short title regulation of furaneol biosynthesis in …...faqr is mainly expressed in 143 the late...

1

Short title 1

Regulation of furaneol biosynthesis in strawberry 2

3

4

Corresponding author details 5

Kunsong Chen 6

College of Agriculture amp Biotechnology Zhejiang University Zijingang Campus 7

Hangzhou 310058 PR China 8

Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology 9

Zhejiang University Zijingang Campus Hangzhou 310058 PR China 10

The State Agriculture Ministry Laboratory of Horticultural Plant Growth 11

Development and Quality Improvement Zhejiang University Zijingang Campus 12


Email akunzjueducn 14

15

Harry J Klee 16

College of Agriculture amp Biotechnology Zhejiang University Zijingang Campus 17


Horticultural Sciences Plant Innovation Center Genetics Institute University of 19

Florida Gainesville FL 32611 20

Email hjkleeufledu 21

22

Guihua Jiang 23

Institute of Horticulture Zhejiang Academy of Agricultural Sciences Hangzhou 24

310021 Zhejiang China 25

Email jgh2004267sinacom 26

27

Plant Physiology Preview Published on July 9 2018 as DOI101104pp1800598

Copyright 2018 by the American Society of Plant Biologists

wwwplantphysiolorgon February 25 2020 - Published by Downloaded from Copyright copy 2018 American Society of Plant Biologists All rights reserved

2

Article title 28

29

An ETHYLENE RESPONSE FACTOR-MYB Transcription Complex Regulates 30

Furaneol Biosynthesis by Activating QUINONE OXIDOREDUCTASE Expression 31

in Strawberry 32

33

34

Yuanyuan Zhang1 Xueren Yin123 Yuwei Xiao1 Zuying Zhang1 Shaojia Li1 35

Xiaofen Liu1 Bo Zhang123 Xiaofang Yang4 Donald Grierson15 Guihua Jiang4 36

Harry J Klee16 Kunsong Chen123 37

1 College of Agriculture amp Biotechnology Zhejiang University Zijingang Campus 38


2 Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology 40


3 The State Agriculture Ministry Laboratory of Horticultural Plant Growth 42



4 Institute of Horticulture Zhejiang Academy of Agricultural Sciences Hangzhou 45


5 Division of Plant and Crop Sciences School of Biosciences University of 47

Nottingham Sutton Bonington Campus Loughborough LE12 5RD UK 48

6 Horticultural Sciences Plant Innovation Center Genetics Institute University of 49


These authors contributed equally to this manuscript 51

52

One-sentence summary 53

Fragaria times ananassa quinone oxidoreductase which catalyses the final step in 54

furaneol biosynthesis in strawberry is transcriptionally upregulated by a complex 55

consisting of an ethylene response factor and MYB during ripening 56

57


3

Footnotes 58

List of author contributions 59

KC HK and GJ conceived the original screening and research plans XrY and 60

YZ designed the experiments YZ YX and ZZ performed the experiments XrY 61

SL XL and XfY provided technical assistance to YZ XrY BZ and KC 62

supervised the experiments YZ and XrY analysed the data YZ and XrY wrote 63

the article with contributions from all the authors DG discussed results and 64

interpretation and supervised the writing and editing of the manuscript 65

66

Funding information 67

This research was supported by the National Key Research and Development 68

Program (2016YFD0400101) the Project of the Science and Technology Department 69

of Zhejiang Province (2016C04001) and the 111 Project (B17039) 70

71

Corresponding author email 72

The authors responsible for distribution of materials integral to the findings presented 73

in this article in accordance with the policy described in the Instructions for Authors 74

(wwwplantphysiolorg) are Kunsong Chen (akunzjueducn) Harry J Klee 75

(hjkleeufledu) and Guihua Jiang (jgh2004267sinacom) 76


4

Abstract 77

4-Hydroxy-25-dimethyl-3(2H)-furanone (HDMF) is a major contributor to the aroma 78

of strawberry (Fragaria times ananassa) fruit and the last step in its biosynthesis is 79

catalysed by Fragaria times ananassa quinone oxidoreductase (FaQR) Here an ethylene 80

response factor (FaERF9) was characterized as a positive regulator of the FaQR 81

promoter Linear regression analysis indicated that FaERF9 transcript levels were 82

significantly correlated with both FaQR transcripts and furanone content in different 83

strawberry cultivars Transient overexpression of FaERF9 in strawberry fruit 84

significantly increased FaQR expression and furaneol production Yeast one-hybrid 85

assays however indicated that FaERF9 by itself did not bind to the FaQR promoter 86

An MYB transcription factor (FaMYB98) identified in yeast one-hybrid screening of 87

the strawberry cDNA library was capable of both binding to the promoter and 88

activating the transcription of FaQR by ~56-fold Yeast two-hybrid assay and 89

bimolecular fluorescence complementation confirmed a direct protein-protein 90

interaction between FaERF9 and FaMYB98 and in combination they activated the 91

FaQR promoter 14-fold in transactivation assays These results indicate that an 92

ERF-MYB complex containing FaERF9 and FaMYB98 activates the FaQR 93

promoter and upregulates HDMF biosynthesis in strawberry 94

95


5

Introduction 96

Strawberry (Fragaria times ananassa) is an economically important globally popular 97

and attractive fruit with nutritional benefits for human health An important feature of 98

its appeal is a unique fragrance with more than 360 different volatile compounds 99

detected in ripe fruit (Meacutenager 2004 Jetti et al 2007) These volatiles include 100

alcohols organic acids aldehydes terpenes lactones esters aromatic hydrocarbons 101

furans and others (Zabetakis and Holden 1997) Sensory evaluation indicates that 102

terpenes confer on strawberry a ldquofresh orangerdquo smell aldehydes generate a ldquoherbal 103

flavourrdquo γ -decalactone is ldquopeach-likerdquo esters are ldquofruityrdquo and 104

4-hydroxy-25-dimethyl-3(2)H-furanone (HDMF) and 105

25-dimethyl-4-methoxy-3(2)H-furanone (DMMF) generate a ldquocaramel-likerdquo aroma 106

