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Stress- and pathway-specific impacts of impaired jasmonoyl-isoleucine (JA-Ile) 1

catabolism on defense signaling and biotic stress resistance in Arabidopsis 2

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Valentin Marquis1†, Ekaterina Smirnova1†, Laure Poirier1, Julie Zumsteg1, Fabian 4

Schweizer2, Philippe Reymond2 and Thierry Heitz1* 5

6 1 Institut de Biologie Moléculaire des Plantes (IBMP) du CNRS, Université de Strasbourg, Strasbourg, 7 France 8 2 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland 9

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† These authors contributed equally to the work 11

12 *Author for correspondence: 13 [email protected] 14 15 16 Short title: Pathway-specific impact of JA-Ile catabolism on induced defense 17 18

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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted June 28, 2019. ; https://doi.org/10.1101/686709doi: bioRxiv preprint

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ABSTRACT 20

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Jasmonate (JA) synthesis and signaling are essential for plant defense upregulation upon 22

herbivore or microbial attacks. Stress-induced accumulation of jasmonoyl-isoleucine (JA-Ile), 23

the bioactive hormonal form triggering major transcriptional changes, is often dynamic and 24

transient, due to the existence of potent removal mechanisms. Two distinct but interconnected 25

JA-Ile turnover pathways have been described in Arabidopsis, either via cytochrome P450 26

(CYP94)-mediated oxidation, or through deconjugation by the amidohydrolases (AH) IAR3 27

and ILL6. Their impact was not well known because of gene redundancy and compensation 28

mechanisms when each pathway was partially impaired. Here we address the consequences of 29

fully blocking either or both pathways on JA homeostasis and defense signaling in three mutant 30

backgrounds: a double iar3 ill6 (2ah) mutant, a triple cyp94b1 b3 c1 mutant (3cyp), and a newly 31

generated quintuple (5ko) mutant deficient in all known JA-Ile-degrading activities. These lines 32

behaved very differently in response to either mechanical wounding, insect attack or fungal 33

infection, highlighting the stress-specific contributions and impacts of JA-Ile catabolic 34

pathways. Deconjugation and oxidative pathways contributed additively to JA-Ile removal 35

upon wounding, but their genetic impairement had opposite impacts on Spodoptera littoralis 36

larvae feeding: 2ah line was more resistant whereas 3cyp was more susceptible to insect attack. 37

In contrast, 2ah, 5ko but not 3cyp overaccumulated JA-Ile upon inoculation by Botrytis cinerea, 38

yet 3cyp was most resistant to the fungus. Despite of building-up unprecedented JA-Ile levels, 39

5ko displayed near WT levels of resistance in both bioassays. Molecular and metabolic analysis 40

indicated that restrained JA-Ile catabolism resulted in enhanced defense and resistance levels 41

only if genes encoding JAZ or JAM negative regulators were not simultaneously 42

overstimulated. Our data demonstrate that despite of acting on a shared hormonal substrate, AH 43

or/and CYP94 deficiency differentially impacts JA homeostasis, responses and tolerance to 44

related biotic stresses. 45

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INTRODUCTION 49

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Jasmonates (JAs) have been recognized for more than two decades as powerful regulators of 51

inducible defense responses protecting plants from damage inflicted by herbivorous insects or 52

microbial pathogens (Campos et al. 2014; Wasternack and Hause 2013). Even before the 53


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identification of jasmonoyl-isoleucine (JA-Ile) as the critical bioactive form in 2007 (Chini et 54

al. 2007; Thines et al. 2007), many derivatives of jasmonic acid (JA) were described, resulting 55

from hydroxylation, conjugation to sugars (Miersch et al. 2008), amino acids or amino 56

cyclopropane carboxylic acid, sulfation (Gidda et al. 2003), decarboxylation and many more 57

(Wasternack and Song 2017). The enzymes responsible for these modifications have not all 58

been identified and the function of these derivatives, if any, are generally unknown. The master 59

regulator jasmonoyl-(L)-isoleucine (JA-Ile) results from a specific conjugation reaction of 60

jasmonic acid (JA) by the enzyme JASMONATE RESISTANT 1 (JAR1). In the core 61

perception and signaling pathways, JA-Ile acts as a ligand promoting the assembly of the co-62

receptor CORONATINE INSENSITIVE 1 (COI1) with distinct JASMONATE ZIM-DOMAIN 63

PROTEIN (JAZ) that otherwise powerfully repress target transcription factors and responses. 64

After assembly, COI1-JAZ is recruited into the E3 ubiquitin ligase SCFCOI1 that tags JAZs for 65

proteolytic degradation, and provides the basis for JA-Ile-responsive gene de-repression (Chini 66

et al. 2007; Thines et al. 2007). Several hundreds to thousands of genes are under this control, 67

and depending on developmental stage, organ, nature of stimulus, and crosstalks with other 68

hormones, a wide array of JAZ-transcription factor combinations provide specificity to the 69

system with only one major hormonal ligand (Chini et al. 2016). Distinct JA-Ile-triggered 70

networks regulate leaf defenses against different types of aggressors and are integrated by 71

separate sets of transcription factors (TF). MYC2, a bHLH type TF, integrates (together with 72

MYC3 and MYC4) simultaneous JA/abscisic acid stimuli and defines a wound/insect-specific 73

branch reflected by induction of markers such as vegetative storage protein (VSP); 74

ERF1/ORA59 TFs integrate concomitant JA/ethylene signals into a microbe-specific branch 75

probed by plant defensin PDF1.2 (Pieterse et al. 2012; Wasternack and Hause 2013). 76

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JA signaling, like any hormonal pathway, needs to be tightly controlled in time and space, 78

particularly because defense upregulation is costly and linked to overall growth inhibition. This 79

antagonism was long thought to be merely imposed by limited resources that need to be re-80

allocated from developmental to defense sinks (Havko et al. 2016), but recent advances have 81

shown that additional hardwire connections control growth-defense tradeoffs (Campos et al. 82

2014; Guo et al. 2018). For appropriate control of JA responses, plants rely on a number of 83

negative feedback mechanisms. In addition to JAZ repressor proteins, other negative regulators 84

were identified that repress JA responses at the promoter level, including JAV (Yan et al. 2018) 85

and the JAM subclass of bHLH (Sasaki-Sekimoto et al. 2013) proteins. Another way to 86

terminate jasmonate action is at the metabolic level by eliminating the active ligand JA-Ile. The 87


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characterization of JA-Ile catabolic pathways has shed light on hormone homeostasis regulation 88

and has also provided additional complexity in the JA metabolic grid. Two distinct enzymatic 89

pathways are known to modify or degrade the JA-Ile hormone. One consists in a two step 90

oxidation of the terminal (w) carbon of the JA moiety by cytochrome P450 monooxygenases 91

of the CYP94 family. In Arabidopsis, CYP94B3 and to a minor extent CYP94B1 generate 92

