA-Ile to alleviate its receptor-binding properties. The characterization of JA-Ile catabolic pathways has This article is protected by copyright. All rights reserved.

shed light on hormone homeostasis regulation and has also provided a framework explaining additional complexity in the JA metabolic grid (Heitz et al., 2016). Two distinct enzymatic pathways are known to modify or degrade JA-Ile. One consists in a two-step oxidation of the terminal (ω) carbon of the JA moiety by cytochrome P450 monooxygenases of the CYP94 family. In Arabidopsis, CYP94B3 and to a minor extent CYP94B1 generate 12OH-JA-Ile as a main product whereas CYP94C1 catalyzes also the full oxidation of JA-Ile to 12COOH-JA-Ile (Figure 1) (Bruckhoff et al., 2016;Heitz et al., 2012;Kitaoka et al., 2011;Koo, Cooke, & Howe, 2011;Koo et al., 2014). The first oxidation product retains weak receptor-assembly capacity and gene-inducing activity [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Koo et al., 2011;[START_REF] Poudel | 12-Hydroxy-jasmonoyl-L-isoleucine is an active jasmonate that signals through CORONATINE INSENSITIVE 1 and contributes to the wound response in Arabidopsis[END_REF], but the second product proved fully inactive [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Koo et al., 2014). The second pathway is a deconjugation of JA-Ile by an amidohydrolase initially reported in Nicotiana attenuata (Woldemariam, Onkokesung, Baldwin, & Galis, 2012) of which the Arabidopsis orthologs IAR3 and ILL6 (Figure 1) [START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]. These enzymes act on JA-Ile and other JA-amino acid conjugates but also on 12OH-JA-Ile generated by CYP94Bs, releasing JA and 12OH-JA, respectively [START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF][START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. The interconnection of the two catabolic pathways generates increased metabolic complexity because many of the above-mentioned JAs derive in fact from JA-Ile catabolism (Heitz et al., 2016;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF].

Expression of both JA-Ile oxidases and amidohydrolases genes is induced by environmental cues and co-regulated with the central regulon defined by JA pathway biosynthetic and signaling genes. Their simultaneous enzymatic action shapes specific jasmonate patterns in different tissues and stress conditions [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Heitz et al., 2012;Koo & Howe, This article is protected by copyright. All rights reserved.

2012; Koo et al., 2014;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]Widemann, Smirnova, Aubert, Miesch, & Heitz, 2016).

Manipulating JA-Ile catabolic routes could be an attractive tool to engineer plants for enhanced pathogen resistance or modulate other JA-dependent processes. Loss-or gain-offunction mutant lines in CYP94 or AH genes have revealed profound impacts on JA metabolism but contrasted consequences on JA responses. Arabidopsis lines ectopically overexpressing CYP94 or AH generally have reduced JA-Ile levels and a metabolic shift towards oxidized and/or cleaved derivatives. As expected, increased turnover is correlated with attenuated JA responses, for example increased sensitivity to Botrytis infection or to insect larvae feeding [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Koo et al., 2011;[START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. In comparison, analysis of single, double, or triple knock out mutant lines has not led to a unified conclusion as to how JA-Ile responses are impacted by gradual impairement of JA-Ile catabolism. Deficiency in CYP94 or AH expression impacts JA homeostasis consistently with their in vitro enzymatic activities. For example, cyp94b3 or b3c1 mutations lead to more JA-Ile accumulation upon leaf wounding, concomitantly to reduced 12OH-JA-Ile and 12COOH-JA-Ile levels (Heitz et al., 2012;Kitaoka et al., 2011;Koo et al., 2011). Double cyp94b3c1 and triple cyp94b1b3c1 mutant plants displayed slightly enhanced defense responses and tolerance to fungal infection [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]. Strikingly, in another study, the double cyp94b1b3 and triple cyp94b1b3c1 mutants exhibited deficient JA responses, questioning the current signaling model [START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. On the other hand, AH-deficient lines have not yet been tested for defense and resistance responses, so the contribution and impacts of this pathway are unknown.

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To overcome gene redundancy and compensation mechanisms between pathways that occur frequently in JA metabolic mutants [START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF][START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF], we introduced in the present study new plant lines with higher order mutations (Figure 1): a double iar3 ill6 mutant (thereafter called 2ah) impaired in the JA-Ile deconjugating pathway; and a quintuple cyp94b1 cyp94b3 cyp94c1 iar3 ill6 (thereafter called 5ko) deficient in all characterized enzymes turning over JA-Ile. We submitted these lines, along with the oxidation-deficient cyp94b1b3c1 mutant (thereafter called 3cyp), to parallel leaf stimulation by mechanical wounding or B. cinerea infection, two environmental cues triggering strong JA metabolism and signaling, but activating distinct transcriptional networks due to different cross-talks with other hormonal pathways (Pieterse et al., 2012). We determined the contribution of each catabolic pathway on JA-Ile turnover for each stress and investigated the impact of modified JA-Ile homeostasis on defense responses and tolerance to aggression by an herbivore insect or by fungal infection (Figure 1). The data highlight new stress-specific impacts of fully impairing either one or both JA-Ile catabolic pathways, where defense and resistance phenotypes are more reflecting the transcriptional behavior of negative feedback signaling effectors than actual JA-Ile levels.