(Pyysalo et al 1979 Larsen and Poll 1992) HDMF and DMMF belong to an 107

uncommon group of chemicals with a 25-dimethyl-3(2H)-furanone structure and 108

such compounds possess special odour properties (Schwab and Roscher 1997) They 109

are found at trace levels in various fruits including raspberry some grape cultivars 110

and tomato They are particularly abundant in strawberry and pineapple (Schwab 111

2013 Wuumlst 2017) HDMF is one of the most important volatiles distinguishing 112

strawberry from other fruits (Larsen and Poll 1992 Schwab and Roscher 1997) and 113

is significantly correlated with perceived strawberry fruit intensity (Schwieterman et 114

al 2014) Thus strawberry is an important model for investigating the regulation of 115

furaneol biosynthesis and knowledge of the factors controlling its synthesis is highly 116

desirable for targeting flavor improvement 117

118

HDMF and its methyl ether DMMF have been extensively investigated in research 119

on flavour analyses chemical synthesis and natural product formation in both 120

microbes and plants (Zabetakis et al 1999 Schwab 2013) The first plant studies on 121

the biosynthesis of HDMF used strawberry fruit to investigate potential precursors of 122

furaneol biosynthesis (Pisarnitskii et al 1992) and D-fructose-16-diphosphate was 123

identified as the natural precursor of HDMF and DMMF (Roscher et al 1998 124

Schwab 1998) The immediate precursor of HDMF in strawberry was shown to be 125


6

4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) and Fragaria times 126

ananassa quinone oxidoreductase (FaQR later renamed as FaEO) was identified as 127

the enzyme responsible for reduction of the α β-unsaturated bond of HMMF to form 128

the aroma-active compound HDMF (Raab et al 2006 Klein et al 2007) An 129

O-methyltransferase FaOMT subsequently generates DMMF by methylation of 130

HDMF (Wein et al 2002) HDMF can also be metabolized to 131

25-dimethyl-4-hydroxy-2H-furan-3-one glucoside (HDMF-glucoside) by a 132

UDP-dependent glycosyltransferase (UGT71K3) and further malonylated into 133

25-dimethyl-4-hydroxy-2H-furan-3-one 6rsquo-O-malonyl-β-D-glucopyranoside (HDMF 134

malonyl-glucoside) in strawberry fruit (Roscher et al 1996 Song et al 2016) The 135

biosynthetic pathway of HDMF in other plants is similar to that in strawberry Genes 136

with homology to FaQR have been identified in tomato (SlEO) and mango (MIEO) 137

(Klein et al 2007 Kulkarni et al 2013) 138

139

As an NADH-dependent enone oxidoreductase protein FaQR exhibits o-quinone 140

oxidoreductase activity (Raab et al 2006) FaQR mRNA accumulates in fruit 141

parenchyma tissues but is absent in vascular tissues and FaQR is mainly expressed in 142

the late stages of fruit growth and ripening paralleling furaneol production in the fruit 143

(Raab et al 2006) FaQR is responsive to environmental stimuli associated with 144

furaneol production and in the dark lsquoSweet Charliersquo strawberry fruits have higher 145

levels of FaQR expression and HDMF production at 25degC than at 15degC (Fu et al 146

2017) in contrast the concentration of furanones and the abundance of FaQR protein 147

under low-temperature storage is significantly lower than those under 148

room-temperature storage (Li et al 2015) Thus the regulation of FaQR is important 149

for manipulating furaneol content Although it is assumed that transcription factors 150

control expressions of genes involved in volatile production in plants no such factors 151

regulating FaQR or other furanone biosynthetic genes have been identified 152

153

Different members of the largely plant-specific AP2ERF gene family (Weirauch and 154

Hughes 2011) have diverse functions in plant growth development and stress 155


7

responses (Agarwal et al 2010 Zhu et al 2010 Licausi et al 2013) In recent years 156

several AP2ERF genes have been reported to regulate aspects of fruit quality (Xie et 157

al 2016) including fruit aroma For example CitAP210 is associated with the 158

synthesis of (+)-valencene in Newhall orange acting via regulation of CsTPS1 (Shen 159

et al 2016) CitERF71 was shown to physically bind to the CsTPS16 promoter and 160

contribute to the synthesis of E-geraniol in citrus fruit (Li et al 2017) These 161

observations raised the possibility that FaQR might also be a target for a member of 162

the AP2ERF family and that ERFs might also control furaneol biosynthesis 163

164

In this study screening of the Fragaria times ananassa AP2ERF gene family led to 165

identification of FaERF9 as an activator of FaQR however it did not bind directly 166

to the promoter A parallel search for proteins that directly bind the FaQR promoter 167

using yeast one-hybrid screening identified a second factor FaMYB98 which was 168

capable of physically interacting with the FaQR promoter FaMYB98 physically 169

interacts with FaERF9 and transient expression assays demonstrated a synergistic 170

effect on FaQR expression and furanone synthesis 171

172

Results 173

Changes in the content of both furanones and the activity of their biosynthetic 174

genes in strawberry fruit 175

lsquoYuexinrsquo fruits at four developmental and ripening stages (G green T turning IR 176

intermediate red R full red) were harvested (Figure 1) and determination of HDMF 177

levels indicated a major increase from about 065 μg per gram fruit at the IR stage to 178

346 μg at the R stage in the apical section implying that furaneol accumulation was 179

closely related to colour change and ripening (Figure 1) Furaneol levels were higher 180

in the apical section than in the basal section HDMF could not be detected at the IR 181

stage in the basal section but the amount increased at the R stage to 142 μg per gram 182

fruit Differences were also found for DMMF with the relative content in the apical 183

section 018 μg per gram at the R stage compared to 008 μg per gram in the basal 184

section at the same stage 185


8

186

Measurement of mRNA levels by RT-qPCR indicated that the transcript levels of both 187