12OH-JA-Ile as a main product whereas CYP94C1 catalyzes the full oxidation of JA-Ile to 93

12COOH-JA-Ile (Figure 1) (Bruckhoff et al. 2016; Heitz et al. 2012; Kitaoka et al. 2011; Koo 94

et al. 2011, 2014). The first oxidation product retains reduced receptor-assembly capacity and 95

gene-inducing activity (Aubert et al. 2015; Koo et al. 2011; Poudel et al. 2019), but the second 96

product proved fully inactive (Aubert et al. 2015; Koo et al. 2014). The second pathway is 97

defined by deconjugating activity, first identified in Nicotiana attenuata (Woldemariam et al. 98

2012) and subsequently characterized in Arabidopsis as mediated by the amidohydrolases IAR3 99

and ILL6 (Figure 1) (Widemann et al. 2013). These enzymes act on JA-Ile and other JA-amino 100

acid conjugates but also on 12OH-JA-Ile generated by CYP94Bs, releasing JA and 12OH-JA 101

from these respective substrates (Widemann et al. 2013; Zhang et al. 2016). The interconnection 102

of the two catabolic pathways generates increased metabolic complexity because many of the 103

above-mentioned JAs derive in fact from JA-Ile catabolism (Heitz et al. 2016; Widemann et al. 104

2013). Both JA-Ile oxidases and amidohydrolases genes are induced by environmental cues and 105

are co-regulated with the central regulon defined by JA pathway biosynthetic and signaling 106

genes. Their simultaneous enzymatic action shapes specific jasmonate patterns in different 107

tissues and stress conditions (Aubert et al. 2015; Heitz et al. 2012; Koo and Howe 2012; Koo 108

et al. 2014; Widemann et al. 2013, 2016). 109

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Manipulating JA-Ile catabolic routes could be an attractive tool to engineer plants for enhanced 111

pathogen resistance or modulate other JA-dependent processes. Loss- or gain-of-function 112

mutant lines in CYP94 or AH genes have revealed profound impacts on JA metabolism but 113

contrasted consequences on JA responses. Arabidopsis lines ectopically overexpressing CYP94 114

or AH generally have reduced JA-Ile levels and a metabolic shift towards oxidized and/or 115

cleaved derivatives. As expected, increased turnover is correlated with attenuated JA responses, 116

for example increased sensitivity to Botrytis infection or to insect larvae feeding (Aubert et al. 117

2015; Koo et al. 2011). In comparison, analysis of single, double, or triple mutant lines has not 118

led to a unified conclusion as to how JA-Ile responses are impacted by gradual impairment of 119

JA-Ile catabolism. Deficiency in CYP94 or AH expression impacts JA homeostasis consistently 120

with their in vitro enzymatic activities. For example, cyp94b3 or b3c1 mutations lead to more 121


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JA-Ile upon wounding, concomitantly to reduced 12OH-JA-Ile and 12COOH-JA-Ile levels 122

(Heitz et al. 2012; Kitaoka et al. 2011; Koo et al. 2011). Double cyp94b3c1 and triple 123

cyp94b1b3c1 mutant plants exhibited slightly enhanced defense responses and tolerance to 124

fungal infection (Aubert et al. 2015). Strikingly, in another study, the double cyp94b1b3 and 125

triple cyp94b1b3c1 mutants exhibited deficient JA-Ile responses, questioning the current 126

signaling model (Poudel et al. 2016). On the other hand, AH-deficient lines have not yet been 127

tested for defense and resistance responses, so the contribution and impacts of this pathway are 128

unknown. 129

To overcome gene redundancy and compensation mechanisms between pathways that occur 130

frequently in JA metabolic mutants (Smirnova et al. 2017; Widemann et al. 2013), we 131

introduced in the present study new plant lines with higher order mutations (Figure 1): a double 132

iar3 ill6 mutant (thereafter called 2ah) impaired in the JA-Ile deconjugating pathway; and a 133

quintuple cyp94b1 cyp94b3 cyp94c1 iar3 ill6 (thereafter called 5ko) deficient in all 134

characterized enzymes turning over JA-Ile. We submitted these lines, along with the oxidation-135

deficient cyp94b1b3c1 mutant (thereafter called 3cyp), to parallel leaf stimulation by 136

mechanical wounding or B. cinerea infection, two environmental cues triggering strong JA 137

metabolism and signaling, but activating distinct transcriptional networks due to different cross-138

talks with other hormonal pathways (Pieterse et al. 2012). We determined the contribution of 139

each catabolic pathway on JA-Ile turnover for each stress and investigated the impact of 140

modified JA-Ile homeostasis on defense responses and tolerance to aggression by an herbivore 141

insect or by fungal infection (Figure 1). The data highlight new stress-specific impacts of fully 142

impairing either one or both JA-Ile catabolic pathways, where defense and aggressor resistance 143

intensities are more correlated (negatively) to the hyper-activation of negative feedback 144

signaling effectors rather than on actual JA-Ile levels. 145

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RESULTS 148

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Jasmonate profiling of higher order catabolic mutants reveals stimulus-specific impacts 150

on hormone homeostasis 151

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Effective inactivation of the respective genes was verified in each mutant line by RT-qPCR 153

using RNA extracted from 1 h-wounded leaves (Supplemental Figure S1 and (Aubert et al. 154


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2015)). The four plant genotypes in the Col-0 ecotype (WT, 2ah, 3cyp, 5ko) were submitted 155

separately to mechanical wounding or to B. cinerea inoculation. Mechanical wounding 156

constitutes a synchronous and severe stimulus, and generates a massive jasmonate pulse where 157

compound-specific dynamics can be followed (Chung et al. 2008; Glauser et al. 2008; Heitz et 158

al. 2016). Therefore, a kinetic study was conducted with tissue collected at 1, 3 and 6 h post-159

wounding (hpw). Necrotic lesions inflicted by B. cinerea infection develop radially with fungal 160

hyphae continuously infecting new tissue, and 3 days post-inoculation (dpi) constitutes an 161

optimal time point for recording biochemical changes and for assessing antifungal resistance. 162

We quantified levels of JA, 12OH-JA, JA-Ile and its catabolites 12OH-JA-Ile and 12COOH-163

JA-Ile in the two biological responses. JA levels were only marginally affected by the mutations 164

(Supplemental Figure S2). Its oxidation product, 12OH-JA, is known to be partially formed via 165

conjugate intermediates (Figure 1) upon wounding (Widemann et al. 2013) and accordingly, 166

was less abundant in all mutants at 3 hpw but was only reduced in 2ah at 6 hpw (Supplemental 167

Figure S2). In contrast, 12OH-JA was less affected by mutations upon infection and was even 168

enhanced in 5ko, likely reflecting the predominant contribution of JAO enzymes directly 169

oxidizing JA in this material (Figure 1) (Smirnova et al. 2017). 170

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Impacts on JA-Ile homeostasis upon wounding 172