MATERIALS AND METHODS

Plant growth and treatments

Arabidopsis thaliana genotypes used were in the Col-0 ecotype and were grown under a 12 h This article is protected by copyright. All rights reserved. light/12 h dark photoperiod in a growth chamber. The individual T-DNA insertion lines were obtained from the Nottingham Arabidopsis Stock Center (NASC). The 3cyp line was obtained by crossing the alleles cyp94b1-1 (SALK_129672), cyp94b3-1 (CS302217) and cyp94c1-1 (SALK_55455) [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]. The 2ah line was obtained after crossing the lines iar3-5 (SALK_069047) and ill6-2 (SALK_024894C). The quintuple 5ko line was obtained by crossing the 3cyp line with a double iar3-5 ill6-1 (GK412-E11).

B. cinerea inoculation and resistance scoring were as described in [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]. For mechanical wounding experiments, 5 or 6 fully expanded leaves were wounded three times across mid-vein with a hemostat. At increasing time points following damage, leaf samples were quickly harvested and flash-frozen in liquid nitrogen before storing at -80°C until analysis.

Exogenous hormone feeding experiments were performed on seedlings as described in [START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF]. Briefly, surface-sterilized seeds were germinated in 24-well microtiter plates under a 16 h light/8 h dark photoperiod at 22°C in a controlled growth chamber in the following medium: 1 x Murashige and Skoog liquid medium, supplemented with 0.5% sucrose and 0.05% MES buffer. Seedlings were grown for 7 days before gentle addition of hormones (amino-cyclopropane carboxylic acid, ACC, Sigma-Aldrich A3903) or abscisic acid (ABA, Olchemim 0132721) from stock solutions to a final concentration of 15 µM. JA-Ile was chemically synthesized as described in Wiedemann et al. (2013). At increasing time points after treatment, seedlings were quickly harvested and flash-frozen in liquid nitrogen before storing at -80 °C until RNA extraction.

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Insect performance assay

Four-week-old Arabidopsis plants were challenged with freshly hatched Spodoptera littoralis larvae (eggs obtained from Syngenta, Stein AG, Switzerland). Five larvae were placed on each of 11 pots, each containing 2 plants. Plants were placed in a transparent plastic box and kept in a growth chamber during the experiment (10 h light/14 h dark, 100 µmol m -2 s -1 of light, 20-22°C and 65% relative humidity). After 8-9 days of feeding, larvae were weighed on a precision balance (Mettler-Toledo, Greifensee, Switzerland). The experiment was performed successively three times (different sampling dates).

RT-qPCR gene expression assays

Total RNA was extracted from plant leaves with TRI reagent (Molecular Research Center).

One microgram of RNA was reverse transcribed using the SuperScript IV reverse transcription system (Thermo Fisher Scientific). Real-time PCR was performed on 10 ng of cDNA as described in Berr et al. (2010) using a LightCycler 480 II instrument (Roche Applied Science). The housekeeping genes EXP (At4g26410) and TIP41 (At4g34270) were used as internal references for qPCR on cDNA derived from infected/wounded or nonstimulated leaves. Measurement of fungal biomass was performed as described in [START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF] except that ACT2 (At3g18780) and UBQ10 (At4g05320) were used as reference genes. Gene-specific primer sequences used for qPCR are listed in Table S1.

Jasmonate and camalexin profiling

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Jasmonates were identified and quantified by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). About 50-100 mg frozen plant material was extracted with 10 volumes (10 µL per mg) of ice-cold extraction solution (MeOH:water:acetic acid 70:29:0.5, v/v/v) containing 9,10-dihydro-JA and 9,10-dihydro-JA-Ile as internal standards for workup recovery. Grinding was performed with a glass-bead Precellys tissue homogenizer (Bertin Instruments, France) in 2 mL screw-capped tubes. After 30 min incubation at 4°C on a rotating wheel, homogenates were cleared by centrifugation before concentration of supernatants under a stream of N2 and overnight conservation at -20°C. After a second centrifugation step, extracts were submitted to quantitative LC-MS/MS analysis on an EvoQ Elite LC-TQ (Bruker) equiped with an electrospray ionisation source (ESI) and coupled to a Dionex UltiMate 3000 UHPLC system (Thermo). Five µL plant extract were injected. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column (100 x 2.1 mm, 1.8 µm; Waters) and pre-column. The mobile phase consisted of (A) water and (B) methanol, both containing 0.1 % formic acid. The run started by 2 min of 95 % A, then a linear gradient was applied to reach 100 % B at 10 min, followed by isocratic run using B during 3 min. Return to initial conditions was achieved in 1 min, with a total run time of 15 min. The column was operated at 35 °C with a flow-rate of 0.30 mL min -1 . Nitrogen was used as the drying and nebulizing gas. The nebulizer gas flow was set to 35 L h -1 , and the desolvation gas flow to 30 L h -1 . The interface temperature was set to 350 °C and the source temperature to 300 °C. The capillary voltage was set to 3.5 kV, the ionization was in positive or negative mode. Low mass and high mass resolution were 2 for the first This article is protected by copyright. All rights reserved.

Statistical analysis

All statistical analysis was performed using InfoStat 2015d (http:// www.infostat.com.ar).

Comparisons of sample means were performed by one-way analysis of variance (P % 0.05 or P % 0.01) and Tukey's post-hoc multiple comparisons tests (P < 0.05 or P < 0.01), and significant differences of means were determined.

