Mar 07, 2019 · Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ferreira-Silva, B., Lavandera, I., Kern, A., Faber, K., and Kroutil, W. (2010). Chemo

Short title Biosynthesis of 2-phenylethanol in poplar 1

Author for contact details Tobias G Koumlllner 3

Separate pathways contribute to the herbivore-induced formation of 5

2-phenylethanol in poplar 6

Jan Guumlnthera Nathalie D Lackusa Axel Schmidta Meret Huberab Heike-Jana 8

Stoumldtlera Michael Reichelta Jonathan Gershenzona Tobias G Koumlllnera 9

aMax Planck Institute for Chemical Ecology Hans-Knoumlll-Strasse 8 D-07745 Jena 12

Germany 13

bPresent address Institute of Plant Biology and Biotechnology University of Muumlnster 14

Schlossplatz 7 48143 Muumlnster 15

E-mail addresses 17

JaG jguenthericempgde 18

AS aschmidticempgde 19

MH hubermuni-muensterde 20

NDL nlackusicempgde 21

HJS Heike-JanaSgmxde 22

MR reichelticempgde 23

JoG gershenzonicempgde 24

TGK koellnericempgde 25

One-sentence summary 27

Biochemical analysis of the aromatic amino acid decarboxylase family in poplar 28

revealed two enzymes involved in the herbivore-induced formation of 2-29

phenylethanol and 2-phenylethylamine 30

FOOTNOTES 33

Plant Physiology Preview Published on March 7 2019 as DOI101104pp1900059

Author contributions 34

TGK JaG and JoG designed the research JaG NDL AS HJS and MR carried out 35

the experimental work JaG NDL MH AS MR and TGK analyzed data TGK and 36

JaG wrote the manuscript All authors read and approved the final manuscript 37

Responsibilities of the Author for Contact 39

It is the responsibility of the author for contact to ensure that all scientists who have 40

contributed substantially to the conception design or execution of the work described 41

in the manuscript are included as authors in accordance with the guidelines from the 42

Committee on Publication Ethics (COPE 43

httppublicationethicsorgresourcesguidelines) It is the responsibility of the author 44

for contact also to ensure that all authors agree to the list of authors and the 45

identified contributions of those authors 46

Funding information 48

The research was funded by the Max-Planck Society 49

Corresponding author E-mail koellnericempgde phone +49 (0) 3641 57 1329 52

fax +49 (0) 3641 57 1302 53

ABSTRACT 54

Upon herbivory the tree species western balsam poplar (Populus trichocarpa) 55

produces a variety of phenylalanine-derived metabolites including 2-56

phenylethylamine 2-phenylethanol and 2-phenylethyl-β-D-glucopyranoside To 57

investigate the formation of these potential defense compounds we functionally 58

characterized aromatic L-amino acid decarboxylases (AADCs) and aromatic 59

aldehyde synthases (AASs) which play important roles in the biosynthesis of 60

specialized aromatic metabolites in other plants Heterologous expression in 61

Escherichia coli and Nicotiana benthamiana showed that all five AADCAAS genes 62

identified in the P trichocarpa genome encode active enzymes However only two 63

genes PtAADC1 and PtAAS1 were significantly upregulated after leaf herbivory 64

Despite a sequence similarity of about 96 PtAADC1 and PtAAS1 showed different 65

enzymatic functions and converted phenylalanine into 2-phenylethylamine and 2-66

phenylacetaldehyde respectively The activities of both enzymes were 67

interconvertible by switching a single amino acid residue in their active sites A 68

survey of putative AADCAAS gene pairs in the genomes of other plants suggests an 69

independent evolution of this function-determining residue in different plant families 70

RNAi-mediated downregulation of AADC1 in grey poplar (P times canescens) resulted in 71

decreased accumulation of 2-phenylethylamine and 2-phenylethyl-β-D-72

glucopyranoside while the emission of 2-phenylethanol was not influenced To 73

investigate the last step of 2-phenylethanol formation we identified and characterized 74

two P trichocarpa short-chain dehydrogenases PtPAR1 and PtPAR2 which were 75

able to reduce 2-phenylacetaldehyde to 2-phenylethanol in vitro In summary 2-76

phenylethanol and its glucoside may be formed in multiple ways in poplar Our data 77

indicate that PtAADC1 controls the herbivore-induced formation of 2-78

phenylethylamine and 2-phenylethyl-β-D-glucopyranoside in planta while PtAAS1 79

likely contributes to the herbivore-induced emission of 2-phenylethanol 80

Key words Populus trichocarpa 2-phenylethanol 2-phenylethylamine aromatic 83

amino acid decarboxylase aldehyde synthase aldehyde reductase plant 84

metabolism gene duplication neofunctionalization 85

INTRODUCTION 86

Many plant secondary or specialized metabolites play major roles in plant defense 87

against herbivores Some accumulate as deterrents or toxins as part of direct plant 88

defense while others are released as volatiles and can attract parasitoids or 89

predators of the herbivores a phenomenon that has been termed indirect defense 90

(Unsicker et al 2009) Still others may serve as internal signals to activate the 91

formation of both direct and indirect defenses (Maag et al 2015) Plant specialized 92

metabolites are highly diverse and belong to different compound classes including 93

terpenoids phenylpropanoids benzenoids amino acid derivatives and fatty acid 94

derivatives While some of these metabolites as for example certain terpenes and 95

phenylpropanoids are ubiquitous among plants others are exclusively produced in 96

specific families or even a single genus (Fahey et al 2001 Bieri et al 2006) 97

2-Phenylethanol and its glucoside 2-phenylethyl-β-D-glucopyranoside are amino 98

acid-derived specialized compounds that are widespread among plants The free 99

alcohol is well known for its pleasant lsquorose-likersquo aroma and has been reported to be a 100

flower fruit and vegetative volatile released from a multitude of plant species of 101

more than 50 families (Knudsen et al 2006) As a bioactive compound 2-102

phenylethanol plays diverse roles in plant-insect interactions It acts for example as 103

an attractant for pollinators such as butterflies bees and beetles (Roy and Raguso 104

1997 Honda et al 1998 Imai et al 1998) and mediates direct and indirect plant 105

defenses (Zhu et al 2005 Galen et al 2011) 106

The biosynthesis of 2-phenylethanol from phenylalanine was first elucidated in the 107

yeast Saccharomyces cerevisiae where it proceeds via the Ehrlich pathway 108

(reviewed in Hazelwood et al 2008) This pathway starts with the transamination of 109

an aromatic or branched-chain amino acid followed by a decarboxylation of the 110

resulting α-keto acid The aldehyde formed is finally reduced to yield 2-111

phenylethanol In plants the biosynthesis of 2-phenylethanol has been extensively 112

investigated in rose petunia and tomato (Sakai et al 2007 Kaminaga et al 2006 113

Tieman et al 2006 Tieman et al 2007) The pathways described in these species 114

also start from phenylalanine and involve the formation and subsequent reduction of 115

2-phenylacetaldehyde however the initial reactions are different from those of the 116

Ehrlich pathway (Figure 1) Tomato for example contains several aromatic amino 117

acid decarboxylases (AADCs) that catalyze the formation of 2-phenylethylamine 118 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

which could be further converted to 2-phenylacetaldehyde by a monoamine oxidase 119

(Tieman et al 2006) In contrast rose and petunia possess a bifunctional aromatic 120

aldehyde synthase (AAS) able to catalyze both phenylalanine decarboxylation and 121

the subsequent oxidative transamination (Kaminaga et al 2006 Sakai et al 2007 122

Farhi et al 2010) The recent identification of an aromatic amino acid transaminase 123

(AAAT) involved in aroma compound formation in melon as well as the 124

characterization of an AAAT and a phenylpyruvate decarboxylase (PPDC) in rose 125

indicate that the Ehrlich pathway might also contribute to the formation of 2-126

phenylethanol and other related alcohols in plants (Gonda et al 2010 Hirata et al 127

2016 Sheng et al 2018) Moreover recent research on poplar suggests another 128

potential route to 2-phenylethanol that involves the P450-catalyzed production of 129

(EZ)-phenylacetaldoxime from phenylalanine (Figure 1 Irmisch et al 2013 Irmisch 130

et al 2014a) 131

AADC and AAS both belong to group II pyridoxal-5rsquo-phosphate (PLP)-dependent 132

enzymes that encompass a large family referred to as the plant aromatic amino acid 133

decarboxylase (AAAD) family (Torrens-Spence et al 2018a Facchini et al 2000) 134

AAADs can be assigned to three major groups depending on their substrate 135

specificity for tryptophan tyrosine or phenylalanine respectively They form mid-136

sized gene families with an average number of about 10 members per plant genome 137

and have been identified in all plant lineages including algae non-vascular land 138

plants gymnosperms and angiosperms (Kumar 2016) Due to their relatively high 139

sequence identities a reliable functional prediction of single AAAD members has 140

been difficult in the past However two recent studies identified amino acid residues 141

that determine the substrate selectivity and catalytic activity of AAAD enzymes The 142

different substrate preferences of tryptophan and tyrosine decarboxylases for 143

example are indicated by a conserved glycine-serine polymorphism in the active site 144

of these enzymes (Torrens-Spence et al 2014a) The difference between AADC and 145

AAS activity is determined by the presence of either a tyrosine or phenylalanine (or 146

other apolar residue) on a catalytic loop proximal to the active site (Torrens-Spence 147

et al 2013 Bertoldi et al 2002 Torrens-Spence et al 2018b) 148

The last step in the formation of 2-phenylethanol common to all of these pathways is 149

the reduction of 2-phenylacetaldehyde This reaction is catalyzed by 2-150

phenylacetaldehyde reductase (PAR) which belongs to the large family of NADPH-151 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

dependent short-chain dehydrogenasereductases (Tieman et al 2007 Chen et al 152

2011) Tomato for example possesses two PARs (LePAR1 and LePAR2) that 153

accept 2-phenylacetaldehyde as substrate While LePAR1 is highly substrate-154

specific LePAR2 can convert a variety of aromatic and aliphatic aldehydes into the 155

respective alcohols (Tieman et al 2007) Such substrate promiscuity has also been 156

reported for another PAR that is involved in 2-phenylethanol formation in roses (Chen 157

et al 2011) 158

In recent years poplar has been established as a model system for studying plant 159

defenses against herbivores in long-living woody plant species (Peters and 160

Constabel 2002 Arimura et al 2004 Ralph et al 2006 Frost et al 2007 Philippe 161

and Bohlmann 2007 Major and Constabel 2008 Danner et al 2011 Irmisch et al 162

2014b) After leaf herbivory the western balsam poplar (P trichocarpa) accumulates 163

various defense compounds and releases a complex volatile blend that mediates 164

direct and indirect defense reactions (Irmisch et al 2013 McCormick et al 2014) 165

The volatile blend is dominated by terpenes green leaf volatiles and nitrogen-166

containing compounds and contains 2-phenylethanol as one of its main components 167

(Irmisch et al 2013) RNAi and labeling experiments indicated that a significant 168

proportion of herbivore-induced 2-phenylethanol seems to be produced via an oxime-169

dependent pathway (Irmisch et al 2013 Irmisch et al 2014a) however whether 170

poplar AAAD enzymes may also contribute to the formation of 2-phenylethanol and 171

related compounds was unclear 172

In order to elucidate the possible contribution of AAAD-dependent pathways to the 173

formation of 2-phenylethanol in poplar we measured the accumulation of pathway 174

intermediates in herbivore-damaged P trichocarpa leaves using liquid 175

chromatography-tandem mass spectrometry (LC-MSMS) RNA-seq experiments and 176

RT-qPCR were used to identify herbivore-induced AAAD candidate genes 177

Characterization of heterologously produced and purified AAAD proteins 178

overexpression in Nicotiana benthamiana and RNAi-mediated downregulation 179

revealed that two candidate enzymes PtAADC1 and PtAAS1 are involved in 180

herbivore-induced phenylalanine metabolism and 2-phenylethanol formation in 181

poplar 182

RESULTS 186

Potential precursors and metabolites of 2-phenylethanol accumulate in 187

herbivore-damaged P trichocarpa leaves 188

In order to elucidate the metabolic processes leading to 2-phenylethanol formation in 189

poplar we measured potential precursors and pathway intermediates in undamaged 190

and gypsy moth (Lymantria dispar) caterpillar-damaged P trichocarpa leaves (Figure 191

2) Phenylalanine and phenylpyruvic acid were present in both undamaged and 192

herbivore-damaged leaves and showed no changes in response to herbivory (Figure 193

2AB) The accumulation of 2-phenylethylamine however was significantly increased 194

upon herbivory and mirrored the herbivore-induced emission of 2-phenylethanol 195

(Figure 2CE) 2-Phenylacetaldehyde could not be detected suggesting a rapid 196

reduction to 2-phenylethanol in poplar 197

It has been shown that 2-phenylethanol can be converted to 2-phenylethyl-β-D-198

glucopyranoside in roses (Hayashi et al 2004) Moreover transgenic poplars 199

overexpressing rose RhPAAS and PAR accumulated large amounts of this glucoside 200

(Costa et al 2013) However whether 2-phenylethyl-β-D-glucopyranoside also 201

occurs in wild type poplars was unclear We could detect 2-phenylethyl-β-D-202

glucopyranoside both in undamaged and herbivore-damaged leaves and the 203

accumulation of this compound was slightly but significantly induced upon herbivory 204

(Figure 2D) 205

Identification of putative aromatic amino acid decarboxylase (AAAD) family 207

genes in P trichocarpa 208

A BLAST analysis with RhPAAS (Farhi et al 2010) as query revealed five putative 209

AAAD genes in the genome of P trichocarpa (Tuskan et al 2006) (Figure 3) 210

Considering the function-determining tyrosine-phenylalanine polymorphism in the 211

active sites of their encoded proteins the genes were named PtAADC1 212

(Potri013G052800) PtAADC2 (Potri016G114300) PtAADC3 (Potri004G036200) 213

PtAAS1 (Potri013G052900) and PtAAS2 (Potri002G255600) (Supplemental Figure 214

1) Two genes PtSDC1 (Potri005G190500) and PtSDC2 (Potri002G069800) that 215

also appeared in the BLAST search were similar to histidine (HDC) and serine (SDC) 216

decarboxylase genes (Figure 3) However since SDC-like proteins from barrelclover 217 httpsplantphysiolorgDownloaded on March 22 2021 - Published by

(Medicago truncatula) and chickpea (Cicer arietinum) have recently been 218

characterized as AAS enzymes (Torrens-Spence et al 2014a) poplar PtSDC1 and 219

PtSDC2 were also chosen as potential AAS gene candidates for further 220

characterization 221

Biochemical characterization of poplar AAAD and serine decarboxylase (SDC) 223

enzymes 224

The complete open reading frames of the candidate genes were amplified from 225

cDNA made from herbivore-damaged leaves of P trichocarpa and cloned into the 226

vector pET100D-TOPO After heterologous expression in Escherichia coli 227

recombinant proteins carrying an N-terminal His6-Tag were affinity purified and 228

incubated with phenylalanine tyrosine or tryptophan as potential substrates and PLP 229

as cofactor Both PtAAS1 and PtAAS2 had aldehyde synthase activity and catalyzed 230

the conversion of phenylalanine to 2-phenylacetaldehyde (Table 1) Formation of 2-231

phenylethylamine however could not be detected (Supplemental Figure 2) While 232

PtAAS1 was highly substrate specific and exclusively accepted phenylalanine 233

PtAAS2 showed activity with all tested aromatic amino acids producing the 234

respective aldehydes (Supplemental Figure 3) Notably the amino acid residue that 235

has been reported to determine the substrate specificity of tyrosine and tryptophan 236

decarboxylases (Torrens-Spence et al 2014b) differed between PtAAS1 (serine 237

360) and PtAAS2 (glycine 358) (Supplemental Figure 1) This amino acid change 238

might explain the observed differences in substrate promiscuity In contrast to 239

