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1 Running Head: Auxin regulates herbivory-induced secondary metabolites 1 Correspondence: Matthias Erb, University of Bern, Institute of Plant Sciences, 2 Altenbergrain 21, 3013 Bern, Switzerland, Tel. +41 31 631 86 68, [email protected] 3 Research area: Signaling and response 4 Plant Physiology Preview. Published on August 2, 2016, as DOI:10.1104/pp.16.00940 Copyright 2016 by the American Society of Plant Biologists www.plantphysiol.org on June 1, 2020 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

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    Running Head: Auxin regulates herbivory-induced secondary metabolites 1

    Correspondence: Matthias Erb, University of Bern, Institute of Plant Sciences, 2 Altenbergrain 21, 3013 Bern, Switzerland, Tel. +41 31 631 86 68, [email protected] 3

    Research area: Signaling and response 4

    Plant Physiology Preview. Published on August 2, 2016, as DOI:10.1104/pp.16.00940

    Copyright 2016 by the American Society of Plant Biologists

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    Auxin is rapidly induced by herbivore attack and regulates a subset of systemic, 5 jasmonate-dependent secondary metabolites 6

    Ricardo A.R. Machado1,2, Christelle A.M. Robert1,2, Carla C.M. Arce1,2,3, Abigail P. Ferrieri1, 7

    Shuqing Xu1, Guillermo H. Jimenez-Aleman1, Ian T. Baldwin1 and Matthias Erb1,2,* 8

    9

    1Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany. 10

    2Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland. 11

    3Departamento de Entomologia, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, 12

    Brazil. 13

    14

    One sentence summary: Herbivory-induced auxin promotes the production of anthocyanins 15 and phenolamides. 16

    17

    18

    This work was supported by the Max Planck Society, a Humboldt Postdoctoral Research 19

    Fellowship (AF), the Brazilian National Council for Research CNPq Grant No. 237929/2012-20

    0 (CA), a Marie Curie Intra European Fellowship Grant No. 328935 (SX), a Marie Curie 21

    Intra European Fellowship Grant No. 273107 (ME), a Swiss National Foundation Fellowship 22

    Grant No. 140196 (CR), a European Research Council advanced Grant No. 293926 (ITB) and 23

    Human Frontier Science Program Grant No. RGP0002/2012 (ITB). 24

    25

    26

    * Author for correspondence (Phone: +41 31 631 8668 E-mail: [email protected])27

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    ABSTRACT 28

    Plant responses to herbivore attack are regulated by phytohormonal networks. To date, the 29

    role of the auxin indole-3-acetic acid (IAA) in this context is not well understood. We 30

    quantified and manipulated the spatiotemporal patterns of IAA accumulation in herbivore-31

    attacked Nicotiana attenuata plants to unravel its role in the regulation of plant secondary 32

    metabolism. We found that IAA is strongly, rapidly and specifically induced by herbivore 33

    attack. IAA is elicited by herbivore oral secretions and fatty acid conjugate elicitors and is 34

    accompanied by a rapid transcriptional increase of auxin biosynthetic YUCCA-like genes. 35

    IAA accumulation starts 30-60 seconds after local induction and peaks within 5 minutes after 36

    induction, thereby preceding the jasmonate (JA) burst. IAA accumulation does not require JA 37

    signaling and spreads rapidly from the wound site to systemic tissues. Complementation and 38

    transport inhibition experiments reveal that IAA is required for the herbivore-specific, 39

    jasmonate-dependent accumulation of anthocyanins and phenolamides in the stems. In 40

    contrast, IAA does not affect the accumulation of nicotine or 7-hydroxygeranyllinalool 41

    diterpene glycosides in the same tissue. Taken together, our results uncover IAA as a rapid 42

    and specific signal that regulates a subset of systemic, jasmonate-dependent secondary 43

    metabolites in herbivore-attacked plants. 44

    45

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    INTRODUCTION 46

    Plants withstand herbivore attack by specifically recognizing the attacker and mounting 47

    appropriate defenses. Induced defense responses are activated by hormone-mediated 48

    signaling cascades (Erb et al., 2012; Wu and Baldwin, 2009), and jasmonates (JA) have 49

    emerged as key regulators in this context (Geyter et al., 2012; Howe and Jander, 2008). As a 50

    consequence, their behavior and mode of action have been studied in great detail (Wasternack 51

    and Hause, 2013). Similarly, other stress-related hormones such as salicylic acid, abscisic 52

    acid and ethylene have been shown to play important roles in the orchestration of plant 53

    defenses against herbivores (Dahl et al., 2007; Winz and Baldwin, 2001; Thaler and Bostock, 54

    2004; Zhang et al., 2013; Kroes et al., 2014). Recent evidence also suggests that hormones 55

    which have traditionally been classified as growth regulators participate in induced defense 56

    responses. Cytokinins for instance modulate wound-induced local and systemic defense 57

    responses (Schäfer et al., 2015), and gibberellins are involved in regulating the plant’s 58

    investment into growth and defense (Li et al., 2015; Hou et al., 2010; Yang et al., 2012). 59

    In contrast to the hormones mentioned above, little is known about the role of auxins in 60

    induced responses against herbivores. Auxins regulate a vast array of plant processes 61

    including growth and development as well as responses to light, gravity, abiotic stress and 62

    pathogen attack (Glick, 2015; Mano and Nemoto, 2012; Yang et al., 2014). Several studies 63

    suggest that the auxin indole-3-acetic acid (IAA) also regulates gall formation by many 64

    herbivores since some gall-forming herbivores contain high levels of IAA (Mapes and 65

    Davies, 2001b, 2001a; Tooker and Moraes, 2011a; Straka et al., 2010; Dorchin et al., 2009; 66

    Yamaguchi et al., 2012; Tanaka et al., 2013), IAA pools and signaling are enhanced in 67

    parasitized plant tissue (Yamaguchi et al., 2012; Tooker and Moraes, 2011b), and direct 68

    applications of IAA can result in the formation of gall-resembling structures (Hamner and 69

    Kraus, 1937; Guiscafrearrillaga, 1949; Schäller, 1968; Bartlett and Connor, 2014; Connor et 70

    al., 2012). In the context of chewing insects, however, our understanding is more limited 71

    (Dafoe et al., 2013). IAA levels seem to remain unaltered in Solidago altissima and Triticum 72

    aestivum attacked by Heliothis virescens caterpillars (Tooker and Moraes, 2011a, 2011b) and 73

    to be reduced in Helicoverpa zea attacked Zea mays (Schmelz et al., 2003) and Manduca 74

    sexta-challenged Nicotiana attenuata leaves (Onkokesung et al., 2010; Woldemariam et al., 75

