identification of a hexenal reductase that modulates the … · 2018. 8. 20. · 2-hexenal or...

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Short title Reduction of green leaf aldehydes 1

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Corresponding Author 3

Kenji Matsui 4

Graduate School of Sciences and Technology for Innovation Yamaguchi University 5

Yamaguchi 753-8515 Japan 6

Telephone +81-83-933-5850 7

Fax +81-83-933-5820 8

Email matsuiyamaguchi-uacjp 9

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Research Area 11

Biochemistry and Metabolism 12

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One-sentence Summary 14

NADPH-dependent cinnamaldehyde and hexenal reductase affects the composition of 15

green leaf volatiles and is involved in indirect plant defenses 16

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Funding 18

This work was supported partly by JSPS KAKENHI Grant Number 16H03283 (to K M) 19

The authors declare that they have no conflict of interest with the contents of this 20

article 21

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Plant Physiology Preview Published on August 20 2018 as DOI101104pp1800632

Copyright 2018 by the American Society of Plant Biologists

httpsplantphysiolorgDownloaded on February 4 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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Research Article 24

25

Identification of a hexenal reductase that modulates the composition of 26

green leaf volatiles 27

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Toshiyuki Tanakaa Ayana Ikedab Kaori Shiojiric Rika Ozawad Kazumi Shikia 31

Naoko Nagai-Kunihiroa Kenya Fujitab Koichi Sugimotoa Katsuyuki T Yamatoe 32

Hideo Dohraf Toshiyuki Ohnishig Takao Koedukaab Kenji Matsuiab1 33

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aDivision of Agricultural Sciences Graduate School of Sciences and Technology for 35

Innovation Yamaguchi University Yamaguchi 753-8515 Japan 36

bDepartment of Biological Chemistry Faculty of Agriculture Yamaguchi University 37


cDepartment of Agriculture Ryukoku University Ooe Otsu Shiga 520-2194 Japan 39

dCenter for Ecological Research Kyoto University Hirano Otsu Shiga 520-2113 Japan 40

eFaculty of Biology-Oriented Science and Technology Kindai University Kinokawa 41

Wakayama 649-6493 Japan 42

fInstrumental Research Support Office Research Institute of Green Science and 43

Technology Shizuoka University Shizuoka 422-8529 Japan 44

gCollege of Agriculture Academic Institute Shizuoka University Shizuoka 422ndash8529 45

Japan 46

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1Address correspondence to matsuiyamaguchi-uacjp 48

The author responsible for the distribution of materials to the findings presented in this 49

article in accordance with the policy described in the Instructions for Authors 50

(wwwplantphysiolorg) is Kenji Matsui (matsuiyamaguchi-uacjp) 51

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Author Contributions 53

KM conceived this project designed experiments and wrote the article TT AI K 54

Shiki NNK KF and KSugimoto purified the enzyme and examined its properties 55

KTY and TK analyzed the data HD and TO determined amino acid sequences of 56 httpsplantphysiolorgDownloaded on February 4 2021 - Published by

Copyright (c) 2020 American Society of Plant Biologists All rights reserved

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the enzyme TT KF and KM analyzed volatiles and examined phenotypes of 57

knockout mutants K Shioriji and RO performed bioassays with wasps 58

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ABSTRACT 62

Green leaf volatiles (GLVs) including six-carbon (C6) aldehydes alcohols and esters 63

are formed when plant tissues are damaged GLVs play roles in direct plant defense at 64

wound sites indirect plant defense via the attraction of herbivore predators and 65

plant-plant communication GLV components provoke distinctive responses in their 66

target recipients and therefore control of GLV composition is important for plants to 67

appropriately manage stress responses Reduction of C6-aldehydes into C6-alcohols is a 68

key step in the control of GLV composition and is also important to avoid toxic buildup 69

of C6-aldehydes However the molecular mechanisms behind C6-aldehyde reduction 70

remain poorly understood In this study we purified an Arabidopsis thaliana 71

NADPH-dependent cinnamaldehyde and hexenal reductase encoded by At4g37980 72

named here CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) CHR 73

T-DNA knockout mutant plants displayed a normal growth phenotype however we 74

observed significant suppression of C6-alcohol production following partial mechanical 75

wounding or herbivore infestation Our data also showed that the parasitic wasp Cotesia 76

vestalis was more attracted to GLVs emitted from herbivore-infested wild-type plants 77

compared to GLVs emitted from chr plants which corresponded with reduced 78

C6-alcohol levels in the mutant Moreover chr plants were more susceptible to 79

exogenous high-dose exposure to (Z)-3-hexenal as indicated by their markedly lowered 80

photosystem II activity Our study shows that reductases play significant roles in 81

changing GLV composition and are thus important in avoiding toxicity from volatile 82

carbonyls and in the attraction of herbivore predators 83

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Key words Green leaf volatiles (Z)-3-Hexen-1-ol Hexenal reductase Carbonyl stress 86

Indirect defense 87

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This article contains supplemental Figs S1-S9 and Tables S1-S2 89

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INTRODUCTION 92

93

Plants form and emit a wide variety of volatile organic compounds Volatiles 94

from flowers attract pollinators and increase the fitness of plants by promoting efficient 95

reproduction and those from fruits attract seed-dispersing animals and help plants to 96

find new habitats (Dudareva et al 2013) Vegetative organs such as leaves stems and 97

roots also produce and emit volatiles and this process is generally induced by various 98

types of biotic and abiotic stresses as a defense response (Pierik et al 2014) 99

Green leaf volatiles (GLVs) are ubiquitously expressed with other groups of 100

volatile compounds such as terpenoids and amino acid derivatives GLVs are 101

derivatives of fatty acids and include six-carbon (C6) aldehydes alcohols and esters 102

(Fig 1 Matsui 2006 Scala et al 2013) In intact and healthy plant tissues GLV levels 103

are generally low but when tissues suffer stresses associated with disruption of cells 104

such as herbivore damage or attack by necrotrophic fungi GLV-forming pathways are 105

rapidly activated to yield large quantities of GLVs at damaged tissues (Matsui 2006 106

Scala et al 2013 Ameye et al 2017) GLVs at damage tissues participate in direct 107

plant defense by preventing the invasion of harmful microbes (Shiojiri et al 2006a 108

Kishimoto et al 2008) GLVs also function as infochemicals that are an indirect plant 109

defense via the attraction of natural predators of infesting herbivores (Kessler amp 110

Baldwin 2001 Shiojiri et al 2006a Shiojiri et al 2006b Joo et al 2018) GLVs have 111

also been associated with plant-plant communication (Sugimoto et al 2013) 112

Because the chemical properties of GLV components are distinct it is 113

assumed that each plays a distinctive role For example C6-aldehydes were shown to be 114

prominently involved in direct defense against the necrotrophic fungal pathogen 115

Botrytis cinerea whereas C6-alcohols and esters were less involved (Kishimoto et al 116

2008) The formyl groups in C6-aldehydes are potently reactive with nucleophiles such 117

as amines and alcohols and contribute to the suppression of pathogen growth When 118

carbon-carbon double bonds are conjugated to the formyl group as in (E)-2-hexenal 119

the αβ-unsaturated carbonyl moiety behaves as a more potent electrophile and is prone 120

to reactions with various biological molecules specifically reactions that eliminate their 121

intrinsic functions (Farmer and Muumlller 2013) Even though (Z)-3-hexenal is not a 122

reactive carbonyl species because the two double bonds are interrupted with one 123

methylene group bis-allylic hydrogen is prone to removal often resulting in 124 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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oxygenation to yield 4-hydroxy-(E)-2-hexenal or 4-oxo-(E)-2-hexenal (Pospiacutesil amp 125

Yamamoto 2017) These oxygenated hexenals are highly reactive carbonyl species and 126

(Z)-3-hexenal has specifically been identified as a potentially reactive compound 127

(Matsui et al 2012) C6-aldehydes were reported to be more effective bacteriostatic 128

chemicals than C6-alcohols and incorporation of a double bond either in the C2 or C3 129

position enhances their activities (Nakamura and Hatanaka 2002) albeit the formyl 130

group is not always a prerequisite for antimicrobial activity (Prost et al 2005) 131

GLVs are either attractive or repulsive to arthropods depending on their 132

composition and on the arthropod species (Wei amp Kang 2011 Scala et al 2013) 133

Females of the parasitic wasp Cotesia glomerata are attracted to (E)-2-hexenal and 134

(Z)-3-hexen-1-yl acetate but not to (Z)-3-hexen-1-ol (Shiojiri et al 2006) Moreover 135

(Z)-3-Hexen-1-yl acetate increased predation rates of a generalist natural predator 136

toward eggs of the herbivore Manduca quinquemaculata on Nicotiana attenuata plants 137

(Kessler and Baldwin 2001) Composition of GLVs elicited by herbivores also differ 138

between dawn and dusk as indicated by Joo et al (2018) who showed that GLV 139

aldehydes and alcohols were less abundant in comparison with GLV esters at dawn 140

whereas GLV ester levels were lower at dusk Higher predation rates on M sexta eggs 141

by Geocoris spp were observed with dawn GLV composition in nature suggesting that 142

the predator distinguished between GLV compositions (Joo et al 2018) 143

Concerning plant-plant communication (Z)-3-hexen-1-ol emitted from 144

Spodoptera litura-infested tomato leaves was shown to be absorbed by neighboring 145

tomato plants to form the defense compound (Z)-3-hexen-1-yl primeveroside (Sugimoto 146

et al 2013) It was also observed that the secretion of extrafloral nectar by lima beans 147

(Phaseolus lunatus) to attract predatory and parasitoid insects (ants and wasps) was 148

significantly induced after exposure of these plants to (Z)-3-hexen-1-yl acetate vapors 149

(Kost amp Heil 2006) These accumulating lines of evidence indicate that composition of 150

GLVs is crucial for plants to aptly cope with enemies and foes and that reduction of 151

C6-aldehydes to C6-alcohol is a crucial step in the control of GLV composition 152

Lipoxygenase (LOX) acts on linoleniclinoleic acids either in their free forms 153

or as acyl groups in glycerolipids to form corresponding fatty acidlipid hydroperoxides 154

which are subsequently cleaved by hydroperoxide lyase (HPL) to form C6-aldehydes 155

such as n-hexanal or (Z)-3-hexenal (Fig 1 Matsui 2006 Scala et al 2013 Mwenda amp 156