FaQR (GenBank AY1588361) and FaOMT (GenBank AF2204912) increased 188

throughout ripening (Figure 1) Comparison of the expression of these genes showed 189

that the transcript levels of FaQR and FaOMT significantly increased as early as in T 190

stage in basal fruit sections and for each gene over half of the peak transcript 191

abundance occurred as early as in IR stage in the apical section while for the basal 192

section it was in the R stage indicating a gradient of gene expression and volatile 193

production from apex to base of the fruit during ripening 194

195

Genome-wide identification of AP2ERF gene family in strawberry 196

Coding sequences of 120 non-redundant FaAP2ERF genes were isolated and 197

identified from lsquoYuexinrsquo strawberry and a systematic nomenclature for the AP2ERF 198

family genes was used to distinguish the members based on the structural features 199

defined previously (Supplemental Table S1) (Shigyo and Ito 2004 Nakano et al 200

2006 Licausi et al 2013) CD-search analysis showed that this superfamily is 201

consisted of 95 ERFs 18 AP2 and 6 RAV family members and 1 soloist member 202

(Supplemental Table S2) Phylogenetic analysis of the Fragaria times ananassa and 203

Arabidopsis AP2ERF members is shown in Supplemental Figure S1 with the 120 204

AP2ERF genes divided into three major groups (ERF AP2 RAV) and a soloist and 205

the ERF family being further divided into 10 groups (groups I to X in Supplemental 206

Table S1) 207

208

We performed a dual-luciferase assay to determine the ability of 116 FaAP2ERF 209

members to activate transcription from the promoter of FaQR The results indicated 210

about 12 members (mainly belonging to the ERF and RAV families) showed a 211

significant activation effect on the FaQR promoter while the remaining members 212

showed very limited effects (Figure 2) The relative activity of only the following 12 213

FaAP2ERF transcripts reached the threshold value set at 2 FaERF8FaERF9 214

(group II) FaERF27FaERF29FaERF31FaERF32 (group IV) 215


9

FaERF37FaERF38 (group V) FaERF62FaERF64 (group VIII) FaERF68 216

(group IX) and FaRAV4 (Figure 2) Of these FaERF9 exhibited the highest 217

activation effect on the FaQR promoter (466-fold) (Figure 2) The strawberry 218

FaERF9 cDNA is 699 bp long and ExPASy Compute pIMw tool analysis indicated 219

that this gene encodes a 232-amino acid protein with a calculated molecular mass of 220

254 KD and a pI of 56 221

222

FaERF9 expression pattern and subcellular localization 223

To further explore the connections between AP2ERFs and furaneol biosynthesis we 224

obtained the transcript profiles of the AP2ERF genes in strawberry fruit samples at 225

different fruit developmental and ripening stages by reverse transcription quantitative 226

PCR (RT-qPCR) excluding genes with very low abundance in the fruit samples The 227

results presented as a heatmap (Supplemental Figure S2) demonstrate that these 228

genes displayed various expression patterns in both the apical and basal sections and 229

by analysing the expression patterns of the members indicated as activators in the 230

dual-luciferase assay we found that only a few genes including FaERF8 FaERF9 231

FaERF27 FaERF32 and FaERF37 were up-regulated during ripening stages and 232

their expression correlated with furaneol accumulation in the fruit (Figure 1) 233

Furthermore the expression patterns of the up-regulated genes in the apical and basal 234

sections showed that one ERF gene FaERF9 not only showed an up-regulation 235

pattern in both sections but its expression in the apical section occurred ahead of that 236

in the basal section (Figure 3) consistent with the actual changes in furaneol content 237

(Figure 1) and the expression pattern of FaQR (Figure 1) The subcellular localization 238

of FaERF9 was predicted to be in the nucleus using the WoLF PSORT program In 239

experimental tests 35S-FaERF9-GFP (green fluorescence protein) showed strong 240

fluorescence in the nucleus and the green signal merged with the red fluorescence 241

signal of mCherry-nucleus-marker in tobacco plants (Figure 3) 242

243

Transient overexpression of FaERF9 in strawberry 244

The expression of FaERF9 in agro-infiltrated fruits was analysed by performing 245


10

RT-qPCR on the seventh day after infiltration by which time they had reached the IR 246

or R stage and the result showed that the transcript levels were significantly higher 247

(52-fold) than in control fruits (Figure 4) The expression of FaQR was examined in 248

the same fruits and the transcript abundance also increased 22-fold The relative 249

content of HDMF in the FaERF9-overexpressing fruits was 008 μg per gram fruit 250

and undetectable in the control fruits (Figure 4) The relative content of DMMF in the 251

overexpressing fruits was also significantly increased to 038 μg per gram fruit 252

compared to 004 μg per gram fruit in the control (Figure 4) These observations 253

provide direct evidence for a regulatory role for FaERF9 in promoting FaQR 254

biosynthesis and activity in ripening strawberry fruit Taken together these results 255

indicate that by activating the transcription of FaQR FaERF9 functions as a positive 256

regulator in furaneol biosynthesis 257

258

Correlation of FaERF9 expression and FaQR transcript profiles 259

GC-MS quantification showed that furanones are distributed in variable levels among 260

different strawberry cultivars (Supplemental Figure S3) The transcript profiles of 261

FaQR and FaERF9 in fruits of different cultivars were measured and linear 262

regression analysis was performed In the cultivars the changes in FaQR transcripts 263

correlated positively with those in FaERF9 transcripts (r = 079 plt001) (Figure 5) 264

In addition FaERF9 transcript levels also correlated with furanone content across 265

cultivars (Figure 5) 266

267

FaERF9 is an indirect regulator of the FaQR promoter and acts via 268

protein-protein interaction with FaMYB98 269

Although the results of the dual-luciferase assay expression analysis transient 270

overexpression and correlation analysis were all consistent with the suggestion that 271

FaERF9 regulates transcript abundance by acting on the FaQR promoter a yeast 272

one-hybrid assay indicated that FaERF9 cannot bind directly to the FaQR promoter 273

(Figure 6) This led us to speculate that activation may occur via a transcription 274

complex involving an additional as-yet unknown regulator which can bind directly to 275