    2012). Moreover, mechanical wounding alone can either increase or decrease IAA levels in 76

    the leaves (Thornburg and Li, 1991; Tanaka and Uritani, 1979; Machado et al., 2013). A 77

    limitation of some of these early studies is that IAA was measured at single time points or 78

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    during the later stages of infestation (Onkokesung et al., 2010; Schmelz et al., 2003, Tooker 79

    and Moraes, 2011a, 2011b), which may have resulted in an incomplete picture of IAA 80

    dynamics under herbivore attack. We recently demonstrated in N. attenuata that IAA is 81

    induced in locally damaged leaves upon simulated M. sexta attack (Machado et al., 2013). 82

    IAA signaling may influence plant responses to herbivore attack by modulating other 83

    hormonal pathways and defenses (Erb et al., 2012). Exogenous IAA for instance reduces the 84

    herbivory-induced accumulation of nicotine and jasmonates (Baldwin et al., 1997; Baldwin, 85

    1989), gene expression of jasmonate-dependent proteinase inhibitors genes (Kernan and 86

    Thornburg, 1989) and vegetative storage proteins (DeWald et al., 1994; Liu et al., 2005). 87

    Conversely, IAA promotes the production of phenolics and flavonoids in root-cell cultures in 88

    a dose-dependent manner (Lulu et al., 2015; Mahdieh et al., 2015) and the auxin homologue 89

    2,4-dichlorophenoxyacetic acid (2,4-D) acts as a strong inducer of defense responses in rice 90

    (Xin et al., 2012; Song, 2014). 91

    In this study, we aimed to understand the spatiotemporal patterns of IAA accumulation in 92

    herbivore-attacked Nicotiana attenuata plants as well as the role of IAA in regulating the 93

    biosynthesis of secondary metabolites. In an earlier study, we found that IAA accumulates 94

    within 1 h following the application of M. sexta oral secretions to wounded leaves. To 95

    understand this pattern in more detail, we first evaluated IAA accumulation dynamics in 96

    several plant organs in response to real and simulated M. sexta attack, including the 97

    application of a specific herbivore elicitor to wounded leaves, at different time points ranging 98

    from 15 seconds to 6 h. Secondly, we analyzed the induction of potential IAA biosynthetic 99

    genes. Lastly, we manipulated IAA accumulation and transport as well as jasmonate 100

    signaling to unravel the impact of M. sexta-induced IAA on systemic jasmonate-dependent 101

    secondary metabolites. Our experiments reveal that IAA is a rapid herbivory-induced signal 102

    that acts in concert with jasmonates to regulate the systemic induction of plant secondary 103

    metabolites.104

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    RESULTS 105

    Real and simulated M. sexta attack induce the accumulation of indole-3-acetic acid 106 (IAA) in the leaves 107

    To investigate the behavior of IAA in herbivore-attacked plants, we measured IAA 108

    concentrations in the leaves of Nicotiana attenuata subjected to either real or simulated M. 109

    sexta attack (Figure 1A to 1D). We observed a significant increase in IAA levels in response 110

    to real M. sexta herbivory 3h after infestation. This effect could be mimicked by leaf 111

    wounding and simultaneous application of either M. sexta oral secretions (W+OS) or the fatty 112

    acid-amino acid conjugate N-linolenoyl-glutamic acid as a specific herbivore elicitor 113

    (W+FAC) (Figure 1A to 1D). Wounding alone led to a delayed and weaker increase in IAA 114

    (Figure 1C). The herbivory-induced accumulation of IAA started 30-60 seconds after 115

    induction (Figure 1B) and occurred independently of the time of day at which the induction 116

    took place (Supplemental Figure 1). Overall, IAA concentrations increased 2-3 fold in 117

    herbivore induced leaves compared to controls. 118

    IAA induction gradually spreads through the shoots of attacked plants 119

    To explore whether IAA also increases in systemic tissues, we induced N. attenuata plants 120

    and measured IAA concentrations in local, treated plant tissues and systemic, untreated plant 121

    tissues at different time points over a 2 h time period. Again, we found a rapid increase in 122

    IAA levels locally upon simulated M. sexta attack (W+OS) which transiently and steadily 123

    spread to systemic, untreated tissues (Figure 2A to 2F). IAA levels slightly increased in 124

    petioles 10 min post treatment, in stems 60 min post treatment and in systemic leaves 120 125

    min post treatment. No significant changes were found in the main and lateral roots (Figure 126

    2A to 2F). 127

    IAA induction in leaves is conserved across different developmental stages 128

    Herbivore-induced jasmonate and ethylene signaling are influenced by plant development 129

    (Diezel et al., 2011a). To test whether plant development specifically influences M. sexta-130

    induced IAA levels, we induced plants by simulated M. sexta attack and measured IAA levels 131

    in the leaves of early rosette, elongated and flowering plants. We found that the herbivore-132

    elicited increase in IAA concentration was independent of plant developmental stage (Figure 133

    3A to 3C). However, the absolute IAA levels and magnitude of induction were strongest in 134

    early rosette plants (Figure 3A to 3C). 135

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    YUCCA-like, IAA-biosynthesis homologues are rapidly upregulated upon herbivore 136 attack 137

    In Arabidopsis thaliana, YUCCA-genes encode for flavin monooxygenase-like proteins that 138

    convert indole-3-pyruvic acid into IAA, a reaction which likely represents the rate-limiting 139

    step in IAA biosynthesis (Mashiguchi et al., 2011) (Figure 4A). We identified YUCCA-like 140

    genes in N. attenuata and measured their transcript levels upon herbivore elicitation. To 141

    achieve this, we first searched the sequence of the Arabidopsis thaliana YUCCA2 gene 142

    (NCBI accession number NM_117399.3) in N. attenuata draft genome (Ling et al., 2015) and 143

    reconstructed the phylogenetic tree of the gene family (Mashiguchi et al., 2011). Our analysis 144

    revealed that the N. attenuata genome contains at least nine YUCCA-like genes that share 145

    high similarity with AtYUCCA2 and contain the four conserved amino acid motifs 146

    characteristic of this gene family (Supplemental Figure 2) (Expósito-Rodríguez et al., 2011; 147

    Expósito-Rodríguez et al., 2007). We designed specific primers and profiled the expression 148

    patterns of these genes upon simulated M. sexta attack. Several YUCCA-like genes were 149

    upregulated in response to simulated M. sexta attack (Figure 4B to 4I). NaYUCCA-like 1, 3, 150