RESULTS

Jasmonate profiling of higher order catabolic mutants reveals stimulus-specific impacts on hormone homeostasis

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Effective inactivation of the respective genes was verified in each mutant line by RT-qPCR using RNA extracted from 1 h-wounded leaves (Figure S1 and [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]). The four plant genotypes in the Col-0 ecotype (wild-type WT,2ah,3cyp,5ko) were submitted separately to mechanical wounding or to B. cinerea inoculation, to activate JA metabolism and responses. Mechanical wounding constitutes a synchronous and severe stimulus, and generates a massive jasmonate pulse where compound-specific dynamics can be followed (Chung et al., 2008;Glauser et al., 2008;Heitz et al., 2012). Therefore, a kinetic study was conducted with tissue collected at 1, 3 and 6 h post-wounding (hpw). In contrast, necrotic lesions inflicted by B. cinerea infection develop radially with fungal hyphae continuously infecting new tissue (an asynchronous stimulus), and 3 days post-inoculation (dpi) constitutes an optimal time point for recording biochemical changes and for assessing antifungal resistance. We quantified levels of JA, 12OH-JA, JA-Ile and its catabolites 12OH-JA-Ile and 12COOH-JA-Ile in the two biological responses. JA was less accumulated in infected 3cyp (Figure S2a,b). Its oxidation product, 12OH-JA, is known to be partially formed via conjugate intermediates (Figure 1) upon wounding [START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF] and accordingly, was less abundant in all mutants at 3 hpw, remaining low in 2ah at 6 hpw (Figure S2). In contrast, 12OH-JA was less affected by the mutations upon infection and was even enhanced in 5ko, in accordance with the predominant contribution of Jasmonic Acid Oxidase (JAO) enzymes directly oxidizing JA in response to Botrytis (Figure 1) [START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF].

Impacts on JA-Ile homeostasis upon wounding

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We next examined the evolution of JA-Ile and its oxidized catabolites. JA-Ile profiles showed the typical pattern (Chung et al., 2008;Glauser et al., 2008;Heitz et al., 2012;Koo et al., 2011) in wounded WT leaves, peaking at 1 hpw and declining thereafter (Figure 2a). In 2ah and 3cyp lines, a significant overaccumulation of JA-Ile was recorded at 1 hpw and was prolonged in 3cyp, extending data from lower order cyp94 mutants described previously (Heitz et al., 2012;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF][START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. In the 5ko line that has both pathways impaired, a huge JA-Ile accumulation was recorded that culminated between 3-6 hpw close to 40-50 nmol g FW -1 . These data indicate that in WT wounded leaves, both AH and CYP94 pathways contribute similarly to JA-Ile turnover and that their simultaneous inactivation synergistically boosts hormone hyperaccumulation at later time points. Profiles of CYP94-generated JA-Ile oxidation products were as expected: 12OH-JA-Ile levels were about double in 2ah compared to WT, were suppressed in 3cyp and of note, were higher in 5ko than in 3cyp at 6 hpw (Figure 2a). 12COOH-JA-Ile was only detected from 3 hpw and evolved similarly to 12OH-JA-Ile in 2ah and 3cyp, but was barely detected in 5ko.

Impacts on JA-Ile homeostasis upon Botrytis infection

The metabolic impacts of mutations were different in response to B. cinerea inoculation. JA-Ile levels were similar to WT in 3cyp, confirming our previous data [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF] (Figure 2b). In contrast, steady-state JA-Ile levels were strongly enhanced in 2ah but no further increase was observed in 5ko. This result indicates that the amidohydrolase pathway is essential for JA-Ile clearance upon fungal infection and that blocking simultaneously the This article is protected by copyright. All rights reserved. oxidative pathway does only marginally impact JA-Ile accumulation. 2ah also considerably enhanced 12OH-JA-Ile and 12COOH-JA-Ile levels, reinforcing initial trend obtained with iar3 or ill6 single mutants [START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]. In 2ah, excess uncleaved JA-Ile is likely oxidized to 12OH-JA-Ile that can no more be deconjugated by IAR3 and ILL6 and part of this enlarged pool is further oxidized to 12COOH-JA-Ile by CYP94C1. Together, these experiments demonstrate stimulus-specific contributions of AH and CYP94 catabolic pathways to JA-Ile turnover and accumulation of downstream derivatives.

Impact of impaired catabolic pathways on defense and resistance responses is not reflecting JA-Ile accumulation in mutant plants

As the mutations altered the steady-state levels of bioactive JA-Ile, the lines were examined for induced expression of typical JA-regulated marker genes for each leaf stress model. In the case of wounding, transcripts of MYC2, an early-responsive TF gene controlling late targets, accumulated similarly at 1 hpw, but declined less than WT in 2ah and 5ko mutants as best visible at 6 hpw (Figure 3a). Two VSP genes were examined as late markers of the anti-insect branch of the JA defense pathway (Pieterse et al., 2012;[START_REF] Wasternack | Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in[END_REF]. In WT, both VSP1 and VSP2 expression peaked at 3 hpw before declining to low levels at 6 hpw (Figure 3a). Expression was globally enhanced in mutant lines compared to WT, but with distinct patterns. VSP1 was strongly enhanced in 2ah at all time points, while in 3cyp and 5ko, persistence in transcript levels was only observed at 6 hpw when WT signal faded. For VSP2, only 2ah displayed enhanced expression at all 3 time points. This indicates that impaired JA-This article is protected by copyright. All rights reserved.