JA-Ile showed the typical pattern (Chung et al. 2008; Glauser et al. 2008; Heitz et al. 2012; Koo 173

et al. 2011) in wounded WT leaves, peaking at 1 hpw and declined thereafter (Figure 2A). In 174

2ah and 3cyp lines, a significant overaccumulation of JA-Ile was recorded at 1 hpw and was 175

prolonged in 3cyp, extending data from mutants of lower order described previously (Heitz et 176

al. 2012; Widemann et al. 2013; Zhang et al. 2016). In the 5ko line that has both pathways 177

impaired, a huge JA-Ile accumulation was recorded that culminated between 3-6 hpw close to 178

40-50 nmol g FW-1. These data show that in wounded leaves, both AH and CYP94 pathways 179

contribute similarly to JA-Ile turnover and that their simultaneous inactivation synergistically 180

boosts hormone hyperaccumulation at later time points. Profiles of CYP94-generated JA-Ile 181

oxidation products were as expected: 12OH-JA-Ile levels were about double in 2ah compared 182

to WT, were suppressed in 3cyp and of note, were higher in 5ko than in 3cyp at 6 hpw (Figure 183

2A). 12COOH-JA-Ile was only detected from 3 hpw and evolved similarly to 12OH-JA-Ile in 184

2ah and 3cyp, but was barely detected in 5ko. 185

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Impacts on JA-Ile homeostasis upon Botrytis infection 189

The metabolic impacts were different in response to B. cinerea inoculation. JA-Ile levels were 190

similar to WT in 3cyp, confirming our previous data (Aubert et al. 2015) (Figure 2B). In 191

contrast, JA-Ile levels were strongly enhanced in 2ah but no further increase was observed in 192

5ko. This result indicates that the amidohydrolase pathway is essential for JA-Ile clearance upon 193

fungal infection and that blocking simultaneously the oxidative pathway does marginally 194

impact JA-Ile accumulation. 2ah also considerably enhanced 12OH-JA-Ile levels and 195

12COOH-JA-Ile, reinforcing initial data obtained with iar3 or ill6 single mutants (Widemann 196

et al. 2013). In 2ah, excess JA-Ile is likely oxidized into 12OH-JA-Ile that can no more be 197

deconjugated by IAR3 and ILL6 and part of this enlarged pool is further oxidized to 12COOH-198

JA-Ile by CYP94C1. Together, these experiments demonstrate stimulus-specific contributions 199

of AH and CYP94 catabolic pathways to JA-Ile turnover and accumulation of downstream 200

derivatives. 201

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Impact of impaired catabolic pathways on defense and resistance responses is not 203

reflecting JA-Ile accumulation in mutant plants 204

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As the mutations altered the steady-state levels of bioactive JA-Ile, the lines were examined for 206

induced expression of typical JA-regulated genes for each leaf stress model. In the case of 207

wounding, transcripts of MYC2, an early-responsive transcription factor (TF) gene controlling 208

late targets, accumulated similarly at 1 hpw, but declined less than WT in mutants at 3 hpw 209

(3cyp and 5ko) and 6 hpw (2ah and 5ko) (Figure 3A). Two VSP genes were examined as late 210

markers of the anti-insect branch of the JA defense pathway (Pieterse et al. 2012; Wasternack 211

and Hause 2013). In WT, both VSP1 and VSP2 expression peaked at 3 hpw before declining to 212

low levels at 6 hpw (Figure 3A). Expression was globally enhanced in mutant lines compared 213

to WT, but with distinct patterns. VSP1 was strongly enhanced in 2ah at all time points, while 214

in 3cyp and 5ko, gain in transcript levels was only observed at 6 hpw when WT signal faded. 215

For VSP2, only 2ah displayed enhanced expression at all 3 time points. This indicates that 216

impaired JA-Ile catabolism results in persistent defense marker expression, but this is not 217

commensurate with JA-Ile accumulation, because 5ko displays lower defense than 2ah. To 218

determine the physiological impacts of such altered hormone and defense profiles, we used an 219

insect feeding assay as a biological readout of signaling. When larvae of Spodoptera littoralis 220

were placed on leaves of the four genotypes for 8 to 9 days, contrasted results were recorded: 221

larvae fed on 2ah plants were consistently lighter than those feeding on WT, indicating stronger 222


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anti-insect resistance (Figure 3B). This opposed to 3cyp, that sustained higher insect 223

development, confirming impaired resistance reported previously by Poudel et al. (2016). 224

Finally, similarly to fungal infection, 5ko displayed WT level of resistance to herbivory. These 225

conclusions were drawn from 3 successive trials (Supplemental Figure S3) with large insect 226

populations. 227

A similar analysis was conducted for the response to B. cinerea infection. The antimicrobial 228

branch of JA-Ile-dependent defense signaling was probed by monitoring ORA59 TF and 229

PDF1.2 marker expression. Surprisingly, both genes exhibited reduced expression in 2ah and 230

5ko at 3 dpi, in contrast to 3cyp that maintained WT levels (Figure 4A). Anti-fungal resistance 231

assay indicated that only 3cyp displayed smaller lesions, in agreement with our previous report 232

(Aubert et al. 2015), while 3cyp and to a lesser extent 2ah supported reduced fungal biomass 233

as estimated from B. cinerea cutinase signal (Figure 4B). Content in camalexin, a major 234

antimicrobial phytoalexin in Arabidopsis, was determined but no significant differences were 235

found between genotypes. Together, the data show that impairing either one or both JA-Ile 236

catabolic pathways has distinct and stress-specific consequences on JA-mediated defense and 237

resistance responses. 238

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Impaired JA-Ile catabolism results in gain in resistance only when negative feedback 240

effectors are not overinduced 241

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Upon parallel investigation of the two leaf defense models, increased JA-Ile accumulation due 243

to impaired catabolism did not correlate systematically with stronger defense or resistance 244

phenotypes. Conversely, enhanced resistance can occur without elevated hormone levels 245

(Figures 2, 3 and 4). These observations suggest that other players may be at work to limit or 246

counteract overinduction of defense responses under deficient JA-Ile turnover. Obvious 247

candidates for such negative regulation are genes encoding JAZ repressors (Browse 2009) or 248

JAM bHLH factors competing with MYC transcription factors (Sasaki-Sekimoto et al. 2013), 249

both classes of genes being JA-Ile-responsive. We selected JAZ genes that were previously 250

described as highly induced and sensitive to catabolic pathway manipulation (Aubert et al. 251

2015; Heitz et al. 2012; Widemann et al. 2013). As shown in Figure 5A, JAZ8, JAZ10, JAM1 252

and JAM2 expression was maximal in all lines at 1 hpw with only slight enhancement in some 253

mutants, but their transcripts were clearly more persistent in 3cyp and 5ko lines at 3 and 6 hpw, 254

particularly for JAZ, compared to 2ah and WT. This result establishes in 3cyp and 5 ko a 255

correlation between hyperaccumulation of JA-Ile (Figure 2), the overinduction of JAZ and JAM 256