    5, 6 and 9 were upregulated 3 min after the application of M. sexta oral secretions and fatty 151

    acid-conjugates (Figure 4B to 4H). The upregulation of NaYUCCA-like 1 and 3 was 152

    maintained for at least one hour (Figure 4G to 4H). The expression of NaYUCCA-like 2, 4, 7 153

    and 8 was not significantly influenced by simulated M. sexta attack (Supplemental Figure 3). 154

    IAA accumulation precedes the JA burst 155

    To investigate the temporal dynamics of IAA and JA accumulation in M. sexta-attacked 156

    plants, we quantified IAA and JA in plants subjected to simulated M. sexta herbivory at 157

    different time points. We found that IAA peaked more rapidly than jasmonic acid in response 158

    to herbivore attack (Figure 5). IAA accumulation commenced within minutes after the onset 159

    of the elicitation and reached its maximum five minutes after induction. JA accumulated in an 160

    equally rapid fashion, but peaked significantly later than IAA (Figure 5). 161

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    Jasmonate signaling is not required for the M. sexta-induced IAA accumulation 162

    Plant responses to attackers are modulated by a complex signaling network consisting of 163

    antagonistic, neutral and synergistic effects (Erb et al., 2012). For example, jasmonate 164

    signaling antagonizes IAA signaling (Chen et al., 2011). To further explore the potential 165

    crosstalk between these two phytohormones, we measured M. sexta-induced IAA in 166

    transgenic plants that are impaired to different degrees in jasmonate signaling, biosynthesis 167

    and/or perception (Table 1). We found that the M. sexta-triggered accumulation of IAA does 168

    not require JA signaling as it was induced in all of the evaluated JA-deficient genotypes 169

    (Figure 6 and supplemental Figure 4). 170

    M. sexta-induced IAA is required for the induction of anthocyanins in the stems 171

    To investigate the impact of IAA on plant secondary metabolites, we sought to manipulate its 172

    perception in planta. Our initial attempts to create transgenic, dexamethasone (DEX) 173

    inducible plants (Schäfer et al., 2013) harboring a silencing construct for the IAA receptor 174

    TIR1 failed, either because of promotor methylation in the F2 crosses (Weinhold et al., 2013) 175

    or because the identified TIR1 homologue was inactive. We therefore took advantage of our 176

    knowledge on systemic IAA accumulation to devise a series of chemical manipulation 177

    experiments. First, we exogenously applied IAA and MeJA at doses that exceed endogenous 178

    levels (Baldwin, 1989; Machado et al., 2013). Second, we inhibited local IAA synthesis with 179

    L-kynurenine (L-Kyn). L-kynurenine is a specific inhibitor of tryptophan aminotransferases 180

    (TATs) which are key enzymes of the indole-3-pyruvic acid pathway that leads to IAA 181

    formation (He et al., 2011). Third, we inhibited IAA transport at the leaf base and petiole of 182

    the induced leaves using 2,3,5-triiodobenzoic acid (TIBA). TIBA inhibits auxin polar 183

    transport by blocking auxin efflux transporter PIN-FORMED PIN1 cycling (Geldner et al., 184

    2001). We observed that within hours following M. sexta attack, N. attenuata stems became 185

    red (Figure 7D, inset), a phenotype that is likely due to anthocyanin accumulation. As IAA 186

    can regulate the production of anthocyanins in plants (Pasqua et al., 2005), we quantitatively 187

    and qualitatively evaluated anthocyanin accumulation in the stems following several 188

    simulated and real herbivory in combination with IAA manipulation. We observed that the 189

    levels of anthocyanins in the stems were strongly induced by real M. sexta attack, an effect 190

    that could be mimicked by wounding and applications of M. sexta oral secretions (W+OS), 191

    but not by wounding alone (W+W) (Figure 7A). Application of IAA or MeJA alone did not 192

    trigger anthocyanin accumulation (Figure 7A). By contrast, the simultaneous application of 193

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    IAA and MeJA (IAA+MeJA) triggered anthocyanin accumulation (Figure 7A). Chemical 194

    inhibition of IAA biosynthesis or transport as well as genetic inhibition of JA biosynthesis led 195

    to the complete disappearance of induced anthocyanin accumulation (Figure 7B and 7C). 196

    Furthermore, we found a positive correlation between anthocyanin contents and red 197

    pigmentation in the stems (Figure 7D). 198

    IAA specifically potentiates the herbivore-induced accumulation of phenolamides in the 199 stems 200

    To investigate the role of IAA in the accumulation of known defensive metabolites in the 201

    stems of N. attenuata (Onkokesung et al., 2012; Heiling et al., 2010; Paschold et al., 2007), 202

    we induced leaves of N. attenuata plants by different simulated and real herbivory treatments 203

    and complemented them with IAA at doses that exceed endogenous levels (Baldwin, 1989; 204

    Machado et al., 2013). The stems of N. attenuata are often attacked by herbivores, including 205

    stem borers (Diezel et al., 2011b; Lee et al., 2016), and are very important for plant fitness 206

    (Machado et al., 2016). We observed a strong upregulation of defensive secondary 207

    metabolites in the stems in response to M. sexta attack (Figure 8A to 8D). Petiole 208

    pretreatments with IAA dramatically increased the accumulation of caffeoylputrescine and 209

    dicaffeoylspermidine in response to real and simulated herbivory as well as MeJA 210

    application. IAA application alone did not induce the metabolites (Figure 8A and 8B). By 211

    contrast, nicotine and 7-hydroxygeranyllinalool diterpene glycosides did not respond to IAA 212

    petiole pretreatments (Figure 8A to 8D). 213

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    DISCUSSION 214

    In this study, we show that auxin is a rapidly and specifically induced regulator of defensive 215

    secondary metabolites in Nicotiana attenuata. Infestation by M. sexta caterpillars induced the 216

    accumulation of IAA levels in local tissues, an effect that could be mimicked by both the 217

    applications of M. sexta oral secretions and the application of the well-known insect elicitor 218

    N-linolenoyl-glutamic acid (Halitschke et al., 2003), and to a lesser extent by mechanical 219

    wounding. These results are in contrast to earlier studies in maize, goldenrod and coyote 220

    tobacco which found either a slight decrease or no changes in IAA levels in response to 221

    herbivore attack (Schmelz et al., 2003; Tooker and Moraes, 2011a; Onkokesung et al., 2010; 222

    Tooker and Moraes, 2011b), but are in agreement with our previous study (Machado et al., 223