Ile catabolism results in persistent defense marker expression, but this is not commensurate to JA-Ile accumulation (Figure 2a), because 5ko displays lower defense than 2ah. To determine the physiological impacts of such altered hormone and defense profiles, we used an insect feeding assay as a biological readout of signaling. When larvae of the generalist herbivore Spodoptera littoralis were placed on leaves of the four genotypes for 8 to 9 days, contrasted results were recorded: larvae fed on 2ah plants were consistently lighter than those feeding on WT, indicating stronger anti-insect resistance (Figure 3b). This opposed to 3cyp, that sustained higher insect development, confirming impaired resistance reported previously by [START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. Surprisingly, 5ko displayed WT level of resistance to herbivory. These conclusions were drawn from 3 successive trials (Figure S3) with large insect populations.

A similar defense-resistance analysis was conducted for the response to B. cinerea infection. The antimicrobial branch of JA-Ile-dependent defense signaling was probed by monitoring ORA59 TF and PDF1.2 defense marker expression. Unexpectedly, both genes exhibited reduced expression in 2ah and 5ko at 3 dpi, in contrast to 3cyp that maintained WT transcript levels (Figure 4a). Anti-fungal resistance assay indicated that only 3cyp displayed smaller lesions, in agreement with our previous report [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF], while 3cyp and to a lesser extent 2ah supported reduced fungal biomass as estimated from B. cinerea cutinase signal (Figure 4b). Of note, 5ko behaved essentially like WT in these infection assays.

Contents in camalexin, a major antimicrobial phytoalexin in Arabidopsis, were determined but no significant differences were found between genotypes. Together, the data show that impairing either one or both JA-Ile catabolic pathways has distinct and stress-specific consequences on JA-mediated defense and resistance responses.

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Impaired JA-Ile catabolism results in gain in resistance only when negative feedback effectors are not overinduced

Upon parallel investigation of the two leaf defense models, increased JA-Ile accumulation due to impaired catabolism did not correlate systematically with stronger defense or resistance phenotypes. Conversely, enhanced antifungal resistance occured in 3cyp without elevated hormone levels (Figures 2 and4). These observations suggest that additional players may be at work to limit or counteract overinduction of defense responses under deficient JA-Ile turnover. Obvious candidates for such negative regulation are genes encoding JAZ repressors (Browse, 2009) or JAM bHLH factors competing with MYC TFs [START_REF] Sasaki-Sekimoto | Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate responses in Arabidopsis[END_REF], or JAV1 repressor (Hu et al., 2013), all classes being JA-Ile-and stress-responsive. We selected JAZ genes that were previously described as highly induced and sensitive to catabolic pathway deregulation [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Heitz et al., 2012;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]. As shown in Figure 5a, JAZ8, JAZ10, JAM1, JAM2 and JAV1 expression was maximal in WT at 1 hpw with only slight enhancement in some mutants, but their transcripts were clearly more persistent in 3cyp and 5ko lines at 3 hpw, particularly for JAZ and JAV1, compared to 2ah that behaved like WT. This result establishes in wounded 3cyp and 5ko leaves a correlation between 1/ hyperaccumulation of JA-Ile (Figure 2), 2/ absence of enhanced defense or insect resistance (Figure 3) and 3/ persistence of JAZ, JAM and JAV1 transcripts (Figure 5). In contrast, despite of enhanced JA-Ile levels upon wounding, 2ah does not display persistent This article is protected by copyright. All rights reserved. expression of these repressors (Figure 5, see 3 and 6 hpw), and this particular pattern correlates with more robust defense (Figure 3a) and reduced insect herbivory (Figure 3b).

In response to B. cinerea infection, JAZ1, JAZ8 and JAM1 transcripts were overinduced in 2ah and 5ko (JAM2 only in 5ko) (Figure 5b), that overaccumulate JA-Ile, correlating with reduced defense (Figure 4a) and WT antifungal resistance (Figure 4b). On the contrary, these repressor genes were not overinduced in 3cyp, the only genotype exhibiting significantly increased antifungal resistance (Figure 4b). JAV1 transcripts overaccumulated in all infected mutants, but not differentially (Figure 5b). In summary, the comparative analysis across responses to two aggressors revealed that enhanced defense and resistance under deficient JA-Ile turnover is achieved only if negative effectors are themselves not over-responding to JA-Ile accumulation. combined with the ET precursor amino-cyclopropyl-carboxylic acid (ACC), or JA combined with ABA (all 15 µM). Kinetic analysis of gene expression was conducted by RT-qPCR. All analyzed genes were found induced by JA to various extents (Figure S4), but were differentially impacted by hormone combinations. VSP1 and PDF1.2 were further stimulated by JA+ABA and JA+ACC treatments, respectively. As expected from known antagonism between anti-insect and antimicrobial branches of JA-defense (Pieterse et al., 2012), PDF1.2 was repressed by JA+ABA treatment, and to a lower extent, VSP1 was repressed by JA+ACC (Figure S4). Surprisingly, only very limited genotype-specific effects were recorded and here, response of defense genes was not mirrored by repressor behavior. In particular, both defense markers exhibited a stronger response in the mutants, best visualized in their respective most potent treatments, with a hyperinduction of VSP1 in 3cyp. When the three repressor gene families were analyzed, the main feature that emerged was an unexpected excessive response of 3cyp for JAZ and JAM1 genes. Remarkably, repressor responses were essentially similar to WT in 2ah for all treatments. Therefore, the hormone combinations applied produced a gene response pattern that is distinct from those arising in mutants submitted to wounding or infection stress.