11

the FaQR promoter 276

277

In parallel with the investigation of the FaAP2ERF genes a cDNA library screening 278

was performed with a yeast one-hybrid assay using the promoter of FaQR as the bait 279

This screen identified several transcription factors that could physically bind to the 280

FaQR promoter but only one MYB could activate transcription from the FaQR 281

promoter (approximately 56-fold) relative to the control (Figure 6) in the 282

dual-luciferase assay This MYB showed 44 amino acid identity with MYB98 283

(GenBank ABA420611) a transcriptional regulator in the synergid cells in 284

Arabidopsis (Kasahara et al 2005) and it was designated as FaMYB98 The 285

specificity of FaMYB98 binding to the FaQR promoter was confirmed by EMSA 286

assay (Figure 7) and increasing the concentration of the cold probe reduced binding 287

In addition mutating the putative binding sites (Goacutemez-Maldonado et al 2004 288

Punwani et al 2007) as shown in Figure 7 eliminated FaMYB98 protein binding 289

When the combined effect of FaERF9 and FaMYB98 was analysed (Figure 8) a 290

synergistic 14-fold activation of the FaQR promoter was observed compared with the 291

activation by either FaERF9 or FaMYB98 which alone promoted transactivation 292

39-fold and 45-fold respectively In addition overexpression of both genes 293

(FaERF9-FaMYB98-SK) in strawberry fruits resulted in higher expression of 294

FaERF9 transcripts and higher furanone content than overexpression of FaERF9 295

(Supplemental Figure S4 Supplemental Table S3) Subcellular localization analysis 296

revealed that FaMYB98 was also localized in the nucleus (Supplemental Figure S5) 297

298

To investigate the potential interactions between FaERF9 and FaMYB98 the 299

DUALhunter system was used (Figure 9) Cells expressing the FaMYB98 and 300

FaERF9 protein pair using either FaMYB98 or FaERF9 as the bait grew on DDO 301

QDO and QDO supplemented with 1 mM 3-AT selective medium indicating 302

interaction between the two proteins This putative protein-protein interaction was 303

confirmed by BiFC assay and the combinations of FaMYB98-YFPNFaERF9-YFPC 304

and FaERF9-YFPNFaMYB98-YFPC exhibited strong coincident signals in the 305


12

nucleus while negative controls produced no detectable fluorescence signal (Figure 306

10) In addition coexpression of FaERF9-YFPNFaERF9-YFPC as well as 307

FaMYB98-YFPNFaMYB98-YFPC also generated signals in the nucleus indicating 308

that FaERF9 and FaMYB98 both could form homodimers or possibly multimers in 309

the nucleus (Figure 10) 310

311

Discussion 312

The AP2ERF superfamily in strawberry 313

The AP2ERF superfamily of plant transcription factors is defined by the AP2ERF 314

domain and comprises the ERF AP2 and RAV families as well as one soloist member 315

(Riechmann et al 2000 Weirauch and Hughes 2011) The completion of multiple 316

plant genome sequences has enabled a genome-scale analysis of the AP2ERF family 317

which in recent years has been systematically studied in diverse fruit species such as 318

tomato grape peach and kiwi fruit (Zhuang et al 2009 Sharma et al 2010 Licausi 319

et al 2010 Yin et al 2010 Pirrello et al 2012 Zhang et al 2012 Zhang et al 320

2016) A few AP2ERF genes have been isolated and characterized in strawberry and 321

CBF orthologs (Owens et al 2002 Koehler et al 2012 Zhang et al 2014) have 322

been identified from different strawberry cultivars in several independent studies 323

Although they have distinct names eg Fragaria times ananassa CBF1 (FaCBF1) 324

FaCBF4 (GenBank HQ9105151) and CBF1 (GenBank EU1172142) according to 325

the phylogenetic tree generated in this study the orthologs of 326

CBF1CBF4CBF2CBF3 in Arabidopsis are similar to each other and to FaERF20 327

(Supplemental Figure S1) (Owens et al 2002 Koehler et al 2012 Zhang et al 328

2014) FaERF20 has 85 sequence similarity with FaCBF1 identified by Owens et 329

al (2002) 93 similarity with FaCBF4 amplified by Koehler et al (2012) and 75 330

similarity with CBF1 cloned by Zhang et al (2014) The AP2ERF genes 331

characterized in other studies (Jia et al 2011 GAO et al 2015 Chai and Shen 2016 332

Mu et al 2016) are also noted in Supplemental Table S1 333

334

Based on the computational analysis we have identified a total of 120 AP2ERF 335


13

genes in octoploid strawberry (Supplemental Table S1) The number of predicted 336

AP2ERF genes (120) is comparable to those observed in other fruits including 337

tomato (112) Chinese plum (116) citrus (126) and peach (131) but lower than those 338

reported for grape (149) (Xie et al 2016) As shown in Supplemental Table S2 79 339

(95 genes) of the AP2ERF genes belong to the ERF family in strawberry constituting 340

the largest sub-family The members of this sub-family can be classified into 10 341

groups (group I to X) according to their sequence similarities to the Arabidopsis ERF 342

genes (Nakano et al 2006) The AP2 family encompasses the same number of genes 343

in Arabidopsis citrus and strawberry (eighteen) (Nakano et al 2006 Xie et al 2014) 344

and there are six RAV genes in strawberry which are highly conserved in dicots such 345

as Vitis vinifera Arabidopsis thaliana and Populus trichocarpa (Licausi et al 2010) 346

347

Role of FaERF9 in regulating the biosynthesis of HDMF a positive regulator 348

acting in an indirect manner 349

The large-scale screening of the AP2ERF superfamily in strawberry led to 350

identification of 11 ERFs and 1 RAV that trans-activated the FaQR promoter more 351

than two-fold with the highest activation caused by FaERF9 which trans-activated 352

the FaQR promoter (Figure 2) as well as up-regulating FaQR expression and furanone 353

production in transient overexpression assays (Figure 4) which has been extensively 354

used to explore the fruit-associated gene functions in strawberry (Han et al 2015 355