    2013). Interestingly, in comparison with our previous study, we observed differences in both 224

    absolute quantities and timing of IAA induction. One possible explanation for these 225

    differences is that plants were grown using different substrates. While sand was used in the 226

    previous study, potting soil was used in the present paper. Given the strong feedback effects 227

    of soil bacteria, soil nutrients and root growth on IAA signaling (Lambrecht et al., 2000; 228

    Kurepin et al., 2015; Tian et al., 2008; Sassi et al., 2012), it is likely that the growth substrate 229

    affected IAA homeostasis and responsiveness in N. attenuata. On the other hand, the absence 230

    of IAA induction reported in earlier studies may be due to the fact that late time points were 231

    measured (Onkokesung et al., 2010; Schmelz et al., 2003; Tooker and Moraes, 2011a), which 232

    may not have captured the rapid and dynamic accumulation of IAA following herbivore 233

    attack. To further investigate these contradicting results, we determined IAA responses in 234

    herbivore attacked maize plants (Maag et al., submitted). We found that IAA levels increased 235

    in an herbivore-specific manner 1-6 h after the onset of the attack. Together, these 236

    experiments suggest that the rapid and transient herbivory-induced accumulation of IAA may 237

    be a conserved plant response to insect attack. 238

    Spatiotemporal IAA profiling revealed that the rapid increase in IAA pools at the site of 239

    attack is followed by a weak and transient increase in auxin pools in systemic tissues. Similar 240

    to what has been observed for other phytohormones, (Koo et al., 2009; Stitz et al., 2011; 241

    VanDoorn et al., 2011), IAA levels increased sequentially in petioles, stems and systemic 242

    leaves. Together with the rapid local induction of YUCCA-like IAA biosynthetic homologues 243

    and the absence of IAA dependent systemic defense induction in transport inhibitor treated 244

    plants, these data suggest that IAA might be synthesized de novo at the site of the attack and 245

    then transported across the plant. Several studies have demonstrated that auxin is a mobile 246

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    signal in plants (Reed et al., 1998; Bhalerao et al., 2002; Jin et al., 2015; van Noorden et al., 247

    2006). Based on the IAA accumulation kinetics, we estimate that herbivory-induced IAA 248

    would need to be transported at a speed of at least 0.29 cm*min-1 to reach the petioles 5-10 249

    minutes after elicitation (based on the fact that IAA accumulates locally 30-60 seconds after 250

    elicitation). This value is at least tenfold greater than typical values of polar auxin transport 251

    velocities (Kramer et al., 2011), but twenty fold slower than wound-induced electrical signals 252

    that trigger systemic JA accumulation (Mousavi et al., 2013). We propose two hypotheses 253

    that may be responsible for the atypical signal propagation speed that we observed. First, it is 254

    possible that IAA is transported to systemic tissues by a combination of both polar and non-255

    polar, phloem-based transport (Friml, 2003). Second, rapid secondary signals, including 256

    electrical potentials, may spread through the plant at high speeds and induce de novo IAA 257

    biosynthesis in systemic tissues. Further experiments with IAA radiotracers (Agtuca et al., 258

    2014) and transient, tissue-specific deactivation of IAA biosynthesis (Koo et al., 2009) would 259

    help to shed further light on the exact mechanisms responsible for the systemic spread of IAA 260

    following herbivore attack. 261

    Impairing key genes of the jasmonate signaling cascade including mitogen-activated protein 262

    kinases, jasmonate biosynthesis and jasmonate perception elements did not impair the 263

    herbivory-induced accumulation of IAA, suggesting that IAA induction does not require JA 264

    signaling. This observation is consistent with the temporal dynamics of herbivory-induced 265

    IAA and JA that we observed. IAA accumulation peaks within 5 minutes after the onset of 266

    the elicitation while JA starts accumulating in an equally rapid fashion, but peaks 267

    significantly later than IAA (Figure 5). 268

    An important aim of our study was to understand whether IAA is involved in the regulation 269

    of induced secondary metabolites in N. attenuata. Because of the systemic accumulation 270

    pattern of IAA and the possibility to block this effect through the local application of 271

    transport inhibitors, we chose to focus on the induction of stem secondary metabolites. The 272

    stem of N. attenuata is vital for its reproduction and can be attacked by a wide variety of 273

    organisms, including vertebrates and invertebrate stem borers (Machado et al., 2016; Diezel 274

    et al., 2011b). We observed that real and simulated M. sexta attack induced anthocyanin 275

    accumulation in the stems, an effect that could not be reproduced by MeJA or IAA treatments 276

    alone, but by the combination of these two hormones. Together with the IAA transport and 277

    biosynthesis inhibitor treatments and the genetic silencing of JA biosynthesis, all of which led 278

    to the disappearance of the anthocyanin response, these results strongly suggest that IAA is 279

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  • 12

    required to activate the JA-dependent accumulation of stem anthocyanins. In A. thaliana, 280

    anthocyanin production is controlled by the MYB75 transcription factor Production of 281

    Anthocyanin Pigment 1 (PAP1) (Shin et al., 2015; Borevitz et al., 2000), which is 282

    transcriptionally upregulated by IAA (Lewis et al., 2011), and postranscriptionally repressed 283

    by jasmonate-ZIM-Domain (JAZ) proteins (Qi et al., 2011). The resulting co-regulation of 284

    MYB transcription factors by IAA and JA provides a potential mechanism for the synergistic 285

    interaction between JA and IAA observed in our study. 286

    In a second set of experiments, we found that IAA also boosts the production of 287

    phenolamides in herbivore-attacked plants. Phenolamide accumulation in N. attenuata is 288

    controlled by the transcription factor MYB8 in a JA-dependent manner (Onkokesung et al., 289

    2012; Paschold et al., 2007). This transcription factor may therefore represent a target for the 290

    integration of IAA and JA signaling. While IAA strongly potentiated the accumulation of 291

    stem phenolamides, it had little effect on the accumulation of other JA-dependent secondary 292

    metabolites, including nicotine and 7-hydroxygeranyllinalool diterpene glycosides (Machado 293

    et al., 2013; Paschold et al., 2007; Jimenez-Aleman et al., 2015; Machado et al., 2016). This 294

    result is consistent with earlier studies showing neutral to negative effects of auxin 295

    application on nicotine accumulation in Nicotiana spp. (Baldwin, 1989; Baldwin et al., 1997; 296

    Shi et al., 2006). The direct application of IAA to wounded tissues can even suppress local 297

    damage-induced JA accumulation (Dahl and Baldwin, 2004; Baldwin et al., 1997; Shi et al., 298