Matsui 2014) A portion of (Z)-3-hexenal is isomerized to (E)-2-hexenal in some plant 157 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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species (Kunishima et al 2016 Spyropoulou et al 2017) and a factor in oral 158

secretions of tobacco hawkmoth (Manduca sexta) catalyzes this isomerization in 159

Nicotiana attenuata resulting in higher attraction of the predator Geocoris spp 160

(Allmann amp Baldwin 2011) Other portions of C6-aldehydes are reduced to 161

corresponding C6-alcohols and portions of C6-alcohols are further converted into 162

corresponding acetates by acetyl-CoA transferase (DrsquoAuria et al 2007) It was assumed 163

that NAD(H)-dependent alcohol dehydrogenase (EC 1111) is involved in these 164

reduction steps (Hatanaka 1993) and this may be the case with completely disrupted 165

plant tissues Accordingly in completely homogenized leaves of Arabidopsis mutants 166

that were deficient in alcohol dehydrogenase (AtADH1 At1g77120) n-hexan-1-ol and 167

3-hexen-1-ol were formed at 62 and 51 of the levels found in parent wild-type 168

plants respectively (Bate et al 1998) In completely homogenized tomato tissues with 169

increased and suppressed expression levels of SlADH2 which is the tomato homolog of 170

AtADH1 n-hexan-1-ol and (Z)-3-hexen-1-ol levels roughly correlated with ADH 171

activities (Speirs et al 1998) In contrast in partially wounded leaf tissues of 172

Arabidopsis (Z)-3-hexenal was formed at disrupted tissues and diffused into the 173

neighboring intact tissues where it was reduced to (Z)-3-hexen-1-ol in an 174

NADPH-dependent manner (Matsui et al 2012) Hence an NADPH-dependent 175

C6-aldehyde reductase is likely accountable for the reduction of C6-aldehydes to 176

C6-alcohols Because plant leaves are commonly damaged only in part such as 177

following consumption by an herbivore such a NADPH-dependent reductase may be 178

ecophysiologically relevant but has not been identified yet In this study we purified 179

and identified a NADPH-dependent reductase preferring (Z)-3-hexenal which we 180

named CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) Involvement of 181

CHR in changing the composition of GLVs was verified using a T-DNA knockout 182

mutant disrupted in the corresponding CHR gene Morover chr mutant lines were 183

employed to assess the role of CHR in indirect defense of Arabidopsis via GLV 184

composition CHR involvement in detoxification of (Z)-3-hexenal was also examined 185

186

187

RESULTS 188

189

Purification of hexenal reductase 190 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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Following exposure to (Z)-3-hexenal vapors intact Arabidopsis leaves absorb 191

the compound into leaves reduce it to (Z)-3-hexen-1-ol and then re-emit it from the 192

leaves to the atmosphere (Matsui et al 2012) Therefore substantial enzyme activity 193

for converting (Z)-3-hexenal to (Z)-3-hexen-1-ol is already present in Arabidopsis 194

leaves under normal growth conditions even without stress treatments and tissue 195

disruptions to trigger GLV formation When crude extracts from Arabidopsis leaves 196

(No-0) were exposed to (Z)-3-hexenal exogenous NADPH was consumed (Fig 4) 197

Moreover the rate of NADH consumption was less than 20 of that found with 198

NADPH under the same reaction conditions Taken with our previous results (Matsui et 199

al 2012) this result indicated that Arabidopsis leaves carry a NADPH-dependent 200

(Z)-3-hexenal reductase 201

This reductase was purified from crude Arabidopsis leaf extracts using 202

ammonium sulfate fractionation followed by three consecutive chromatography steps 203

(Table I) During these steps reductase activity was identified as a single peak with a 204

substantial yield and SDS-PAGE analyses of fractions that were separated using a 205

HiTrap DEAE column in the third chromatography step indicated that a protein of 38 206

kDa correlated with the observed metabolic activity (Fig 2A) In the presence of 207

(Z)-3-hexenal the purified enzyme consumed NADPH most actively at pH 75 (Fig 208

2B) When a mixture of (Z)-3-hexenal and (E)-2-hexenal (154 and 46 microM respectively) 209

was used as a substrate in the absence of cofactor formation of the corresponding 210

alcohol was hardly detected (Fig 2C) Addition of NADH slightly facilitated reduction 211

of hexenals whereas NADPH resulted in more extensive formation of (Z)-3-hexen-1-ol 212

and (E)-2-hexen-1-ol (Fig 2D) However n-hexanal formation through reduction of the 213

carbon-carbon double bond in (Z)-3-hexenal and (E)-2-hexenal was barely increased 214

indicating that the enzyme has little double bond reductase activity 215

216

Identification of C6-aldehyde reductase gene 217

Internal amino acid sequences from the purified protein showed complete 218

identity with Arabidopsis CINNAMYL ALCOHOL DEHYDROGENASE 7 (AtCAD7 219

At4g37980 Supplemental Fig S1) The AtCAD7 gene is also known as 220

ELICITOR-ACTIVATED GENE 3 (ELI3 Kiedrowski et al 1992) and the protein 221

comprises 357 amino acids and has a molecular weight of 38245 Da close to that of the 222

purified enzyme estimated in SDS-PAGE analyses (Fig 2A) A MotifFinder search 223 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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(httpwwwgenomejptoolsmotif) of the Pfam database showed that the protein 224

contains ADH_N and ADH_zinc_N motifs Cytosolic localization was also predicted 225

based on the amino acid sequence using SUBA3 (httpsubaplantenergyuwaeduau) 226

A recombinant protein encoded by the open reading frame of At4g37980 was 227

expressed as a carboxy-terminal His-tagged protein in E coli and was then purified 228

(Supplemental Fig S2) With straight chain saturated aldehydes ranging from one to ten 229

carbons in length the recombinant enzyme displayed highest activity with n-heptanal 230

(Fig 3) followed by n-hexanal n-octanal and then n-pentanal The enzyme showed 231

little activity toward formaldehyde and acetaldehyde Among unsaturated aldehydes 232

examined in this study (Z)-3-hexenal was the best substrate followed by (E)-2-hexenal 233

and (E)-2-pentenal and the six-carbon ketone n-hexan-2-one was a poor substrate The 234

enzyme exhibited relatively high activities with aromatic aldehydes such as 235

2-furaldehyde benzaldehyde and cinnamaldehyde but little activity was observed with 236

sinapyl aldehyde and coniferyl aldehyde which are intermediates in the lignin 237

biosynthesis pathway and are good substrates for AtCAD4 and AtCAD5 (Kim et al 238

2004) Activity profiles of enzyme from Arabidopsis leaves with seven representative 239

aldehydes were similar to those observed with the recombinant enzyme (Supplemental 240

Fig S3) The enzyme also obeyed Michaelis-Menten kinetics with (Z)-3-hexenal 241

n-heptanal and cinnamaldehyde (Supplemental Fig S4) and Km values were estimated 242

to be 327 840 and 846 microM respectively (Table II) Based on properties of the 243

enzyme encoded by At4g37980 we renamed the gene CINNAMALDEHYDE AND 244

HEXENAL REDUCTASE (CHR) 245

246

Involvement of CHR in GLV composition 247

To examine in vivo functions of CHR an Arabidopsis strain with a T-DNA 248

insertion in the third exon of At4g37980 (Salk 001773) was prepared (Supplemental Fig 249

S5) T-DNA-disrupted strains with the Col-0 background grew normally and did not 250

differ from the parent strain (Col-0 Supplemental Fig S5C) but because Arabidopsis 251

Col-0 has a 10-bp deletion in the HPL gene its ability to form GLV aldehydes is 252

impaired (Duan et al 2005) Thus we crossed the T-DNA-disrupted line (chr hpl) with 253

Arabidopsis No-0 carrying active HPL (CHR HPL) and a strain that was homozygous 254

for the At4g37980 T-DNA insertion and the functional HPL gene (chr HPL) was 255

selected from the F2 generation for further analysis (Supplementary Fig S5) In the 256 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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presence of NADPH enzymatic reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in crude 257

homogenates from chr hpl and chr HPL plants was suppressed to approximately 14 of 258

that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants (Fig 4) Activities with NADH in 259

extracts from chr hpl and chr HPL plants however were not significantly different 260

from that for Col-0 (CHR hpl) and No-0 (CHR HPL) plants 261

Intact leaves from No-0 (CHR HPL) or mutant (chr HPL) plants hardly 262

emitted volatile compounds but partial mechanical wounding promoted a GLV burst in 263

these leaves (Fig 5) The resulting n-hexanal and (Z)-3-hexenal quantities did not differ 264

significantly between No-0 (CHR HPL) and the mutant (chr HPL) whereas the amounts 265

of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol were significantly lower from the 266

mutant (chr HPL) plants Moreover the amount of 1-penten-3-ol which was formed 267

depending on LOX but not on HPL (Mochizuki et al 2016) was relatively unaffected 268

by the presence of functional CHR Because total amounts of GLVs differed between 269

these two genotypes we investigated whether their abilities to form C6-aldehydes 270

through LOX and HPL also differ After completely disrupting the leaves of No-0 (CHR 271

HPL) and mutant (chr HPL) plants the GLV pathway was mostly arrested after the HPL 272

step that forms C6-aldehydes and reduction reactions hardly proceeded largely due to 273

NADPH deficiencies (Matsui et al 2012) Moreover formation of (Z)-3-hexenal 274

1-penten-3-ol (E)-2-hexenal and (Z)-2-penten-1-ol was observed from both genotypes 275

and (Z)-3-hexen-1-ol n-hexan-1-ol and (Z)-3-hexen-1-yl acetate were detected at low 276

levels in both No-0 (CHR HPL) and mutant (chr HPL) leaves (Supplemental Fig S6) 277

Quantities of (Z)-3-hexenal that were formed after complete disruption of mutant leaves 278

were slightly higher than that for No-0 leaves suggesting that the ability to form 279

(Z)-3-hexenal from lipidsfatty acids was not the prominent cause of lowered 280

(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate production after partial mechanical 281

wounding of chr HPL leaves 282

283

CHR is involved in indirect defenses 284

We observed flight responses of the parasitoid wasp C vestalis to the volatiles 285

emitted from Arabidopsis leaves that had been infested for 24 h with larvae of the 286

diamond back moth P xylostella In experiments with No-0 (CHR HPL) C vestalis 287

females preferred P xylostella-damaged plants over intact plants but did not show any 288

preference when Col-0 (CHR hpl) plants were used (Fig 6A) No preference was 289 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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observed with the mutant (chr HPL) either and numbers of wasps that landed on P 290

xylostella-infested plants and intact plants did not differ significantly Examinations of 291

wasp preferences also showed significant preferences for herbivore-infested No-0 (CHR 292