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genes (Figure 5), near-WT defense induction and WT (5ko) or deficient (3cyp) levels of insect 257

resistance (Figure 3) compared to WT. In contrast, despite of enhanced JA-Ile levels upon 258

wounding, 2ah does not display persistent expression of these repressors (Figure 5, 3 and 6 259

hpw), and this correlates with more robust defense and reduced insect herbivory (Figure 3B). 260

In response to B. cinerea infection, JAZ1, JAZ8 and JAM1 transcripts were overinduced in 2ah 261

and 5ko (JAM2 only in 5ko) (Figure 5B), that overaccumulate JA-Ile, correlating with reduced 262

defense (Figure 4A) and WT antifungal resistance (Figure 4B). On the contrary, these genes 263

were not overinduced in 3cyp, the only genotype exhibiting significantly increased antifungal 264

resistance (Figure 4B). Therefore, the comparative analysis showed that elevated defense and 265

resistance under deficient JA-Ile turnover is induced only if negative effectors are themselves 266

not over-responding to JA-Ile accumulation. 267

To determine if JA-Ile catabolism mutations affected also basal gene expression in leaves 268

before stress, we analyzed transcript levels of the same target genes in untouched leaves in a 269

separate set of plants. Although higher fluctuations were recorded within a given genetic 270

background than for stimulated tissues, two trends emerged: MYC2 and ORA59 TFs transcripts 271

were significantly upregulated in 3cyp and 5ko lines, but levels of their respective targets VSP 272

and PDF1.2 were equal to or lower than WT in these lines (Supplemental Figure S4). 273

Strikingly, all 5 analyzed genes encoding JAZ and JAM negative regulators were expressed at 274

slightly but significantly higher levels in mutant (mostly 3cyp and 5ko) compared to WT. This 275

result indicates that severe or even total block in JA-Ile catabolism does not increase basal 276

expression of typical defense marker genes in Arabidopsis leaves. 277

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DISCUSSION 279

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Hormonal compounds must be timely and spatially controlled to ensure coordinated 281

physiological responses. Catabolic pathways have been characterized for all major plant 282

receptor-binding hormonal ligands and are integral components of hormone homeostasis and 283

action. Since their relatively recent discovery, JA-Ile catabolic pathways have been the focus 284

of intense biochemical and physiological studies that disclosed their relationship with JA 285

signaling. As a common theme in hormone catabolism (Kawai et al. 2014; Mizutani and Ohta 286

2010), an oxidative inactivation pathway was characterized, with CYP94 enzymes defining a 287

two-step JA-Ile oxidation process. In contrast to most hormones for which conjugation 288

corresponds to inactivation or generation of storage forms (Piotrowska and Bajguz 2011), JA 289


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requires conjugation to the amino acid isoleucine as the critical activation step. Consistently, 290

deconjugation by the amidohydrolases IAR3 and ILL6 was characterized in Arabidopsis as a 291

second JA-Ile removal pathway, acting in a JAR1 reverse reaction. 292

Understanding the functions of JA-Ile catabolism potentially offers avenues to tailor increased 293

or on-demand defense signaling in diverse situations, and was addressed repetitively by loss-294

of-function studies in the two catabolic pathways (Aubert et al. 2015; Heitz et al. 2012; Kitaoka 295

et al. 2011; Koo et al. 2011, 2014; Luo et al. 2016; Poudel et al. 2016; Woldemariam et al. 296

2012). In the present work, we conducted a comprehensive analysis of selected genotypes that 297

allowed to evaluate the respective impact of each biochemical pathway on alterations in JA 298

metabolism, defense regulation and resistance to biotic stress. The data clarify some former 299

unexpected results and fill significant knowledge gaps by establishing novel pathway- and 300

stress-specific features. It is remarkable that all mutant lines, including the newly generated 5ko 301

defective in all known JA-Ile-catabolizing enzymes, were undistinguishable from WT for 302

growth under standard conditions. This comes along with the observation that CYP94 and 303

IAR3/ILL6 deficiency does not increase basal JA-Ile accumulation or elevate defense gene 304

expression in resting plants. We conclude that abolition of JA-Ile catabolism does neither turn 305

on constitutive JA responses nor provoke growth inhibition. 306

307

Wounding/insect stress and infection by a necrotroph both induce strong JA metabolism and 308

signaling (Campos et al. 2014), but due to different interactions with other hormonal networks 309

(Pieterse et al. 2012), the transcriptome and defensive outcome display only partial overlap. 310

Previous reports showed that single mutations in either the oxidative or cleavage pathway were 311

sufficient to overaccumulate JA-Ile upon leaf wounding (Heitz et al. 2012; Kitaoka et al. 2011; 312

Koo et al. 2011). This feature was extended in 3cyp or 2ah lines fully impaired in one or the 313

other pathway, but here we demonstrate that their simultaneous deficiency raises JA-Ile to 314

remarkably high levels, indicating that upon massive and rapid JA-Ile biosynthesis triggered by 315

mechanical wounding, no additional enzymatic pathway can efficiently turnover and limit 316

accumulation of JA-Ile. In response to B. cinerea infection, a comparable JA-Ile 317

overaccumulation was recorded in 2ah and 5ko lines, but in marked contrast 3cyp infected 318

leaves accumulated WT levels of JA-Ile, suggesting that in this background, the activity of the 319

AH pathway is sufficient to prevent abnormal hormone build-up. The CYP94 oxidation 320

pathway is however active in infected WT plants, because oxidized derivatives are readily 321

detected, albeit less abundantly than upon wounding. A preponderance of the AH pathway upon 322

infection is supported by the strongly enhanced JA-Ile levels in 2ah and 5ko lines. This indicates 323


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that CYP94 cannot substitute for AH deficiency and that IAR3/ILL6 enzymes are essential for 324

proper JA-Ile homeostasis upon necrotroph attack. 325

326

We next investigated the signaling output of perturbed JA-Ile homeostasis in the two leaf 327

defense models and found that transcript levels of canonical regulatory and defense markers 328

and resistance phenotypes were overall poorly correlated with changes in JA-Ile levels in 329

mutant lines. Upon wounding, 3cyp was closest to WT in term of VSP induction, contrasting 330

with the deficient defense performance of this genotype. Both AH-deficient lines gained a more 331

persistent VSP1/2 expression, more pronounced for VSP2 in 5ko. This behavior was 332

accompanied by a lower weight gain in S. littoralis larvae in 2ah, but not in 5ko, despite of its 333

massive JA-Ile content. The situation with antimicrobial resistance also revealed a strong 334

distortion of the usually linear relationship between hormone, defense and resistance levels, as 335

the high JA-Ile-accumulating lines 2ah and 5ko exhibited depressed defense signaling and WT 336