Medina-Puche et al 2015 Vallarino et al 2015 Carvalho et al 2016 Song et al 356

2016 Wang et al 2018) FaERF9 transcripts accumulate in the fruit apical section 357

before the basal section (Figure 3) a finding consistent with the actual changes in 358

furaneol content (Figure 1) and the expression of FaQR (Figure 1) The association 359

between FaERF9 transcripts and accumulation of furanones were also established 360

with different strawberry cultivars The correlation between FaERF9 and FaQR 361

expression as well as furanone content among cultivars supports a positive role of 362

FaERF9 in regulating furanone content (Figure 5) These results indicate that 363

FaERF9 transcript abundance may be a good indicator of the abundance of these 364

important flavour volatiles in fruits of different cultivars This should be considered as 365


14

a method for high-throughput variety screening to replace direct measurement of 366

volatiles 367

368

FaERF9 is a member of the ERF group II family and is most closely related to the 369

Arabidopsis genes At1g44830 (ATERF014) At1g21910 (DREB26) and At1g77640 370

(Supplemental Figure S1) At1g44830 was observed to be strongly up-regulated in a 371

comprehensive microarray analysis of the root transcriptome following NaCl 372

exposure (Jiang and Deyholos 2006) At1g21910 (DREB26) was functionally 373

characterized as a trans-activator localized in the nucleus exhibiting tissue-specific 374

expression and participating in plant developmental processes as well as biotic andor 375

abiotic stress signalling (Krishnaswamy et al 2011) However none of these genes 376

have been reported as furanone regulators In strawberry another five genes 377

(FaERF6 FaERF7 FaERF8 FaERF10 and FaERF11) also belong to the same 378

group with FaERF8 also showing somewhat weaker activation activity towards the 379

FaQR promoter than FaERF9 The fact that FaERF9 could not bind directly to the 380

promoter of FaQR (Figure 6) indicates that it might function as an indirect regulator 381

of FaQR and furaneol biosynthesis 382

383

A FaERF9 and FaMYB98 complex is involved in regulating FaQR and furaneol 384

biosynthesis 385

The finding that FaERF9 could interact with FaMYB98 protein (Figures 9 and 386

Figure 10) and that FaMYB98 could physically bind to the FaQR promoter (Figure 6 387

and Figure 7) provides evidence for a regulatory mechanism whereby FaERF9 388

regulates FaQR and furaneol production as part of a complex with an MYB 389

transcription factor Since co-overexpression of FaERF9 and FaMYB98 resulted in 390

much higher activation activity towards FaQR we speculate that FaERF9 may 391

recruit FaMYB98 homologs in tobacco to activate the FaQR promoter (Figure 8) 392

FaOMT is another key gene for furanone biosynthesis and a potential target for 393

transcriptional activation However FaERF9 did not significantly activate the 394

FaOMT promoter in a dual-luciferase assay and the transcript level of FaOMT in the 395


15

FaERF9-overexpressing fruits showed no significant difference from that in the 396

control (Supplemental Figure S6 Supplemental Figure S7) The FaOMT promoter 397

was significantly induced by FaMYB98 but no synergistic effect of FaERF9 and 398

FaMYB98 on the promoter was observed (Supplemental Figure S6) In previous 399

studies of the regulation of plant volatile compounds MYBs have been characterized 400

as being involved mainly in the regulation of the benzenoidphenylpropanoid pathway 401

that produces floral volatiles in petunia and eugenol in ripe strawberry fruit (Verdonk 402

et al 2005 Dal Cin et al 2011 Colquhoun et al 2011 Spitzer-Rimon et al 2012 403

Medina-Puche et al 2015) but their role in formation of volatiles from other 404

pathways has not been reported Our study demonstrates a role for MYB in the 405

synthesis of furaneol a carbohydrate-derived volatile compound and reveals the 406

synergistic interactions between an MYB and ERF in a transcription complex (Figure 407

8 Figure 9 and Figure 10) 408

409

Recent research has provided evidence for interactions between AP2ERF and MYB 410

transcription factors in regulating other aspects of fruit quality including texture 411

(EjAP2-1 and EjMYB Zeng et al 2015) and colour (PyERF3 and PyMYB114 Yao 412

et al 2017) A group VIII ERF has also recently been shown to interact with an MYB 413

to regulate floral scent production in petunia (Liu et al 2017) In strawberry fruit 414

DOF in combination with an MYB has been reported to be involved in the regulation 415

of eugenol production and the identification of this second transcription factor (DOF) 416

suggests that it is a good target in breeding for improved flavour (Molina-Hidalgo et 417

al 2017 Tieman 2017) Considering its important contribution to flavour in 418

strawberry and many other highly valued fruit crop species (pineapple tomato mango 419

etc) very little is known about regulation of furaneol synthesis Here we established 420

the role of FaERF9 in the regulation of HDMF synthesis by direct protein-protein 421

interactions with FaMYB98 to synergistically trans-activate FaQR The functional 422

identification of this complex enhances our knowledge of the regulation of volatile 423

compound synthesis and identifies a new AP2ERF-MYB complex Further 424

examination of the other ERFs that stimulated transcription from the FaQR promoter 425


16

(Figure 2) may provide additional information about the complexity of this regulation 426

Overall this study provides important clues for understanding the transcriptional 427

regulation of HDMF synthesis and identifies specific transcription factors that are 428

likely to be good targets for molecular breeding for improved flavour 429

430

Conclusions 431

Screening of the AP2ERF superfamily in strawberry (Fragaria times ananassa) 432

identified candidate members capable of activating the FaQR promoter An ERF 433

transcription factor was identified as a positive regulator of HDMF synthesis in 434

strawberry fruit and the mechanism of activation was shown to involve an ERF-MYB 435

transcription complex that regulates transcription of FaQR leading to the biosynthesis 436

of HDMF the characteristic volatile compound which has a major influence on 437

strawberry aroma 438

439

Materials and Methods 440

441

Plant Materials and Growth conditions 442

The strawberry (Fragaria times ananassa cv lsquoYuexinrsquo an octoploid cultivar) fruit used in 443

this study were bred by Zhejiang Academy of Agricultural Sciences (Haining 444

Zhejiang) Fruits at various developmental and ripening stages including G (green) T 445