PtAAS1 and PtAAS2 PtAADC1 PtAADC2 and PtAADC3 showed no aldehyde 240

synthase activity but were able to catalyze the decarboxylation of different aromatic 241

amino acids The Km and kcat values revealed that PtAADC1 and PtAADC2 most 242

likely act as phenylalanine decarboxylases in planta while PtAADC3 is likely a 243

tyrosine decarboxylase (Table 1) To test for potential aromatic amino acid 244

transaminase activity of poplar AAAD candidates we incubated the enzymes with 245

phenylalanine in the presence of the ammonia acceptor α-ketoglutarate However no 246

formation of phenylpyruvic acid could be observed 247

To determine if the putative serine decarboxylases PtSDC1 and PtSDC2 exhibit 248

decarboxylase activity towards aromatic amino acids as previously described for 249

SDC-like enzymes in M truncatula and C arietinum (Torrens-Spence et al 2014a) 250 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

the enzymes were heterologously expressed and tested with phenylalanine tyrosine 251

tryptophan histidine and serine as potential substrates Both enzymes converted 252

serine into ethanolamine but showed no activity with aromatic amino acids and 253

histidine (Supplemental Figure 4) indicating that PtSDC1 and PtSDC2 likely function 254

as SDCs in planta Thus they were not further considered in this study 255

PtAAS1 and PtAADC1 are interconvertible by the change of one amino acid 257

PtAAS1 and PtAADC1 differ in a characteristic amino acid residue that has been 258

described as function-dictating residue in AADC and AAS enzymes from other plants 259

(Torrens-Spence et al 2013 Bertoldi et al 2002 Torrens-Spence et al 2018b) To 260

demonstrate that this tyrosine-phenylalanine polymorphism also determines the 261

decarboxylase and aldehyde synthase activity of PtAADC1 and PtAAS1 262

respectively we constructed the mutant enzymes PtAADC1 (Y338F) and PtAAS1 263

(F338Y) by in vitro mutagenesis and incubated them with phenylalanine as substrate 264

As expected both mutations led to a complete interconversion of the wild type 265

activities While PtAADC1 (Y338F) produced exclusively 2-phenylacetaldehyde 266

PtAAS1 (F338Y) had only decarboxylase activity and produced 2-phenylethylamine 267

(Figure 4A) 268

The tyrosine-phenylalanine-determined shift in AADCAAS enzyme activity 270

evolved independently in different plant families 271

Within the poplar AADCAAS clade PtAAS1 PtAADC1 and PtAADC2 form a small 272

subfamily with high nucleotide sequence identity of about 96-98 (Figure 3) While 273

PtAADC2 was found to be located on chromosome 16 PtAADC1 and PtAAS1 were 274

located close to each other on chromosome 13 (Figure 4B) This and the fact that 275

PtAADC1 and PtAAS1 have no other open reading frames between them in the 276

genomic sequence suggest a recent gene duplication that was subsequently followed 277

by neofunctionalization through the mutation of the function-determining amino acid 278

A survey of putative AADCAAS sequences in other available plant genomes 279

revealed the presence of comparable AADCAAS pairs in at least ten species from 280

diverse plant families including monocotyledonous and dicotyledonous plants 281

(Supplemental Figures 5 and 6) The putative AADC and AAS genes in each of these 282

pairs were highly similar to each other and grouped in a species-specific manner 283 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

indicating an independent evolution of the tyrosine-phenylalanine shift in different 284

plant families or even genera 285

Functional analysis of PtAADC1 and PtAAS1 in planta 287

To verify the different enzymatic functions of the characterized poplar phenylalanine-288

using AAAD enzymes in a heterologous plant system PtAADC1 and PtAAS1 were 289

chosen for transient expression in N benthamiana Plants overexpressing PtAAS1 290

showed a significant emission of 2-phenylethanol while wild type plants and plants 291

expressing either eGFP or PtAADC1 produced only trace amounts of this compound 292

if any (Figure 5A) In addition PtAAS1 plants accumulated 2-phenylethyl-β-D-293

glucopyranoside (Figure 5E) The overexpression of PtAADC1 however resulted in 294

the accumulation of 2-phenylethylamine tyramine tryptamine and 2-phenylethyl-β-295

D-glucopyranoside (Figure 5B-E) 2-Phenylacetaldehyde 4-hydroxy-296

phenylacetaldehyde and indole-3-acetaldehyde could not be detected in the tested 297

lines Altogether the exclusive emission of 2-phenylethanol from PtAAS1 plants and 298

the different accumulation levels of the aromatic amines in PtAADC1-overexpressing 299

plants corresponded well to the enzymatic activities and kinetic parameters that were 300

determined in vitro (Table 1 Figure 5) Moreover the results suggest that the 301

reactive aldehydes can be rapidly converted to less reactive alcohols or other 302

compounds in vivo even in a heterologous plant system 303

Expression of PtAADC1 and PtAAS1 is induced in response to herbivory in P 305

trichocarpa leaves 306

In order to study the expression of poplar AAAD genes we sequenced the 307

transcriptomes of herbivore-damaged and undamaged P trichocarpa leaves and 308

mapped the quality-trimmed sequence reads to the P trichocarpa genome version 309

v30 (httpsphytozomejgidoegov Tuskan et al 2006) All AAAD genes were 310

found to be expressed in poplar leaves (Figure 6) However the transcript 311

accumulation of PtAADC1 and PtAAS1 was highly upregulated in herbivore-312

damaged leaves in comparison to undamaged control leaves with fold changes of 313

294 and 124 respectively (Figure 6) Expression of PtAADC2 and PtSDC2 was also 314

increased in response to the herbivore treatment but showed lower fold increases 315

(64 and 16 respectively) PtAAS2 showed a significant reduction in gene 316 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

expression upon herbivory (fold change -15) while expression of PtAADC3 and 317

PtSDC1 were not influenced by the treatment (Figure 6) The RNA-Seq results for 318

poplar AAAD genes were confirmed by RT-qPCR (Supplemental Figure 7) The 319

highly similar genes PtAAS1 PtAADC1 and PtAADC2 could not be targeted by 320

specific primers and were thus amplified using a universal primer pair Extensive 321

sequencing of cloned amplicons that encompassed several polymorphisms including 322

the function-determining codon allowed a reliable assignment of the amplicons to 323

specific genes The RT-qPCR results confirmed the upregulation of PtAAS1 and 324

PtAADC1 gene expression in response to herbivory (Supplemental Table 2) 325

Transcripts of PtAADC2 however could not be detected in control and herbivore-326

damaged leaves Notably total expression of PtAAS1 in herbivore-damaged leaves 327

(925 of sequenced amplicons) was over ten times higher than PtAADC1 (75 of 328

sequenced amplicons) 329

RNAi-mediated downregulation of PtAADC1 in P times canescens affects the 331

formation of 2-phenylethylamine and 2-phenylethyl-β-D-glucopyranoside 332

To investigate the function of herbivore-induced AAAD genes in poplar we aimed to 333

generate RNAi lines with reduced AADC1AAS1 gene expression by transformation 334

of a DNA fragment complementary to AADC1 AADC2 and AAS1 Since there is no 335

established transformation method for P trichocarpa we chose grey poplar (P times 336

canescens) for transformation a species commonly used for the generation of 337

transgenic poplar trees (Lepleacute et al 1992) Grey poplar is a hybrid species derived 338

from a cross between silver poplar (P alba) and quaking aspen (P tremula) and 339

BLAST searches with the different P trichocarpa AAAD genes as query and the 340

genomes of P times canescens P alba and P tremula 341

(httpaspendbugaeduindexphpdatabasesspta-717-genome Zhou et al 2015 342

Xue et al 2015) as templates revealed that all three species possess putative 343

AADC1 AADC2 and AAS1 orthologues (Supplemental Figure 8) Interestingly the 344

function-dictating tyrosine of P trichocarpa PtAADC2 was changed into 345

phenylalanine in P times canescens PcanAADC2 and in P tremula PtremAADC2 346

suggesting that both orthologues likely function as aldehyde synthases However 347

sequencing of cloned qPCR fragments that were amplified with a primer pair specific 348

for AADC1 AADC2 and AAS1 from cDNA made from herbivore-damaged P times 349 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

canescens leaves revealed exclusively PcanAADC1 amplicons (Supplemental Figure 350

9) This indicates that the two aldehyde synthase genes PcanAAS1 and PcanAADC2 351

are likely not expressed in herbivore-damaged leaves of P times canescens 352

Populus times canescens PcanAADC1 RNAi lines as well as wild type trees and trees 353

carrying an empty vector were subjected to L dispar herbivory and the emission of 2-354

phenylethanol and the accumulation of 2-phenylethylamine and 2-phenylethyl-β-D-355

glucopyranoside in herbivory-induced leaves were measured Downregulation of 356

PcanAADC1 in the transgenic lines led to a significantly reduced accumulation of 357

herbivore-induced 2-phenylethylamine and 2-phenylethyl-β-D-glucopyranoside 358

(Figure 7A Supplemental Figure 10) The emission of 2-phenylethanol however 359

remained constant in comparison to wild type and empty vector trees Expression 360

levels of PcanAAS2 were not affected by the RNAi approach (Supplemental Figure 361

10) Thus the observed metabolic changes in the transgenic lines are most likely 362

caused by the downregulation of PcanAADC1 gene expression 363

One of the most characteristic volatiles released from herbivore-damaged poplars is 364

the nitrile benzyl cyanide (McCormick et al 2014) Previous studies showed that this 365

compound is mainly formed from phenylalanine via (EZ)-phenylacetaldoxime 366

through the action of two P450s from the CYP79 and CYP71 families (Irmisch et al 367

2013 Irmisch et al 2014a) Since 2-phenylethylamine has been proposed as 368

alternative substrate for benzyl cyanide (Tieman et al 2006) we measured the 369

emission of the volatile nitrile in herbivore-damaged PcanAADC1 RNAi lines 370

Downregulation of PcanAADC1 expression and the resulting decrease in 2-371

phenylethylamine accumulation did not influence benzyl cyanide emission 372

(Supplemental Figure 11) suggesting that this nitrile is exclusively produced via the 373

oxime-dependent pathway in poplar 374

Poplar aldehyde reductases PtPAR1 and PtPAR2 convert 2-376

phenylacetaldehyde into 2-phenylethanol in vitro 377

The findings that 2-phenylacetaldehyde could neither be detected in herbivore-378

damaged poplar leaves nor in PtAAS1-overexpressing N benthamiana plants 379

suggest a rapid and highly efficient conversion of the aldehyde into the 380

corresponding alcohol in planta likely catalyzed by a NADPH-dependent reductase 381

(PAR) as already reported in rose and tomato (Tieman et al 2007 Chen et al 382 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

2011) To identify putative poplar PARs we performed a TBLASTN analysis with the 383

tomato gene LePAR1 (Tieman et al 2007) as query and the P trichocarpa genome 384

as template A dendrogram of putative poplar reductases and characterized 385

reductases from other plants was generated and the five poplar genes that grouped 386

together with either LePAR1 (PtPAR1 PtPAR2) or aliphatic aldehyde reductases 387

(PtAAR1 PtAAR2 PtAAR3) were considered for further biochemical characterization 388

(Supplemental Figure 12) Heterologously expressed and purified enzymes were 389

tested with a variety of aromatic and aliphatic aldehydes as potential substrates in 390

the presence of the cosubstrate NADPH PtPAR1 and PtPAR2 exhibited enzyme 391

activity with 2-phenylacetaldehyde and produced 2-phenylethanol (Table 2 392

Supplemental Figure 13) However they were also able to reduce other aromatic and 393

aliphatic aldehyde substrates In contrast PtAAR1 PtAAR2 and PtAAR3 were only 394

active with a few aliphatic aldehydes and produced no 2-phenylethanol (Table 2 395

Supplemental Figure 13) Gene expression analysis revealed that all five aldehyde 396

reductase genes were constitutively expressed in P trichocarpa leaves 397

(Supplemental Figure 14) Their transcript accumulation was not influenced by the 398

herbivore treatment 399

DISCUSSION 402

In response to herbivore feeding poplar produces several aromatic metabolites 403

including 2-phenylethanol 2-phenylethyl-β-D-glucopyranoside and 2-404

phenylethylamine that may function in direct and indirect plant defense and defense 405

signaling Aromatic amino acid decarboxylases (AADC) and aromatic aldehyde 406

synthases (AAS) have been shown to be involved in the biosynthesis of 407

phenylalanine-derived specialized compounds in other plants (Kaminaga et al 2006 408

Tieman et al 2007 Hirata et al 2012) We aimed to study AADCAAS enzymes in 409

P trichocarpa to elucidate their roles in the herbivore-induced metabolism of 410

phenylalanine 411

PtAAS1 likely contributes to the formation of herbivore-induced 2-413

phenylethanol in P trichocarpa 414

Aromatic aldehyde synthases are bifunctional enzymes that catalyze the 415

decarboxylation and subsequent oxidative deamination of aromatic amino acids In 416

roses and petunia AAS enzymes have been shown to produce 2-417

phenylacetaldehyde as substrate for the formation of the corresponding alcohol 2-418

phenylethanol (Kaminaga et al 2006 Sakai et al 2007 Farhi et al 2010) P 419

trichocarpa contains two putative AAS (Figure 3) and biochemical characterization of 420

the recombinant enzymes confirmed their aldehyde synthase activity (Table 1) While 421

PtAAS1 accepted exclusively phenylalanine as a substrate PtAAS2 showed activity 422

with phenylalanine tyrosine and tryptophan However the similar Km values of 423

PtAAS1 and PtAAS2 for phenylalanine suggest that both enzymes catalyse the 424

formation of 2-phenylacetaldehyde in vivo (Table 1) In contrast to PtAAS2 which 425

was constitutively expressed transcript accumulation of PtAAS1 was highly 426

upregulated in herbivore-damaged leaves of P trichocarpa and mirrored the 427

herbivore-induced emission of 2-phenylethanol (Figures 2 and 6) This indicates that 428

PtAAS1 likely contributes to the formation of 2-phenylethanol upon herbivory by 429

providing the precursor 2-phenylacetaldehyde 430

Recent studies demonstrated that the herbivore-induced production of 2-431

phenylethanol in poplar is also mediated by an alternative pathway that involves the 432

formation of (EZ)-phenylacetaldoxime (Irmisch et al 2013 Irmisch et al 2014 433

Figure 1) This oxime is biosynthesized from phenylalanine only upon herbivory by 434

the P450 enzyme CYP79D6v3 Downregulation of CYP79D6v3 in P times canescens led 435

to decreased emission of herbivore-induced 2-phenylethanol while N benthamiana 436

plants overexpressing CYP79D6v3 produced the alcohol suggesting a role of (EZ)-437

phenylacetaldoxime as an alternative precursor for 2-phenylethanol in planta (Irmisch 438

et al 2013) Indeed it has been proposed that (EZ)-phenylacetaldoxime can be 439

converted to 2-phenylacetaldehyde by a transoximase activity (Soumlrenssen et al 440