    2006). From these results, it is evident that IAA does not simply enhance JA signaling, but 299

    that it specifically modulates a plant’s defensive network. Thereby, IAA signaling may help 300

    plants to mount specific, fine-tuned responses to different attackers. 301

    The ecological function of an upregulation of anthocyanin and phenolamide compounds in 302

    the stems upon M. sexta attack remains an open question. The current literature however 303

    provides interesting insights in this context. Trichobaris stem weevils prefer to feed and 304

    perform better on defenseless, jasmonate-deficient plants in a species-specific manner: T. 305

    compacta grows better on nicotine-impaired N. attenuata plants while T. mucorea is not 306

    affected by nicotine but by other, yet unknown, jasmonate-dependent defenses (Diezel et al., 307

    2011b; Lee et al., 2016). It is therefore possible that the IAA-triggered potentiation of 308

    jasmonate-dependent secondary metabolite accumulation in the stems may reduce the 309

    performance of stem feeders. To disentangle the specific effects that IAA signaling has in this 310

    context requires the development of IAA-signaling impaired genotypes and represents an 311

    interesting prospect of this study. 312

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    In conclusion, this study identifies IAA as a rapid and specific signal that regulates a 313

    biologically relevant subset of herbivory-induced secondary metabolites. Current models on 314

    plant defense signaling networks in plant-herbivore interactions can now be expanded to 315

    include auxins as potentially important defense hormones. 316

    METHODS 317

    Plant genotypes, germination and planting conditions 318

    Wild-type N. attenuata Torr. Ex. Watson plants of the 31th inbred generation derived from 319

    seeds collected at the Desert Inn Ranch in Utah in 1988 and all genetically engineered plant 320

    genotypes were germinated on Gamborg’s B5 medium as described (Krügel et al., 2002). 321

    Nine to ten days later, seedlings were transferred to Teku pots (Pöppelmann GmbH & Co. 322

    KG, Lohne, Germany) for 10-12 days before transferring them into 1 L pots filled with either 323

    sand (to facilitate the harvesting of belowground tissues) or soil. All plants were grown at 45-324

    55% relative humidity and 23-25 °C during days and 19-23 °C during nights under 16 h of 325

    light (6am-10pm). Plants planted in soil were watered every day by a flood irrigation system. 326

    Plants planted in sand were watered twice a day. The characteristics of the transgenic plants 327

    used in this study are presented in table 1. 328

    Auxin and jasmonate measurements 329

    Phytohormone measurements were conducted as described earlier (Machado et al., 2013; 330

    Machado et al., 2015). Briefly, plant tissues were harvested, flash frozen and stored at -80°C. 331

    After grinding, 100 mg of plant tissue per sample were extracted with 1 mL ethyl acetate: 332

    formic acid (99.5:0.5 v/v) containing the following phytohormone standards: 40ng of 9,10-333

    D2-9,10-dihydrojasmonic acid (JA), 8 ng of jasmonic acid-[13C6] isoleucine (JA-Ile) and 20 334 ng of D5-indole-3-acetic-acid (IAA). All samples were then vortexed for 10 min and 335

    centrifuged at 14.000 rpm for 20 min at 4 °C. Supernatants were evaporated to dryness in a 336

    centrifugal vacuum concentrator (Eppendorf 5301, Eppendorf, Hamburg, Germany) at room 337 temperature. The remaining pellets were resuspended in 50 μL methanol: water (70:30) and 338 dissolved using an ultrasonic cleaner (Branson 1210, Branson Ultrasonics, 339 Danbury, Connecticut, USA) for 5 min. Samples were then analyzed using liquid 340 chromatography (Agilent 1260 Infinity Quaternary LC system, Agilent Technologies, Santa 341 Clara, California, USA) coupled to a triple quadrupole mass spectrometer (API 5000 342 LC/MS/MS, Applied Biosystems, Foster City, California, USA). 343

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    IAA levels in herbivore attacked plants 344

    IAA levels were determined in local, treated leaves of plant subjected to real or simulated M. 345

    sexta attack. Plants were infested by placing 3 first-instar larvae on one fully developed 346

    rosette leaf (n=3). Caterpillars were removed, and attacked leaves were harvested. M. sexta 347

    attack was simulated by rolling a pattern wheel over the leaves on each side of the midvein. 348

    Three fully developed rosette leaves were wounded and the resulting wounds were 349

    immediately treated with either 1:5 (v/v) water-diluted M. sexta oral secretions (W+OS), with 350

    pure water (W+W) or with fatty acid-amino acid conjugates (FACs; N-linolenoyl-glutamic 351

    acid) as described (Xu et al., 2015; Machado et al., 2013). Intact plants were used as controls 352

    (n=5). 353

    M. sexta-induced auxin levels in different plant tissues 354

    Forty-day-old elongating plants were subjected to simulated M. sexta attack as described 355

    above. Five, 10, 30, 60 and 120 min after elicitation, treated leaves and their untreated 356

    petioles, as well as stems, systemic leaves (young leaves directly above treated leaves), and 357

    main and lateral roots were harvested. The same plant tissues were collected from untreated 358

    control plants at each time point (n=5). 359

    M. sexta-induced auxin levels at different developmental stages 360

    IAA levels were measured at three developmental stages: early rosette (32 days after 361

    germination, DAG), elongating (39 DAG) and flowering (46 DAG). Tissues were harvested 362

    at three time points after elicitation as described above: 0.5, 1 and 3h (n=5). 363

    Identification and expression profiling of YUCCA-like genes 364

    YUCCA genes encode for flavin monooxygenase-like proteins that convert indole-3-pyruvic 365

    acid into indole-3-acetic acid (IAA), a catalytic reaction that is currently seen as the limiting 366

    step of IAA biosynthesis (Mashiguchi et al., 2011). To identify YUCCA-like genes in N. 367

    attenuata, we searched the Arabidopsis thaliana YUCCA2 gene sequence (NCBI accession 368

    number NM_117399.3) in the N. attenuata draft genome (Ling et al., 2015) using BLAST (E-369

    value200) and reconstructed the phylogenetic tree of the gene family. We 370

    then designed specific primers (Supplemental Table 1) for each gene using Primique 371

    (Fredslund and Lange, 2007) and profiled gene expression patterns upon simulated M. sexta 372

    attack by quantitative real-time PCR (qPCR)(n=3). Total RNA was extracted by the TRIZOL 373