(turning) IR (intermediate red) and R (full red) were harvested (Figure 1) and 446

transported to the laboratory within 2 h Thirty-six fruits of uniform size and free of 447

visible defects were selected at each stage with twelve fruits in each biological 448

replicate After removing the calyces fruits were then divided into the basal and 449

apical sections which were sampled separately (Figure 1) They were rapidly cut into 450

pieces frozen immediately in liquid nitrogen and stored at -80deg C until use Red ripe 451

fruits of different strawberry cultivars (Fragaria times ananassa octoploid cultivars) 452

including lsquoYuelirsquo lsquoBenihoppersquo lsquoMengxiangrsquo lsquoAmaoursquo lsquo10-1-4rsquo lsquoAkihimersquo lsquoSweet 453

Charliersquo lsquo07-1-4rsquo lsquoDarselectrsquo lsquoYuexinrsquo and lsquoXuemeirsquo were also provided by Zhejiang 454

Academy of Agricultural Sciences For each cultivar the fruits of three biological 455


17

replicates were sampled frozen in liquid nitrogen and stored at -80deg C for further use 456

457

Tobacco plants (Nicotiana benthamiana) used for dual-luciferase assays and 458

subcellular localization analysis in this study were grown in a growth chamber with a 459

lightdark cycle of 16 h8 h at 24degC 460

461

Detection of DMMF and HDMF 462

Automated HS-SPME-GC-MS was performed to detect both furanones in lsquoYuexinrsquo 463

strawberry fruit (Zorrilla-Fontanesi et al 2012) Samples were ground into a powder 464

under liquid nitrogen for analysis 465

466

For the detection of DMMF (Figure 1) 05 g (fresh weight) of each sample was 467

weighed in the vial to which was added 1 mL of EDTA solution (100 mM pH 75) 1 468

mL of CaCl2 solution (mv = 20) and 20 microL of 2-octanol as the internal standard 469

(007 μgμL) The mixture was homogenized and the vial closed and placed on the 470

sample tray for analysis by CombiPAL autosampler (CTC Analytic CA USA) The 471

fruit volatiles were sampled by headspace solid-phase microextraction with a 65-μm 472

polydimethylsiloxane-divinylbenzene fibre (PDMS-DVB) Initially vials were 473

pre-incubated at 50degC for 10 min under continuous agitation (500 rpm) then volatiles 474

were extracted for 30 min at the same temperature and agitation speed After that the 475

fibre was desorbed in the GC injection port for 5 min in splitless mode The 7890A 476

GC chromatograph was equipped with a DB-5ms column (60 m times 250 μm times 1 μm 477

JampW Scientific Folsom CA USA) with helium as the carrier gas at a constant flow 478

of 12 mLmin The GC was programmed at an initial temperature of 35degC for 2 min 479

with a ramp of 5degC min up to 250degC and held for 5 min The injection port interface 480

and MS source temperatures were 250degC 260degC and 230degC respectively The 481

ionization potential was set at 70 eV recorded by a 5975B mass spectrometer (Agilent 482

Technologies JampW Scientific Folsom CA USA) and the scanning speed was 7 483

scanss The same method was used to analyse HDMF and DMMF in fruits from 484

different cultivars (Supplemental Figure S3) except for the sample preparation 485


18

procedure (Araguumlez et al 2013) briefly 05 g (fresh weight) of powdered fruit was 486

incubated at 30degC in a water bath for 5 min then 2 mL of NaCl (mv = 20) and 20 487

microL of 2-octanol as the internal standard (007 μgμL) were added and homogenized 488

489

For the detection of HDMF (Figure 1) and both furanones (Figure 4 and Supplemental 490

Table S3) a liquid-injection system equipped with CTC Pal ALS was used One gram 491

(fresh weight) of powdered fruit and 2 mL of NaCl (mv = 20) were homogenized in 492

a 10-mL tube and 300 μL of CH2Cl2 and 20 microL of 2-octanol (0766 μgμL) as the 493

internal standard were added to extract the volatiles the mixture was then 494

homogenized again The tubes were stored at room temperature for 20 min and 495

centrifuged at 12000 rpm for 5 min The subnatant was pipetted into a clean 15-mL 496

tube in which 20 mg of anhydrous sodium sulphate was added the tube was left 497

without shaking for half an hour Then 150 μL of the solution was transferred into a 498

GC vial and placed on the sample tray as described above Each sample (1 μL) was 499

injected using the autosampler and the analysis was accomplished by a 7890A 500

GC-MS system (Agilent Technologies JampW Scientific Folsom CA USA) equipped 501

with a DB-WAX column (30 m times 025 mm times 025 μm JampW) Helium (12 mLmin) 502

was used as a carrier gas The injection port (splitless injection mode) interface and 503

MS source temperatures were as described above The oven temperature was set at 504

60degC for 4 min then increased to 180degC at a rate of 4degCmin and maintained for 30 505

min DMMF was identified by comparison of electron ionization mass spectra and 506

retention time data with the data from the NISTEPANIH mass spectral library 507

(NIST-08 and Flavor) HDMF (Figure 1) was identified and qualified by comparison 508

with injected standard (Sigma) The quantitative analysis of both furanones (Figure 4 509

Supplemental Table S3 and Supplemental Figure S3) was determined using the peak 510

area of the internal standard as a reference based on total ion chromatogram (TIC) 511

512

RNA isolation and Reverse Transcription Quantitative PCR (RT-qPCR) 513

Total RNA from strawberry samples was isolated in this study using the CTAB 514

method (Chang et al 1993) Genomic DNA contamination was eliminated by 515


19

TURBO Dnase (Ambion) 1 μg of RNA was used to obtain cDNA using an IScriptTM 516

cDNA Synthesis Kit (Bio-Rad) The synthesized cDNA was diluted with water (120) 517

and 2 μL of diluted cDNA was used as the template for RT-qPCR Reactions were 518

performed in a total volume of 20 μL consisting of 10 μL of SYBR PCR supermix 519