2018) or even directly to 2-phenylethanol by an alcohol dehydrogenase (Ferreira-441

Silva et al 2010) The enzymatic machinery behind these reactions however is still 442

not known Since the Km value of CYP79D6v3 for phenylalanine (Km = 744 microM) was 443

similar to that of PtAAS1 (Km = 460 microM) we hypothesize that both enzymes and 444

pathways contribute to the herbivore-induced formation of 2-phenylethanol in P 445

trichocarpa Notably the related poplar species P times canescens lacked expression of 446

AAS1 It is thus likely that P times canescens produces the alcohol exclusively via the 447 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

oxime-dependent pathway upon herbivory This assumption is strengthened by the 448

fact that the knockdown of AADC1 in P times canescens had no effect on the herbivore-449

induced emission of 2-phenylethanol (Figure 7) ruling out a potential contribution of 450

this enzyme to the formation of volatile 2-phenylethanol 451

PtAADC1 generates 2-phenylethylamine which can be metabolized into 2-453

phenylethyl-β-D-glucopyranoside in vivo 454

Plant aromatic amines and derived metabolites play important roles as signalling and 455

defence compounds and are usually produced by AADC enzymes (reviewed in 456

Facchini et al 2000) We identified and characterized three AADCs in P trichocarpa 457

that accepted various aromatic amino acids as substrates and converted them into 458

the corresponding aromatic amines in vitro Kinetic characterization revealed that 459

PtAADC1 and PtAADC2 preferred phenylalanine as substrate while PtAADC3 460

preferred tyrosine (Table 1) N benthamiana plants overexpressing PtAADC1 461

produced 2-phenylethylamine and minor amounts of tyramine and tryptamine 462

confirming the kinetic parameters determined in vitro RNAi-mediated knockdown of 463

AADC1 in P times canescens led to decreased levels of herbivore-induced 2-464

phenylethylamine and 2-phenylethyl-β-D-glucopyranoside while the emission of 2-465

phenylethanol was not influenced (Figure 7) These results indicate I) that PtAADC1 466

produces 2-phenylethylamine upon herbivory in planta and II) that this compound is 467

further metabolized most likely via 2-phenylacetaldehyde and 2-phenylethanol into 2-468

phenylethyl-β-D-glucopyranoside The presence of separate pathways to herbivore-469

induced 2-phenylethanol and 2-phenylethyl-β-D-glucopyranoside (both constitutive 470

and induced) suggests that these two metabolites have different functions in the 471

plant even if the metabolic fate of 2-phenylethyl-β-D-glucopyranoside is eventually to 472

be hydrolyzed to 2-phenylethanol Since emission of the free alcohol was not 473

influenced by the AADC1 knockdown we propose that the AADC-dependent 2-474

phenylethanol pathway in contrast to the AAS-mediated route is tightly associated 475

with a glucosyltransferase as part of a protein complex The formation of such 476

complexes or metabolons which can prevent the release of unstable or toxic 477

pathway intermediates has been described for the cyanogenic glycoside pathway in 478

sorghum (Sorghum bicolor) where a glucosyltransferase is also included (Nielsen et 479

Constitutive 2-phenylethyl-β-D-glucopyranoside might be produced via PtAAS2 482

or the Ehrlich pathway 483

Undamaged leaves of P trichocarpa were found to accumulate significant amounts 484

of 2-phenylethyl-β-D-glucopyranoside (Figure 2) Since PtAAS2 was the only AAAD 485

gene with a considerable constitutive expression we hypothesize that PtAAS2 486

provides 2-phenylacetaldehyde as substrate for the formation of 2-phenylethyl-β-D-487

glucopyranoside in undamaged poplar leaves (Figure 2) However the accumulation 488

of phenylpyruvic acid in these leaves indicates that the Ehrlich pathway might also 489

contribute to the formation of 2-phenylethyl-β-D-glucopyranoside An aromatic amino 490

acid transaminase has recently been reported in melon Its characterization and gene 491

expression pattern suggest that the catabolism of aromatic amino acids into aroma 492

volatiles likely proceeds via an initial transamination rather than decarboxylation or 493

decarboxylationdeamination in this species (Gonda et al 2010) Moreover roses 494

were shown to possess both the AADC-dependent pathway and the phenylpyruvic 495

acid-dependent pathway for the formation of floral 2-phenylethanol While the former 496

pathway is active throughout the whole year the latter is induced exclusively during 497

hot seasons and has been discussed as an adaptation to summer environmental 498

conditions (Hirata et al 2016) The presence of multiple 2-phenylethanol pathways 499

in poplar may also enable the tree to react to different environmental stresses by 500

producing either 2-phenylethanol or 2-phenylethyl-β-D-glucopyranoside 501

independently 502

PtAAS1 and PtAADC1 have evolved from a common ancestor by gene 504

duplication and neofunctionalization 505

The emergence of large gene families such as terpene synthases cytochrome P450 506

monooxygenases UDP-glucosyltransferases methyltransferases and acyl 507

transferases that encode enzymes with diverse substrate specificity regiospecificity 508

or catalytic activity created the basis for the chemical diversity of specialized 509

metabolism in plants (Leong and Last 2017) These gene families evolved through 510

repeated duplications of single genes chromosomes or even whole genomes 511

followed by neofunctionalization of resulting gene copies (Moghe and Kruse 2018) It 512

has been shown that single amino acid changes can dramatically alter the specificity 513 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

or activity of enzymes involved in specialized metabolism (eg Koumlllner et al 2004 514

Irmisch et al 2015 Johnson et al 2001 Junker et al 2013 Fan et al 2017) 515

suggesting that novel enzyme functions may arise in relatively short time periods by 516

the accumulation of only a few nucleotide mutations PtAADC1 and PtAAS1 provide 517

an example of rapid evolutionary changes They form a gene cluster on chromosome 518

13 of the P trichocarpa genome (Tuskan et al 2006) and their high similarity to each 519

other indicates a recent tandem gene duplication event However PtAADC1 and 520

PtAAS1 have already come to encode different enzymatic activities catalysing the 521

decarboxylation and decarboxylationoxidation of phenylalanine respectively Recent 522

studies reported a tyrosinephenylalanine switch that determines the catalytic activity 523

of AADC and AAS enzymes respectively in different species (Bertoldi et al 2002 524

Torrens-Spence et al 2013) In vitro mutagenesis experiments performed in our 525

study confirmed the role of this residue as a function-dictating element in PtAADC1 526

and PtAAS1 (Figure 4) Interestingly a survey of putative AADCAAS genes in a set 527

of sequenced vascular plants (httpwwwphytozomenet) revealed similar 528

AADCAAS gene pairs that differed in the function-dictating residue in at least ten 529

species from monocotyledonous and dicotyledonous plants (Supplemental Figure 5) 530

Although none of these enzymes has been characterized so far their sequence 531

similarities with already published AADCAAS from other plants suggest similar 532

catalytic functions (Supplemental Figure 6) The presence of the putative AADCAAS 533

gene pairs indicates an independent occurrence of the tyrosinephenylalanine-534

mediated activity switch of AADCAAS enzymes in single genera or even single 535

species of the angiosperms The multiple evolution of this trait as well as its 536

conservation in numerous plant lineages suggest different but important functions of 537

AADCAAS enzyme products in plant metabolism 538

PtPAR1 and PtPAR2 may participate in the biosynthesis of 2-phenylethanol 540

The last reaction of the various 2-phenylethanol pathways is the reduction of 2-541

phenylacetaldehyde catalyzed by 2-phenylacetaldehyde reductase (Watanabe et al 542

2002 Hirata et al 2012 Figure 1) We identified a group of five genes in the 543

genome of P trichocarpa that showed significant sequence similarity to the recently 544

reported PAR genes from tomato and damask rose (Tieman et al 2007 Chen et al 545

2011) Heterologous expression and enzyme characterization revealed PAR activity 546 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

for two candidates PtPAR1 and PtPAR2 However both enzymes also accepted a 547

variety of other aromatic and aliphatic aldehydes as substrates and converted them 548

into the corresponding alcohols (Table 2 Supplemental Figure 12) Substrate 549

promiscuity has been described for tomato LePAR2 and rose PAR1 (Tieman et al 550

2007 Chen et al 2011) as well as for short-chain dehydrogenasesreductases 551

involved in other metabolic processes (Oberschall et al 2000 Yamaguchi et al 552

2011 Sengupta et al 2015 Jain et al 2016) Since PtPAR1 and PtPAR2 showed 553

high constitutive expression that remained constant upon herbivory (Supplemental 554

Figure 12) they might contribute to the constitutive as well as herbivore-induced 555

formation of 2-phenylethanol and its glucoside in planta However we cannot rule out 556

that other members of the large short-chain dehydrogenasereductase family of 557

poplar (Supplemental Figure 10) are also involved in the biosynthesis of these 558

compounds 559

Herbivory-induced emission of 2-phenylethanol and accumulation of 2-561

phenylethylamine might serve as defensive weapons or signals in P 562

trichocarpa 563

Herbivore-induced volatiles play important roles in plant defence They can 564

negatively impact the attacking herbivore directly by toxicity or deterrence Or they 565

can act as indirect defences by functioning as cues for natural enemies of the 566

herbivore 2-Phenylethanol has been shown to be involved in both processes 567

Florivorous ants for example that feed on flowers of the alpine skypilot (Polemonium 568

viscosum) are strongly repelled by higher concentrations of 2-phenylethanol (Galen 569

et al 2011) On the other hand the aphid-feeding lacewing Chrysoperla carnea is 570

attracted by 2-phenylethanol released from host plants of the aphid (Zhu et al 571

2005) suggesting a function of this compound in indirect defense We propose that 572

2-phenylethanol might also be involved in direct or indirect defense in poplar The 573

volatile blend emitted from L dispar-infested poplar leaves has been shown to attract 574

Glyptapanteles liparidis parasitoids that oviposit on L dispar caterpillars (McCormick 575

et al 2014) and 2-phenylethanol one of the prominent blend components could 576

influence the behavior of the parasitic wasp Additionally 2-phenylethanol has been 577

shown to act as antimicrobial agent (Zhu et al 2011 Liu et al 2014) and thus might 578

protect poplar plants against pathogens associated with herbivores (Chung et al 579 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

2013 Zhu et al 2014) Alternatively 2-phenylethanol could also act as an internal 580

plant signal to indicate the presence of herbivore damage to other organs Other 581

herbivore-induced natural products especially volatiles have been shown to play 582

roles as defence signals (Maag et al 2015) In woody plants such as poplar where 583

transport through the vascular system involves long distances between branches 584

volatile signals may be important in providing more rapid information about the 585

location of herbivory (Frost et al 2007) 586

Aromatic amines and related condensation products such as hydroxycinnamic acid 587

amides are known to be of central importance in defence responses upon wounding 588

and pathogen attack (Facchini et al 2002 Macoy et al 2015) They have been 589

shown to accumulate after tissue damage in a variety of plants including for example 590

Arabidopsis tomato pepper and potato (Muroi et al 2009 Campos et al 2014 591

Newman et al 2001 Schmidt et al 1998) In potato hydroxycinnamic acid amides 592

are induced by Phytophthora infestans infection and elicitor-treated potato cell 593

cultures also accumulate significant amounts of these compounds (Schmidt et al 594

1998 Schmidt et al 1999) Similar effects have been observed in chitosan-treated 595

potato suspension cultures suggesting that insect feeding might also trigger related 596

plant responses (Villegas and Brodelius 1990) Whether aromatic amines 597

biosynthesized by the herbivore-induced enzyme PtAADC1 might also act as 598

substrates for other defence compounds such as hydroxycinnamic acid amides in 599

poplar should be investigated in future research 600

MATERIALS AND METHODS 603

Plant treatment 604

Western balsam poplar (Populus trichocarpa) (genotype Muhle Larsen) trees were 605

grown from monoclonal stem cuttings in a greenhouse (24 degC 60 relative humidity 606

16 h8 h lightdark cycle) in a 11 mixture of sand and soil (Klasmann potting 607

substrate Klasmann-Deilmann Geeste Germany) until they reached 1 m in height 608

Wildtype and transgenic grey poplar (P times canescens clone INRA 717-1B4) plants 609

were amplified by micropropagation as described by Behnke et al (2007) Saplings 610

of around 10 cm high were repotted to soil (Klasmann potting substrate) and 611

propagated in a controlled environment chamber for one month (day 22 degC night 18 612 httpsplantphysiolorgDownloaded on March 22 2021 - Published by

degC 65 rel humidity 16 h8 h lightdark cycle) before they were transferred to the 613

green house 614

For herbivore treatment of poplar plants were enclosed in a PET bag 615

(ldquoBratschlauchrdquo Toppits Germany) with (herbivory) or without (control) 10 gypsy 616

moth (Lymantria dispar) caterpillars (larval stage L4) for 24 hours (start of the 617

treatment ~ 4 pm of day 1 end of the treatment ~ 4 pm of day 2) 618

Lymantria dispar egg batches were provided by Hannah Nadel United States 619

Department of Agriculture- Animal and Plant Health Inspection Service (Buzzardrsquos 620

Bay MA USA) After hatching the caterpillars were reared on an artificial diet (gypsy 621

moth diet MP Biomedicals Illkirch France) until L4 stage Caterpillars were starved 622

in single cups for 1 day prior to the experiment 623

RNA extraction and cDNA synthesis 625

Poplar leaf material was harvested immediately after the herbivore treatment flash-626

frozen in liquid nitrogen and stored at -80 degC until further processing After grinding 627

the frozen leaf material in liquid nitrogen to a fine powder with a Vibratory Micro Mill 628

(Pulverisette-0 Fritsch Germany) total RNA was isolated using an InviTrap Spin 629

Plant RNA kit (Stratec Berlin Germany) according to manufacturerrsquos instructions 630

RNA concentration was assessed using a spectrophotometer (NanoDrop 2000c 631

Thermo Scientific Wilmington DE USA) Prior to cDNA synthesis 1 microg of RNA was 632

DNase treated using 1 U of DNase (Thermo Fisher Scientific 633

httpswwwthermofishercom) Single-stranded cDNA was prepared using 634

SuperScriptTM III reverse transcriptase and oligo (dT20) primers (Thermo Fisher 635

Scientific) 636

Identification and cloning of putative AAAD genes 638

Putative AAAD genes were identified using a BLAST search against the P 639

trichocarpa genome (Tuskan et al 2006) version 30 640

(httpwwwphytozomenetpoplar) with rose RhPAAS (GenBank Accession 641

ABB04522) as template The complete ORF of the resulting candidates PtAAS1 642

PtAAS2 PtAADC1 PtAADC2 and PtAADC3 were amplified from cDNA made from 643

L dispar-damaged P trichocarpa leaves using a Phusionreg High Fidelity DNA 644

Polymerase (New England Biolabs) and inserted into the pCRtrade-Blunt II-TOPOreg 645 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

vector (Thermo Fisher Scientific) Primer sequence information is available in 646