HPL) plants over herbivore-infested mutant (chr HPL) plants Validating experiments 293

showed similar areas of consumption by P xylostella on leaves of CHR HPL and chr 294

HPL plants (Supplemental Fig S7) 295

Quantities of volatiles emitted at 24 h after the onset of P xylostella 296

infestation of mutant (chr HPL) and No-0 (CHR HPL) plants were quite low and the 297

volatiles (Z)-3-hexenal and (Z)-3-hexen-1-ol were detected under the experimental 298

conditions employed here with the selected ion monitoring mode of GC-MS In these 299

analyses (Z)-3-hexen-1-yl acetate contents were below the detection limit (lt 08 ng in 300

glass volatile collection jar) Quantities of (Z)-3-hexenal emitted from the two 301

genotypes at 24 h after the onset of P xylostella infestation were also almost the same 302

whereas that of (Z)-3-hexen-1-ol was significantly lower from infested mutant (chr 303

HPL) plants than from infested No-0 (CHR HPL) plants (Fig 6B) 304

305

Involvement of CHR in detoxification of hexenal 306

The chlorophyll fluorescence parameter FvFm was used in a bioassay as an 307

indicator of cell deterioration When Col-0 (CHR hpl) plants were exposed to 308

(Z)-3-hexenal vapor at 133 or 267 nmol cm-3

FvFm remained relatively unchanged 309

FvFm decreased significantly when Col-0 plants were exposed to 400 nmol cm-3

310

(Z)-3-hexenal (Fig 7A) as we observed previously (Matsui et al 2012) Mutant plants 311

(chr hpl) treated with 133 nmol cm-3

(Z)-3-hexenal maintained normal FvFm values 312

however FvFm decreased significantly after 4 h of treatment with 267 nmol cm-3 313

(Z)-3-hexenal Following treatment with 400 nmol cm-3

(Z)-3-hexenal FvFm of the 314

mutant plants decreased more quickly than that of Col-0 (CHR hpl) and after 175 h 315

FvFm of the mutant plants (chr hpl) was significantly lower than that of Col-0 (CHR 316

hpl) Both Col-0 (CHR hpl) and the mutant (chr hpl) plants showed no visible 317

symptoms after treatment with 133 nmol cm-3

(Z)-3-hexenal (Fig 7B) However 318

treatment with 267 nmol cm-3

(Z)-3-hexenal caused the leaves of mutant plants to wilt 319

slightly and their curl at their margins and 400 nmol cm-3

(Z)-3-hexenal treatment 320

caused obvious damage in both genotypes with more serious effects seen in the mutant 321

plants 322 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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323

324

DISCUSSION 325

In this study we confirmed that At4g37980 which had been previously designated 326

CAD7 encodes an NADPH-dependent aldehyde reductase that prefers aliphatic 327

aldehydes of five to eight carbons in length and simple aromatic aldehydes as substrates 328

Crude extracts from leaves of the At4g37980 T-DNA knockout mutant had significantly 329

lower enzyme activities to oxidize NADPH and reduce (Z)-3-hexenal Furthermore 330

knockout mutant plants with active HPL formed significantly lower concentrations of 331

(Z)-3-hexen-1-ol and (Z)-3-hexen-1-yl acetate after partial mechanical wounding of 332

leaves (Z)-3-Hexen-1-ol formation after herbivore infestation was also suppressed in 333

knockout mutant plants with active HPL These results indicate that the enzyme 334

encoded by At4g37980 converts (Z)-3-hexenal to (Z)-3-hexen-1-ol during GLV-bursts 335

that are elicited by partial wounding of Arabidopsis leaves Accordingly we renamed 336

the gene CINNAMALDEHYDE AND HEXENAL REDUCTASE (CHR) 337

CHR was originally designated a member (AtCAD7) of the cinnamyl alcohol 338

dehydrogenase (CAD) gene family which comprises nine putative AtCAD genes 339

[AtCAD1ndash9 according to the classification by Kim et al (2004)] (Sibout et al 2003 340

Kim et al 2004) Kim et al (2004) showed that recombinant CHR (AtCAD7) activities 341

against intermediates of the lignin biosynthetic pathway such as p-coumaryl aldehyde 342

caffeyl aldehyde coniferyl aldehyde 5-hydroxyconiferyl aldehyde or sinapyl aldehyde 343

were considerably lower than those of recombinant AtCAD4 or AtCAD5 enzymes 344

AtCAD4 and 5 were later confirmed to be CADs of the lignin biosynthesis pathway in 345

floral stems of Arabidopsis as indicated by lignin biosynthetic activities in 346

corresponding knockout mutant lines (Sibout et al 2005) In agreement Klason lignin 347

contents in T-DNA knockout mutants of CHR (AtCAD7) were similar to those in 348

corresponding wild-type Arabidopsis (Eudes et al 2006) Taken with these 349

observations this study indicates that CHR has limited involvement in lignin 350

biosynthesis and rather that its primary role is to convert (Z)-3-hexenal to 351

(Z)-3-hexen-1-ol during GLV-bursts 352

BLASTP searches were performed against NCBI Protein Reference 353

Sequences and those with BLAST scores of more than 250 and with biochemically 354

confirmed functions were chosen A phylogenetic tree was then constructed with these 355 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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sequences and those of AtCAD 1ndash9 (Fig 8) We focused on two clades of the tree and 356

the first of these the CAD clade comprises Oryza sativa OsCAD2 (Zhang et al 2006) 357

Populus tremula times Populus alba PtrCAD1 (Van Acker et al 2017) Arabidopsis 358

AtCAD4 and 5 (Sibout et al 2005 Anderson et al 2015) Zea mays ZmCAD2 (Chen 359

et al 2012) and Sorghum bicolor SbCAD2 (Saballos et al 2009 Sattler et al 2009) 360

In vivo functions of all of these as CADs of the lignin synthesis pathway have been 361

confirmed in expression studies of respective mutant and wild-type genes Arabidopsis 362

CHR was located in another clade with oxidoreductases of terpenoid biosynthesis 363

including Catharanthus roseus tetrahydroalstonin synthase (Stavrinides et al 2015) C 364

roseus tabersonin 3-reductase (Qu et al 2015) C roseus 8-hydroxygeraniol 365

dehydrogenase (Krithika et al 2015) Ocimum basilicum geraniol dehydrogenase 366

(Iijima et al 2006) and Zingiber officinale geraniol dehydrogenase (Iijima et al 2014) 367

as proteins with biochemically confirmed functions Populus tremuloides sinapyl 368

alcohol dehydrogenase (Bomati et al 2005) and AtCAD6 and 8 (Kim et al 2004) 369

which were thought to be involved in the lignin biosynthesis were also located within 370

the clade however their in vivo functions have not been completely confirmed and for 371

example AtCAD8 knockout mutants showed no differences in lignin contents compared 372

with that in corresponding wild-type plants even though recombinant AtCAD8 had 373

slight activities against intermediates of the lignin biosynthetic pathway (Eudes et al 374

2006) Accordingly we propose that the genes in the CAD clade are true CADs and are 375

involved in monolignol synthesis whereas those encoding oxidoreductases of the 376

specialized metabolism (ORSM) clade are less involved in monoliginol synthesis but 377

take primary roles as oxidoreductases that contribute to the biosynthesis of specialized 378

metabolites In further studies TBLASTN searches were performed using CHR as a 379

query with the whole genome sequences of six representative plant species (Populus 380

trichocarpa Glycine max Solanum lycopersicum Zea mays Oryza sativa and 381

Marchantia polymorpha) Subsequent phylogenetic tree analysis of genes with BLAST 382

scores of more than 250 also gave a tree with two clades comprising ORSM family and 383

true CAD family members (Supplementary Fig S8) These observations imply that 384

ancestral genes for oxidoreductases functionally diversified into specialized metabolism 385

and lignin biosynthesis warranting detailed studies of in vitro and in vivo functions of 386

the present genes 387

Formerly NADH-dependent alcohol dehydrogenase (ADH EC 1111) was 388 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


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thought to be involved in the reduction of (Z)-3-hexenal to (Z)-3-hexen-1-ol in the GLV 389

biosynthetic pathway (Lai et al 1982 Bicsak et al 1982 Dolferus amp Jacobs 1984) 390

Accordingly several studies examined the effects of mutations or genetic manipulation 391

of these ADH genes on volatiles Overexpression or antisense-suppression of SlADH2 in 392

tomatoes led to higher or lower amounts of C6-alcohols respectively formed after 393

complete tissue disruption (Speirs et al 1998 Prestage et al 1999) Given that ADH 394

from tomato fruits reduced C6-aldehydes in vitro (Bicsak et al 1982) and that SlADH2 395

was highly expressed in fruits (Van Der Straeten et al 1991) SlADH2 is likely 396

involved in the reduction of C6-aldehydes at least in completely disrupted tissue GLVs 397

that formed from completely disrupted leaves of Arabidopsis adh1 mutants showed 398

reduced amounts (about 50 of that in the parental line) of C6-alcohols and quantities 399

of (E)-2-hexenal were approximately 10-fold higher than those detected in wild-type 400

plants (Bate et al 1998) Overexpression of VvADH2 in grapevines also resulted in 401

increased 2-hexenal and hexan-1-ol contents of leaves that were disrupted through 402

freezethaw treatment and subsequent buffer extraction whereas antisense-suppression 403

did not affect the concentrations of these metabolites (Tesniere et al 2006) These 404

results suggest that NADH-dependent ADH is at least partly involved in the reduction 405

of C6-aldehydes to C6-alcohols in totally disrupted plant tissues As we reported 406

previously C6-aldehydes are mostly formed in disrupted tissues after partial mechanical 407

wounding of leaves and diffuse into neighboring intact tissues where reduction 408

proceeds to form C6-alcohols in a NADPH-dependent manner (Matsui et al 2012) 409

This study indicates that CHR is largely involved in the reduction of C6-aldehydes to 410

C6-alcohols in partially wounded Arabidopsis leaf tissue Accordingly the reduction of 411

C6-aldehydes into C6-alcohols in totally and partially disrupted Arabidopsis tissue is 412

performed by different enzymes such as NADH-dependent alcohol dehydrogenases and 413