B. cinerea resistance. In contrast, 3cyp, while maintaining WT JA-Ile levels, was able to better 337

defend against the fungus, as reported earlier (Aubert et al. 2015). In this case, the normal 338

behavior of the ORA59-PDF1.2 module or camalexin levels do not seem to account for the 339

better tolerance. Nevertheless, this result shows that more JA-dependent resistance can arise 340

without an obvious over-accumulation of JA-Ile. 341

342

The JA-Ile regulatory network is complex and sets in motion cascades of positive and negative 343

transcriptional regulators (Hickman et al. 2017). We hypothesized that known elements 344

defining negative feedback loops may counteract some of the effects of chronic JA-Ile 345

overaccumulation to restrain defense induction. This notion was introduced earlier for some 346

JAZ genes in single or double mutants of one pathway, but its relevance was difficult to assess 347

in single biological situations analyzed separately (Aubert et al. 2015; Heitz et al. 2012; Koo et 348

al. 2011). Here we analyzed expression of JAZ and JAM, a second type of negative regulator 349

inhibiting transcription of JA-Ile- and MYC-regulated target genes (Liu et al. 2019; Sasaki-350

Sekimoto et al. 2013) in the different pathway/genotype/stress combinations. We found a 351

seemingly robust correlation between the overinduction of several JAZ and JAM genes and the 352

absence of enhanced expression of defense response under impaired JA-Ile turnover (Figure 6). 353

We selected JAZ genes that were previously found to be sensitive to altered JA-Ile homeostasis 354

(Aubert et al. 2015; Heitz et al. 2012). JAZ8, JAZ10, JAM1 and JAM2 persisted longer than in 355

WT in 3cyp and 5ko after wounding, but for unknown reasons behaved like WT in 2ah, 356

correlating with sustained VSP expression and better tolerance to feeding by S. littoralis larvae 357


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in this latter line. In the case of B. cinerea infection, a similar logic was respected, but in 358

different genotypes. Both 2ah and 5ko displayed significantly higher expression of either JAZ 359

or JAM or both genes, but this was not observed in 3cyp. This correlated well with the former 360

lines showing low defense and WT necrotic lesion sizes, and 3cyp maintaining WT defense and 361

being more resistant to infection. It should be noted that in 3cyp, ORA59-PDF1.2 as well as 362

camalexin accumulation behaved like in WT, so other defense determinants must account for 363

the observed phenotypes. 364

Together these results demonstrate that the two JA-Ile catabolic pathways contribute 365

differentially to JA-Ile removal upon two distinct leaf stresses, and further variations may be 366

expected in other organs or under other environmental constraints. 367

368

The impact of rewiring genetically JA-Ile catabolic capacity may vary between plant species 369

and needs further investigations. For example, the oxidative or deconjugation pathway were 370

silenced transiently (CYP94) or stably (JIH) in Nicotiana attenuata, and in two studies, clearly 371

enhanced JA-Ile-dependent defenses and insect resistance were described (Luo et al. 2016; 372

Woldemariam et al. 2012). We showed that it is possible to genetically extend the half-life and 373

amplitude of JA-Ile burst in Arabidopsis, but this has contrasted consequences on signaling. 374

The precise mechanism underlying control of signaling when JA-Ile catabolism is impaired 375

remains unknown. Stress-specific “sensing” of excess JA-Ile on JAZ or JAM promoters may be 376

mediated by the different interactions engaged between JA signaling and other hormonal 377

pathways, such as the synergy with ethylene upon necrotroph attack, or with abscisic acid upon 378

wounding or herbivory (Pieterse et al., 2012). The observation that blocking either catabolic 379

pathway acting on the same substrate has distinct signaling and physiological outcomes 380

suggests that signaling consequences of JA metabolism are not mediated solely through the 381

control of JA-Ile levels. Modification of abundance of enzymatic products may also contribute 382

to alter the signaling process(s). A recent paper reported that in addition to JA-Ile, 12OH-JA-383

Ile was an active jasmonate signaling through COI1 and contributing to wound responses 384

(Poudel et al. 2019). Such an interpretation would be consistent with our description of 385

metabolic and defensive features of 2ah in wound/insect responses, but not with 3cyp in the 386

Botrytis assay. In addition, one must keep in mind that IAR3 also accepts auxin conjugates as 387

substrates, which may also be at the basis of hormonal cross-talk and alter the signaling output 388

(Widemann et al. 2013; Zhang et al. 2016). The only cases where restrained catabolism ends-389

up in enhanced tolerance to fungal or insect attack is when JAZ and/or JAM repressor transcripts 390

are not overstimulated to reinforce negative feedback loops. The factors determining when 391


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negative regulators take control remain unknown but their elucidation will be of major 392

importance to maximize the potential output of JA-regulated defenses. This provides the ground 393

for future work at the protein, promoter and chromatin levels to determine how inhibitory 394

mechanisms could be at work. 395

396

397

METHODS 398

399

Plant growth and treatments 400

401

Arabidopsis thaliana genotypes used were in the Col0 ecotype and were grown under a 12 h 402

light/12 h dark photoperiod in a growth chamber. The individual T-DNA insertion lines were 403

obtained from the Nottingham Arabidopsis Stock Center (NASC). The 3cyp line was obtained 404

by crossing the alleles cyp94b1-1 (SALK_129672), cyp94b3-1 (CS302217) and cyp94c1-1 405

(SALK_55455) (Aubert et al. 2015). The 2ah line was obtained after crossing the lines iar3-5 406

(SALK_069047) and ill6-2 (SALK_024894C). The quintuple 5ko line was obtained by crossing 407

the 3cyp line with a double iar3-5 ill6-1 (GK412-E11). 408

B. cinerea inoculation and resistance scoring were as described in (Aubert et al. 2015). For 409

mechanical wounding experiments, 5 or 6 fully expanded leaves were wounded three times 410

across mid-vein with a hemostat. At increasing time points following damage, leaf samples 411

were quickly harvested and flash-frozen in liquid nitrogen before storing at -80°C until analysis. 412

Insect performance assay 413

Four-week-old Arabidopsis plants were challenged with freshly hatched Spodoptera littoralis 414

larvae (eggs obtained from Syngenta, Stein AG, Switzerland). Five larvae were placed on each 415

of 11 pots, each containing 2 plants. Plants were placed in a transparent plastic box and kept in 416

a growth chamber during the experiment (10 h light/14 h dark, 100 µmol m-2 s-1 of light, 20-417

22°C and 65% relative humidity). After 8-9 days of feeding, larvae were weighed on a precision 418

balance (Mettler-Toledo, Greifensee, Switzerland). The experiment was performed 419

successively three times (different sampling dates). 420

421

RT-qPCR gene expression assays 422


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Total RNA was extracted from plant leaves with TRI reagent (Molecular Research Center). 423

One microgram of RNA was reverse transcribed using the SuperScript IV reverse transcription 424

system (Thermo Fisher Scientific). Real-time PCR was performed on 10 ng of cDNA as 425

described in Berr et al. (2010) using a LightCycler 480 II instrument (Roche Applied Science). 426