Supplemental Table 3 647

Identification and cloning of putative PAR genes 649

For the identification of putative PAR and AAR genes a BLAST search was carried 650

out as described above with LePAR1 (Gene Bank Accession EF6134921) as query 651

The complete ORFs of the resulting candidates PtAAR1 PtAAR2 PtAAR3 PtPAR1 652

and PtPAR2 were amplified from cDNA made from L dispar-damaged P trichocarpa 653

leaves using a Phusionreg High Fidelity DNA Polymerase (New England Biolabs) and 654

inserted into the pET100D-TOPOreg vector (Thermo Fisher Scientific) Primer 655

sequence information is available in Supplemental Table 3 656

Phylogenetic analysis and amino acid alignment 658

Nucleotide sequence alignments were constructed with the Muscle (codon) algorithm 659

(gap open -29 gap extend 0 hydrophobicity multiplier 15 clustering method 660

upgmb) implemented in MEGA6 (Tamura et al 2013) Phylogenetic trees were 661

generated with MEGA6 using the Maximum Likelihood method (modelmethod 662

Kimura-2-parameter model substitutions type nucleotide rates among sites uniform 663

rates gapsmissing data treatment partial deletion site coverage cutoff 80) An 664

amino acid alignment of poplar AAAD enzymes characterized in this study together 665

with Arabidopsis AtAAS and AtTyrDC (Gutensohn et al 2011 Lehmann and 666

Pollmann 2009) was constructed and visualized with BioEdit 667

(httpwwwmbioncsuedubioeditbioedithtml) and the ClustalW algorithm 668

RNA-Sequencing and RT-qPCR analysis 670

Total RNA was extracted from leaf material as described above TruSeq RNA-671

compatible libraries were prepared and PolyA enrichment was performed before 672

sequencing the eight transcriptomes of P trichocarpa (four trees (biological 673

replicates) for each the control and herbivory treatment) on an IlluminaHiSeq 2500 674

sequencer (Max Planck Genome Centre Cologne Germany) with 18 Mio reads per 675

library 100 base pair single end Trimming of the obtained Illumina reads and 676

mapping to the poplar gene model version 30 677

(httpsphytozomejgidoegovpzportalhtml) were performed with the program CLC 678 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

Genomics Workbench (Qiagen Bioinformatics) (mapping parameter length fraction 679

07 similarity fraction 09 max number of hits 25) Empirical analysis of digital gene 680

expression (EDGE) implemented in the program CLC Genomics Workbench was 681

used for gene expression analysis 682

For validation of the RNA-sequencing results quantitative real-time PCR was carried 683

out with specific primers for each gene Therefore cDNA was prepared as described 684

above and diluted 110 with sterile water For the amplification of gene fragments 685

with a length of about 150-300 bp primer pairs were designed having a Tm ge 60 degC 686

a GC content between 40 - 55 and a primer length in the range of 20 - 25 nt (see 687

Supplemental Table 3 for primer information) Primer pair efficiency was tested by 688

measuring Ct values in stepwise diluted cDNA Amplicon identity was validated via 689

sequencing of cloned fragments Samples were run in triplicate using Brilliant III 690

SYBR Green qPCR Master Mix (Agilent) The following PCR conditions were applied 691

for all reactions initial incubation at 95 degC for 3 min followed by 40 cycles of 692

amplification (95 degC for 20 s and 60 degC for 20 s) SYBR Green fluorescence was 693

measured at the end of each amplification cycle Melting curves were recorded within 694

a range of 55 to 95 degC at the end of a total of 40 cycles All samples were run on a 695

Bio-Rad CFX Connecttrade Real-Time PCR Detection System (Bio-Rad Laboratory 696

Hercules CA USA) in 96-well Hard-Shellreg PCR plates with 6 biological replicates 697

The relative normalized expression compared to the housekeeping gene ubiquitin 698

(Irmisch et al 2013) was analyzed using the Bio-Rad CFX Manager 31 699

Biochemical characterization of poplar AAAD enzymes 701

For heterologous expression in Escherichia coli the complete ORFs of the candidate 702

AAAD genes were subcloned into the pET100D-TOPOreg vector and expressed in E 703

coli BL21(DE3) cells (Thermo Fisher Scientific) as N-terminal His6-Tag fusion 704

proteins Cells were grown in TB medium (12 wv tryptone 24 wv yeast 705

extract 05 (vv) glycerol adjusted to pH 72 via potassium buffer 50 microgmL 706

carbenicillin) at 32 degC and 220 rpm until an OD600 of 04 was reached Expression 707

was induced by adding 04 mM IPTG and the cultures were grown overnight at 18degC 708

under continuous shaking at 220 rpm Cells were harvested by centrifugation (5000 times 709

g 10 min 4 degC) the supernatant was decanted and the pellet was resuspended in 710

chilled lysis buffer (20 mM imidazole pH 72 100 mM NaCl 50 mM KH2PO4 2 711 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

(vv) glycerol 10 mM MgCl2 03 mgmL lysozyme 100 uL Benzonase (Thermo 712

Fisher Scientific) 1 gL proteinase inhibitor HP (Serva wwwservade)) The cells 713

were frozen in liquid nitrogen and thawed in a water bath at 25 degC a total of 5 times 714

and additionally disrupted by a 3 times 30 s treatment with a sonicator (Bandelin 715

UW2070 Berlin Germany 70 intensity) The resulting slurry was pelleted via 716

centrifugation (12000 times g 20 min 4 degC) and the supernatant was transferred to a 717

new reaction tube Soluble protein was purified from the supernatant using a Poly-718

Prepreg Chromatography gravity flow column (Bio-Rad www-bio-radcom) with 500 microL 719

HisPurtrade Cobalt Resin (Thermo Fischer Scientific) according to the manufacturerrsquos 720

instructions (equilibration buffer 100 mM NaCl 50 mM Tris-HCl 2 vv glycerol 20 721

mM imidazole pH 72 washing buffer 100 mM NaCl 50 mM Tris-HCl 2 vv 722

glycerol 40 mM imidazole pH 72 elution buffer 100 mM NaCl 50 mM Tris-HCl 2 723

vv glycerol 150 mM imidazole pH 72) The enzyme-containing eluate was desalted 724

via an Illustra NAP-10 gravity flow column (GE Healthcare) into a storage buffer (50 725

mM Tris-HCl 01 mM EDTA pH 80) and immediately mixed 11 with a PLP-726

containing buffer (50 mM Tris-HCl 02 mM PLP 01 mM EDTA pH 80) Protein 727

concentration was determined via a PCA-enhanced Biuret assay as stated by the 728

manufacturer (Rotireg-Quant universal Karl Roth GmbH Germany) Protein purity and 729

identity was confirmed via SDS-PAGE and colloidal Coomassie staining (Rotireg-Blue 730

Karl Roth GmbH Germany) The relative abundance of the enzymes defined by 731

densitometry (GS-900 Calibrated Densitometer Bio-Rad Germany) was calculated 732

for the determination of kcat values 733

Enzyme assays were performed in closed glass tubes containing approximately 1 microg 734

of purified enzyme and 1 mM of substrate in a total volume of 200 microL of PLP buffer 735

The reaction was carried out for 30 minutes under shaking at 300 rpm and 32 degC and 736

was stopped by adding 800 microl of 100 methanol After placing on ice for 30 minutes 737

the denatured enzymes were removed by centrifugation (4000 times g 4 degC 10 min) 738

and the supernatant was transferred into a glass vial or a 96 Deep-Well Agilent 41 739

mm plate for LC-MSMS analysis 740

For the determination of the kinetic parameters assays were carried out in triplicate 741

and with varying substrate concentrations The enzyme concentration and the 742

incubation times were chosen so that the reaction velocity was linear during the 743

Biochemical characterization of poplar PAR enzymes 746

The complete ORFs of the candidate AAR and PAR genes were cloned into the 747

pET100D-TOPOreg vector and expressed in E coli BL21(DE3) cells (Thermo Fisher 748

Scientific) as N-terminal His6-Tag fusion proteins as described above for AADC and 749

AAS enzymes Soluble protein was purified from the supernatant as described by 750

Irmisch et al (2015) Protein purity and identity was confirmed via SDS-PAGE and 751

colloidal Coomassie staining (Rotireg-Blue Karl Roth GmbH Germany) Enzyme 752

assays were performed in closed glass tubes containing approximately 1 microg of 753

purified enzyme 1 mM of substrate and 1 mM of NADPH in a total volume of 100 microL 754

of reaction buffer (10 glycerol (vv) 10 mM TrisHCl pH = 75 1 mM dithiothreitol) 755

and were carefully overlaid with 150 microL hexane and incubated for 15 hours After 756

incubation enzyme assays were thoroughly mixed the hexane phase was 757

transferred to a new glass vessel and 1 microL was analyzed as described in Irmisch et 758

al (2015) 759

Site-directed mutagenesis of PtAAS1 and PtAADC1 761

For site-directed mutagenesis of PtAAS1 and PtAADC1 specific primers possessing 762

the desired mutated codon were designed after the method described by Ho et al 763

1989 (Supplemental Table 3) The vectors pET100D-TOPOregPtAADC1 and 764

pET100D-TOPOregPtAAS1 were used as templates for a Phusionreg High Fidelity DNA 765

PCR The elongation time was adapted to the total length of the template vector 766

according to the instructions of the supplier The double-nicked vector was directly 767

transformed into chemically competent E coli cells (TOP10 One Shot Thermo Fisher 768

Scientific) and screened for successful mutagenesis by sequencing of plasmids 769

purified from single colonies Plasmids containing the desired mutations were 770

transformed into E coli BL21(DE3) cells (Thermo Fisher Scientific) and heterologous 771

expression protein purification and activity tests were performed as described 772

above 773

Transient Expression of PtAADC1 and PtAAS1 in Nicotiana benthamiana 775

For expression in N benthamiana the coding regions of PtAADC1 and PtAAS1 were 776

amplified from the respective pET100D-TOPOreg vectors and inserted into the 777 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

pCAMBiA2300U vector as described in Irmisch et al (2013) The pCAMBiA 2300U 778

vector and vectors carrying eGFP and p19 were kindly provided by the group of D 779

Werck-Reichhart Strasbourg France After verification of the sequence integrity 780

pCAMBiA vectors were separately transferred into Agrobacterium tumefaciens strain 781

C58p90 Protein expression was confirmed by fluorescence analysis (microscope) of 782

N benthamiana plants transformed with eGFP Five milliliters of an overnight culture 783

(220 rpm 28 degC) were used to inoculate 200 mL of Luria-Bertani medium (50 microgmL 784

kanamycin 25 microgmL rifampicin 25 microgmL gentamicin) for overnight growth The 785

following day the cultures were centrifuged (4000 times g 5 min) and the cells were 786

resuspended in infiltration buffer (10 mM MES 10 mM MgCl2 and 100 microM 787

acetosyringone pH 56) to reach a final OD600 of 05 After shaking for at least 2 h at 788

room temperature the cultures carrying PtAADC1 PtAAS1 or eGFP were mixed 789

with an equal volume of cultures carrying pBINp19 For transformation 3- to 4-week-790

old N benthamiana plants were dipped upside down in an Agrobacterium solution 791

and vacuum was applied to infiltrate the leaves Infiltrated plants were placed in a 792

room without direct sunlight Three days after transformation plants were placed 793

under low direct light (LED 40) for three more days Volatiles were measured on 794

the sixth day after transformation For volatile collection plants were separately 795

placed in gas-tight 3-liter glass desiccators Charcoal-filtered air was pumped into the 796

desiccators at a flow rate of 1 Lmin and the air left the desiccators through a filter 797

packed with 30 mg of PorapaqQ (ARS Gainesville FL USA) Volatiles were 798

collected for 6 h (10 AM to 4 PM) The volatile compounds were desorbed from the 799

filters and analyzed by gas chromatography-mass spectrometry (GC-MS) and gas 800

chromatographyndashflame ionization detection (GC-FID) as described below Plants 801

were harvested after the volatile collection ground in liquid nitrogen and stored at -802

80 degC until further analysis 803

Vector construction and transformation of poplar 805

The construction of the binary vector was described by Leveacutee et al (2009) The 806

transformation of the P times canescens clone INRA 7171-B4 followed a protocol 807

published by Meilan and Ma (2007) To target only AADC1AAS1 mRNA a fragment 808

between position 455 and 765 of the coding sequence of PtAADC1 was selected 809

Transgenic RNAi poplar plants were amplified by micropropagation as described by 810 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

Behnke et al (2007) To test the level of transgenicity RT-qPCR analysis was done 811

on wild type vector control (pCAMBIA) and RNAi plants 812

Chemical conversion of PtAAS enzyme products into aldoximes 814

For the identification of reactive aromatic aldehydes PtAAS enzymes were incubated 815

with single amino acids as substrates overlaid with 400 microl of pure toluol and 816

incubated for 1 hour at 32 degC The aromatic aldehydes produced by the enzymes 817

were extracted into the toluol phase and transferred to a new glass vial The toluol 818

phase was incubated with an aqueous solution of 100 mM hydroxylamine (pH = 70) 819

at 25 degC for one hour as described earlier (Irmisch et al 2014 Hofmann et al 2018) 820

to form the corresponding aldoxime The toluol phase was then transferred to a fresh 821

glass vial and dried down under a continuous flow of nitrogen The pellet was 822

dissolved in 200 microl methanol and analyzed via LC-MSMS as described in Irmisch et 823

al (2013) 824

LC-MSMS analysis of plant methanol extracts 826

Metabolites were extracted from ground plant material (poplar or N benthamiana) 827

with methanol (101 vw) Analytes were separated using an Agilent 1200 HPLC 828

system on a Zorbax Eclipse XDB-C18 column (50 times 46 mm 18 microm (Agilent 829

Technologies)) Mobile phase consisted of formic acid (005 vv) in water (A) and 830

acetonitrile (B) The column temperature was maintained at 25 degC HPLC parameters 831

are given in Supplemental Table 4 832

The liquid chromatography was coupled to an API-5000 tandem mass spectrometer 833

(Applied Biosystems Foster City CA USA) equipped with a Turbospray ion source 834

(ion spray voltage 5500 eV turbo gas temperature 700 degC nebulizing gas 70 psi 835

curtain gas 35 psi heating gas 70 psi collision gas 2 psi) Multiple reaction 836

monitoring (MRM) was used to monitor a parent ion rarr product ion reaction for each 837

analyte Detailed parameters are given in Supplemental Table 5 Identification and 838

quantification of compounds were performed using standard curves made from 839

authentic and commercially available standards of 2-phenylethylamine tyramine 840

tryptamine 2-phenylethyl-β-D-glucopyranoside phenylpyruvic acid and 841

phenylalanine (Supplemental Table 6) Relative quantification of ethanolamine 842

(Supplemental Figure 4) was carried out under the conditions mentioned in 843 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

Supplemental Tables 4 and 5 Ethanolamine was identified via an authentic standard 844

(Supplemental Table 6) 845

GC-MSFID analysis of plant volatiles PAR and AAR products 847

A Hewlett-Packard model 6890 gas chromatograph (GC) was employed with He 848

(MS) or H2 (FID) as carrier gas at 2 mLmin splitless injection (injector temperature 849