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    method, followed by DNase-I treatment (Fermentas, St. Leon-Rot, Germany) according to 374

    the manufacturer’s instructions. Five micrograms of total RNA were reverse-transcribed 375

    using oligo (dT)18 and the SuperScript-II Reverse Transcriptase kit (Invitrogen). The 376

    obtained cDNA was used for gene expression profiling with SYBR Green I following the 377

    manufacturer’s protocol, and the ∆Ct method was used for transcript evaluation. The 378

    housekeeping gene actin was used as reference. Gene expression levels were determined 3, 5 379

    and 60 minutes after elicitation. 380

    Characterization of the YUCCA-like gene family 381

    The YUCCA-like gene family sequences were aligned by Clustal W (Thompson et al., 1994) 382

    in BioEdit (Hall, 1999) and the occurrence of the already described conserved amino acid 383

    motifs characteristic of the flavin monooxygenase gene family was determined (Expósito-384

    Rodríguez et al., 2011; Expósito-Rodríguez et al., 2007). 385

    OS-induced auxin and jasmonate kinetics 386

    Rosette leaves of wild type plants were subjected to simulated M. sexta attack (W+OS) as 387

    described and harvested 5, 45 and 90 min after elicitation (n=5). Phytohormone 388

    measurements were carried out as described. 389

    M. sexta-induced auxin levels in jasmonate and signaling impaired genotypes 390

    Three rosette leaves of rosette-stage plant genotypes impaired in salicylic acid-induced and 391

    wound-induced mitogen-activated protein kinases (irSIPK, irWIPK, respectively), jasmonic 392

    acid biosynthesis (irGLA, irAOS, irAOC, irOPR3), jasmonic acid-isoleucine biosynthesis 393

    (irJAR4/6), jasmonate perception (irCOI1) and wild type, empty vector (EV) were subjected 394

    to M. sexta simulated attack as described. 45 min after elicitation, the leaves were harvested 395

    and analyzed for IAA, jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) (n=5). These 396

    transgenic plant genotypes were selected as they are impaired at different layers of the 397

    jasmonate signaling cascade: early regulatory elements (irSIPK, irWIPK), jasmonate 398

    biosynthesis (irGLA, irAOS, irAOC, irOPR3), hormone activation (irJAR4/6) and hormone 399

    perception (irCOI1), and their main characteristics are listed in table 1. 400

    Stem anthocyanin quantifications 401

    To determine the role of IAA in M. sexta induced stem anthocyanin accumulation, we carried 402

    out three experiments. First, we measured anthocyanins in the stem of plants whose rosette 403

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    leaves were either left intact (Control), wounded and treated with water (W+W), wounded 404

    and treated with M. sexta oral secretions (W+OS), subjected to real, continuous M. sexta 405

    attack (M. sexta), treated with the natural auxin IAA (IAA), methyl jasmonic acid (MeJA) or 406

    with both IAA and MeJA (IAA+MeJA) dissolved in lanoline paste (n=5). Simulated M. sexta 407

    attack treatments were carried out as described above. Hormonal treatments were carried out 408

    as described below. In the second experiment, we measured stem anthocyanins in plants 409

    whose petioles were treated (petiole pretreatment) with the IAA biosynthesis inhibitor L-410

    kynurenine (L-Kyn) (He et al., 2011), the IAA transport inhibitor 2,3,5-triiodobenzoic acid 411

    (TIBA) (Hertel et al., 1983; Goldsmith, 1982; Rubery, 1979) or with the natural auxin indole-412

    3-acetic acid (IAA) prior to eliciting the plants by simulated M. sexta attack (W+OS) (n=12). 413

    One hour prior to the simulated M. sexta attack treatments, approximately 2 µg of L-Kyn, 414

    TIBA or IAA, or 150 µg MeJA dissolved in lanoline paste were applied to the petioles. 415

    Applied doses were selected according to previous studies (Baldwin, 1989; Morris et al., 416

    1973; Kang et al., 2006; He et al., 2011) (n=12). In a third experiment, we measured changes 417

    in stem anthocyanin levels upon simulated M. sexta herbivory in jasmonate-deficient irAOC 418

    and empty vector (EV) controls (n=10). Simulated and real M. sexta attack treatments were 419

    carried out as described. For all the experiments, the stems were harvested five days after 420

    treatments, and the anthocyanin content of the outer layer (epidermis, cortex, phloem and 421

    cambium) was determined 5 cm above the shoot-root junction as described (Steppuhn et al., 422

    2010). 423

    Stem secondary metabolite quantifications 424

    To further explore the regulatory role of IAA in secondary metabolite production, we induced 425

    the leaves of N. attenuata plants using real and simulated M. sexta attack treatments. Plants 426

    were either pretreated with IAA in lanolin paste or with pure lanolin as controls as described 427

    above. Petiole pretreatments with IAA were carried out one hour prior to induction. Five days 428

    after induction, the stems were harvested and secondary metabolites were measured as 429

    described (Gaquerel et al., 2010; Ferrieri et al., 2015)(n=5). 430

    Statistics 431

    All data were analyzed by ANOVA using Sigma Plot 12.0 (Systat Software Inc., San Jose, 432

    CA, USA). Normality and equality of variance were verified using Shapiro–Wilk and 433

    Levene’s tests, respectively. Holm–Sidak post hoc tests were used for multiple comparisons. 434

    Datasets from experiments that did not fulfill the assumptions for ANOVA were natural log-, 435

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    root square- or rank-transformed before analysis. Correlation between jasmonate and IAA 436 levels, and stem coloration index and stem anthocyanin content were evaluated by Pearson 437

    product moment test. 438

    ACKNOWLEDGEMENTS 439

    All experimental work of this study was supported by the Max Planck Society. We would 440

    also like to thank the members of the Department of Molecular Ecology and the glasshouse 441

    team of the MPI-CE for their help. Special thanks go to Mareike Schirmer and Mareike 442

    Schmidt for technical support and to Wenwu Zhou, Martin Schäfer and Michael Reichelt for 443

    their valuable help with the auxin measurements. CAMR was supported by a Swiss National 444

    Foundation Fellowship (grant no. 140196), CCMA by the Brazilian National Council for 445

    Research (grant no. 237929/2012-0), APF by an Alexander von Humboldt Postdoctoral 446

    Fellowship, SX by a Marie Curie Intra European Fellowship (grant no. 328935), ITB by a 447

    European Research Council advanced (grant no. 293926) and by a Human Frontier Science 448

    Program (grant no. RGP0002/2012), and ME by an SNF early post doc fellowship (grant no. 449

    134930) and a Marie Curie Intra European Fellowship (grant no. 273107). 450

    AUTHOR CONTRIBUTIONS 451

    Designed the research: RARM, ME, ITB. Carried out the experimental work: RARM, 452