Exogenous hormone treatments do not phenocopy genotype-specific responses of JA-

Ile catabolic mutants

Jasmonate

Impaired JA-Ile catabolism impacts basal JA homeostasis and differentially regulates resting levels of target transcripts

We finally examined if impaired JA-Ile catabolism also affected basal JA homeostasis and target transcript levels in absence of imposed stress, by surveying these parameters in This article is protected by copyright. All rights reserved.

untouched leaves of a separate set of adult plants. JA profiling revealed significantly elevated JA levels in resting 3cyp and to a lesser extents in 5ko (Figure S5a). In addition, JA-Ile was clearly overaccumulated in CYP94-deficient lines, along with reduced abundance of its oxidation products. Due to high biological fluctuation in unstimulated leaves, statistical significance was difficult to ascertain for these latter, but both 12OH-JA-Ile and 12COOH-JA-Ile were undetected in 3cyp as expected.

At the level of gene expression, MYC2 and ORA59 TFs transcripts were significantly upregulated in 3cyp and 5ko lines (Figure S5b), but levels of their respective targets VSP and PDF1.2 were similar to or lower than WT in mutant lines. Consistently, genes encoding JAZ, JAM and JAV1 negative regulators had their transcripts accumulating to higher levels in 3cyp and 5ko, i.e. the lines with elevated JA-Ile contents. We conclude that 3cyp rather than 2ah restrains basal JA-Ile turnover and that non-cleared JA-Ile primarily upregulates negative feedback mechanisms, that in turn lower JA-Ile-dependent basal defenses.

DISCUSSION

Hormonal compounds must be timely and spatially controlled to ensure coordinated physiological responses. Catabolic pathways have been characterized for all major plant receptor-binding hormonal ligands and are integral components of hormone homeostasis and action. Since their recent discovery, JA-Ile catabolic pathways have been the focus of intense biochemical and physiological studies that disclosed their intricate relationships with JA signaling. As a common theme in plant hormone catabolism (Kawai, Ono, & Mizutani, 2014; This article is protected by copyright. All rights reserved.

Mizutani & Ohta, 2010), an oxidative inactivation pathway was characterized, with CYP94 enzymes defining a two-step JA-Ile metabolisation process. In contrast to most hormones for which conjugation corresponds to inactivation or generation of storage forms [START_REF] Piotrowska | Conjugates of abscisic acid, brassinosteroids, ethylene, gibberellins, and jasmonates[END_REF], JA requires conjugation to the amino acid isoleucine as the critical activation step. Consistently, deconjugation by the amidohydrolases IAR3 and ILL6 was characterized in Arabidopsis as a second JA-Ile removal pathway, acting in a JAR1 reverse reaction.

Understanding the functions of JA-Ile catabolism potentially offers avenues to tailor increased or on-demand defense signaling in diverse situations, and was addressed repetitively by lossof-function studies in the two catabolic pathways [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Bruckhoff et al., 2016;Heitz et al., 2012;Kitaoka et al., 2011;Koo et al., 2011;Koo et al., 2014;Luo et al., 2016;[START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]Woldemariam et al., 2012;[START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. However, due to the separate use of different biological systems and of partially impaired mutant lines, the possible logic underlying seemingly contradictory results was out of reach. In the present work, we conducted a comprehensive analysis of selected genotypes that allowed to evaluate the respective impact of each biochemical pathway on alterations in JA metabolism, defense regulation and resistance to biotic stress. The data clarify some former discrepancies and fill significant knowledge gaps by establishing novel pathway-and stress-specific features. either the oxidative or cleavage pathway were sufficient to overaccumulate JA-Ile upon leaf wounding (Heitz et al., 2012;Kitaoka et al., 2011;Koo et al., 2011;Koo et al., 2014;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]. This feature was extended in 3cyp or 2ah lines fully impaired in one or the other pathway, but here we demonstrate that their simultaneous deficiency raises JA-Ile to much higher levels, indicating that upon massive and rapid JA-Ile biosynthesis triggered by mechanical wounding, no additional enzymatic pathway can efficiently turnover and limit accumulation of JA-Ile. In response to B. cinerea infection, a comparable JA-Ile hyperaccumulation was recorded in 2ah and 5ko lines, but in marked contrast 3cyp infected leaves accumulated WT levels of JA-Ile, suggesting that in this latter background, the activity of the AH pathway is sufficient to prevent abnormal hormone build-up. The CYP94 oxidation pathway is however active in infected WT plants, because oxidized JA-Ile derivatives are readily detected, albeit less abundantly than upon wounding. The metabolic preponderance of the AH pathway upon infection suggests that CYP94 cannot substitute for AH deficiency and that IAR3/ILL6 enzymes are essential for proper JA-Ile homeostasis upon necrotrophic attack. Together these results demonstrate that the two JA-Ile catabolic pathways contribute differentially to JA-Ile removal upon two distinct leaf stresses.

We next investigated the signaling output of perturbed JA-Ile catabolism in the two leaf defense models and found that transcript levels of canonical regulatory and defense markers and resistance phenotypes were overall poorly correlated with perturbed JA-Ile levels in mutant lines. Upon wounding, 3cyp was closest to WT in terms of VSP induction, contrasting with the deficient insect defense performance of this genotype. Both AH-deficient lines gained a highly persistent VSP1/2 expression, particularly marked for VSP2 in 2ah. This This article is protected by copyright. All rights reserved.

behavior was accompanied by a lower weight gain by S. littoralis larvae in 2ah, but not in 5ko, despite of its massive JA-Ile content. The situation with antimicrobial resistance also revealed a strong distortion of the usually linear relationship between hormone, defense and resistance levels, as the high JA-Ile-accumulating lines 2ah and 5ko exhibited reduced ORA59/PDF1.2 defense signaling and WT B. cinerea resistance. In contrast, 3cyp, while maintaining JA-Ile levels similar to WT, was able to better defend against the fungus, as reported earlier [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]. In this line, the behavior of the ORA59-PDF1.2 module or camalexin levels were normal and do not seem to account for the better tolerance.