230 degC injection volume 1 microL) and a DB-5MS column (Agilent Santa Clara USA 30 850

m times 025 mm times 025 microm) The GC oven temperature was held for 2 min and then 851

increased to 225 degC with a gradient of 5 degCmin held for another 2 min and then 852

further increased to 250 degC with 100 degCmin and a hold for 1 min The coupled mass 853

spectrometer (MS) was a Hewlett-Packard model 5973 with a quadrupole mass 854

selective detector (transfer line temperature 230 degC source temperature 230 degC 855

quadrupole temperature 150 degC ionization potential 70 eV scan range of 40-400 856

atomic mass units) Quantification was performed with the trace of a flame ionization 857

detector (FID) operated at 300 degC For analysis of PAR and AAR reaction products 1 858

microl of the hexane phase covering the enzyme assays was injected into GC-MS and 859

analyzed as described above 2-Phenylethanol and 2-phenylacetaldehyde were 860

identified via authentic standards (Supplemental Table 6) 861

TDU-GC-MS analysis of PtAAS enzyme products 863

PtAAS enzyme products were collected from the headspace of the assays with 864

PDMS silicone tubes as described by Kallenbach et al (2014) For the determination 865

of Km values enzyme assays as described above were incubated with 866

preconditioned PDMS silicone tubes in an air-tight vial for 30 minutes After the 867

incubation PDMS tubes were immediately analyzed using thermal desorption GC-868

MS For preparing a standard curve 2-phenylacetaldehyde was dissolved in 100 869

ethanol and added in specific amounts to the buffer system used for the enzyme 870

assays As an enzyme control 1 microg of BSA was added per assay and total volume of 871

the assay was set to 200 microl 872

A Shimadzu thermal desorption unit TD-20 combined with a GC-MS-QP2010 Ultra 873

gas chromatograph was employed with He as carrier gas Thermal desorption of 874

PDMS tubes was carried out with a sample flow of 60 mLmin a desorption 875

temperature of 200 degC a valve temperature of 250 degC and a transfer line 876 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

temperature of 230 degC The trap tube was cooled (-20 degC) and upon completed 877

desorption heated to 230 degC to release the sample onto the GC column The gas 878

chromatograph was operated at a column flow rate of 15 mLmin (helium) split 879

injection (split ratio 20) and a ZB-5MS column (Zebron Phenomenex California 880

USA 30 m times 025 mm times 025 microm) The GC oven temperature was set to 45 degC held 881

for 3 min then increased to 180 degC with a gradient of 6 degCmin finally increased to 882

300 degC with a 100 degCmin increment and held for another 23 min The coupled mass 883

spectrometer with a quadrupole mass selective detector was operated in Scan mode 884

selective for 33 ndash 350 atomic mass units (interface temperature 250 degC source 885

temperature 230 degC ionization potential 70 eV) 886

Statistical analysis 888

Statistical analysis was carried out as described in the figure legends for the 889

respective experiments Studentacutes t-tests Mann-Whitney Rank Sum tests Kruskal-890

Wallis One Way ANOVA and Tukey tests were performed with SigmaPlot 120 891

EDGE tests were performed with CLC Genomics Workbench Data visualization was 892

done with SigmaPlot 120 and R Studio Version 099903 (R Development Core 893

Team httpwwwR-projectorg) 894

ACCESSION NUMBERS 897

Sequence data for PtAAS1 (MK440562) PtAAS2 (MK440561) PtAADC1 898

(MK440568) PtAADC2 (MK440567) PtAADC3 (MK440566) PtSDC1 (MK440558) 899

PtSDC2 (MK440569) PtPAR1 (MK440560) PtPAR2 (MK440559) PtAAR1 900

(MK440565) PtAAR2 (MK440564) and PtAAR3 (MK440563) can be found in the 901

GenBank under the corresponding identifiers Raw reads of the RNAseq experiment 902

were deposited in the NCBI Sequence Read Archive (SRA) under the accession 903

PRJNA516861 904

Supplemental Data 906

Supplemental Figure S1 Amino acid alignment of PtAAS and PtAADC enzymes 907

from Populus trichocarpa and characterized AtAAS and AtTyrDC from Arabidopsis 908

Supplemental Figure S2 PtAAS1 and PtAAS2 do not release 2-phenylethylamine 910

Supplemental Figure S3 Detection of poplar AAS products via the conversion to 911

corresponding aldoximes 912

Supplemental Figure S4 PtSDC1 and PtSDC2 convert L-serine to ethanolamine 913

Supplemental Figure S5 Phylogeny and comparison of plant AAAD gene clusters 914

Supplemental Figure S6 Dendrogram analysis of putative AADCAAS gene pairs 915

and characterized AADC AAS and GluSerHis-decarboxylase genes from other 916

plants 917

Supplemental Figure S7 Transcript accumulation of PtAAS1 PtAADC1 PtAADC2 918

PtAAS2 and PtAADC3 in caterpillar-damaged (herb) and undamaged (ctr) leaves of 919

Populus trichocarpa 920

Supplemental Figure S8 Dendrogram analysis of AAAD genes from Populus 921

trichocapra P times canescens P tremula and P alba 922

Supplemental Figure S9 Transcript accumulation of AAS1 AADC1 and AADC2 in 923

gypsy moth (Lymantria dispar) caterpillar-damaged Populus times canescens leaves 924

Supplemental Figure S10 Transcript accumulation of PcanAADC1 (A) and 925

PcanAAS2 (B) in caterpillar-damaged leaves of control and AADC1AADC2AAS1 926

RNAi lines of Populus times canescens 927

Supplemental Figure S11 The RNAi-mediated knockdown of PcanAADC1 in 928

Populus times canescens does not influence the herbivore-induced emission of benzyl 929

cyanide 930

Supplemental Figure S12 Dendrogram analysis of putative reductases from poplar 931

and characterized reductases from other plants 932

Supplemental Figure S13 PtPAR1 and PtPAR2 convert 2-phenylacetaldehyde into 933

2-phenylethanol in vitro 934

Supplemental Figure S14 Transcript accumulation of putative aldehyde reductase 935

genes in Lymantria dispar-damaged and undamaged Populus trichocarpa leaves 936

Supplemental Table S1 Accession numbers of genes used for phylogenetic 937

analysis 938

Supplemental Table S2 Expression of PtAAS1 PtAADC1 and PtAADC2 in 939

undamaged and herbivore-damaged Populus trichocarpa leaves 940

Supplemental Table S3 Oligonucleotides used for isolation and RT-qPCR analysis 941

Supplemental Table S4 HPLC gradients used for separation and analysis of 943

metabolites 944

Supplemental Table S5 MSMS parameters used for LC-MSMS analysis 945

Supplemental Table S6 Compounds used as substrates for enzyme assays and as 946

standards for LC-MSMS and GC-MS quantification 947

ACKNOWLEDGEMENTS 950

We thank Tamara Kruumlgel Danny Kessler and all the MPI-CE gardeners for their help 951

with rearing the poplar and Nicotiana benthamiana plants D Werck-Reichhart 952

Strasbourg France is thanked for kindly providing the pCAMBIA vectors and for the 953

nice introduction to the USER cloning system We thank Tabea Kroumlber Daniel Veit 954

Natascha Rauch and Marion Staumlger for excellent technical assistance Additionally 955

we thank Sybille B Unsicker (MPI-CE) and her group members for rearing L dispar 956

caterpillars Alexander Haverkamp is thanked for help with the data visualization 957

This research was funded by the Max Planck Society 958

TABLES 961

Table 1 Kinetic parameters of Populus trichocarpa PtAAS1 PtAAS2 PtAADC1 962

PtAADC2 and PtAADC3 Enzymes were heterologously expressed in Escherichia 963

coli purified and incubated with phenylalanine tyrosine or tryptophan Kinetic 964

parameters are shown as means plusmn SE (n = 3) Volatile phenylacetaldehyde was 965

analyzed via TDU-GC-MS while all other compounds were measured using LC-966

MSMS Compounds marked with asterisks were chemically converted into the 967

corresponding aldoximes before LC-MSMS analysis (see method section) 968

Enzyme Substrate Product Km

(mM) V

(nmolmicrog-1

PtAAS1

Phe PHA 046 plusmn 002 - - - Phe PEA - - - - Tyr 4-OH-PHA - - - - Trp IAAld - - - -

PtAAS2 Phe PHA 069 plusmn 039 - - - Phe PEA - - - -

Tyr 4-OH-PHA detected - - - Trp IAAld detected - - -

PtAADC1

Phe PHA - - - - Phe PEA 017 plusmn 003 430 plusmn 12 039 23 Tyr TyrA 20 plusmn 02 854 plusmn 39 008 004 Trp TrpA 30 plusmn 04 77 plusmn 05 0007 0002

PtAADC2

Phe PHA - - - - Phe PEA 019 plusmn 001 089 plusmn 001 081 42 Tyr TyrA 12 plusmn 02 148 plusmn 7 014 011 Trp TrpA - - - -

PtAADC3

Phe PHA - - - - Phe PEA 23 plusmn 02 67 plusmn 02 014 0003

Tyr TyrA 0043 plusmn 0004 361 plusmn 006 0003 008

Trp TrpA - - - - 969

Table 2 Enzymatic activity of poplar aldehyde reductases Enzymes were 971

heterologously expressed in Escherichia coli purified and tested with different 972

aldehydes as potential substrates in the presence of NADPH Enzyme products were 973

extracted with hexane and analyzed using GC-MS - no activity detected + activity 974

detected 975

Substrate Product PtPAR1 PtPAR2 PtAAR1 PtAAR2 PtAAR3 citronellal citronellol + + + + +

geranial geraniol + + - - -

nonanal nonanol + + + - -

decanal decanol + + + - +

hexanal hexanol + + - - -

(E)-2-hexenal (E)-2-hexenol + + - - -

benzaldehyde benzyl alcohol + + - - -

2-phenylacetaldehyde 2-phenylethanol + + - - - 976

FIGURE LEGENDS 978

PHA phenylacetaldehyde PEA 2-phenylethylamine 4-OH-PHA 4-hydroxy phenylacetaldehyde IAAld indole-3-acetaldehyde TyrA tyramine TrpA tryptamine

Figure 1 Proposed pathways for the biosynthesis of 2-phenylethanol in poplar 979

AAAT aromatic amino acid transaminase AADC aromatic amino acid 980

decarboxylase CYP79 cytochrome P450 family 79 enzyme AAS aromatic 981

aldehyde synthase PPDC phenylpyruvic acid decarboxylase MAO monoamine 982

oxidase TOX transoximase PAR 2-phenylacetaldehyde reductase UGT UDP-983

glucuronosyltransferase β-Glu β-glucosidase Solid lines indicate well-established 984

reactionsenzymes while dashed lines indicate reactions hypothesized in the 985

literature 986

Figure 2 Herbivore-damaged leaves of Populus trichocarpa accumulate and 988

release phenylalanine-derived metabolites The accumulation of L-phenylalanine 989

(A) phenylpyruvic acid (B) 2-phenylethylamine (C) and 2-phenylethyl-β-D-990

glucopyranoside (D) and the emission of 2-phenylethanol (E) were analyzed in 991

Lymantria dispar damaged (herb) and undamaged control (ctr) leaves Plant material 992

was extracted with methanol and analyzed via LC-MSMS (A - D) Volatiles were 993

collected for 8 hours and analyzed via GC-FID (E) Means plusmn SE are shown (A - D n 994

= 10 E n = 9) Asterisks indicate statistical significance as assessed by Studentacutes t-995

test or Mann-Whitney Rank Sum Tests L-phenylalanine (P = 0530 t = -064) 996

phenylpyruvic acid (P = 0772 t = -0294) 2-phenylethylamine (P lt 0001 T = 55) 2-997

phenylethyl-β-D-glucopyranoside (P = 0011 T = 71) 2-phenylethanol (P = 0005 T = 998

585) DW dry weight ns not significant nd not detected 999

Figure 3 Dendrogram analysis of aromatic amino acid decarboxylase family 1001

genes from Populus trichocarpa and characterized AADC AAS and 1002

GluSerHis-decarboxylase genes from other plants Substrates of each clade are 1003

indicated in parentheses The tree was inferred by using the maximum likelihood 1004

method and n = 1000 replicates for bootstrapping Bootstrap values are shown next 1005

to each node The tree is drawn to scale with branch lengths measured in the 1006

number of substitutions per site Genes described in this study are shown in red and 1007

bold with corresponding gene identifiers (italics) Accession numbers are given in 1008

Figure 4 PtAAS1 and PtAADC1 are interconvertible by the mutation of a single 1011

amino acid and are likely derived by tandem gene duplication (A)The 1012

recombinant proteins PtAAS1 PtAADC1 and the corresponding mutant enzymes 1013

PtAAS1 (F338Y) and PtAADC1 (Y338F) were incubated with L-phenylalanine in the 1014

presence of PLP Enzyme products were analyzed using GC-MS (2-1015

phenylacetaldehyde) and LC-MSMS (2-phenylethylamine) (B) PtAADC1 and 1016

PtAAS1 form a gene cluster on chromosome 13 Both genes are oriented in the 1017

same direction and there are no coding regions between them Sequence data were 1018

retrieved from the P trichocarpa genome assembly version 30 (wwwphytozomenet 1019

Tuskan et al 2006) 1020

Figure 5 Overexpression of PtAAS1 PtAADC1 and PtAADC3 in Nicotiana 1022

benthamiana alters the emission of 2-phenylethanol (A) and the accumulation 1023

of 2-phenylethylamine (B) tyramine (C) tryptamine (D) and 2-phenylethanol-β-1024

D- glucopyranoside (E) N benthamiana plants were infiltrated with Agrobacterium 1025

tumefaciens carrying either PtAAS1 or PtAADC1 Five days after infiltration the 1026

headspaces of plants were collected and analyzed via GC-MS and GC-FID 1027

Additionally plants were harvested extracted with methanol and metabolite 1028

accumulation was analyzed via LC-MSMS Different letters above each bar indicate 1029

statistically significant differences as assessed by Kruskal-Wallis One Way ANOVA 1030

and Tukey tests 2-phenylethanol (H = 19867 P le 0001) 2-phenylethylamine (H = 1031

1694 P le 0001) tyramine (H = 18267 P le 0001) tryptamine (H = 18645 P le 1032

0001) 2-phenylethyl-β-D-glucopyranoside (H = 216 P le 0001) Means plusmn SE are 1033

shown (n = 6) FW fresh weight 1034

Figure 6 Transcript accumulation of AAAD and SDC genes in Lymantria 1036

dispar-damaged and undamaged Populus trichocarpa leaves Gene expression 1037

in herbivore-damaged (herb) and undamaged (ctr) leaves was analyzed by Illumina 1038

sequencing and mapping the reads to the transcripts of the P trichocarpa genome 1039

version v30 Expression was normalized to RPKM Significant differences in EDGE 1040

tests are visualized by asterisks Means plusmn SE are shown (n = 4) PtAAS1 (P = 1041

823213E-12 weighted difference (WD) = 486618E-05) PtAAS2 (P = 0039452261 1042

WD = -111092E-05) PtAADC1 (P = 899025E-09 WD = 156932E-05) PtAADC2 (P 1043 httpsplantphysiolorgDownloaded on March 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

PtSDC1 (P = 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012 WD = 1045

108813E-05) 1046

Figure 7 RNAi-mediated knockdown of AADC1 in Populus times canescens Sterile 1048