NADPH-dependent CHR respectively Further studies are expected to extend these 414

findings into other plant species 415

GLVs are employed by some herbivores to find food and by some predators 416

and parasitoids to find their prey (Matsui 2006 Scala et al 2013) Arthropod behaviors 417

vary depending on each component of GLVs For example C glomerata is a parasitoid 418

of Pieris rapae larvae and was attracted by (Z)-3-hexen-1-yl acetate and (E)-2-hexenal 419

but comparatively not by (Z)-3-hexen-1-ol (Shiojiri et al 2006b) Higher (E)(Z) ratios 420

of GLVs formed by Manduca sexta-infested Nicotiana attenuata resulted in greater egg 421 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


15

predation by Geocoris spp (Allmann amp Baldwin 2011) (E)-2-Hexenal was more 422

attractive to C vestalis a parasitoid of Plutella xylostella larvae and (Z)-3-hexen-1-ol 423

was favored under certain conditions (Yang et al 2016) In this context it is assumed 424

that adjustment of GLV composition is beneficial for plants allowing management of 425

both beneficial and harmful arthropods We demonstrated that CHR in Arabidopsis 426

plants is involved in adjustment of GLV composition and showed that (Z)-3-hexen-1-ol 427

production in response to infestations of P xylostella larvae was lower in knockout 428

mutant (chr HPL) plants than that in wild-type (CHR HPL) plants The volatiles emitted 429

from the P xylostella-infested No-0 (CHR HPL) plants were also sufficient to attract C 430

vestalis However C vestalis failed to distinguish between mutant (chr HPL) 431

Arabidopsis plants that were infested with P xylostella larvae and intact mutant plants 432

C vestalis also failed to distinguish between infested and intact plants that lacked GLV 433

formation (Col-0 CHR hpl) These observations indicate that (Z)-3-hexen-1-ol is 434

required for C vestalis to find its prey on Arabidopsis plants suggesting that CHR plays 435

a role in this Arabidopsis plant-herbivore-carnivore system 436

We previously reported that exogenous (Z)-3-hexanal vapor was harmful to 437

Arabidopsis plants (Matsui et al 2012) likely because the aldehyde moiety is potently 438

reactive and is readily oxidized to 4-hydroxyoxo-(E)-2-hexenal which is highly 439

reactive with biological molecules (Farmer amp Mueller 2013) Herein we showed that 440

exposure of mutant (chr hpl) Arabidopsis plants to (Z)-3-hexenal vapor led to 441

significant malfunctions of photosynthesis likely due to their inability to reduce 442

(Z)-3-hexenal Particularly high concentrations of (Z)-3-hexenal were used in the vapor 443

phase in this study specifically 267 and 400 nmol cm-3

at which a difference in 444

susceptibility between the mutant (chr hpl) and Col-0 (CHR hpl) plants was observed 445

However the local concentration of (Z)-3-hexenal in the liquid phase of disrupted 446

tissues can reach up to ca 15 micromol cm-3

(Matsui et al 2012 Mochizuki et al 2016) 447

therefore intact leaf tissues adjacent to disrupted tissues should cope with high 448

concentrations of (Z)-3-hexenal Thus the ability of CHR to reduce (Z)-3-hexenal is one 449

of the systems required to avoid the toxicity of potentially harmful GLV-aldehydes at 450

least under the assay conditions employed in this study Nevertheless ecophysiological 451

significance of (Z)-3-hexenal-detoxification by CHR should be confirmed by further 452

studies These may include direct determination of (Z)-3-hexenal concentration in intact 453

leaf tissues adjacent to tissues disrupted by partial wounding direct determination of the 454 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


16

amount of (Z)-3-hexenal that is taken into intact Arabidopsis leaf tissues from the vapor 455

phase or investigation of the precise mechanism through which (Z)-3-hexenal has 456

harmful effects on leaf tissues 457

458

459

EXPERIMENTAL PROCEDURES 460

Plant materials 461

Arabidopsis thaliana (L) Heynh ecotype Col-0 (CHR hpl) No-0 (CHR HPL) and 462

T-DNA tagged mutant (chr hpl background Col-0 Salk_001773) were obtained from 463

the Arabidopsis Biological Resource Center (ABRC Columbus OH USA) Because 464

wild-type Col-0 has a deletion in its HPL gene and lacks the ability to form green leaf 465

volatiles (Duan et al 2005) a chr hpl mutant with the Col-0 background 466

(Salk_001773) was crossed with No-0 and homozygotes with the T-DNA-disrupted 467

CHR gene (chr mutant) and the active HPL gene (chr HPL) were isolated from the F2 468

population using genomic PCR analyses with the primers listed in Supplementary Table 469

S1 Plants were germinated in soil (Tanemaki Baido Takii Seeds Kyoto Japan) in 470

plastic pots (6 cm id) were incubated at 4degC in the dark for 2ndash3 days and were then 471

grown in a chamber at 22degC under fluorescent lights (60 micromol m-2

s-1

) with a 14-h 472

light10-h dark photoperiod 473

474

475

Enzyme assays 476

Hexenal reductase activity was monitored according to the oxidation of NADPH by 477

continuously following decreases in absorption at 340 nm using a photometer 478

(UV-160A Shimadzu Kyoto Japan) at 25degC Oxidation of NADH was also monitored 479

using absorption at 340 nm (Z)-3-Hexenal was dispersed in 1 (wv) Tween 20 to a 480

concentration of 40 mM using a tip-type ultrasonic disruptor (UD-211 Tomy Seiko 481

Tokyo Japan) Suspensions were kept under nitrogen gas at minus20degC until use In typical 482

assays 10-microL aliquots of 40 mM (Z)-3-hexenal in 1 (wv) Tween 20 were mixed with 483

the enzyme in 50 mM Tris-HCl (pH 80 1 mL total) Slight changes in background 484

absorption at 340 nm likely reflected instability of the aqueous emulsion and were 485

recorded for 2ndash4 min prior to starting the reaction with the addition of 10-microL aliquots of 486

10 mM NADPH (Oriental Yeast) The aldehydes used for determinations of substrate 487 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


17

specificity (Fig 2) are listed in Supplementary Fig S9 and all except sinapaldehyde 488

and coniferaldehyde were suspended in 1 (wv) Tween 20 as 10 mM stock solutions 489

Sinapaldehyde and coniferaldehyde were dissolved in dimethyl sulfoxide to prepare 20 490

mM stock solutions At 1 (vv) dimethylsulfoxide showed little effect on CHR 491

activity with (Z)-3-hexenal To estimate Km and Vmax values SndashV plots were 492

constructed and analyzed using Hyper 32 (version 100) 493

Enzyme activities were also evaluated using GC-MS analyses of the products 494

Briefly enzymes were placed in 22-mL glass vials (Perkin Elmer Waltham MA USA) 495

with 10-microL aliquots of 20 mM (Z)-3-hexenal [suspended in 1 (wv) Tween 20] and 496

20-microL aliquots of 10 mM NADPH in buffer containing 50 mM Tris-HCl (pH 80 total 497

volume 1 mL) Vials were then tightly closed with rubber septums and mixtures were 498

incubated at 28degC for 5 min Reactions were terminated by adding 1 mL of saturated 499

CaCl2 solution Vials were then sealed tightly with a butyl stopper and a crimp-top seal 500

(National Scientific Rockwood TN USA) and solid phase microextraction (SPME) 501

fibers (5030 m DVBCarboxenPDMS Supelco Bellefonte PA USA) were exposed 502

to headspaces of vials for 30 min at 60degC Fibers were then inserted into the port of a 503

GC-MS (QP-5050 Shimadzu Kyoto Japan) instrument equipped with a 025 mm times 30 504

m Stabiliwax column (film thickness 025 microm Restek Bellefonte PA USA) The 505

column temperature was programmed as follows 40degC for 1 min increasing by 15degC 506

min-1

to 180degC then 180degC for 1 min The carrier gas (He) was delivered at a flow rate 507

of 1 mL min-1

The glass insert was an SPME Sleeve (Supelco) and splitless injections 508

were performed with a sampling time of 1 min To remove all compounds from the 509

matrix the fiber was held in the injection port for 10 min Injector and interface 510

temperatures were 200C and 230C respectively The mass detector was operated in 511

electron impact mode with an ionization energy of 70 eV Compounds were identified 512

using retention indexes and MS profiles of corresponding authentic specimens and 513

quantitative analyses were performed with standard curves that were generated for each 514

compound using aqueous suspensions containing Tween 20 Standard solutions were 515

mixed at various ratios and were then mixed with 1-mL aliquots of saturated CaCl2 516

solution in a glass vial Volatiles were analyzed using SPME-GC-MS as described 517

above and calibration curves were constructed for each compound 518

519



18

Leaves (350 g fresh weight FW) were harvested from Arabidopsis (No-0) plants that 521

were grown for 40ndash60 days and were frozen in liquid nitrogen and then homogenized 522

in 05 L of 01 M Tris-HCl (pH 80) containing 4 mM dithiothreitol and 1 mM 523

phenylmethane sulfonyl fluoride using a juicer-mixer (TM837 TESCOM Tokyo 524

Japan) Debris was removed by squeezing juices through two layers of bleached cotton 525

cloth and the remaining juice was centrifuged at 10000 rpm (R12-2 rotor Hitachi Koki 526

Tokyo Japan) at 4degC for 20 min to afford crude extract as the supernatant The crude 527

extract was then fractionated in ammonium sulfate (40ndash60 saturation) and was 528

dissolved in 30 saturated ammonium sulfate in Buffer A containing 10 mM Tris-HCl 529

(pH 80) and 4 mM dithiothreitol 530

After removing insoluble materials by centrifuging at 10000 rpm (T15A36 531

rotor Hitachi Koki) at 4degC for 10 min supernatants were applied to a Toyopearl Butyl 532

650M column (28 times 18 cm Tosoh Yamaguchi Japan) equilibrated with 30 saturated 533

ammonium sulfate in Buffer A The column was then washed with 02 L of equilibration 534

buffer and enzyme was eluted using a linear gradient of 30ndash0 saturated ammonium 535

sulfated in Buffer A (total 03 L) Fractions with substantial activity were combined 536

desalted using a PD-10 column (GE Healthcare Life Sciences Little Chalfont UK) and 537

equilibrated with Buffer A and were then applied to Bio-Scale Mini CHT 40-microm 538

Cartridges (5 mL Bio-Rad Laboratories Hercules CA USA) equilibrated with 10 mM 539

potassium phosphate buffer (pH 80) containing 4 mM dithiothreitol After washing 540

columns with 30 mL of the same buffer enzymes were eluted with a linear gradient of 541