(Bio-Rad) 1 μL of each primer (10 μM) 6 μL of DEPC-H2O and 2 μL of diluted 520

cDNA on CFX96 instrument (Bio-Rad) (Yin et al 2012) The oligonucleotide 521

primers for RT-qPCR analysis of genes including the AP2ERF FaQR and FaOMT 522

were designed according to the coding sequences of genes and the specificity was 523

verified before use (Min et al 2012) NCBIPrimer-BLAST was applied to design the 524

primers Relative expression levels were normalized to that of an internal controlmdashthe 525

interspacer 26S-18S strawberry RNA housekeeping gene FaRIB413 526

(Zorrilla-Fontanesi et al 2012) To investigate the differential expression of genes 527

during fruit development and ripening stages relative expression of each gene at the 528

R stage in the basal fruit section was set as 1 using the 2minus∆∆119862119879 method For transient 529

overexpression assay relative expression of control fruits with an empty vector (SK) 530

was set as 1 The primers used in RT-qPCR are listed in Supplemental Table S4 531

532

Gene isolation and analysis 533

Multiple database searches were performed to identify members of the strawberry 534

(Fragaria times ananassa) AP2ERF superfamily in the published strawberry databases 535

and annotations of Fragaria times ananassa and Fragaria times vesca by using the 536

AP2ERF DNA binding domain as a query sequence and 120 genes were identified 537

with AP2ERF domain(s) Based on the BLAST result primers (listed in 538

Supplemental Table S5) were used to amplify the coding full-length cDNAs of 539

AP2ERF genes The deduced amino acid sequences of AP2ERF proteins in 540

Arabidopsis thaliana used for construction of a phylogenetic tree were obtained from 541

the Arabidopsis Information Resource (TAIR) Alignment of the proteins was 542

performed using the neighbour-joining method in ClustalX (v181) and the 543

phylogenetic tree was constructed using FigTree (v131) 544

545


20

FaMYB98 was obtained by yeast one-hybrid screening sequencing and the full-length 546

sequences were predicted by BLAST Primers were designed (listed in Supplemental 547

Table S5) to amplify the full-length coding sequence 548

549

The promoter of FaQR was cloned by Genome Walking as previously described (Yin 550

et al 2010) and conserved cis-element motifs in the promoter were manually 551

searched 552

553

Conserved domains within the FaAP2ERF proteins and FaMYB98 were analysed by 554

CD-search (httpswwwncbinlmnihgovStructurecddwrpsbcgi) and cellular 555

locations of proteins including FaERF9 and FaMYB98 were predicted with the 556

WoLF PSORT program (httpswwwgenscriptcomwolf-psorthtml) 557

558

Dual-Luciferase Assays 559

In accordance with a previous protocol (Yin et al 2010) the full-length cDNAs of 560

FaAP2ERF or FaMYB98 transcription factors were cloned into pGreen II 0029 561

62-SK vector and the promoter of FaQR was cloned into pGreen II 0800-LUC vector 562

A luciferase gene from Renillia (REN) driven by a 35S promoter in the LUC vector 563

acted as a positive control The above constructs were transiently expressed in 564

Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration (strain 565

GV3101) and the activity of the TFs on the promoter was measured on the third day 566

after infiltration indicated by the ratio of enzyme activities of firefly luciferase (LUC) 567

and renilla luciferase (REN) using a Modulus Luminometer (Promega Madison WI 568

USA) To determine the activity of a specific transcription factor towards the 569

promoter we mixed the Agrobacterium culture with 1 mL of TF and 100 μL of 570

promoter and the LUCREN value of the empty vector SK on the promoter was set as 571

1 as a calibrator To test the combined effect of two transcription factors on the 572

promoter to the Agrobacterium culture mixture we added 05 mL of each TF and 100 573

μL of promoter the effect of the mixtures that contained each transcription factor (05 574

mL) and empty vector SK (05 mL) was also tested on the promoter as a control 575


21

576

Subcellular localization analysis 577

35S-FaERF9-GFP and 35S-FaMYB98-GFP were transiently expressed in tobacco (N 578

benthamiana) leaves by Agrobacterium infiltration (EHA105) using the same method 579

as described in the dual-luciferase assay above The transiently infected leaves were 580

imaged on the third day after infiltration using a Nikon A1-SHS confocal laser 581

scanning microscope The excitation wavelength for GFP fluorescence was 488 nm 582

and fluorescence was detected at 490ndash520 nm The primers used for GFP construction 583

are listed in Supplemental Table S6 584

585

Transient overexpression in strawberry fruit 586

Transient overexpression of FaERF9 and an overexpression binary vector (pGreen II 587

0029 62-SK-FaERF9-FaMYB98) were performed in strawberry fruit using the 588

FaERF9-SK construct described above for the dual-luciferase assays and the binary 589

vector constructed according to Liu et al 2013 The transfection of fruit was carried 590

out at the G stage (Hoffmann et al 2006) The FaERF9-SK construct and binary 591

vector were individually electroporated into Agrobacterium tumefaciens GV3101 and 592

the Agrobacterium culture suspended in infiltration buffer was infiltrated into the 593

whole fruit using a syringe As a control treatment fruits were infiltrated with 594

Agrobacterium carrying an empty vector SK under the same transfection conditions 595

Fruits were left attached to the plants and harvested on the seventh day after 596

infiltration Fruits of three biological replicates were sampled for further analysis 597

598

Yeast one-hybrid assay 599

In order to identify proteins that bind to the FaQR promoter the Matchmaker Gold 600