Populus times canescens calli were transformed via Agrobacterium tumefaciens 1049

Nontransgenic trees (WT) empty vector lines (EV-2-4) and RNAi lines (RNAi-1-4) 1050

were subjected to Lymantria dispar feeding 2-Phenylethanol was collected from the 1051

headspace and analyzed using GC-MSFID 2-Phenylethylamine and 2-phenylethyl-1052

β-D-glucopyranoside were extracted with methanol from ground plant material and 1053

analyzed via LC-MSMS Biological replicates (nb) and technical replicates (nt) of EV 1054

lines and RNAi lines were used to test for statistical differences WT nb = 4 EV nb = 1055

3 nt = 5 RNAi nb = 4 nt = 5 Asterisks indicate statistical significance as assessed 1056

by Studentacutes t-test or Mann-Whitney Rank Sum Tests 2-Phenylethylamine (P lt 1057

0001 t = 7940) 2-phenylethyl-β-D-glucopyranoside (P = 0011 T = 547) 2-1058

phenylethanol (P = 0509 T = 404) Medians plusmn quartiles and outliers are shown 1059

Each data point is represented by a circle FW fresh weight ns ndash not significant 1060

COMPETING INTERESTS 1064

The authors declare that they have no competing interests 1065

LITERATURE CITED 1067

Arimura GI Huber DPW and Bohlmann J (2004) Forest tent caterpillars 1068 (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles 1069 in hybrid poplar (Populus trichocarpa x deltoides) cDNA cloning functional 1070 characterization and patterns of gene expression of (-)-germacr Plant J 37 603ndash616 1071

Behnke K Ehlting B Teuber M Bauerfeind M Louis S Haumlnsch R Polle A 1072 Bohlmann J and Schnitzler JP (2007) Transgenic non-isoprene emitting poplars 1073 donrsquot like it hot Plant J 51 485ndash499 1074

Bertoldi M Gonsalvi M Contestabile R and Voltattorni CB (2002) Mutation of 1075 tyrosine 332 to phenylalanine converts dopa decarboxylase into a decarboxylation-1076 dependent oxidative deaminase J Biol Chem 277 36357ndash36362 1077

Bieri S Brachet A Veuthey JL and Christen P (2006) Cocaine distribution in wild 1078 Erythroxylum species J Ethnopharmacol 103 439ndash447 1079

Campos L Lisoacuten P Loacutepez-Gresa MP Rodrigo I Zacareacutes L Conejero V and 1080 Belleacutes JM (2014) Transgenic Tomato Plants Overexpressing Tyramine N -1081 Hydroxycinnamoyltransferase Exhibit Elevated Hydroxycinnamic Acid Amide Levels and 1082

Enhanced Resistance to Pseudomonas syringae Mol Plant-Microbe Interact 27 1083 1159ndash1169 1084

Chen XM Kobayashi H Sakai M Hirata H Asai T Ohnishi T Baldermann S 1085 and Watanabe N (2011) Functional characterization of rose phenylacetaldehyde 1086 reductase (PAR) an enzyme involved in the biosynthesis of the scent compound 1087 2-phenylethanol J Plant Physiol 168 88ndash95 1088

Chung SH Rosa C Scully ED Peiffer M Tooker JF Hoover K Luthe DS 1089 and Felton GW (2013) Herbivore exploits orally secreted bacteria to suppress plant 1090 defenses Proc Natl Acad Sci 110 15728ndash15733 1091

Costa MA Marques J V Dalisay DS Herman B Bedgar DL Davin LB and 1092 Lewis NG (2013) Transgenic hybrid poplar for sustainable and scalable production of 1093 the commodityspecialty chemical 2-phenylethanol PLoS One 8 1094

Danner H Boeckler GA Irmisch S Yuan JS Chen F Gershenzon J Unsicker 1095 SB and Koumlllner TG (2011) Four terpene synthases produce major compounds of 1096 the gypsy moth feeding-induced volatile blend of Populus trichocarpa Phytochemistry 1097 72 897ndash908 1098

Facchini PJ Hagel J and Zulak KG (2002) Hydroxycinnamic acid amide metabolism 1099 physiology and biochemistry Can J Bot 80 577ndash589 1100

Facchini PJ Huber-Allanach KL and Tari LW (2000) Plant aromatic L-amino acid 1101 decarboxylases Evolution biochemistry regulation and metabolic engineering 1102 applications Phytochemistry 54 121ndash138 1103

Fahey JW Zalcmann a T and Talalay P (2001) The chemical diversity and distribution 1104 of glucosinolates and isothiocyanates amoung plants Phytochemistry 56 5ndash51 1105

Fan P Miller AM Liu X Jones AD and Last RL (2017) Evolution of a flipped 1106 pathway creates metabolic innovation in tomato trichomes through BAHD enzyme 1107 promiscuity Nat Commun 8 1ndash13 1108

Farhi M Lavie O Masci T Hendel-Rahmanim K Weiss D Abeliovich H and 1109 Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional 1110 complementation in yeast Plant Mol Biol 72 235ndash245 1111

Ferreira-Silva B Lavandera I Kern A Faber K and Kroutil W (2010) Chemo-1112 promiscuity of alcohol dehydrogenases reduction of phenylacetaldoxime to the alcohol 1113 Tetrahedron 66 3410ndash3414 1114

Frost CJ Appel M Carlson JE De Moraes CM Mescher MC and Schultz JC 1115 (2007) Within-plant signalling via volatiles overcomes vascular constraints on systemic 1116 signalling and primes responses against herbivores Ecol Lett 10 490ndash8 1117

Galen C Kaczorowski R Todd SL Geib J and Raguso RA (2011) Dosage-1118 Dependent Impacts of a Floral Volatile Compound on Pollinators Larcenists and the 1119 Potential for Floral Evolution in the Alpine Skypilot Polemonium viscosum Am Nat 177 1120 258ndash272 1121

Gonda I Bar E Portnoy V Lev S Burger J Schaffer AA Tadmor Y Gepstein 1122 S Giovannoni JJ Katzir N and Lewinsohn E (2010) Branched-chain and 1123 aromatic amino acid catabolism into aroma volatiles in Cucumis melo L fruit 61 1111ndash1124 1123 1125

Hayashi S Yagi K Ishikawa T Kawasaki M Asai T Picone J Turnbull C 1126 Hiratake J Sakata K Takada M Ogawa K and Watanabe N (2004) Emission 1127 of 2-phenylethanol from its β-D-glucopyranoside and the biogenesis of these 1128 compounds from [2H8] L-phenylalanine in rose flowers Tetrahedron 60 7005ndash7013 1129

Hazelwood LA Daran JM Van Maris AJA Pronk JT and Dickinson JR (2008) 1130 The Ehrlich pathway for fusel alcohol production A century of research on 1131 Saccharomyces cerevisiae metabolism (Applied and Environmental Microbiology (2008) 1132 74 8 (2259-2266)) Appl Environ Microbiol 74 3920 1133

Hirata H Ohnishi T Ishida H Tomida K Sakai M Hara M and Watanabe N 1134 (2012) Functional characterization of aromatic amino acid aminotransferase involved in 1135 2-phenylethanol biosynthesis in isolated rose petal protoplasts J Plant Physiol 169 1136

444ndash451 1137 Hirata H Ohnishi T Tomida K Ishida H Kanda M Sakai M Yoshimura J 1138

Suzuki H Ishikawa T Dohra H and Watanabe N (2016) Seasonal induction of 1139 alternative principal pathway for rose flower scent Sci Rep 6 1140

Ho SN Hunt HD Horton RM Pullen JK and Pease LR (1989) Site-directed 1141 mutagenesis by overlap extension using the polymerase chain reaction Gene 77 51ndash1142 59 1143

Hofmann F Rausch T and Hilgenberg W (1981) Preparation of radioactively labelled 1144 indole-3-acetic acid precursors J Label Compd Radiopharm 18 1491ndash1495 1145

Honda K Ocircmura H and Hayashi N (1998) Identification of Floral Volatiles From 1146 Ligustrum japonicum that Stimulate Flower-Visiting by Cabbage Butterfly Pieris rapae 1147 J Chem Ecol 24 2167ndash2180 1148

Imai T Maekawa M Tsuchiya S and Fujimori T (1998) Field attraction of Hoplia 1149 communis to 2-phenylethanol a major volatile component from host flowers Rosa spp 1150 J Chem Ecol 24 1491ndash1497 1151

Irmisch S Clavijo McCormick A Boeckler GA Schmidt A Reichelt M 1152 Schneider B Block K Schnitzler J-P Gershenzon J Unsicker SB and 1153 Kollner TG (2013) Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 1154 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar 1155 Defense Plant Cell 25 4737ndash4754 1156

Irmisch S Clavijo McCormick A Guumlnther J Schmidt A Boeckler GA 1157 Gershenzon J Unsicker SB and Koumlllner TG (2014a) Herbivore-induced poplar 1158 cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which 1159 repel a generalist caterpillar Plant J 80 1095ndash1107 1160

Irmisch S Jiang Y Chen F Gershenzon J and Koumlllner TG (2014b) Terpene 1161 synthases and their contribution to herbivore-induced volatile emission in western 1162 balsam poplar (Populus trichocarpa) BMC Plant Biol 14 1ndash16 1163

Irmisch S Muumlller AT Schmidt L Guumlnther J Gershenzon J and Koumlllner TG 1164 (2015) One amino acid makes the difference The formation of ent-kaurene and 16α-1165 hydroxy-ent-kaurane by diterpene synthases in poplar BMC Plant Biol 15 262 1166

Jain D Khandal H Paul J and Debasis K (2016) A pathogenesis related-10 protein 1167 CaARP functions as aldo keto reductase to scavenge cytotoxic aldehydes Plant Mol 1168 Biol 90 171ndash187 1169

Johnson ET Ryu S Yi H Shin B Cheong H and Choi G (2001) Alteration of a 1170 single amino acid changes the substrate specificity of dihydroflavonol 4-reductase Plant 1171 J 25 325ndash333 1172

Junker A Fischer J Sichhart Y Brandt W and Draumlger B (2013) Evolution of the 1173 key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase Front 1174 Plant Sci 4 1ndash11 1175

Kallenbach M Oh Y Eilers EJ Veit D Baldwin IT and Schuman MC (2014) A 1176 robust simple high-throughput technique for time-resolved plant volatile analysis in field 1177 experiments Plant J 78 1060ndash1072 1178

Kaminaga Y et al (2006) Plant phenylacetaldehyde synthase is a bifunctional 1179 homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation J 1180 Biol Chem 281 23357ndash23366 1181

Knudsen JT Eriksson R and Gershenzon J (2006) Diversity and Distribution of 1182 Floral Scent The Botanical Review 72 1ndash120 1183

Koumlllner TG Schnee C Gershenzon J and Degenhardt J (2004) The Variability of 1184 Sesquiterpenes Emitted from Two Zea mays Cultivars Is Controlled by Allelic Variation 1185 of Two Terpene Synthase Genes Encoding Stereoselective Multiple Product Enzymes 1186 Plant Cell 16 1115 LP-1131 1187

Kumar R (2016) Evolutionary Trails of Plant Group II Pyridoxal Phosphate-Dependent 1188 Decarboxylase Genes Front Plant Sci 07 1ndash8 1189

Laursen T et al (2016) Characterization of a dynamic metabolon producing the defense 1190 httpsplantphysiolorgDownloaded on March 22 2021 - Published by

compound dhurrin in sorghum Science 354 890ndash893 1191 Lehmann T and Pollmann S (2009) Gene expression and characterization of a stress-1192

induced tyrosine decarboxylase from Arabidopsis thaliana FEBS Lett 583 1895ndash1900 1193 Leong BJ and Last RL (2017) Promiscuity impersonation and accommodation 1194

evolution of plant specialized metabolism Curr Opin Struct Biol 47 105ndash112 1195 Lepleacute JC Bonadeacute-Bottino M Augustin S Pilate G Lecirc Tacircn VD Delplanque A 1196

Cornu D and Jouanin L (1995) Toxicity to Chrysomela tremulae (Coleoptera 1197 Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor Mol 1198 Breed 1 319ndash328 1199

Leveacutee V Major I Levasseur C Tremblay L MacKay J and Seacuteguin A (2009) 1200 Expression profiling and functional analysis of Populus WRKY23 reveals a regulatory 1201 role in defense New Phytol 184 48ndash70 1202

Liu P Cheng Y Yang M Liu Y Chen K Long CA and Deng X (2014) 1203 Mechanisms of action for 2-phenylethanol isolated from Kloeckera apiculata in control of 1204 Penicillium molds of citrus fruits BMC Microbiol 14 1ndash14 1205

Major IT and Constabel CP (2008) Functional Analysis of the Kunitz Trypsin Inhibitor 1206 Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against 1207 Herbivores Plant Physiol 146 888ndash903 1208

Maag D Erb M Koumlllner TG and Gershenzon J (2015) Defensive weapons and 1209 defensive signals in plants Some metabolites serve both roles BioEssays 37 167-174 1210

Macoy DM Kim WY Lee SY and Kim MG (2015) Biotic stress related functions of 1211 hydroxycinnamic acid amide in plants J Plant Biol 58 156ndash163 1212

McCormick AC Boeckler GA Koumlllner TG Gershenzon J and Unsicker SB 1213 (2014) The timing of herbivore-induced volatile emission in black poplar (Populus nigra) 1214 and the influence of herbivore age and identity affect the value of individual volatiles as 1215 cues for herbivore enemies BMC Plant Biol 14 1216

Meilan R and Ma C (2007) Poplar (Populus spp) BT - Agrobacterium Protocols Volume 1217 2 In K Wang ed (Humana Press Totowa NJ) pp 143ndash151 1218

Moghe GD and Kruse LH (2018) The study of plant specialized metabolism 1219 Challenges and prospects in the genomics era Am J Bot 105 959ndash962 1220

Muroi A Ishihara A Tanaka C Ishizuka A Takabayashi J Miyoshi H and 1221 Nishioka T (2009) Accumulation of hydroxycinnamic acid amides induced by 1222 pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis 1223 thaliana Planta 230 517ndash527 1224

Newman M a von Roepenack-Lahaye E Parr A Daniels MJ and Dow JM 1225 (2001) Induction of hydroxycinnamoyl-tyramine conjugates in pepper by Xanthomonas 1226 campestris a plant defense response activated by hrp gene-dependent and hrp gene-1227 independent mechanisms Mol Plant Microbe Interact 14 785ndash792 1228

Nielsen KA Tattersall DB Jones PR and Moslashller BL (2008) Metabolon formation 1229 in dhurrin biosynthesis Phytochemistry 69 88ndash98 1230

Oberschall A Deaacutek M Toumlroumlk K Sass L Vass I Kovaacutecs I Feheacuter A Dudits D 1231 and Horvaacuteth G V (2000) A novel aldosealdehyde reductase protects transgenic 1232 plants against lipid peroxidation under chemical and drought stresses Plant J 24 437ndash1233 446 1234

Peters DJ and Constabel CP (2002) Molecular analysis of herbivore-induced 1235 condensed tannin synthesis Cloning and expression of dihydroflavonol reductase from 1236 trembling aspen (Populus tremuloides) Plant J 32 701ndash712 1237

Philippe RN and Bohlmann J (2007) Poplar defense against insect herbivores Can J 1238 Bot 85 1111ndash1126 1239