10ndash400 mM potassium phosphate buffer (pH 80) supplemented with 4 mM 542

dithiothreitol 543

Fractions with substantial activity were combined concentrated using an 544

Amicon Ultra-15 (Merck Darmstadt Germany) and were then desalted in a PD-10 545

column with Buffer A Active fractions were applied to HiTrap DEAE Sepharose 546

Fast-Flow resin (1 mL GE Healthcare Life Sciences) equilibrated with Buffer A After 547

washing with 10 mL of Buffer A enzyme was eluted with a linear gradient of 0ndash05 M 548

NaCl in Buffer A Hexenal reductase activity was determined in every fraction and 549

SDS-PAGE analyses revealed a protein band that correlated well with enzyme activity 550

(see Fig 1A) 551

After purification the protein band of 38 kDa excised from the SDS-PAGE 552

gel was digested in-gel using trypsin Trypsin-digested peptides were then analyzed 553 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


19

using HPLC-MSMS and the protein was identified LC-MSMS analyses were 554

performed using a linear ion trap time-of-flight mass spectrometer (LITndashTOF MS 555

NanoFrontier eLD Hitachi High-Technologies Co Tokyo Japan) coupled to a 556

nanoflow HPLC instrument (NanoFrontier nLC Hitachi High-Technologies 557

Corporation) Trypsin-digested peptides were then separated using a MonoCap C18 558

Fast-flow column (005 times 150 mm GL Science Tokyo Japan) and were eluted with a 559

linear gradient of 2ndash40 (vv) solvent B over 60 min at a flow rate of 200 nL min-1

560

Solvent A was 2 (vv) acetonitrile and 01 (vv) formic acid and solvent B was 98 561

(vv) acetonitrile with 01 (vv) formic acid The eluent was ionized using a 562

nano-electrospray ionization source equipped with an uncoated SilicaTip (New 563

Objective Woburn MA USA) and was analyzed using LITndashTOF MS Mass spectra 564

were obtained in positive ion mode at scan mass range of mz 100ndash2000 MSMS 565

spectra were generated by collision-induced dissociation in the linear ion trap To 566

identify the protein MS and MSMS data were converted to a MGF file using 567

NanoFrontier eLD Data Processing software (Hitachi High-Technologies Co) and were 568

then analyzed using the MASCOT MSMS Ions Search (httpwwwmatrixsciencecom) 569

with the following parameters database NCBInr enzyme trypsin missed cleavages 3 570

taxonomy all entries fixed modifications carbamidomethyl (C) variable modifications 571

oxidation (HW) and oxidation (M) peptide tolerance 02 Da MSMS tolerance 02 572

Da Peptide charge 1+ 2+ and 3+ and Instrument ESI-TRAP 573

574

cDNA cloning 575

Total RNA was extracted from mature Arabidopsis (Col-0) leaves using FavorPrep Plant 576

Total RNA Purification Mini Kits (Favorgen Biotech Ping-Tung Taiwan) DNA was 577

degraded using DNA-free Kits (Ambion Thermo Fischer Scientific Waltham MA 578

USA) and cDNA was synthesized using SuperScript VILO cDNA Synthesis Kits 579

(Invitrogen) Subsequently CHR cDNA was PCR-amplified using the primers 580

5prime-acatATGGGAAAGGTTCTTGAGAAG-3prime and 581

5prime-gaagcttAGGAGTTGGCTTCATCGTGT-3prime which were designed according to the 582

sequence of the gene (At4g37980) The resulting PCR products were cloned into pGEM 583

T-easy vectors (Promega Madison WI USA) for sequencing The PCR product was 584

then sub-cloned into the NdeI-HindIII site of the pET24a vector (Merck) and the 585

resulting plasmid was transfected into Escherichi coli Rosetta2 cells (Merck) Cells 586 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


20

were grown in Luria broth supplemented with kanamycin (50 microg mL-1

) and 587

chloramphenicol (30 microg mL-1

) at 37degC until the optical density at 600 nm reached 06ndash588

08 After chilling cultures on ice for 15 min isopropyl -D-1-thiogalactosylpyranoside 589

was added to a concentration of 05 mM and cells were then cultured at 16degC for 16 h 590

Cells were recovered by centrifugation at 5000 times g for 5 min at 4degC and were 591

resuspended in 25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol 592

045 M KCl 01 mM phenylmethane sulfonyl fluoride and 50 microg mL-1

lysozyme After 593

incubating on ice for 15 min with intermittent swirling suspended cells were disrupted 594

with a tip-type ultrasonic disruptor (UD-211 Tomy Seiko Tokyo Japan) Lysates were 595

then cleared with centrifugation at 10000 times g for 10 min at 4degC and were directly 596

applied to a His-Accept (Nacalai Tesque Kyoto Japan) column pre-equilibrated with 597

25 mM Tris-HCl (pH 75) containing 01 (vv) 2-mercaptoethanol and 045 M KCl 598

After washing the column in the same buffer the recombinant Arabidopsis CHR 599

enzyme was eluted with the same buffer supplemented with 50 mM imidazole 600

601

Volatiles from leaves 602

Above-ground parts of 37-day-old Arabidopsis plants with fully developed leaves were 603

cut off with a razor blade weighed and then carefully placed in glass jars (200 mL 63 604

mm id times 70 mm) Cut surfaces were immersed in distilled water in microtube caps to 605

avoid desiccation The leaves were left intact or were partially (125 of total leaf area) 606

wounded using forceps without injuring the mid vein The jar was then tightly closed 607

and an SPME fiber (5030 microm DVBCarboxenPDMS Supelco) was exposed to the 608

headspace of the jar for 30 min at 25degC Volatiles collected on the fiber were analyzed 609

using GC-MS under the conditions used in enzyme assays Molecular ion 610

chromatograms with mz of 56 (for n-hexanal n-hexan-1-ol) 69 [for (Z)-3-hexenal] 611

and 82 [(Z)-3-hexen-1-yl acetate (Z)-3-hexen-1-ol and (E)-2-hexen-1-ol] were used for 612

quantification Calibration curves were constructed from GC-MS analyses under the 613

same conditions by placing known amounts of authentic compounds [suspended in 614

01 (wv) Tween 20] onto the tips of cotton swabs in the glass jar To analyze volatiles 615

formed in thoroughly disrupted leaf tissues leaves (about 60 mg FW) were 616

homogenized in 1-mL aliquots of 50 mM MES-KOH (pH 55) for 1 min using a mortar 617

and pestle To determine volatiles in intact leaves buffer was replaced with solution 618

containing 1 g mL-1

CaCl2 to arrest enzyme activities Homogenates were then 619 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


21

transferred into tubes by rinsing the mortar with 05-mL aliquots of the same buffer 620

twice Volatile compounds in homogenates were extracted with 2 mL of methyl 621

tert-butyl ether containing 1 microg mL-1

of nonanyl acetate GC-MS analyses were then 622

performed as described above 623

To collect volatiles three pots with CHR HPL (No-0) and chr HPL plants that 624

had been infested for 24 h with P xylostella larvae were placed in a glass jar (85 cm 625

diameter times 11 cm) with a tight cover and SPME fibers were exposed to headspaces for 626

30 min at 25degC Volatiles were then analyzed using GC-MS under the conditions 627

described above but in the selected ion monitoring (SIM) mode with mz 6910 628

[C6H10O-CHO]+ and 8210 [C6H12O-H2OC8H14O2-C2H4O2]

+ 629

630

Flight responses of C vestalis 631

P xylostella larvae were collected from crucifer crops in a field in Kyoto City Japan 632

and were reared in a climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) 633

with a 16-h photoperiod on leaves of Komatsuna (Brassica rapa var perviridis) C 634

vestalis from parasitized host larvae were collected from a field in Kyoto Japan Adult 635

parasitoids were fed honey and were maintained in plastic cages (25 times 35 times 30 cm) in a 636

climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) with a 16-h photoperiod 637

for 3 d to ensure mating Females were then individually transferred to glass tubes of 20 638

mm diameter and 130 mm length containing honey and were kept in a 639

climate-controlled room (18 plusmn 2degC 50ndash70 relative humidity) under continuous 640

darkness Females were never kept for more than 10 d At least 1 h before the start of 641

each experiment oviposition-inexperienced female wasps were transferred to another 642

climate-controlled room (25 plusmn 2degC 50ndash70 relative humidity) and were exposed to 643

continuous light 644

Two groups of three pots containing 4ndash5-week-old non-flowering Arabidopsis 645

plants with fully developed leaves were positioned about 25 cm apart in a cage (25 times 35 646

times 30 cm) with three windows covered by a nylon gauze and a door for introducing 647

plants and wasps To determine the effects of P xylostella infestation on CHR HPL 648

(No-0) CHR hpl (Col-0) and chr hpl Arabidopsis plants five second-instar larvae of P 649

xylostella were placed onto one group (three pots) of plants After 24 h larvae and feces 650

were removed and the cleaned plants were used as infested plants The other group of 651

plants was left for 24 h without larvae Ten wasps were then released halfway between 652 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


22

the two groups of plants and the first landing by each wasp on a plant was recorded as 653

its choice of the two groups Upon landing on a plant wasps were immediately removed 654

from the cage using an insect aspirator If the wasp did not land on any of the plants 655

within 30 min it was recorded as no-choice The trial was repeated for four times and 656

plants were replaced for each trial Preferences of female C vestalis for volatiles 657

emitted from CHR HPL (No-0) and chr HPL that had been infested with P xylostella 658

for 24 h were also assessed 659

660

Susceptibility to (Z)-3-hexenal vapor 661

Maximum quantum yields of photosystem II (PSII) were estimated from chlorophyll 662

fluorescence measurements using a pulse amplitude modulated (PAM) fluorometer 663

(Mini-PAM photosynthesis yield analyzer Walz Effeltrich Germany) PSII yields 664

were calculated as ratios of FVFM (Maxwell amp Johnson 2000) The saturation pulse 665

duration was 08 s and was applied with an intensity of approximately 8300 mmol m-2

666

s-1

Four plants (Col-0 and chr hpl 39 days old) were placed inside a glass cylinder 667

(2800 cm3) and 112-microL aliquots of CH2Cl2 containing 033 067 or 10 M 668

(Z)-3-hexenal were applied to cotton swabs that were placed 5 cm above plant canopies 669

The flask was then tightly sealed with a glass plate and was incubated at 22degC in the 670

light (60 micromol m-2

s-1

) For each plant fluorescence measurements were conducted at 671

the midpoint of four fully matured leaves immediately before treatment or at 4 or 175 h 672

after the onset of treatment The plants were dark-acclimated for at least 15 min before 673