The housekeeping genes EXP (At4g26410) and TIP41 (At4g34270) were used as internal 427

references for qPCR on cDNA derived from infected/wounded or non-stimulated leaves. 428

Measurement of fungal biomass was performed as described in (Smirnova et al. 2017) except 429

that ACT2 (At3g18780) and UBQ10 (At4g05320) were used as reference genes. Gene-specific 430

primer sequences used for qPCR are listed in Supplemental Table 1. 431

432

Jasmonate and camalexin profiling 433

434

Jasmonates were identified and quantified by ultra high performance liquid chromatography 435

coupled to tandem mass spectrometry (UHPLC-MS/MS). About 50-100 mg frozen plant 436

material was extracted with 10 volumes (10 µL per mg) of ice-cold extraction solution 437

(MeOH:water:acetic acid 70:29:0.5, v/v/v) containing 9,10-dihydro-JA and 9,10-dihydro-JA-438

Ile as internal standards for workup recovery. Grinding was performed with a glass-bead 439

Precellys tissue homogenizer (Bertin Instruments, France) in 2 mL screw-capped tubes. After 440

30 min incubation at 4°C on a rotating wheel, homogenates were cleared by centrifugation 441

before concentration of supernatants under a stream of N2 and overnight conservation at -20°C. 442

After a second centrifugation step, extracts were submitted to quantitative LC-MS/MS analysis 443

on an EvoQ Elite LC-TQ (Bruker) equiped with an electrospray ionisation source (ESI) and 444

coupled to a Dionex UltiMate 3000 UHPLC system (Thermo). Five µL plant extract were 445

injected. Chromatographic separation was achieved using an Acquity UPLC HSS T3 column 446

(100 x 2.1 mm, 1.8 µm; Waters) and pre-column. The mobile phase consisted of (A) water and 447

(B) methanol, both containing 0.1 % formic acid. The run started by 2 min of 95 % A, then 448

a linear gradient was applied to reach 100 % B at 10 min, followed by isocratic run using B 449

during 3 min. Return to initial conditions was achieved in 1 min, with a total run time of 15 450

min. The column was operated at 35 °C with a flow-rate of 0.30 mL min-1. Nitrogen was used 451

as the drying and nebulizing gas. The nebulizer gas flow was set to 35 L h-1, and the desolvation 452

gas flow to 30 L h-1. The interface temperature was set to 350 °C and the source temperature to 453

300 °C. The capillary voltage was set to 3.5 kV, the ionization was in positive or negative mode. 454

Low mass and high mass resolution were 2 for the first mass analyzer and 2 for the second. 455


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15

Data acquisition was performed with the MS Workstation 8 for the mass spectrometry and the 456

liquid chromatography was piloted with Bruker Compass Hystar 4.1 SR1 software. The data 457

analysis was performed with the MS Data Review software. Absolute quantifications were 458

achieved by comparison of sample signal with dose-response curves established with pure 459

compounds and recovery correction based on internal standard signal. The transitions were, in 460

negative mode: JA 209.3>59.3; JA-Ile 322.3>130.2; 12OH-JA-Ile 338.3>130.2; 12COOH-JA-461

Ile 352.2>130.1; 12OH-JA 225.2>59.3; in positive mode: camalexin 201.0>59.3. 462

463

Statistical analysis 464

All statistical analysis was performed using InfoStat 2015d (http:// www.infostat.com.ar). 465

Comparisons of sample means were performed by one-way analysis of variance (P % 0.05 or 466

P % 0.01) and Tukey’s post-hoc multiple comparisons tests (P < 0.05 or P < 0.01), and 467

significant differences of means were determined. 468

469

LEGENDS TO FIGURES 470

Figure 1. Positions of the impaired enzymatic steps in JA-Ile catabolic mutants and 471

simplified view of analyzed signaling and defense responses 472

Left box: Necrotroph fungus infection or mechanical wounding/insect attack trigger jasmonic 473

acid (JA) biosynthesis. Some JA is conjugated to isoleucine by JAR1 enzyme to form bioactive 474

jasmonoyl-isoleucine (JA-Ile). JA-Ile is turned over by a two-step oxidation to 12OH-JA-Ile 475

and 12COOH-JA-Ile by the cytochrome P450 enzymes CYP94B3/B1 and CYP94C1, or by 476

conjugate cleavage mediated by the amidohydrolases (AH) IAR3 and ILL6. These AH also 477

cleave 12OH-JA-Ile to release 12OH-JA. Red crosses indicate impaired JA-Ile catabolic steps 478

in higher order mutants utilized in this study: oxidation-deficient (triple cyp94b1 b3 c1 mutant, 479

3cyp), deconjugation-deficient (double iar3 ill6, 2ah) or deficient for both pathways (quintuple 480

mutant, 5ko). Right box: in absence of JA-Ile, JAZ repressors block transcription of target 481

genes. Upon biosynthesis, JA-Ile promotes the assembly of the COI1-JAZ co-receptor which 482

is recruited into the SCFCOI1 E3 ubiquitin ligase directing proteolytic degradation of JAZ 483

repressors. Transcription factors (TFs) like MYC2 or ORA59 become activated in association 484

with mediator 25 subunit (MED25) and allow the transcription of numerous stimulus-specific 485


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16

defense responses. The anti-insect branch was probed by the MYC2-VSP genes, and the 486

antimicrobial branch by the ORA59 and PDF1.2 genes. Among JA-Ile targets are also genes 487

encoding JAZ repressors or JAM proteins that compete with MYC2 and ORA59, to attenuate 488

signaling. In mutant plants impaired for JA-Ile catabolism, overinduction of this negative 489

feedback loop may occur and prevent hyper-stimulation of JA-Ile-dependent defenses and 490

associated enhanced resistance. 491

Figure 2. JA-Ile and oxidized catabolites accumulation in 2ah, 3cyp and 5ko mutants after 492

mechanical wounding or Botrytis cinerea Infection. 493

Six-week-old plants were wounded 3 times across leaf mid-vein (A) or drop-inoculated on two 494

sites per leaf with a suspension containing 2.5x106 fungal spores mL-1 (B). Treated leaves were 495

harvested at 1, 3 and 6 hours post wounding (hpw) or 3 days post inoculation (dpi) on WT 496

(black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) plants. Jasmonates were 497

extracted and quantified by LC–MS/MS. JA-Ile, 12OH-JA-Ile and 12COOH-JA-Ile levels were 498

expressed in nmol g-1 fresh weight (FW). Histograms represent the mean ± SEM of three 499