Nevertheless, this result shows that improved JA-dependent resistance can arise without an apparent over-accumulation of JA-Ile in a whole-leaf analysis.

The JA-Ile regulatory network is complex and sets in motion cascades of positive and negative transcriptional regulators (Hickman et al., 2017). We hypothetized that known elements defining negative feedback loops may counteract effects of chronic JA-Ile overaccumulation to restrain defense induction. This notion was introduced earlier for some JAZ genes in single or double mutants of one pathway, but its relevance was difficult to assess in single biological situations investigated in a limited set of genetic backgrounds [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Heitz et al., 2012;Koo et al., 2011;[START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF]. Here, we surveyed three gene families of characterized transcriptional repressors in the JA pathway in the different pathway/genotype/stress combinations: in addition to several JAZ, we also analyzed expression of JAM, a second type of negative regulator inhibiting transcription of JA-Ile-and MYC-regulated genes (Liu et al., 2019;[START_REF] Sasaki-Sekimoto | Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate responses in Arabidopsis[END_REF], and JAV1, that directs This article is protected by copyright. All rights reserved. a calcium-sensitive complex repressing JA responses (Yan et al., 2018). We found a robust negative correlation spanning both pathosystems between the behavior of several JAZ, JAM and JAV1 genes and the amplitude and timing of defense response under impaired JA-Ile turnover (summarized in Figure 6). We selected JAZ genes that were previously found to be sensitive to altered JA-Ile homeostasis [START_REF] Aubert | CYP94-mediated jasmonoyl-isoleucine hormone oxidation shapes jasmonate profiles and attenuates defence responses to Botrytis cinerea infection[END_REF]Heitz et al., 2012). JAZ8, JAZ10, JAM1 and JAM2, and JAV1 expression persisted longer than in WT in 3cyp and 5ko after wounding, but strikingly behaved like WT in 2ah, correlating in this latter line with more sustained VSP expression and poorer feeding by S. littoralis larvae. In the case of B. cinerea infection, a similar logic was respected, but in different genotypes. Both 2ah and 5ko displayed significantly higher than WT expression of either JAZ or JAM or both repressor genes, but remarkably, this was not observed in 3cyp. This pattern correlated well with the former lines showing low defense and WT necrotic lesion sizes, and 3cyp maintaining WT defense and being more resistant to infection. In 3cyp, ORA59-PDF1.2 as well as camalexin accumulation behaved like in WT, so other defense determinants must account for the better resistance. In summary, we established that impairing the JA-Ile catabolic pathways has contrasted signaling consequences in Arabidopsis: a gain in defensive/resistance capacity is recorded compared to WT only when JAZ/JAM type repressor genes are not themselves overinduced, and this does not necessarily require higher hormone accumulation. In most situations, impaired JA-Ile turnover triggers a syndrome of negative feedback that likely prevents excessive signaling and that can even result in lower-than-WT defense [START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF].

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The impacts of rewiring genetically JA-Ile catabolic capacity may vary between plant species and needs further investigations. For example, the oxidative or deconjugation pathway were silenced transiently (CYP94) or stably (JIH) in Nicotiana attenuata, and enhanced JA-Iledependent defenses and insect resistance were described in two studies (Luo et al., 2016;Woldemariam et al., 2012). We showed that it is possible to genetically extend the half-life and amplitude of JA-Ile pulses in Arabidopsis, but the precise mechanism underlying control of signaling when JA-Ile catabolism is impaired remains unknown. Emerging evidence indicates that distinct COI1-JAZ co-receptors have different JA-Ile detection thresholds (Chini et al., 2016;[START_REF] Takaoka | A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants[END_REF] and modified JA-Ile homeostasis may selectively alter the output of some of these sensors. The mechanism directing conditional feedback hyperinduction of JAZ/JAM genes is currently unknown. JA signaling is embedded in highly hierarchized hormonal networks, such as synergy with ET upon necrotroph attack, or with ABA upon wounding or herbivory, and these variable integration nodes may impact the outcome (Pieterse et al., 2012). We addressed the possible influence of these hormonal interactions by submitting young seedlings to JA treatment combined with either the ET precursor ACC or with ABA. Although a consistent but unexplained enhanced induction of both defense and repressor genes was observed in 3cyp, this hormone feeding experiment could not reproduce the genotype-specific responses arising after wounding or infection stress. Therefore, these latter cannot be explained by a simple ET vs ABA dichotomy in the integration of JA responses.