PAM analysis 674

675

Phylogenetic analysis 676

Amino acid sequences of selected proteins were initially aligned using the multiple 677

sequence alignment program MUSCLE (Edgar 2004) at EMBL-EBI The resulting 678

alignments were inspected and manually edited to remove gaps and unconserved 679

regions using the alignment editing program AliView 121 (Larsson 2014) 680

Phylogenetic trees were constructed using the phylogeny analysis program MrBayes 681

326 (Huelsenbeck and Ronquist 2001) under the WAG model 682

683

ACCESSION NUMBERS 684

Sequence data from this article can be found in the GenBankEMBL data libraries under 685 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


23

accession numbers NP_195511 686

687

688

689

690

691


24

SUPPLEMENTAL DATA 692

693

The following supplemental materials are available 694

695

Supplemental Figure S1 Amino acid sequences as determined using MASCOT 696

697

Supplemental Figure S2 SDS-PAGE analysis of recombinant Arabidopsis CHR 698

protein with a His-tag following isolation from E coli 699

700

Supplemental Figure S3 Substrate specificity of CHR purified from Arabidopsis 701

leaves 702

703

Supplemental Figure S4 SndashV plots of recombinant Arabidopsis CHR with 704

(Z)-3-hexenal n-heptanal and cinnamaldehyde 705

706

Supplemental Figure S5 CHR and HPL loci and characterization of wild-type and 707

mutant plants 708

709

Supplemental Figure S6 Formation of GLVs after complete disruption of Arabidopsis 710

leaves 711

712

Supplemental Figure S7 Consumption of leaves by P xylostella larvae 713

714

Supplemental Figure S8 Phylogenetic tree of Arabidopsis CHR and related genes 715

716

Supplemental Figure S9 Carbonyls used as substrates for enzyme assays 717

718

Supplemental Table S1 Primer sequences used for genotyping 719

720

Supplemental Table S2 A list of genes used for constructing phylogenetic tree in Fig 721

8 722

723

Acknowledgements 724 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


25

In this research we used the supercomputer of ACCMS Kyoto University Japan for 725

phylogenetic analyses The authors are thankful to Mr Brian St Aubin at Michigan 726

State University for English proofreading 727

728

FIGURE LEGENDS 729

Figure 1 The Green leaf volatile synthetic pathway in plants 730

Linolenic acid or linolenoyl residues of lipids are oxygenated by lipoxygenase (LOX) to 731

yield corresponding hydroperoxides These are cleaved by hydroperoxide lyase (HPL) 732

to form six-carbon aldehydes and twelve carbon -oxo carboxylic acids (or esters when 733

lipids are the starting material) A portion of (Z)-3-hexenal is spontaneously or 734

enzymatically isomerized to form (E)-2-hexenal and a portion of (Z)-3-hexenal is 735

reduced by reductases to (Z)-3-hexen-1-ol Further acetylation leads to the formation of 736

(Z)-3-hexen-1-yl acetate 737

738

Figure 2 Purification and properties of hexenal reductase 739

A protein profile of the fractions collected using HiTrap DEAE chromatography Equal 740

volumes of fractions were loaded onto each lane and proteins were stained with 741

Coomassie Blue (Z)-3-Hexenal reductase activities of each fraction (from fraction 5 to 742

11) are shown The 38-kDa protein used in amino acid sequence analyses is indicated 743

with a white arrowhead Mr molecular weight marker B pH-activity profile of the 744

partially purified enzyme C GC-MS chromatograms of products formed from 745

(Z)-3-hexenal in the absence or presence of the cofactors NADH or NADPH D 746

quantities of (Z)-3-hexen-1-ol from C are presented as averages plusmn SE (n = 4) Because 747

(Z)-3-hexen-1-ol was not formed in the absence of cofactor the bars corresponding to 748

activity are indicated by nd not detected 749

750

Figure 3 Substrate specificity of recombinant Arabidopsis CHR expressed in E coli 751

Data are presented as averages plusmn SE (n = 4) 752

753

Figure 4 NADPH consumption depending on (Z)-3-hexenal in wild-type and mutant 754 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


26

Arabidopsis leaves 755

Activities were determined in four biological replicates (n = 4) and are presented as 756

averages plusmn SE Different letters indicate significant differences as identified using two 757

way ANOVA and Tukey post-hoc tests P lt 005 758

759

Figure 5 Formation and emission of volatiles from partially wounded Arabidopsis 760

leaves 761

A Representative chromatograms of volatiles from intact leaves (lower chromatogram 762

of each genotype) and partially wounded leaves (upper chromatogram of each 763

genotype) Wild-type (No-0 CHR HPL) and mutant (chr HPL) signals are shown in 764

black and red respectively The inset shows a representative image of mutant (chr hpl) 765

plant after partial wounding B Quantities of green leaf volatiles from intact and 766

partially wounded wild-type and mutant leaves Data are averages plusmn SE (n = 4) 767

Different letters represent significant differences as identified using two way ANOVA 768

and Tukeyrsquos post-hoc tests P lt 001 ns no significant difference 769

770

Figure 6 Role of CHR in C vestalis flight responses and in volatile emission following 771

P xylostella infestation 772

A Flight responses of C vestalis to P xylostella-infested (gray) and uninfested (white) 773

Arabidopsis CHR HPL (No-0) chr HPL and CHR hpl (Col-0) plants (40 individuals 774

above dashed line) and to P xylostella-infested chr HPL and CHR HPL (No-0) plants 775

(60 individuals below dashed line) NS no significantly difference 005 gt PTotal gt 776

001 (replicated G-test) C vestalis that did not choose either plant (lsquoNo choicersquo 777

subjects) were not included in the statistical analysis B (Z)-3-Hexenal and 778

(Z)-3-hexen-1-ol emissions from wild-type (No-0 CHR HPL) and mutant (chr HPL) 779

plants after 24-h infestation with P xylostella larvae Quantities of volatiles in 780

headspaces of glass vials containing three plants were collected after a 24-h infestation 781

period using solid phase microextraction (SPME) fibers for 30 min Data are presented 782

as averages plusmn SE (n = 3) P lt 001 as identified using Studentrsquos t-test Quantities of 783

(Z)-3-hexen-1-yl acetate were below the detection limit (lt 08 ng in the pot) 784

785

Figure 7 Changes in photosystem II activity in Arabidopsis wild-type (Col-0 CHR hpl) 786

and mutant (chr hpl) leaves during exposure to (Z)-3-hexenal 787 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


27

A Above-ground parts of Arabidopsis wild-type (white circles) and mutant (black 788

circles) plants were exposed to 0 133 267 or 400-nmol cm-3

(Z)-3-hexenal vapor in a 789

closed glass jars and photosystem II activity was estimated using the chlorophyll 790

fluorescence parameter FVFM Data are averages plusmn SE (n = 8) Differences were 791

identified using two-way ANOVA and Tukeyrsquos post-hoc test Different letters indicate 792

significant differences P lt 005 B Representative images of Col-0 (CHR hpl) and 793

mutant (chr hpl) plants at 175 h after the onset of exposure to 0 133 267 or 794

400-nmol cm-3

(Z)-3-hexenal vapor 795

796

Figure 8 Phylogenetic analysis of cinnamyl alcohol dehydrogenases (CADs) and 797

oxidoreductases involved in specialized metabolism (ORSMs) 798

Proteins are listed with BLASTP analyses that were performed using Arabidopsis CHR 799

as a query Proteins with BLAST scores of more than 250 were chosen and those 800

studied at least at the protein level were selected to construct the tree (Supplementary 801

Table S2) In vivo functions of the CADs in lignin synthesis have been characterized in 802

mutant expression studies for the gene indicated by blue stars The scale bar represents 803

009 amino acid substitutions per site 804

805


28

Table I Summary of the (Z)-3-hexenal reductase purification process from Arabidopsis leaves 806

Purification step Total protein Total activity Specific

activity

Purification Yield

mg nkat nkatmg -Fold

Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557

Butyl-Toyopearl 837 900 108 173 847

Hydroxyapatite 028 212 662 119 199

HiTrap DEAE 0031 394 788 209 037

807

808


29

Table II Km and Vmax values of recombinant Arabidopsis CHR with (Z)-3-hexenal n-heptanal and 809

cinnamaldehyde 810

Substrate Km (microM) Vmax (nkat mg-1

)

(Z)-3-Hexenal 327 plusmn 3090 347 plusmn 1280

n-Heptanal 840 plusmn 3451 286 plusmn 5253

Cinnamaldehyde 846 plusmn 7172 587 plusmn 1030

811

812

813

814


30

References 815

Allmann S Baldwin IT (2010) Insects betray themselves in nature to predators by 816

rapid isomerization of green leaf volatiles Science 329 1075-1078 817

Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC 818

Audenaert K (2017) Green leaf volatile production by plants a meta-analysis New 819

Phytol doi101111nph14671 820

Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch 821

M Chapple C (2015) Manipulation of guaiacyl and syringyl monomer biosynthesis 822

in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin 823

biosynthesis and modified cell wall structure Plant Cell 27 2195-2209 824

Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative 825

differences in C6-volatile production from the lipoxygenase pathway in an alcohol 826

dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104 827

Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase 828

purification and substrate specificity Arch Biochem Biophys 216 605-615 829

Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in 830

Populus tremuloides sinapyl alcohol dehydrogenase Plant Cell 17 1598-1611 831

Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S 832

Kumpatla S Zheng P (2012) Transposon insertion in a cinnamyl alcohol 833

dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol 834

Biol 80 289-297 835

DrsquoAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization 836

of a BAHD acyltransferase responsible for producing the green leaf volatile 837

(Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207 838

Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C 839

(2006) Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the 840

downstream lipoxygenase pathway in the tobacco hypersensitive response Plant 841

Physiol 140 1484-1493 842

Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis 843

thaliana (L) Heynh genetical and biochemical characterization Biochem Genet 22 844

817-838 845

Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 846

(hydroperoxide lyase) gene expression differentially affect hexenal signaling in the 847 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


31

Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 848

1529-1544 849

Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and 850

metabolic engineering of plant volatile organic compounds New Phytol 198 16-32 851

Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 852

throughput Nucl Acids Res 32 1792-1797 853

Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence 854

for a role of AtCAD1 in lignification of elongating stems of Arabidopsis thaliana 855