Ralph S et al (2006) Genomics of hybrid poplar (Populus trichocarpa times deltoides) 1240 interacting with forest tent caterpillars (Malacosoma disstria) Normalized and full-length 1241 cDNA libraries expressed sequence tags and a cDNA microarray for the study of 1242 insect-induced defences Mol Ecol 15 1275ndash1297 1243

Roy BA and Raguso RA (1997) Olfactory versus visual cues in a floral mimicry system 1244 httpsplantphysiolorgDownloaded on March 22 2021 - Published by

Oecologia 109 414ndash426 1245 Sakai M Hirata H Sayama H Sekiguchi K Itano H Asai T Dohra H Hara M 1246

and Watanabe N (2007) Production of 2-phenylethanol in roses as the dominant floral 1247 scent compound from L-phenylalanine by two key enzymes a PLP-dependent 1248 decarboxylase and a phenylacetaldehyde reductase Biosci Biotechnol Biochem 71 1249 2408 1250

Schmidt A Grimm R Schmidt J Scheel D Strack D and Rosahl S (1999) 1251 Cloning and expression of a potato cDNA encoding hydroxycinnamoyl-CoA  Tyramine 1252 N-(hydroxycinnamoyl)transferase J Biol Chem 274 4273ndash4280 1253

Schmidt A Scheel D and Strack D (1998) Elicitor-stimulated biosynthesis of 1254 hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum Planta 1255 205 51ndash55 1256

Sengupta D Naik D and Reddy AR (2015) Plant aldo-keto reductases (AKRs) as 1257 multi-tasking soldiers involved in diverse plant metabolic processes and stress defense 1258 A structure-function update J Plant Physiol 179 40ndash55 1259

Sheng L Zeng Y Wei T Zhu M Fang X Yuan X Luo Y and Feng L (2018) 1260 Cloning and Functional Verification of Genes Related to 2-Phenylethanol Biosynthesis 1261 in Rosa rugosa Genes 9 576 1262

Soslashrensen M Neilson EHJ and Moslashller BL (2017) Oximes Unrecognized 1263 Chameleons in General and Specialized Plant Metabolism Mol Plant 11 95ndash117 1264

Tamura K Stecher G Peterson D Filipski A and Kumar S (2013) MEGA6 1265 Molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 2725ndash2729 1266

Tieman D Taylor M Schauer N Fernie AR Hanson AD and Klee HJ (2006) 1267 Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor 1268 volatiles 2-phenylethanol and 2-phenylacetaldehyde Proc Natl Acad Sci 103 8287ndash1269 8292 1270

Tieman DM Loucas HM Kim JY Clark DG and Klee HJ (2007) Tomato 1271 phenylacetaldehyde reductases catalyze the last step in the synthesis of the aroma 1272 volatile 2-phenylethanol Phytochemistry 68 2660ndash2669 1273

Torrens-Spence MP Liu P Ding H Harich K Gillaspy G and Li J (2013) 1274 Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination 1275 Activities of Plant Aromatic Amino Acid Decarboxylases J Biol Chem 288 2376ndash1276 2387 1277

Torrens-Spence MP Lazear M Von Guggenberg R Ding H and Li J (2014a) 1278 Investigation of a substrate-specifying residue within Papaver somniferum and 1279 Catharanthus roseus aromatic amino acid decarboxylases Phytochemistry 106 37ndash43 1280

Torrens-Spence MP von Guggenberg R Lazear M Ding H and Li J (2014b) 1281 Diverse functional evolution of serine decarboxylases identification of two novel 1282 acetaldehyde synthases that uses hydrophobic amino acids as substrates BMC Plant 1283 Biol 14 247 1284

Torrens-spence MP Chiang Y Smith T Vicent MA and Wang Y (2018a) 1285 Structural basis for independent origins of new catalytic machineries in plant AAAD 1286 proteins bioRxiv 404970 1287

Torrens-spence MP Li F Carballo V and Weng J (2018b) Complete Pathway 1288 Elucidation and Heterologous Reconstitution of Rhodiola Salidroside Biosynthesis Mol 1289 Plant 11 205ndash217 1290

Tuskan GA et al (2006) The Genome of Black Cottonwood Populus trichocarpa (Torr amp 1291 Gray) Science 313 1596ndash1605 1292

Unsicker SB Kunert G and Gershenzon J (2009) Protective perfumes the role of 1293 vegetative volatiles in plant defense against herbivores Curr Opin Plant Biol 12 479ndash1294 485 1295

Villegas M and Brodelius PE (1990) Hydroxycinnamoyltransferase in Plant Cell 1296 Suspension Cultures Enzyme 414ndash420 1297

Xue LJ Alabady MS Mohebbi M and Tsai CJ (2015) Exploiting genome variation 1298 httpsplantphysiolorgDownloaded on March 22 2021 - Published by

to improve next-generation sequencing data analysis and genome editing efficiency in 1299 Populus tremula times alba 717-1B4 Tree Genet Genomes 11 1300

Watanabe S Hayashi K Yagi K Asai T Mactavish H Picone J Turnbull C and 1301 Watanabe N (2002) Biogenesis of 2-phenylethanol in rose flowers Incorporation of 1302 [2H8] L -phenylalanine into 2-phenylethanol and its β-D-glucopyranoside during flower 1303 opening of Rosa acuteHoh-Junacute and Rosa damascena Mill Biosci Biotechnol Biochem 66 1304 943ndash947 1305

Yamauchi Y Hasegawa A Taninaka A Mizutani M and Sugimoto Y (2011) 1306 NADPH-dependent reductases involved in the detoxification of reactive carbonyls in 1307 plants J Biol Chem 286 6999ndash7009 1308

Zhou X Jacobs TB Xue LJ Harding SA and Tsai CJ (2015) Exploiting SNPs 1309 for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4-1310 coumarate CoA ligase specificity and redundancy New Phytol 208 298ndash301 1311

Zhu F Poelman EH and Dicke M (2014) Insect herbivore-associated organisms affect 1312 plant responses to herbivory New Phytol 204 315ndash321 1313

Zhu J Obrycki JJ Ochieng SA Baker TC Pickett JA and Smiley D (2005) 1314 Attraction of two lacewing species to volatiles produced by host plants and aphid prey 1315 Naturwissenschaften 92 277ndash281 1316

Zhu YJ Zhou HT Hu YH Tang JY Su MX Guo YJ Chen QX and Liu B 1317 (2011) Antityrosinase and antimicrobial activities of 2-phenylethanol 2-1318 phenylacetaldehyde and 2-phenylacetic acid Food Chem 124 298ndash302 1319

Figure 1 Proposed pathways for the biosynthesis of 2-phenylethanol in poplar

AAAT aromatic amino acid transaminase AADC aromatic amino acid decarboxylase

CYP79 cytochrome P450 family 79 enzyme AAS aromatic aldehyde synthase PPDC

phenylpyruvic acid decarboxylase MAO monoamine oxidase TOX transoximase PAR

2-phenylacetaldehyde reductase UGT UDP-glucuronosyltransferase β-Glu β-

glucosidase Solid lines indicate well-established reactionsenzymes while dashed lines

indicate reactions hypothesized in the literature

phenylpyruvic acid

2-phenylethylamine

2-phenylacetaldehyde

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

(EZ)-phenylacetaldoxime

2-phenylethyl-β-D-glucopyranoside

2-phenylethanol

L-phenylalanine

UGT β-Glu

Figure 2 Herbivore-damaged leaves of Populus trichocarpa accumulate and

release phenylalanine-derived metabolites The accumulation of L-phenylalanine

(A) phenylpyruvic acid (B) 2-phenylethylamine (C) and 2-phenylethyl-β-D-

glucopyranoside (D) and the emission of 2-phenylethanol (E) were analyzed in

Lymantria dispar damaged (herb) and undamaged control (ctr) leaves Plant material

was extracted with methanol and analyzed via LC-MSMS (A - D) Volatiles were

collected for 8 hours and analyzed via GC-FID (E) Means plusmn SE are shown (A - D n

= 10 E n = 9) Asterisks indicate statistical significance as assessed by Studentacutes t-

test or Mann-Whitney Rank Sum Tests L-phenylalanine (P = 0530 t = -064)

phenylpyruvic acid (P = 0772 t = -0294) 2-phenylethylamine (P lt 0001 T = 55) 2-

phenylethyl-β-D-glucopyranoside (P = 0011 T = 71) 2-phenylethanol (P = 0005 T =

585) DW dry weight ns not significant nd not detected

ctr herb

2-phenylethylamine C

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

Figure 3 Dendrogram analysis of aromatic amino acid decarboxylase family

genes from Populus trichocarpa and characterized AADC AAS and GluSerHis-

decarboxylase genes from other plants Substrates of each clade are indicated in

parentheses The tree was inferred by using the maximum likelihood method and n =

1000 replicates for bootstrapping Bootstrap values are shown next to each node The

tree is drawn to scale with branch lengths measured in the number of substitutions per

site Genes described in this study are shown in red and bold with corresponding gene

identifiers (italics) Accession numbers are given in Supplemental Table 1

CaTDC1

CaTDC2

PtAAS1 (Potri013G052900)

PtAADC1 (Potri013G052800)

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

PtSDC2 (Potri002G069800)

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

decarboxylase (AADC)

tive a

Retention time (min)

A 2-phenylacetaldehyde

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

Figure 4 PtAAS1 and PtAADC1 are interconvertible by the mutation of

a single amino acid and are likely derived by tandem gene duplication

(A)The recombinant proteins PtAAS1 PtAADC1 and the corresponding

mutant enzymes PtAAS1 (F338Y) and PtAADC1 (Y338F) were incubated

with L-phenylalanine in the presence of PLP Enzyme products were

analyzed using GC-MS (2-phenylacetaldehyde) and LC-MSMS (2-

phenylethylamine) (B) PtAADC1 and PtAAS1 form a gene cluster on

chromosome 13 Both genes are oriented in the same direction and there are

no coding regions between them Sequence data were retrieved from the P

trichocarpa genome assembly version 30 (wwwphytozomenet Tuskan et

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

Figure 5 Overexpression of PtAAS1 PtAADC1 and PtAADC3 in Nicotiana

benthamiana alters the emission of 2-phenylethanol (A) and the accumulation of

2-phenylethylamine (B) tyramine (C) tryptamine (D) and 2-phenylethanol-β-D-

glucopyranoside (E) N benthamiana plants were infiltrated with Agrobacterium

tumefaciens carrying either PtAAS1 or PtAADC1 Five days after infiltration the

headspaces of plants were collected and analyzed via GC-MS and GC-FID Additionally

plants were harvested extracted with methanol and metabolite accumulation was

analyzed via LC-MSMS Different letters above each bar indicate statistically significant

differences as assessed by Kruskal-Wallis One Way ANOVA and Tukey tests 2-

phenylethanol (H = 19867 P le 0001) 2-phenylethylamine (H = 1694 P le 0001)

tyramine (H = 18267 P le 0001) tryptamine (H = 18645 P le 0001) 2-phenylethyl-β-

D-glucopyranoside (H = 216 P le 0001) Means plusmn SE are shown (n = 6) FW fresh

weight

(microgg

2-phenylethylamine B

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

tyramine C 2-phenylethanol A

Figure 6 Transcript accumulation of AAAD and SDC genes in

Lymantria dispar-damaged and undamaged Populus

trichocarpa leaves Gene expression in herbivore-damaged

(herb) and undamaged (ctr) leaves was analyzed by Illumina

sequencing and mapping the reads to the transcripts of the P

trichocarpa genome version v30 Expression was normalized to

RPKM Significant differences in EDGE tests are visualized by

asterisks Means plusmn SE are shown (n = 4) PtAAS1 (P = 823213E-

12 weighted difference (WD) = 486618E-05) PtAAS2 (P =

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

202404E-06) PtAADC3 (P = 1 WD = -435895E-08) PtSDC1 (P

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

Figure 7 RNAi-mediated knockdown of AADC1 in Populus times canescens Sterile

Populus times canescens calli were transformed via Agrobacterium tumefaciens Nontransgenic

trees (WT) empty vector lines (EV-2-4) and RNAi lines (RNAi-1-4) were subjected to

Lymantria dispar feeding 2-Phenylethanol was collected from the headspace and analyzed

using GC-MSFID 2-Phenylethylamine and 2-phenylethyl-β-D-glucopyranoside were

extracted with methanol from ground plant material and analyzed via LC-MSMS Biological

replicates (nb) and technical replicates (nt) of EV lines and RNAi lines were used to test for

statistical differences WT nb = 4 EV nb = 3 nt = 5 RNAi nb = 4 nt = 5 Asterisks indicate

statistical significance as assessed by Studentacutes t-test or Mann-Whitney Rank Sum Tests 2-

Phenylethylamine (P lt 0001 t = 7940) 2-phenylethyl-β-D-glucopyranoside (P = 0011 T =

547) 2-phenylethanol (P = 0509 T = 404) Medians plusmn quartiles and outliers are shown Each

data point is represented by a circle FW fresh weight ns ndash not significant

2-phenylethylamine 2-phenylethyl-β-D-glucopyranoside

(micro

2-phenylethanol

(micro

Parsed CitationsArimura GI Huber DPW and Bohlmann J (2004) Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnalemissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides) cDNA cloning functional characterization andpatterns of gene expression of (-)-germacr Plant J 37 603ndash616

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Bertoldi M Gonsalvi M Contestabile R and Voltattorni CB (2002) Mutation of tyrosine 332 to phenylalanine converts dopadecarboxylase into a decarboxylation-dependent oxidative deaminase J Biol Chem 277 36357ndash36362

Bieri S Brachet A Veuthey JL and Christen P (2006) Cocaine distribution in wild Erythroxylum species J Ethnopharmacol 103439ndash447

Campos L Lisoacuten P Loacutepez-Gresa MP Rodrigo I Zacareacutes L Conejero V and Belleacutes JM (2014) Transgenic Tomato PlantsOverexpressing Tyramine N -Hydroxycinnamoyltransferase Exhibit Elevated Hydroxycinnamic Acid Amide Levels and EnhancedResistance to Pseudomonas syringae Mol Plant-Microbe Interact 27 1159ndash1169

Chen XM Kobayashi H Sakai M Hirata H Asai T Ohnishi T Baldermann S and Watanabe N (2011) Functionalcharacterization of rose phenylacetaldehyde reductase (PAR) an enzyme involved in the biosynthesis of the scent compound 2-phenylethanol J Plant Physiol 168 88ndash95

Chung SH Rosa C Scully ED Peiffer M Tooker JF Hoover K Luthe DS and Felton GW (2013) Herbivore exploits orallysecreted bacteria to suppress plant defenses Proc Natl Acad Sci 110 15728ndash15733

Costa MA Marques J V Dalisay DS Herman B Bedgar DL Davin LB and Lewis NG (2013) Transgenic hybrid poplar forsustainable and scalable production of the commodityspecialty chemical 2-phenylethanol PLoS One 8

Danner H Boeckler GA Irmisch S Yuan JS Chen F Gershenzon J Unsicker SB and Koumlllner TG (2011) Four terpenesynthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa Phytochemistry 72897ndash908

Facchini PJ Hagel J and Zulak KG (2002) Hydroxycinnamic acid amide metabolism physiology and biochemistry Can J Bot 80577ndash589