    CCMA, APF, CAMR, GHJA, SX. Analyzed data: RARM, ME, ITB. Wrote the first draft of 453

    the paper: RARM, ME. Revised the paper: ME, RARM, ITB, APF, CCMA, GHJA, SX, 454

    CAMR. All authors read and approved the final manuscript. 455

    456

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    Table 1. Characteristics of the inverted repeat (ir) transgenic lines used in the present study 457

    Genotype Gene silenced/overexpressed Impaired function Phenotype Reference

    irSIPK Salicylic acid-induced

    mitogen activated protein kinase Early

    jasmonate signalling

    Reduced levels of jasmonates

    Meldau et al., 2009

    irWIPK Wound-induced

    mitogen activated protein kinase

    irGLA1 Glycerolipase A1

    Jasmonate biosynthesis

    Bonaventure et al., 2011

    irAOS Allene oxide synthase

    Kallenbach et al., 2012 irAOC Allene oxide cyclase

    irOPR3 12-oxo-phytodienoic acid reductase

    irJAR4/6 JA-Ile synthetase Reduced levels of JA-Ile Wang et al.,

    2008

    irCOI1 Coronatine-insensitive 1 JA-Ile perception Reduced JA-Ile

    perception Paschold et al.,

    2007

    458

    TABLE LEGENDS 463

    Table 1. Characteristics of the inverted repeat (ir) transgenic lines used in the present study 464

    FIGURE LEGENDS 465

    Figure 1. Indole-3-acetic acid (IAA) is induced specifically and rapidly by real and simulated 466 M. sexta attack. Average (±SE) IAA levels in leaves that are attacked by M. sexta caterpillars 467

    (A), treated with M. sexta oral secretions (B, C) or treated with an herbivore elicitor (D) 468 (n=5). Different letters indicate significant differences between treatments (P < 0.05). 469

    Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded and M. 470

    sexta oral secretion-treated plants, W+FACs: wounded and fatty acid-amino acid conjugate-471

    treated plants. 472

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    Figure 2. Herbivory induces IAA both locally and systemically. Average (±SE) IAA levels 473 following simulated M. sexta attack in local, treated leaves (A) and in untreated petioles (B), 474 stem (C), systemic leaves (D), main root (E), and lateral roots (F) (n=5). Asterisks indicate 475 significant differences between treatments within plant tissues and time points (*: P < 0.05; 476

    ***: P < 0.001). Control: intact plants, W+OS: wounded and M. sexta oral secretion-treated 477

    plants. 478

    Figure 3. IAA induction in leaves occurs across different developmental stages. Average 479 (±SE) IAA levels in local treated leaves following simulated M. sexta attack at the early 480

    rosette (A), elongated (B) and flowering stage (C) (n=5). Different letters indicate significant 481 differences between treatments within developmental stages and time points (P < 0.05). 482

    Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded and M. 483

    sexta oral secretion-treated plants. 484

    Figure 4. YUCCA-like genes are upregulated in response to simulated M. sexta herbivory. 485 (A) Schematic representation of YUCCA-mediated conversion of indole-3-pyruvic acid into 486 IAA. Average (±SE) transcript abundance relative to control of YUCCA-like 3 (B), YUCCA-487 like 5 (C), YUCCA-like 6 (D) and YUCCA-like 9 (E) in treated leaves three minutes after 488 elicitation, and YUCCA-like 1 (F) and YUCCA-like 3 (G) 5 and 60 min following simulated 489 M. sexta attack (n=3). Different letters indicate significant differences between treatments (P 490

    < 0.05). Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded 491

    and M. sexta oral secretion-treated plants, W+FACs: wounded and fatty acid-amino acid 492

    conjugate-treated plants. 493

    Figure 5. Manduca sexta-induced IAA peaks earlier than jasmonic acid (JA). Left Y-axis: 494 average (±SE) leaf IAA levels in response to M. sexta attack. Right Y-axis: average (±SE) 495

    leaf JA levels in response to M. sexta attack. Closed squares: IAA levels upon W+OS 496

    treatments; closed triangles: IAA levels in control, untreated plants. Grey squares: JA levels 497

    upon W+OS treatments; grey triangles: jasmonic acid (JA) levels in control, untreated plants 498

    (n=5). Different letters indicate significant differences between treatments for individual 499

    metabolites (P < 0.05). IAA. Time: P = 0.015, treatment: P < 0.001, Time* treatment: P = 500

    0.638; JA. Time: P < 0.001, treatment: P < 0.001, Time* treatment: P < 0.001). Control: 501

    intact plants, W+OS: wounded and M. sexta oral secretion-treated plants. 502

    Figure 6. Jasmonate signaling is not required for the M. sexta-induced accumulation of IAA. 503 (A) Average (±SE) IAA levels in local, treated leaves of wild type plants (empty vector; EV) 504

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    and plant genotypes impaired in early JA signaling, jasmonate biosynthesis and/or JA-Ile 505

    perception 45 minutes after elicitation (n=5). Different letters indicate significant differences 506

    between treatments within each genotype (P < 0.05). C: control, intact plants; W: wounded 507

    and water-treated plants, OS: wounded and M. sexta oral secretions-treated plants. 508

    Figure 7. Manduca sexta-induced IAA and JA act synergistically to trigger anthocyanin 509 accumulation in the stems. (A) Average (±SE) stem anthocyanin content 5 days following 510 either simulated or continuous M. sexta attack, exogenous application of methyl jasmonate 511

    (MeJA) and/or IAA (n=5). (B) Average (±SE) stem anthocyanin content 5 days following 512 simulated M. sexta attack and petiole-pretreatments with either IAA, the IAA biosynthesis 513

    inhibitor L-kynurenine (L-Kyn) or the IAA transport inhibitor TIBA (2,3,5-triiodobenzoic 514

    acid) (n=12). (C) Average (±SE) stem anthocyanin contents following simulated M. sexta 515 attack of wild type and JA-impaired irAOC plants (n=10). (D) Correlation between stem 516 anthocyanin content and stem coloration. Inset: Photograph of the red stem phenotype. 517

    Asterisks indicate significant differences between treatments and control (A), between 518 simulated herbivory treatments within petiole pretreatments (B) and between treatments 519 within genotypes (C) (*: P < 0.05; **: P < 0.01; ***: P < 0.001). The correlation between 520 stem coloration index and stem anthocyanin content was evaluated by a Pearson product 521

    moment test. Leaf treatments. Control: intact plants; W+W: wounded and water-treated 522

    plants, W+OS: wounded and M. sexta oral secretion-treated plants, M. sexta: plants subjected 523

    to actual M. sexta attack, IAA: rosette leaves treated with indole-3-acetic acid, MeJA: rosette 524

    leaves treated with methyl jasmonic acid, IAA+MeJA: rosette leaves treated with IAA and 525