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It is remarkable that all studied mutant lines, including the newly generated 5ko defective in all known JA-Ile-catabolizing enzymes, were undistinguishable from WT in their vegetative growth under standard conditions (Figure S6). This raises the question of the homeostasis of JAs and the regulation of target genes in non-stimulated leaves in these genotypes. Metabolic and molecular analysis revealed that although untouched leaves harbor a much quieter JA metabolism/signaling, a similar negative feedback operates before stress. Basal levels of stress markers are prone to fluctuations among replicates due to unintended stress during plant cultivation, but increases in steady-state JA/JA-Ile levels could be measured, particularly in 3cyp (and 5ko), and those were correlated with higher MYC2 and ORA59 transcripts (Figure S5). Hence, the action of these TFs must be somehow blocked because their target transcripts were lower than in WT, while all repressor transcripts were upregulated in mutants. These observations indicate that JA-Ile catabolism is needed on trace hormone levels to ensure optimal regulation and responsiveness to further stress. This situation is in marked contrast with the metabolic sink defined upstream of JA-Ile formation by JA oxidases (JAOs, Figure 1). The single mutation of JAO2 enhances basal JA-Ile-dependent defense and strongly increases pathogen resistance (Heitz, Smirnova, Marquis, & Poirier, 2019;[START_REF] Smirnova | Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection[END_REF].

The fact that blocking either of two catabolic pathways acting on the same JA-Ile substrate has distinct signaling and physiological outcomes suggests that signaling is not mediated solely through the control of JA-Ile steady-state levels. Modified abundance of its catabolites may also contribute to alter the signaling process(s). Recent papers reported that in addition to This article is protected by copyright. All rights reserved. 2012). Jasmonoyl-Lisoleucine hydrolase 1 (JIH1) regulates jasmonoyl-L-isoleucine levels and attenuates plant defenses against herbivores. Plant J, 72( 5), 758-767. doi:10.1111/j.1365-313X.2012.05117.x. Yan, C., Fan, M., Yang, M., Zhao, J., Zhang, W., Su, Y., . . . Xie, D. (2018)

FIGURE LEGENDS

This article is protected by copyright. All rights reserved. SEM from 3 independent trials (presented in Figure S3). The number of total larvae on each genotype is indicated within the bars. Different letters indicate significant differences at P < 0.05 (linear mixed model). This article is protected by copyright. All rights reserved. Lesions diameter (mm)

FIGURE S1

Marquis et al.

FIGURE S2

PDF1.2 ORA59

Relative expression Relative expression

  mass analyzer and 2 for the second. Data acquisition was performed with the MS Workstation 8 for the mass spectrometry and the liquid chromatography was piloted with Bruker Compass Hystar 4.1 SR1 software. The data analysis was performed with the MS Data Review software. Absolute quantifications were achieved by comparison of sample signal with doseresponse curves established with pure compounds and recovery correction based on internal standard signal. The transitions were, in negative mode: JA 209.3>59.3; JA-Ile 322.3>130.2; 12OH-JA-Ile 338.3>130.2; 12COOH-JA-Ile 352.2>130.1; 12OH-JA 225.2>59.3; in positive mode: camalexin 201.0>59.3.

  -dependent responses are known to be largely specified by crosstalk with other hormones. Particularly, JA and ABA signaling pathways interact cooperatively in the upregulation of transcriptional wound responses whereas JA and ET act synergistically in the induction of antimicrobial defenses (Pieterse et al., 2012). We hypothetized that stressspecific responses in JA-Ile catabolic mutants could reflect different interactions of the manipulated JA pathway with ET and/or ABA pathways. To investigate this possibility, WT, 2ah and 3cyp 7-days old seedlings were treated exogenously with either JA alone, or JA This article is protected by copyright. All rights reserved.

  Wounding/insect stress and infection by a necrotroph both induce strong JA metabolism and signaling (Campos et al., 2014), but due to different interactions with other hormonal networks (Pieterse et al., 2012), the subsequent transcriptomes and defensive outcomes display only partial overlap. Previous reports showed that single mutations in members of This article is protected by copyright. All rights reserved.

  hormone turnover and generate 12-hydroxyjasmonic acid upon wounding in Arabidopsis leaves. J Biol Chem, 288(44), 31701-31714. doi:10.1074/jbc.M113.499228 Widemann, E., Smirnova, E., Aubert, Y., Miesch, L., & Heitz, T. (2016). Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana. Plants (Basel), 5(1). doi:10.3390/plants5010004. Woldemariam, M. G., Onkokesung, N., Baldwin, I. T., & Galis, I. (

FIGURE 1 .

 1 FIGURE 1. Positions of the impaired enzymatic steps in JA-Ile catabolic mutants and

FIGURE 2 .FIGURE 3 .

 23 FIGURE 2. JA-Ile and oxidized catabolites accumulation in 2ah, 3cyp and 5ko mutants after

FIGURE 4 .

 4 FIGURE 4. Expression profiles (a) of JA-Ile-dependent defense genes and (b) resistance

FIGURE 5 .

 5 FIGURE 5. Expression profiles of JA-Ile dependant repressor genes in response to wounding

FIGURE 6 .

 6 FIGURE 6. Relationships between impaired JA-Ile catabolism, JA-Ile levels,

  FIGURE 1

  FIGURE S3

  FIGURE S5

TABLE S1 .