Planta 225 23-39 856

Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated 857

signaling Ann Rev Plant Biol 64 429-450 858

Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 859

34 1201-1218 860

Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic 861

trees Bioinformatics 17 754-755 862

Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation 863

of citral in the glands of sweet basil Arch Biochem Biophys 448 141-149 864

Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent 865

aroma compound in ginger rhizome (Zingiber officinale) molecular cloning and 866

characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534 867

Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) 868

Herbivore-induced volatile blends with both ldquofastrdquo and ldquoslowrdquo components provide 869

robust indirect defence in nature Funct Ecol 32 136-149 870

Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 871

7 improvements in performance and usability Mol Biol Evol 30 722-780 872

Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile 873

emissions in nature Science 291 2141-2144 874

Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) 875

Rapid activation of a novel plant defense gene is strictrly dependent on the 876

Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684 877

Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L 878

B Kang C Lewis N G (2004) Functional reclassification of the putative 879

cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad 880 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


32

Sci USA 101 1455-1460 881

Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal 882

activities of C6-aldehydes are important constitutents for defense responses in 883

Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132 884

Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in 885

neighbouring plants J Ecol 94 619-628 886

Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV 887

(2015) Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus 888

roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258 889

DOI101038srep08258 890

Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) 891

Identification of (Z)-3(E)-2-hexenal isomerases essential to the production of the 892

leaf aldehyde in plants J Biol Chem 191 14023-14033 893

Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three 894

non-allelic alcohol dehydrogenase isozymes in maize Biochim Biophys Acta 706 895

9-18 896

Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large 897

data sets Bioinformatics 30 3276-3278 898

Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin 899

metabolism Curr Opin Plant Biol 9 274-280 900

Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential 901

metabolisms of green leaf volatiles in injured and intact parts of a wounded leaf meet 902

distinct ecophysiological requirements PLoS ONE 7 e36433 903

doi101371journalpone0036433 904

Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp 905

Bot 51 659-668 906

Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the 907

production of green leaf volatiles by lipase-dependent and independent pathway 908

Plant Biotechnol 31 445-452 909

Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent 910

antibacterial action that act on both Gram-negative and Gram-positive bacterial J 911

Agric Food Chem 50 7639-7644 912

Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant 913 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


33

community perspective Plant Cell Environ 37 1845-1853 914

Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation 915

products Biochim Biophys Acta 1861 457-466 916

Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G 917

Esquerreacute-Tugayeacute MT Rosahl S Castresana C Hamberg M Fournier J (2005) 918

Evaluation of the antimicrobial activities of plant oxylipins supports their 919

involvement in defense against pathogen Plant Physiol 139 1902-1913 920

Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) 921

Completion of the seven-step pathway from tabersonine to the anticancer drug 922

precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 923

6224-6229 924

Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis 925

of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L) 926

Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795 927

Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF 928

(2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is 929

responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595 930

Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf 931

volatiles a plantrsquos multifunctional weapon against herbivores and pathogens Int J 932

Mol Sci 14 17781-17811 933

Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G 934

Horiuchi J Nishioka T Matsui K Takabayash J (2006a) Changing green leaf 935

volatile biosynthesis in plants an approach for improving plant resistance against 936

both herbivores and pathogens Proc Natl Acad Sci USA 10316672-16676 937

Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J 938

(2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host 939

searching behavior of two parasitoid species Cotesia glomerata and Cotesia 940

plutellae J Chem Ecol 32 969-979 941

Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin 942

L (2003) Expression pattern of two paralogs encoding cinnamyl alcohol 943

dehydrogenases in Arabidopsis Isolation and characterization of the corresponding 944

mutants Plant Physiol 132 848-860 945

Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) 946 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


34

CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD are the primary genes 947

involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 948

2059-2076 949

Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic 950

manipulation of alcohol dehydrogenase levels in ripening tomato fruit affects the 951

balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058 952

Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG 953

Haring MA Schuurink RC Allmann S (2017) Identification and characterization 954

of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 955

DOI103389fpls201701342 956

Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OrsquoConnor SE 957

(2015) Unlocking the diversity of alkaloids in Catharanthus roseus nuclear 958

localization suggests metabolic channeling in secondary metabolism Chem Biol 22 959

336-341 960

Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki 961

R Md Alamgir K Akitake S Nobuke T Galis I Aoki K Shibata D Takabayashi J 962

(2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested 963

neighbors reveals a mode of plant odor reception and defense Proc Natl Acad Sci 964

USA 111 7144-7149 965

Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 966

molecular evolutionary genetics analysis version 60 Mol Biol Evol 30 967

2725-2729 968

Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans 969

F Kim H Santoro N Foster C Goeminne G Leacutegeacutee F Lapierre C Pilate G Ralph G 970

Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher 971

saccharification upon deficiency in the dehydrogenase CAD1 Plant Physiol 175 972

1018-1039 973

Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol 974

dehydrogenase Expression during fruit ripening and under hypoxic conditions 975

FEBS Lett 123 39-42 976

Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal 977

Behav 6 369-371 978

Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of 979 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


35

acetylated derivatives and a terpenoid in maize Phytochemistry 67 34-42 980

Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z 981

(2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional 982

cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983 983

984


36

985










Parsed CitationsAllmann S Baldwin IT (2010) Insects betray themselves in nature to predators by rapid isomerization of green leaf volatiles Science329 1075-1078

Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Ameye M Allman S Verwaeren J Smagghe G Haesaert G Schuuring RC Audenaert K (2017) Green leaf volatile production byplants a meta-analysis New Phytol doi101111nph14671


Anderson NA Tobimatsu Y Ciesielski PN Ximenes E Ralph J Donohoe BS Ladisch M Chapple C (2015) Manipulation of guaiacyl andsyringyl monomer biosynthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis andmodified cell wall structure Plant Cell 27 2195-2209


Bate NJ Riley JCM Thomspon JE Rothstein SJ (1998) Quantitative and qualitative differences in C6-volatile production from thelipoxygenase pathway in an alcohol dehydrogenase mutant of Arabidopsis thaliana Physiol Plant 104 97-104


Bicsak TA Kann LR Reiter A Chase Jr T (1982) Tomato alcohol dehydrogenase purification and substrate specificity Arch BiochemBiophys 216 605-615


Bomati EK Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenasePlant Cell 17 1598-1611


Chen W VanOpdorp N Fitzl D Tewari J Friedemann P Greene T Thompson S Kumpatla S Zheng P (2012) Transposon insertion in acinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize Plant Mol Biol 80 289-297


DAurea JC Pichersky E Schaub A Hansel A Gershenzon J (2007) Characterization of a BAHD acyltransferase responsible forproducing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabiopsis thaliana Plant J 49 194-207


Davoine C Falletti O Douki T Iacazio G Ennar N Montillet JL Triantaphylideacutea C (2006) Adducts of oxylipin electrophiles toglutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response Plant Physiol 1401484-1493


Dolferus R Jacobs M (1984) Polymorphism of alcohol dehydrogenase in Arabidopsis thaliana (L) Heynh genetical and biochemicalcharacterization Biochem Genet 22 817-838


Duan H Huang MY Palacio K Schuler MA (2005) Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affecthexenal signaling in the Columbia and Landsberg erecta ecotypes of arabidopsis Plant Physiol 139 1529-1544


Dudareva N Klempien A Muhlemann JK Kaplan I (2013) Biosynthesis function and metabolic engineering of plant volatile organiccompounds New Phytol 198 16-32


Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucl Acids Res 32 1792-1797Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Eudes A Pollet B Sibout R Do CT Seacuteguin A Lapierre C Jouanin L (2006) Evidence for a role of AtCAD1 in lignification of elongatingstems of Arabidopsis thaliana Planta 225 23-39


Farmer EE Mueller MJ (2013) ROS-mediated lipid peroxidation and RES-activated signaling Ann Rev Plant Biol 64 429-450



Hatanaka A (1993) The biogeneration of green orour by green leaves Phytochemistry 34 1201-1218Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Huelsenbeck JP Ronquist F (2001) MRBAYES Bayesian inference of phylogenetic trees Bioinformatics 17 754-755Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Iijima Y Wang G Fridman E Pichersky E (2006) Analysis of the enzymatic formation of citral in the glands of sweet basil Arch BiochemBiophys 448 141-149


Iijima Y Koeduka T Suzuki H Kubota K (2014) Biosynthesis of geranial a potent aroma compound in ginger rhizome (Zingiberofficinale) molecular cloning and characterization of geraniol dehydrogenase Plant Biotechnol 31 525-534


Joo Y Schuman MC Goldberg JK Kim SG Yon F Bruumltting C Baldwin IT (2018) Herbivore-induced volatile blends with both fast andslow components provide robust indirect defence in nature Funct Ecol 32 136-149


Kato K Standley D M (2013) MAFFT multiple sequence alignment software version 7 improvements in performance and usabilityMol Biol Evol 30 722-780


Kessler A Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature Science 291 2141-2144Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kiedrowski S Kawalleck P Hahlbrock K Somssich I E Dangl J L (1992) Rapid activation of a novel plant defense gene isstrictrly dependent on the Arabidopsis RPM1 disease resistance EMBO J 11 4677-4684


Kim S-J Kim M-R Bedgar D L Moinuddin S G A Cardenas C L Davin L B Kang C Lewis N G (2004) Functionalreclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis Proc Natl Acad Sci USA 101 1455-1460


Kishimoto K Matsui K Ozawa R Takabayashi J (2008) Direct fungicidal activities of C6-aldehydes are important constitutents fordefense responses in Arabidopsis against Botrytis cinerea Phytochemistry 69 2127-2132


Kost C Heil M (2006) Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants J Ecol 94 619-628Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Krithika R Lal Srivastava P Rani B Kolet SP Chopade M Soniya M Thulasiram HV (2015) Characterization of 10-hydroxygeranioldehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis Sci Rep 5 8258DOI101038srep08258


Kunishima M Yamauchi Y Mizutani M Kuse M Takikawa H Sugimoto Y (2016) Identification of (Z)-3(E)-2-hexenal isomerasesessential to the production of the leaf aldehyde in plants J Biol Chem 191 14023-14033


Lai YK Chandlee JM Scandalios JG (1982) Purification and characterization of three non-allelic alcohol dehydrogenase isozymes inmaize Biochim Biophys Acta 706 9-18


Larsson A (2014) AliView a fast and lightweight alignment viewer and editor for large data sets Bioinformatics 30 3276-3278Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title