    MeJA. Petiole pretreatments. Petioles treated with either pure lanoline paste (Lanoline), L-526

    kynurenine (L-Kyn), 2,3,5-triiodobenzoic acid (TIBA) or indole-3-acetic acid (IAA) 527

    dissolved in lanoline 1h prior to leaf treatments. 528

    Figure 8. IAA specifically potentiates the herbivore-induced, systemic production of 529 phenolamides. Average (±SE) caffeoylputrescine (A), dicaffeoylspermidine (B), nicotine (C) 530 and diterpene glycoside (D) levels in the stems 5 days following simulated or real M. sexta 531 attack and petiole pretreatments with IAA (n=5). Asterisks indicate significant differences 532

    between petiole pretreatments within simulated M. sexta attack treatments (*: P < 0.05; **: P 533

    < 0.01; ***: P < 0.001). Petiole pretreatments. Control: petioles treated with pure lanoline 534

    paste 1h prior to leaf treatments. IAA: petioles treated with IAA dissolved in lanoline 1h prior 535

    to leaf treatments. Leaf treatments. Control: intact plants; W+W: wounded and water-treated 536

    plants, W+OS: wounded and M. sexta oral secretion-treated plants, M. sexta: plants subjected 537

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  • 21

    to actual M. sexta attack, MeJA: rosette leaves treated with methyl jasmonic acid dissolved in 538

    lanoline paste. 539

    SUPPLEMENTAL DATA 540

    Supplemental Figure 1. IAA is induced locally in response to simulated M. sexta herbivory 541 independently of time of day. 542

    Supplemental Figure 2. The N. attenuata genome contains nine YUCCA-like genes. 543

    Supplemental Figure 3. Gene expression patterns of YUCCA-like genes upon simulated M. 544 sexta attack. 545

    Supplemental Figure 4. Jasmonate signaling is not required for the M. sexta-induced 546 accumulation of IAA. 547

    Supplemental Table 1. Sequence of primers used for quantitative PCR analysis. 548

    549

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    Figure 1. Indole-3-acetic acid (IAA) is induced specifically and rapidly by real and simulated M. sextaattack. Average (±SE) IAA levels in leaves that are attacked by M. sexta caterpillars (A), treated with M.sexta oral secretions (B, C) or treated with an herbivore elicitor (D) (n=5). Different letters indicatesignificant differences between treatments (P < 0.05). Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded and M. sexta oral secretion-treated plants, W+FACs: wounded and fattyacid-amino acid conjugate-treated plants.

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    Figure 2. Herbivory induces IAA both locally and systemically. Average (±SE) IAA levels followingsimulated M. sexta attack in local, treated leaves (A) and in untreated petioles (B), stem (C), systemicleaves (D), main root (E), and lateral roots (F) (n=5). Asterisks indicate significant differences betweentreatments within plant tissues and time points (*: P < 0.05; ***: P < 0.001). Control: intact plants,W+OS: wounded and M. sexta oral secretion-treated plants.

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    10

    0.5 1 3

    Time: P = 0.002Treatment: P < 0.001T*T: P < 0.001

    b

    a

    c

    b

    a

    c

    Elongated

    0

    2

    4

    6

    0.5 1 3

    Time: P = 0.049Treatment: P < 0.001T*T: P = 0.414

    a

    b

    a

    a

    ab

    b

    Flowering

    IAA

    (ng/

    gFW

    )

    A B C

    Figure 3. IAA induction in leaves occurs across different developmental stages. Average (±SE) IAAlevels in local treated leaves following simulated M. sexta attack at the early rosette (A), elongated (B)and flowering stage (C) (n=5). Different letters indicate significant differences between treatments withindevelopmental stages and time points (P < 0.05). Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded and M. sexta oral secretion-treated plants.

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  • 0

    5

    10

    0123

    0

    2

    4

    Control 5 60Time after W+OS treatment (min)

    YUCCA-like 3

    YUCCA-like 9

    a

    YUCCA-like 3

    a

    b b

    a a

    b bP < 0.001

    P < 0.001

    a

    b

    c

    Fold

    cha

    nge

    YUCCA-mediated oxidative decarboxylation

    Indole-3-pyruvic acid Indole-3-acetic acid

    A

    B

    00.5

    11.5

    YUCCA-like 5

    a ab b

    P < 0.001

    C

    E

    G

    0

    1

    2

    YUCCA-like 6P = 0.001 b

    a

    b

    a

    D

    P < 0.001

    Figure 4. YUCCA-like genes are upregulated in response to simulated M. sexta herbivory. (A)Schematic representation of YUCCA-mediated conversion of indole-3-pyruvic acid into IAA. Average(±SE) transcript abundance relative to control of YUCCA-like 3 (B), YUCCA-like 5 (C), YUCCA-like 6(D) and YUCCA-like 9 (E) in treated leaves three minutes after elicitation, and YUCCA-like 1 (F) andYUCCA-like 3 (G) 5 and 60 min following simulated M. sexta attack (n=3). Different letters indicatesignificant differences between treatments (P < 0.05). Control: intact plants, W+W: wounded and water-treated plants, W+OS: wounded and M. sexta oral secretion-treated plants, W+FACs: wounded and fattyacid-amino acid conjugate-treated plants.

    012345

    Control 5 60

    YUCCA-like 1

    a

    b

    c

    F

    P < 0.001

    Time after W+OS treatment (min)

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  • IAA

    (ng/

    gFW

    ) JA (ng/gFW)

    0

    400

    800

    1200

    1600

    0

    10

    20

    30

    40

    0 45 90

    IAA Control

    a

    ba

    b

    A

    b

    a

    A

    B BJA Control

    Time after treatment (min)

    Figure 5. Manduca sexta-induced IAA peaks earlier than jasmonic acid (JA). Left Y-axis: average (±SE)leaf IAA levels in response to M. sexta attack. Right Y-axis: average (±SE) leaf JA levels in response toM. sexta attack. Closed squares: IAA levels upon W+OS treatments; closed triangles: IAA levels incontrol, untreated plants. Grey squares: JA levels upon W+OS treatments; grey triangles: jasmonic acid(JA) levels in control, untreated plants (n=5). Different letters indicate signif