(wounding) or four (B. cinerea) biological replicates. Columns labeled with different letters 500

indicate a significant difference between genotypes at each time point as determined by one-501

way ANOVA with Tukey post-hoc test, P < 0.05. 502

Figure 3. Expression profiles of JA-Ile-dependent genes in response to mechanical 503

wounding and impact on insect feeding in JA-Ile catabolic mutants. 504

(A) Expression profiles of jasmonate-dependent MYC2, VSP1 and VSP2 marker genes in 505

response to wounding in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green 506

bars) mutants. Relative expression of each target gene at 1, 3 or 6 hours post wounding (hpw) 507

is represented. Gene expression was determined by real-time PCR using gene-specific primers 508

and normalized using EXP and TIP41 reference genes. Transcript quantification was performed 509

on three biological replicates analyzed in duplicate. Histograms represent mean expression ± 510

SEM. Columns labeled with different letters indicate a significant difference between 511

genotypes at each time point as determined by one-way ANOVA with Tukey posthoc test, P < 512

0.05. 513

(B) Insect feeding assay. S. littoralis larvae were placed on leaves of 4-week-old plants. After 514

8-9 days, larval weight was determined. Histograms represent mean ± SEM from 3 independent 515

trials (presented in Supplemental Figure S3). The number of total larvae on each genotype is 516


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17

indicated within the bars. Different letters indicate significant differences at P < 0.05 (linear 517

mixed model). 518

Figure 4. Expression profiles of JA-Ile-dependent defense genes and resistance levels in 519

response to B. cinerea infection in 2ah, 3cyp and 5ko mutants. 520

(A) Expression profiles of jasmonate-regulated ORA59 and PDF1.2 genes in response to 521

infection by B. cinerea in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green 522

bars) mutants. Relative expression of each target gene at 3 day post infection (dpi) is 523

represented. Gene expression was determined by real-time PCR using gene-specific primers 524

and normalized using EXP and TIP41 reference genes. Transcript quantification was performed 525

on three biological replicates analyzed in duplicate. Histograms represent mean expression ± 526

SEM. Columns labeled with different letters indicate a significant difference between 527

genotypes at each time point as determined by one-way ANOVA with Tukey posthoc test, P < 528

0.05. 529

(B) Disease resistance assessment. Two sites per leaf were inoculated across the main vein with 530

5 µL of spore suspension containing 2.5x106 spores mL-1. Left panel: representative leaves of 531

each genotype were detached and photographed at 3 dpi. Middle panel: histograms represent 532

the mean lesion diameters ± SEM of about 100 lesion sites from 10 to 15 plants for each 533

genotype. Right panel: evaluation of fungal growth by real-time qPCR with B. cinerea cutinase-534

specific primers on genomic DNA extracted from 3-day- infected leaves. Quantification was 535

performed on three biological replicates analyzed in duplicate. Columns labeled with different 536

letters indicate a significant difference as determined by one-way ANOVA with Tukey post-537

hoc test (P < 0.01). 538

Figure 5. Expression profiles of JA-Ile dependant negative feedback genes in response to 539

wounding and infection in 2ah, 3cyp and 5ko mutants. 540

Expression profiles of jasmonate-dependent genes in response to wounding (A, JAZ8, JAZ10, 541

JAM1 and JAM2) or infection by Botrytis cinerea (B, JAZ1, JAZ8, JAM1 and JAM2) in WT 542

(black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative 543

expression of each target gene at 1, 3 or 6 hours post wounding (hpw) and 3 day post infection 544

(dpi) is represented. Gene expression was determined by real-time PCR using gene-specific 545

primers and normalized using EXP and TIP41 reference genes. Transcript quantification was 546

performed on three biological replicates analyzed in duplicate. Histograms represent mean 547


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18

expression ± SEM. Columns labeled with different letters indicate a significant difference 548

between genotypes at each time point as determined by one-way ANOVA with Tukey posthoc 549

test, P < 0.05. 550

Figure 6. Relationships between impaired JA-Ile catabolism, JA-Ile levels, 551

defense/repressor gene induction, and resistance to attackers. 552

The data show distinct circuitry for the two leaf responses analyzed. The signs -, 0, or + indicate 553

lower, equal, or increased response respectively, in stimulated mutant genotypes compared to 554

WT. The two catabolic pathways have differential contributions to JA-Ile turnover in response 555

to wounding or infection. JA-Ile accumulation is enhanced to different extents, except in 556

infected 3cyp, and this has variable consequences on defense amplitude and aggressor 557

resistance. The trend emerging is that impaired hormone catabolism does, with or without JA-558

Ile overaccumulation, result in better defense/resistance, only if negative effectors like JAZ or 559

JAM are themselves not overinduced. 560

561

ACKNOWLEDGMENTS 562

J. Browse (WSU, Pullman, USA) for providing a segregating population of iar3-5 ill6-2 line. 563

564

FUNDING 565

This work was supported by basic funding of CNRS, grant ANR-12-BSV8-005 (Jasmonox) from the 566 Agence Nationale de la Recherche to ES, and IdEx-2014-208e Interdisciplinary grant to LP from 567 Université de Strasbourg and CNRS. VM is recipient of a predoctoral fellowship from the Université de 568 Strasbourg and the Ministère de l’Enseignement Supérieur et de la Recherche. This research was 569 supported by a grant from the Swiss National Science Foundation (31003A_169278 to P.R.). 570 571

572

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684


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O

OHO

OH

O

OHO

O

NHO

O

HO

O

NHO

O

HO

OH

O

NHO

O

HO

OH

O

Figure 1. Positions of the impaired enzymatic steps in JA-‐Ile catabolic mutants and simplified view of analyzedsignaling and defense responsesLeft box: Necrotroph fungus infection or mechanical wounding/insect attack trigger jasmonic acid (JA) biosynthesis.Some JA is conjugated to isoleucine by JAR1 enzyme to form bioactive jasmonoyl-‐isoleucine (JA-‐Ile). JA-‐Ile is turnedover by a two-‐step oxidation to 12OH-‐JA-‐Ile and 12COOH-‐JA-‐Ile by the cytochrome P450 enzymes CYP94B3/B1 andCYP94C1, or by conjugate cleavage mediated by the amidohydrolases (AH) IAR3 and ILL6. These AH also cleave12OH-‐JA-‐Ile to release 12OH-‐JA. Red crosses indicate impaired JA-‐Ile catabolic steps in higher order mutants utilizedin this study: oxidation-‐deficient (triple cyp94b1 b3 c1 mutant, 3cyp), deconjugation-‐deficient (double iar3 ill6, 2ah)or deficient for both pathways (quintuple mutant, 5ko). Right box: in absence of JA-‐Ile, JAZ repressors blocktranscription of target genes. Upon biosynthesis, JA-‐Ile promotes the assembly of the COI1-‐JAZ co-‐receptor which isrecruited into the SCFCOI1 E3 ubiquitin ligase directing proteolytic degradation of JAZ repressors. Transcriptionfactors (TFs) like MYC2 or ORA59 become activated in association with mediator 25 subunit (MED25) and allow thetranscription of numerous stimulus-‐specific defense responses. The anti-‐insect branch was probed by the MYC2-‐VSP genes, and the antimicrobial branch by the ORA59 and PDF1.2 genes. Among JA-‐Ile targets are also genesencoding JAZ repressors or JAM proteins that compete with MYC2 and ORA59, to attenuate signaling. In mutantplants impaired for JA-‐Ile catabolism, overinduction of this negative feedback loop may occur and prevent hyper-‐stimulation of JA-‐Ile-‐dependent defenses and associated enhanced resistance.