Matsui K (2006) Green leaf volatiles hydroperoxide lyase pathway of oxylipin metabolism Curr Opin Plant Biol 9 274-280Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Matsui K Sugimoto K Mano J Ozawa R Takabayashi J (2012) Differential metabolisms of green leaf volatiles in injured and intactparts of a wounded leaf meet distinct ecophysiological requirements PLoS ONE 7 e36433 doi101371journalpone0036433


Maxwell K Johnson GN (2000) Chlorophyll fluorescence ndash a practical guide J Exp Bot 51 659-668Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Mwenda CM Matsui K (2014) The importance of lipoxygenase control in the production of green leaf volatiles by lipase-dependent andindependent pathway Plant Biotechnol 31 445-452


Nakamura S Hatanaka A (2002) Green-leaf-derived C6-aroma compounds with potent antibacterial action that act on both Gram-negative and Gram-positive bacterial J Agric Food Chem 50 7639-7644


Pierik R Ballareacute CL Dicke M (2014) Ecology of plant volatiles taking a plant community perspective Plant Cell Environ 37 1845-1853Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Pospiacutesil P Yamamoto Y (2017) Damage to photosystem II by lipid peroxidation products Biochim Biophys Acta 1861 457-466Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Prost I Dhondt S Rothe G Vicente J Rodriguez MJ Kist N Carbonne F Griffiths G Esquerreacute-Tugayeacute MT Rosahl S Castresana CHamberg M Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense againstpathogen Plant Physiol 139 1902-1913


Qu Y Easson MLAE Froese J Simionescu R Hudlicky T De Luca V (2015) Completion of the seven-step pathway from tabersonine tothe anticancer drug precursor vindoline and its assembly in yeast Proc Natl Acad Sci USA 112 6224-6229


Saballos A Ejeta G Sanchez E Kang C Vermerris W (2009) A genome-wide analysis of the cinnamyl alcohol dehydrogenase family insorghum [Sorghum bicolor (L) Moench] identifies SbCAD2 as the brown midrib6 gene Genetics 181 783-795


Sattler SE Saathoff AJ Haas EJ Palmer NA Funnell-Harris DI Sarath G Pedersen JF (2009) A nonsense mutation in a cinnamyl alcoholdehydrogenase gene is responsible for the sorghum brown midrib6 phenotype Plant Physiol 150 584-595


Scala A Allman S Mirabella R Haring MA Schuuring RC (2013) Greenn leaf volatiles a plants multifunctional weapon againstherbivores and pathogens Int J Mol Sci 14 17781-17811


Shiojiri K Kishimoto K Ozawa R Kugimiya S Urashimo S Arimura G Horiuchi J Nishioka T Matsui K Takabayash J(2006a) Changing green leaf volatile biosynthesis in plants an approach for improving plant resistance against both herbivores andpathogens Proc Natl Acad Sci USA 10316672-16676


Shiojiri K Ozawa R Matsui K Kishimoto K Kugimiya S Takabayashi J (2006b) Role of the lipoxygenaselyase pathway of host-food plants in the host searching behavior of two parasitoid species Cotesia glomerata and Cotesia plutellae J Chem Ecol 32 969-979


Sibout R Eudes A Pollet B Goujon T Mila I Granier F Seacuteguin A Lapierre C Jouanin L (2003) Expression pattern of two paralogsencoding cinnamyl alcohol dehydrogenases in Arabidopsis Isolation and characterization of the corresponding mutants Plant Physiol132 848-860



Sibout R Eudes A Mouille G Pollet B Lapierre C Jouanin L Seacuteguin A (2005) CINNAMYL ALCOHOL DEHYDROGENASE-C and ndashD arethe primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis Plant Cell 17 2059-2076


Speirs J Lee E Holt K Yong-Duk K Scott NS Loveys B Schuch W (1998) Genetic manipulation of alcohol dehydrogenase levels inripening tomato fruit affects the balanace of some flavor aldehydes and alcohols Plant Physiol 117 1047-1058


Spyropouloe EA Dekker HL Steemers L van Maarseveen JH de Koster CG Haring MA Schuurink RC Allmann S (2017)Identification and characterization of (3Z)(2E)-hexenal isomerases from cucumber Front Plant Sci 8 1342 DOI103389fpls201701342


Stavrinides A Tatsis EC Foureau E Caputi L Kellner F Courdavault V OConnor SE (2015) Unlocking the diversity of alkaloids inCatharanthus roseus nuclear localization suggests metabolic channeling in secondary metabolism Chem Biol 22 336-341


Sugimoto K Matsui K Iijima Y Akakabe Y Muramoto S Ozawa R Uefune M Sasaki R Md Alamgir K Akitake S Nobuke T Galis I AokiK Shibata D Takabayashi J (2014) Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode ofplant odor reception and defense Proc Natl Acad Sci USA 111 7144-7149


Tamura K Stecher G Peterson D Filipski A Kumar S (2013) MEGA6 molecular evolutionary genetics analysis version 60 MolBiol Evol 30 2725-2729


Van Acker R Deacutejardin A Desmet S Hoengenaert L Vanholme R Morreel K Laurans F Kim H Santoro N Foster C Goeminne GLeacutegeacutee F Lapierre C Pilate G Ralph G Boerjan W (2017) Different routes for conifer- and sinapaldehyde and higher saccharificationupon deficiency in the dehydrogenase CAD1 Plant Physiol 175 1018-1039


Van Der Straeten D Pousada RAR Gielen J Van Montagu M (1991) Tomato alcohol dehydrogenase Expression during fruit ripeningand under hypoxic conditions FEBS Lett 123 39-42


Wei J Kang L (2011) Roles of (Z)-3-hexenol in plant-insect interactions Plant Signal Behav 6 369-371Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yan ZG Wang CZ (2006) Wound-induced green leaf volatiles cause the release of acetylated derivatives and a terpenoid in maizePhytochemistry 67 34-42


Zhang K Qian Q Huang Z Wang Y Li M Hong L Zeng D Gu M Chu C Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes aprimarily multifunctional cinnamyl-alcohol dehydrogenase in rice Plant Physiol 140 972-983



Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

2

Research Article 24

25

Identification of a hexenal reductase that modulates the composition of 26

green leaf volatiles 27

28

29

30

Toshiyuki Tanakaa Ayana Ikedab Kaori Shiojiric Rika Ozawad Kazumi Shikia 31

Naoko Nagai-Kunihiroa Kenya Fujitab Koichi Sugimotoa Katsuyuki T Yamatoe 32

Hideo Dohraf Toshiyuki Ohnishig Takao Koedukaab Kenji Matsuiab1 33

34

aDivision of Agricultural Sciences Graduate School of Sciences and Technology for 35

Innovation Yamaguchi University Yamaguchi 753-8515 Japan 36

bDepartment of Biological Chemistry Faculty of Agriculture Yamaguchi University 37


cDepartment of Agriculture Ryukoku University Ooe Otsu Shiga 520-2194 Japan 39

dCenter for Ecological Research Kyoto University Hirano Otsu Shiga 520-2113 Japan 40

eFaculty of Biology-Oriented Science and Technology Kindai University Kinokawa 41

Wakayama 649-6493 Japan 42

fInstrumental Research Support Office Research Institute of Green Science and 43

Technology Shizuoka University Shizuoka 422-8529 Japan 44

gCollege of Agriculture Academic Institute Shizuoka University Shizuoka 422ndash8529 45

Japan 46

47

1Address correspondence to matsuiyamaguchi-uacjp 48

The author responsible for the distribution of materials to the findings presented in this 49

article in accordance with the policy described in the Instructions for Authors 50

(wwwplantphysiolorg) is Kenji Matsui (matsuiyamaguchi-uacjp) 51

52

Author Contributions 53

KM conceived this project designed experiments and wrote the article TT AI K 54

Shiki NNK KF and KSugimoto purified the enzyme and examined its properties 55

KTY and TK analyzed the data HD and TO determined amino acid sequences of 56 httpsplantphysiolorgDownloaded on February 4 2021 - Published by


3



59

60

61


4

ABSTRACT 62






















84

85


Indirect defense 87

88


90

91


5

INTRODUCTION 92

93

































6



































7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

3



59

60

61


4

ABSTRACT 62






















84

85


Indirect defense 87

88


90

91


5

INTRODUCTION 92

93

































6



































7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

4

ABSTRACT 62






















84

85


Indirect defense 87

88


90

91


5

INTRODUCTION 92

93

































6



































7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

5

INTRODUCTION 92

93

































6



































7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

6



































7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

7





























186

187

RESULTS 188

189



8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

8


























216









9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

9























246












10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

10



























283








11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

11
















305







310



















12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

12

323

324

DISCUSSION 325
































13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

13



































14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

14



































15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

15





































16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

16




458

459


Plant materials 461












s-1

) with a 14-h 472


474

475

Enzyme assays 476













17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

17




















min-1


of 1 mL min-1












519



18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

18























dithiothreitol 543








(see Fig 1A) 551




19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

19








560














574

cDNA cloning 575













20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

20


) and 587








lysozyme After 593








601


















containing 1 g mL-1



21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

21












+ 629

630
























22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

22








660







666

s-1






s-1




PAM analysis 674

675








683




23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

23


687

688

689

690

691


24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

24


693


695


697



700


leaves 702

703



706


mutant plants 708

709


leaves 711

712


714


716


718


720


8 722

723



25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

25




728

FIGURE LEGENDS 729









738












750



753



26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

26





759


leaves 761









770















785




27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

27









400-nmol cm-3


796









805


28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

28



activity

Purification Yield


Crude extract 1707 1062 166 1 100

Ammonium

sulfate

1634 592 260 058 557



HiTrap DEAE 0031 394 788 209 037

807

808


29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

29


cinnamaldehyde 810


)




811

812

813

814


30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

30

References 815




















Biol 80 289-297 835







Physiol 140 1484-1493 842



817-838 845




31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

31


1529-1544 849







Planta 225 23-39 856




34 1201-1218 860






















32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

32

Sci USA 101 1455-1460 881









DOI101038srep08258 890






9-18 896










Bot 51 659-668 906









33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Parsed Citations

33











6224-6229 924









Mol Sci 14 17781-17811 933















34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

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Parsed Citations

34



2059-2076 949







DOI103389fpls201701342 956




336-341 960





USA 111 7144-7149 965



2725-2729 968





1018-1039 973



FEBS Lett 123 39-42 976


Behav 6 369-371 978



35





984


36

985



















































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

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Figure 6

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Parsed Citations

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36

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Article File

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Parsed Citations

Article File

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Figure 2

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Parsed Citations

identification of a hexenal reductase that modulates the … · 2018. 8. 20. · 2-hexenal or...

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