Facchini PJ Huber-Allanach KL and Tari LW (2000) Plant aromatic L-amino acid decarboxylases Evolution biochemistryregulation and metabolic engineering applications Phytochemistry 54 121ndash138

Fahey JW Zalcmann a T and Talalay P (2001) The chemical diversity and distribution of glucosinolates and isothiocyanatesamoung plants Phytochemistry 56 5ndash51

Fan P Miller AM Liu X Jones AD and Last RL (2017) Evolution of a flipped pathway creates metabolic innovation in tomatotrichomes through BAHD enzyme promiscuity Nat Commun 8 1ndash13

Farhi M Lavie O Masci T Hendel-Rahmanim K Weiss D Abeliovich H and Vainstein A (2010) Identification of rosephenylacetaldehyde synthase by functional complementation in yeast Plant Mol Biol 72 235ndash245

Ferreira-Silva B Lavandera I Kern A Faber K and Kroutil W (2010) Chemo-promiscuity of alcohol dehydrogenases reduction ofphenylacetaldoxime to the alcohol Tetrahedron 66 3410ndash3414

Frost CJ Appel M Carlson JE De Moraes CM Mescher MC and Schultz JC (2007) Within-plant signalling via volatilesovercomes vascular constraints on systemic signalling and primes responses against herbivores Ecol Lett 10 490ndash8

Galen C Kaczorowski R Todd SL Geib J and Raguso RA (2011) Dosage-Dependent Impacts of a Floral Volatile Compound onPollinators Larcenists and the Potential for Floral Evolution in the Alpine Skypilot Polemonium viscosum Am Nat 177 258ndash272

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Hazelwood LA Daran JM Van Maris AJA Pronk JT and Dickinson JR (2008) The Ehrlich pathway for fusel alcoholproduction A century of research on Saccharomyces cerevisiae metabolism (Applied and Environmental Microbiology (2008) 74 8(2259-2266)) Appl Environ Microbiol 74 3920

Hirata H Ohnishi T Ishida H Tomida K Sakai M Hara M and Watanabe N (2012) Functional characterization of aromatic aminoacid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts J Plant Physiol 169 444ndash451

Hirata H Ohnishi T Tomida K Ishida H Kanda M Sakai M Yoshimura J Suzuki H Ishikawa T Dohra H and Watanabe N(2016) Seasonal induction of alternative principal pathway for rose flower scent Sci Rep 6

Ho SN Hunt HD Horton RM Pullen JK and Pease LR (1989) Site-directed mutagenesis by overlap extension using thepolymerase chain reaction Gene 77 51ndash59

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Irmisch S Clavijo McCormick A Guumlnther J Schmidt A Boeckler GA Gershenzon J Unsicker SB and Koumlllner TG (2014a)Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalistcaterpillar Plant J 80 1095ndash1107

Pubmed Author and Title

Irmisch S Jiang Y Chen F Gershenzon J and Koumlllner TG (2014b) Terpene synthases and their contribution to herbivore-induced volatile emission in western balsam poplar (Populus trichocarpa) BMC Plant Biol 14 1ndash16

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Jain D Khandal H Paul J and Debasis K (2016) A pathogenesis related-10 protein CaARP functions as aldo keto reductase toscavenge cytotoxic aldehydes Plant Mol Biol 90 171ndash187

Johnson ET Ryu S Yi H Shin B Cheong H and Choi G (2001) Alteration of a single amino acid changes the substratespecificity of dihydroflavonol 4-reductase Plant J 25 325ndash333

Junker A Fischer J Sichhart Y Brandt W and Draumlger B (2013) Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase Front Plant Sci 4 1ndash11

Kallenbach M Oh Y Eilers EJ Veit D Baldwin IT and Schuman MC (2014) A robust simple high-throughput technique fortime-resolved plant volatile analysis in field experiments Plant J 78 1060ndash1072

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Leong BJ and Last RL (2017) Promiscuity impersonation and accommodation evolution of plant specialized metabolism CurrOpin Struct Biol 47 105ndash112

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McCormick AC Boeckler GA Koumlllner TG Gershenzon J and Unsicker SB (2014) The timing of herbivore-induced volatileemission in black poplar (Populus nigra) and the influence of herbivore age and identity affect the value of individual volatiles as cuesfor herbivore enemies BMC Plant Biol 14

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Muroi A Ishihara A Tanaka C Ishizuka A Takabayashi J Miyoshi H and Nishioka T (2009) Accumulation of hydroxycinnamicacid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana Planta 230517ndash527

Newman M a von Roepenack-Lahaye E Parr A Daniels MJ and Dow JM (2001) Induction of hydroxycinnamoyl-tyramineconjugates in pepper by Xanthomonas campestris a plant defense response activated by hrp gene-dependent and hrp gene-independent mechanisms Mol Plant Microbe Interact 14 785ndash792

Nielsen KA Tattersall DB Jones PR and Moslashller BL (2008) Metabolon formation in dhurrin biosynthesis Phytochemistry 69 88ndash98

Oberschall A Deaacutek M Toumlroumlk K Sass L Vass I Kovaacutecs I Feheacuter A Dudits D and Horvaacuteth G V (2000) A novel aldosealdehydereductase protects transgenic plants against lipid peroxidation under chemical and drought stresses Plant J 24 437ndash446

Peters DJ and Constabel CP (2002) Molecular analysis of herbivore-induced condensed tannin synthesis Cloning and expressionof dihydroflavonol reductase from trembling aspen (Populus tremuloides) Plant J 32 701ndash712

Philippe RN and Bohlmann J (2007) Poplar defense against insect herbivores Can J Bot 85 1111ndash1126Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Ralph S et al (2006) Genomics of hybrid poplar (Populus trichocarpa times deltoides) interacting with forest tent caterpillars(Malacosoma disstria) Normalized and full-length cDNA libraries expressed sequence tags and a cDNA microarray for the study ofinsect-induced defences Mol Ecol 15 1275ndash1297

Roy BA and Raguso RA (1997) Olfactory versus visual cues in a floral mimicry system Oecologia 109 414ndash426Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Sakai M Hirata H Sayama H Sekiguchi K Itano H Asai T Dohra H Hara M and Watanabe N (2007) Production of 2-phenylethanol in roses as the dominant floral scent compound from L-phenylalanine by two key enzymes a PLP-dependentdecarboxylase and a phenylacetaldehyde reductase Biosci Biotechnol Biochem 71 2408

Schmidt A Grimm R Schmidt J Scheel D Strack D and Rosahl S (1999) Cloning and expression of a potato cDNA encodinghydroxycinnamoyl-CoAthinsp Tyramine N-(hydroxycinnamoyl)transferase J Biol Chem 274 4273ndash4280

Schmidt A Scheel D and Strack D (1998) Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension culturesof Solanum tuberosum Planta 205 51ndash55

Sengupta D Naik D and Reddy AR (2015) Plant aldo-keto reductases (AKRs) as multi-tasking soldiers involved in diverse plantmetabolic processes and stress defense A structure-function update J Plant Physiol 179 40ndash55

Sheng L Zeng Y Wei T Zhu M Fang X Yuan X Luo Y and Feng L (2018) Cloning and Functional Verification of GenesRelated to 2-Phenylethanol Biosynthesis in Rosa rugosa Genes 9 576

Soslashrensen M Neilson EHJ and Moslashller BL (2017) Oximes Unrecognized Chameleons in General and Specialized PlantMetabolism Mol Plant 11 95ndash117

Tamura K Stecher G Peterson D Filipski A and Kumar S (2013) MEGA6 Molecular evolutionary genetics analysis version 60Mol Biol Evol 30 2725ndash2729

Tieman D Taylor M Schauer N Fernie AR Hanson AD and Klee HJ (2006) Tomato aromatic amino acid decarboxylasesparticipate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde Proc Natl Acad Sci 103 8287ndash8292

Tieman DM Loucas HM Kim JY Clark DG and Klee HJ (2007) Tomato phenylacetaldehyde reductases catalyze the last stepin the synthesis of the aroma volatile 2-phenylethanol Phytochemistry 68 2660ndash2669

Torrens-Spence MP Liu P Ding H Harich K Gillaspy G and Li J (2013) Biochemical Evaluation of the Decarboxylation andDecarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases J Biol Chem 288 2376ndash2387

Torrens-Spence MP Lazear M Von Guggenberg R Ding H and Li J (2014a) Investigation of a substrate-specifying residuewithin Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases Phytochemistry 106 37ndash43

Torrens-Spence MP von Guggenberg R Lazear M Ding H and Li J (2014b) Diverse functional evolution of serinedecarboxylases identification of two novel acetaldehyde synthases that uses hydrophobic amino acids as substrates BMC Plant Biol14 247

Torrens-spence MP Chiang Y Smith T Vicent MA and Wang Y (2018a) Structural basis for independent origins of new catalyticmachineries in plant AAAD proteins bioRxiv 404970

Torrens-spence MP Li F Carballo V and Weng J (2018b) Complete Pathway Elucidation and Heterologous Reconstitution ofRhodiola Salidroside Biosynthesis Mol Plant 11 205ndash217

Tuskan GA et al (2006) The Genome of Black Cottonwood Populus trichocarpa (Torr amp Gray) Science 313 1596ndash1605Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Unsicker SB Kunert G and Gershenzon J (2009) Protective perfumes the role of vegetative volatiles in plant defense againstherbivores Curr Opin Plant Biol 12 479ndash485

Villegas M and Brodelius PE (1990) Hydroxycinnamoyltransferase in Plant Cell Suspension Cultures Enzyme 414ndash420Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Xue LJ Alabady MS Mohebbi M and Tsai CJ (2015) Exploiting genome variation to improve next-generation sequencing dataanalysis and genome editing efficiency in Populus tremula times alba 717-1B4 Tree Genet Genomes 11

Watanabe S Hayashi K Yagi K Asai T Mactavish H Picone J Turnbull C and Watanabe N (2002) Biogenesis of 2-phenylethanol in rose flowers Incorporation of [2H8] L -phenylalanine into 2-phenylethanol and its β-D-glucopyranoside during floweropening of Rosa acuteHoh-Junacute and Rosa damascena Mill Biosci Biotechnol Biochem 66 943ndash947

Yamauchi Y Hasegawa A Taninaka A Mizutani M and Sugimoto Y (2011) NADPH-dependent reductases involved in thedetoxification of reactive carbonyls in plants J Biol Chem 286 6999ndash7009

Zhou X Jacobs TB Xue LJ Harding SA and Tsai CJ (2015) Exploiting SNPs for biallelic CRISPR mutations in the outcrossingwoody perennial Populus reveals 4-coumarate CoA ligase specificity and redundancy New Phytol 208 298ndash301

Zhu F Poelman EH and Dicke M (2014) Insect herbivore-associated organisms affect plant responses to herbivory New Phytol204 315ndash321

Zhu J Obrycki JJ Ochieng SA Baker TC Pickett JA and Smiley D (2005) Attraction of two lacewing species to volatilesproduced by host plants and aphid prey Naturwissenschaften 92 277ndash281

Zhu YJ Zhou HT Hu YH Tang JY Su MX Guo YJ Chen QX and Liu B (2011) Antityrosinase and antimicrobial activitiesof 2-phenylethanol 2-phenylacetaldehyde and 2-phenylacetic acid Food Chem 124 298ndash302

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Author contributions 34

TGK JaG and JoG designed the research JaG NDL AS HJS and MR carried out 35

the experimental work JaG NDL MH AS MR and TGK analyzed data TGK and 36

JaG wrote the manuscript All authors read and approved the final manuscript 37

Responsibilities of the Author for Contact 39

It is the responsibility of the author for contact to ensure that all scientists who have 40

contributed substantially to the conception design or execution of the work described 41

in the manuscript are included as authors in accordance with the guidelines from the 42

Committee on Publication Ethics (COPE 43

httppublicationethicsorgresourcesguidelines) It is the responsibility of the author 44

for contact also to ensure that all authors agree to the list of authors and the 45

identified contributions of those authors 46

Funding information 48

The research was funded by the Max-Planck Society 49

Corresponding author E-mail koellnericempgde phone +49 (0) 3641 57 1329 52

fax +49 (0) 3641 57 1302 53

ABSTRACT 54

INTRODUCTION 86

et al 2014a) 131

et al 2011) 158

poplar 182

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

ABSTRACT 54

INTRODUCTION 86

et al 2014a) 131

et al 2011) 158

poplar 182

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

INTRODUCTION 86

et al 2014a) 131

et al 2011) 158

poplar 182

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

et al 2014a) 131

et al 2011) 158

poplar 182

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

et al 2011) 158

poplar 182

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

RESULTS 186

(Figure 2D) 205

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

enzymes 224

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

(Figure 4A) 268

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

DISCUSSION 402

phenylalanine 411

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

independently 502

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

compounds 559

trichocarpa 563

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

Plant treatment 604

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

green house 614

Scientific) 636

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2015) 759

above 773

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

al (2013) 824

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

PRJNA516861 904

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

plants 917

cyanide 930

analysis 938

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

metabolites 944

TABLES 961

(mM) V

(nmolmicrog-1

PtAAS1

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

PtAADC1

PtAADC2

PtAADC3

Trp TrpA - - - - 969

detected 975

FIGURE LEGENDS 978

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

literature 986

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

= 0031269756 WD = 202404E-06) PtAADC3 (P = 1 WD = -435895E-08) 1044

108813E-05) 1046

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

phenylpyruvic acid

2-phenylethylamine

AAS PAR

AAAT PPDC

AADC MAO

TOX CYP79

2-phenylethanol

L-phenylalanine

UGT β-Glu

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

ctr herb

(microgg

2-phenylethyl-β-D-

glucopyranoside D

ctr herb

(microgg

E 2-phenylethanol

ctr herb 0

L-phenylalanine

(microg

ctr herb

phenylpyruvic acid

(microgg

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

CaTDC1

CaTDC2

AtTyrDC

RhAADC

RhPAAS

PcTyrDC2

PcTyrDC4

PsTyrDC9

TfTyrDC13

PhPAAS

LeAADC1A

LeAADC1B

LeAADC2

AtGAD1

AtGAD2

PhPPY-AT

AADCaromatic

aldehyde synthase

(PheTyr)

amino acid

decarboxylase

(SerHis)

amino acid

decarboxylase

aromatic amino acid

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

tive a

106 108 110 112

14 PtAAS1

PtAAS1

(F338Y)

PtAADC1

(Y338F)

PtAAS1

(F338Y)

PtAADC1

(Y338F) R

(cps x

2-phenylethylamine

10 30 50 70

al 2006)

chromosome 13

PtAADC1 PtAAS1

coding region

untranslated region

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Parsed Citations

weight

(microgg

2-phenylethyl-β-

D-glucopyranoside

tryptamine D

0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

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2-phenylethanol

(micro

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0039452261 WD = -111092E-05) PtAADC1 (P = 899025E-09

WD = 156932E-05) PtAADC2 (P = 0031269756 WD =

= 0576261262 WD = 161294E-06) PtSDC2 (P = 0015936012

WD = 108813E-05)

(micro

2-phenylethanol

(micro

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Documents

Mar 07, 2019 · Pubmed: Author and Title Google Scholar: Author Only Title Only Author and Title Ferreira-Silva, B., Lavandera, I., Kern, A., Faber, K., and Kroutil, W. (2010). Chemo