JA JA-Ile

12OH-JA 12OH-JA-Ile

12COOH-JA-Ile

SCFCOI1 signalling

JAO2JAO3JAO4

Stimulus

JAR1

CYP94B3CYP94B1

3cyp

CYP94C1

IAR3/ILL6

IAR3/ILL6

5ko

5ko2ah

5ko2ah

3cyp5ko

JA-Ile

TFJAZ

COI1COI1

Transcription

JAZ

JAM

JAZ

JAMJAZ

Defense cascadeNegative feedback

PDF1.2 VSP1 VSP2

Antimicrobial

Repression

JAZ

JAZ

Anti-insect

Physiological responses

MED25TF

Jasmonatemetabolism Resistance

?

Infection Wounding Herbivory

ORA59 MYC2


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JA-‐Ile

12OH-‐JA-‐Ile

12CO

OH-‐JA-‐Ile

Figure 2. JA-‐Ile and oxidized catabolites accumulation in 2ah, 3cyp and 5ko mutants after mechanicalwounding or Botrytis cinerea Infection.Six-‐week-‐old plants were wounded 3 times across leaf mid-‐vein (A) or drop-‐inoculated on two sites perleaf with a suspension containing 2.5x106 fungal spores mL-‐1 (B). Treated leaves were harvested at 1, 3and 6 hours post wounding (hpw) or 3 days post inoculation (dpi) on WT (black bars), 2ah (yellow bars),3cyp (blue bars) and 5ko (green bars) plants. Jasmonates were extracted and quantified by LC–MS/MS.JA-‐Ile, 12OH-‐JA-‐Ile and 12COOH-‐JA-‐Ile levels were expressed in nmol g-‐1 fresh weight (FW). Histogramsrepresent the mean ± SEM of three (wounding) or four (B. cinerea) biological replicates. Columns labeledwith different letters indicate a significant difference between genotypes at each time point asdetermined by one-‐way ANOVA with Tukey post-‐hoc test, P < 0.05.

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https://doi.org/10.1101/686709

Figure 3. Expression profiles of JA-‐Ile-‐dependent genes in response to mechanical wounding and impact oninsect feeding in JA-‐Ile catabolic mutants.(A) Expression profiles of jasmonate-‐dependent MYC2, VSP1 and VSP2marker genes in response to wounding in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative expression of each target gene at 1, 3 or 6 hours post wounding (hpw) is represented. Gene expression was determined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 reference genes. Transcript quantification was performed on three biological replicates analyzed in duplicate. Histograms represent mean expression ± SEM. Columns labeled with different letters indicate a significant difference between genotypes at each time point as determined by one-‐way ANOVA with Tukey posthoc test, P < 0.05.(B) Insect feeding assay. S. littoralis larvae were placed on leaves of 4-‐week-‐old plants. After 8-‐9 days, larval weight was determined. Histograms represent mean ± SEM from 3 independent trials (presented in Supplemental 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).

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https://doi.org/10.1101/686709

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Figure 4. Expression profiles of JA-‐Ile-‐dependent defense genes and resistance levels in response to B.cinerea infection in 2ah, 3cyp and 5komutants.(A) Expression profiles of jasmonate-‐regulated ORA59 and PDF1.2 genes in response to infection by B.cinerea in WT (black bars), 2ah (yellow bars), 3cyp (blue bars) and 5ko (green bars) mutants. Relativeexpression of each target gene at 3 day post infection (dpi) is represented. Gene expression wasdetermined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 referencegenes. Transcript quantification was performed on three biological replicates analyzed in duplicate.Histograms represent mean expression ± SEM. Columns labeled with different letters indicate a significantdifference between genotypes at each time point as determined by one-‐way ANOVA with Tukey posthoctest, P < 0.05.(B) Disease resistance assessment. Two sites per leaf were inoculated across the main vein with 5 µL ofspore suspension containing 2.5x106 spores mL-‐1. Left panel: representative leaves of each genotype weredetached and photographed at 3 dpi. Middle panel: histograms represent the mean lesion diameters ±SEM of about 100 lesion sites from 10 to 15 plants for each genotype. Right panel: evaluation of fungalgrowth by real-‐time qPCR with B. cinerea cutinase-‐specific primers on genomic DNA extracted from 3-‐day-‐infected leaves. Quantification was performed on three biological replicates analyzed in duplicate.Columns labeled with different letters indicate a significant difference as determined by one-‐way ANOVAwith Tukey post-‐hoc test (P < 0.01).

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https://doi.org/10.1101/686709

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Figure 5. Expression profiles of JA-‐Ile dependant negative feedback genes in response to wounding andinfection in 2ah, 3cyp and 5komutants.Expression profiles of jasmonate-‐dependent genes in response to wounding (A, JAZ8, JAZ10, JAM1 andJAM2) or infection by Botrytis cinerea (B, JAZ1, JAZ8, JAM1 and JAM2) in WT (black bars), 2ah (yellowbars), 3cyp (blue bars) and 5ko (green bars) mutants. Relative expression of each target gene at 1, 3 or 6hours post wounding (hpw) and 3 day post infection (dpi) is represented. Gene expression wasdetermined by real-‐time PCR using gene-‐specific primers and normalized using EXP and TIP41 referencegenes. Transcript quantification was performed on three biological replicates analyzed in duplicate.Histograms represent mean expression ± SEM. Columns labeled with different letters indicate asignificant difference between genotypes at each time point as determined by one-‐way ANOVA withTukey posthoc test, P < 0.05.

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https://doi.org/10.1101/686709

Figure 6. Relationships between impaired JA-‐Ile catabolism, JA-‐Ile levels, defense/repressorgene induction, and resistance to attackers.The data show distinct circuitry for the two leaf responses analyzed. The signs -‐, 0, or +indicate lower, equal, or increased response respectively, in stimulated mutant genotypescompared to WT. The two catabolic pathways have differential contributions to JA-‐Ileturnover in response to wounding or infection. JA-‐Ile accumulation is enhanced to differentextents, except in infected 3cyp, and this has variable consequences on defense amplitudeand aggressor resistance. The trend emerging is that impaired hormone catabolism does,with or without JA-‐Ile overaccumulation, result in better defense/resistance, only if negativeeffectors like JAZ or JAM are themselves not overinduced.

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3cyp oxidation + 0 ++ *+*2ah deconjugation + ++ 0 +5ko both +++ + ++ 0

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B.#cinerea&infection

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https://doi.org/10.1101/686709

stress- and pathway-specific impacts of impaired …57! cyclopropane carboxylic acid, sulfation...

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