 S1 Primers used in the study

	qPCR
	Gene/allele Locus	Primer3name	Sequence (5'38>33')
	EXP$	At4g26410 At4g26410)qPCR_F	gagctgaagtggcttcaatgac
	TIP4.1$	At4g26410)qPCR_R At4g34270 TIP41_LP FIGURE S6	Marquis et al. ggtccgacatacccatgatcc gtgaaaactgttggagagaagcaa
			TIP41_RP	tcaactggataccctttcgca
	MYC2$	At1g32640 E49)MYC2)L	gccgaaggaatacacgcaat
			E50)MYC2)R	cgggttgtgaacgggcta
	VSP1$	At5g24780 VSP1)F	ccgtcaatgtttggatctttg
			VSP1)R	gctgtgttctcggtcccata
	VSP2$	At5g24770 VSP2_qPCR_for	ggtgcccgcaaattgcaaagacta
			VSP2_qPCR_rev	ggttgatgctccggtccctaacca
	ORA59$	At1g06160 ORA59_qPCR_for_new	ttcgacgttgacatcttctcc
			ORA59_qPCR_rev_new	tcttgcgtcataacaacactctg
	PDF1.2$	At5g44420 E39)PDF1.2)L	cacccttatcttcgctgctctt
			E40)PDF1.2)R	tacacttgtgtgctgggaagac
	JAZ1$	At1g19180 JAZ1)F	ttctgagttcgtcggtagcc
			JAZ1)R	cacgtctgtgagaagctaggc
	JAZ8$	At1g30135 JAZ8)F	aatgtgtttttcttcagatgttaccc
			JAZ8)R	ttctctgcttgcgatcgatatt
	JAZ10$	At5g13220 JAZ10)F	cgctcctaagcctaagttcca
			JAZ10)R	tttcgaaatcgcaccttgaat
	JAM1$	At2g46510 JAM1_qPCR_for	ggagctcacgcgtatcctaa
			JAM1_qPCR_rev	ggattcgaagaagcagcaac
	JAM2$	At1g01260 JAM2_qPCR_for	gaggccaatcaacgtgaagg
			JAM2_qPCR_rev	tcccctcctccttctccatc
	CYP94B1$	At5g63450 CYP94B1LqPCRLF62	caatgaggctttacccaccag
			CYP94B1LqPCRLR62	aaatgtcgtcgtttgctgcat
	CYP94B3$	At3g48520 CYP94B3LqPCRLF62	tggcttacacgaaggcttgtc
			CYP94B3LqPCRLR62	agtcccacgaaactggaggat
	CYP94C1$	At2g27690 CYP94C1LqPCRLF63	ggcccggattacgaagagttt
			CYP94C1LqPCRLR63	ggccggaacttaccttcgtt
	IAR3$	At1g51760 IAR3)0,4F	gttgctttaagggctgatatgg
			IAR3)N69047)R	accgagaagcatcgtagtgtga
	ILL6$	At1g44350 ILL6)GK212E12)F	gtgtcccatatccatccaacgg
			ILL6)GK212E12)R	agactaatgaccgcggaagaag
	Actin2$	At3g18780 Act_for	cttgcaccaagcagcatgaa
			Act_rev	ccgatccagacactgtacttcctt
	UBQ10$	At4g05320 UBQ10_for	ggccttgtataatccctgatgaataag
			UBQ10_rev	aaagagataacaggaacggaaacatagt
	Cutinase	B.c Z69264 CutA)L	gatgtgacggtcatctttgccc
			CutA)R	agatttgagagcggcgagg
	T8DNA3 genotyping
	Gene/allele		Primer3name	Sequence (5'38>33')
			LBb1.3L(SALK)	attttgccgatttcggaac
			GABI8409LB	atattgaccatcatactcattgc
	cyp94b1L1		94B1)LP	tcgaatcacattgctctctcc
			94B1)RP	gggaattcactttcgaaatcc
	cyp94b3L1		CYP94B3)0,72F	gaacgtgggaagcgagaggaagc
			BO1BG68	tggtttggttctcactgttcac
	cyp94c1L1		SALK_055455LLP	tgtctttttggaaagtagcacc
			SALK_055455LRP	gattccacggcctaaaagatc
	iar3L5 iar3L7		Salk_069047)LP	gttctccacgtgcgttatagc
			Salk_069047)RP	aaaaagccacactgttccatg
	ill6L1		GK412E11)LP	gactatgcttcttggtgctgc
			GK412E11)RP	cgcacctcttgaatacgtttc
	ill6L2		SALK_024894)LP	gactatgcttcttggtgctgc
			SALK_024894)RP	cgcacctcttgaatacgtttc
			7

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Pathway-specific impact of JA-Ile catabolism on defense Funding information CNRS, grants ANR-12-BSV8-005 from the Agence Nationale de la Recherche and IdEx-2014-208e Interdisciplinary grant from Université de Strasbourg and CNRS, Université de

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Strasbourg and the Ministère de l'Enseignement Supérieur et de la Recherche to T.H. Swiss National Science Foundation grant number 31003A_169278 to P.R.

doi:10.1093/jxb/erv190.

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Browse, J. (2009). Jasmonate passes muster: a receptor and targets for the defense hormone.

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Nature

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wound responses (Jimenez-Aleman et al., 2019;[START_REF] Poudel | 12-Hydroxy-jasmonoyl-L-isoleucine is an active jasmonate that signals through CORONATINE INSENSITIVE 1 and contributes to the wound response in Arabidopsis[END_REF]. Such an interpretation would be consistent with metabolic and defensive features of 2ah in wound/insect responses, but not with 3cyp in the Botrytis assay. In addition, one must keep in mind that IAR3 also accepts auxin conjugates as substrates, which may also be at the basis of hormonal cross-talk and alter signaling [START_REF] Widemann | The amidohydrolases IAR3 and ILL6 contribute to jasmonoyl-isoleucine[END_REF][START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF]. Elucidating the factors determining when negative regulators take control will be of major importance to maximize the potential output of JA-regulated defenses. This provides the ground for future work at the protein, promoter and chromatin levels to determine how inhibitory mechanisms could be at work.

Conflict of interest

The authors declare having no conflict of interests.