Yeast One-hybrid Library Screening System (Clontech USA) was used The sequence 601

of the FaQR promoter was cloned into the pAbAi vector and the construct was 602

integrated into the genome of the Y1HGold yeast strain A mixture of total RNA from 603

strawberry fruit at four stages (G T IR R) was used to construct the prey cDNA 604

library (TaKaRa) The background AbAr expression of Y1HGold FaQR-pAbAi strain 605


22

was tested according to the system user manual and then screening for protein-DNA 606

interactions was carried out Furthermore the full length of each transcription factor 607

was separately cloned into pGADT7 AD vector to confirm the screening results The 608

relationship between FaERF9 and FaQR promoter was also examined individually 609

Primers used in this assay are listed in Supplemental Table S6 610

611

Expression and purification of FaMYB98 recombinant protein 612

A 405-bp region spanning the DNA-binding domain of FaMYB98 was amplified 613

from FaMYB98 cDNA and cloned into a pET6timesHN vector (Clontech Palo CA USA) 614

to generate the recombinant N-terminal His-tagged protein the primers used are listed 615

in Supplemental Table S6 The resulting construct was sequenced and introduced into 616

Escherichia coli strain Rosetta 2(DE3)pLysS (Novagen) Ten millilitres of the 617

overnight culture was combined with 500 mL of an LB liquid medium (100 μgmL 618

ampicillin and 34 μgmL chloramphenicol) as described (Li et al 2017) Isopropyl 619

β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and 620

the bacterial culture was incubated at 18degC at 150 rpm for 18 h Then the cells were 621

harvested using a centrifuge and resuspended in 1times PBS buffer (Sangon Biotech) 622

after which they were disrupted by sonication and the supernatant was purified using 623

a HisTALONTM Gravity Column (Clontech) according to the user manual The PD-10 624

Desalting column (GE Healthcare) was then used for buffer change and sample 625

clean-up of proteins according to the gravity protocol SDS-PAGE was performed and 626

the protein was visualized using Coomassie brilliant blue 627

628

Electrophoretic mobility shift assay (EMSA) 629

A Lightshift Chemiluminescent EMSA kit (Thermo) was used to perform EMSA 630

experiments according to the manufacturerrsquos instructions Single-strand 631

oligonucleotides were synthesized and biotinylated by GeneBio Biotech (Hangzhou 632

China) The details of the EMSA assay are provided in Ge et al (2017) The probes 633

used for EMSAs are listed in Supplemental Table S6 634

635


23

636

Yeast two-hybrid assay 637

The DUALhunter system (Dualsystems Biotech Switzerland) was used to investigate 638

the interaction between FaERF9 and FaMYB98 Full-length coding sequences of 639

FaERF9 and FaMYB98 were respectively cloned into pDHB1 bait vector and 640

pPR3-N prey vector sequences were verified and the bait plasmid was 641

co-transformed with prey plasmid into NMY51 in the combinations below 642

FaMYB98-pDHB1pPR3-N FaMYB98-pDHB1FaERF9-pPR3-N 643

FaMYB98-pDHB1pOst1-NubI FaERF9-pDHB1pPR3-N 644

FaERF9-pDHB1FaMYB98-pPR3-N and FaERF9-pDHB1pOst1-NubI (Li et al 645

2017) Transformed cells were spread onto the following plates DDO (SD 646

medium-Trp-Leu) QDO (SD medium-Trp-Leu-His-Ade) and QDO+3-AT (QDO 647

medium supplemented with 1 mM 3-amino-124-triazole) The control plasmid 648

pOst1-NubI was used to determine whether the bait was functional the pPR3-N prey 649

vector plasmid was used to test whether the bait displayed non-specific background 650

and positive interactions were indicated by the growth of yeast on QDO and 651

QDO+3-AT plates The Primers used in the DUALhunter assay are listed in 652

Supplemental Table S6 653

654

Bimolecular fluorescence complementation (BiFC) assays 655

Full-length FaERF9 and FaMYB98 respectively were cloned into sequences 656

encoding the N- and C- fragments of YFP protein (Lv et al 2014) All constructs 657

were transiently expressed in tobacco (N benthamiana) leaves by Agrobacterium 658

infiltration (EHA105) The YFP fluorescence of transfected leaves was imaged 40 h 659

after infiltration by using a Nikon A1-SHS confocal laser scanning microscope The 660

excitation wavelength for YFP was 488 nm and fluorescence was detected at 520ndash661

540 nm Primers used are listed in Supplemental Table S6 662

663

Statistics 664

A Studentrsquos two-tailed t test ( plt005 plt001 and plt0001) was used to 665


24

determine significant differences between two groups in this study Microsoft Excel 666

was used to perform linear regressive analysis significant differences were 667

determined by SPSS Statistics 200 and scatter plots were prepared with Origin80 668

Least significant difference (LSD) at the 5 level was calculated using Microsoft 669

Excel Figures were prepared with Origin80 (Microcal Software Inc Northampton 670

MA USA) The online tool MetaboAnalyst 36 (httpwwwmetaboanalystca) was 671

used to analyse the transcript abundance of the AP2ERF genes 672

673

Accession numbers 674

Sequence data from this article have been deposited in GenBank under the accession 675

numbers MH332903-MH333022 for AP2ERF genes in Fragaria times ananassa and 676

MH333023 for FaMYB98 GenBank numbers for FaQR and FaOMT were 677

AY1588361 (Raab et al 2006) and AF2204912 (Wein et al 2002) 678

679

Supplemental Material 680

Supplemental Figure S1 Phylogenetic analysis of FaAP2ERF and Arabidopsis 681

AP2ERF proteins 682

Supplemental Figure S2 Analysis of FaAP2ERF gene expression patterns in 683

strawberry fruit during development and ripening 684

Supplemental Figure S3 Contents of furanones in fruit of strawberry cultivars 685

Supplemental Figure S4 Relative expression of FaERF9 in FaERF9-FaMYB98- 686

and FaERF9-overexpressing fruits 687

Supplemental Figure S5 Subcellular localization of FaMYB98 688

Supplemental Figure S6 The combined activation effects of FaERF9FaMYB98 on 689

the FaOMT promoter 690

Supplemental Figure S7 Relative expression of FaOMT in 691

FaERF9-overexpressing fruits 692

Supplemental Table S1 AP2ERF superfamily genes in Fragaria times ananassa 693

Supplemental Table S2 Classification of the AP2ERF superfamily members in 694

Fragaria times ananassa 695


25

Supplemental Table S3 Furanone content in FaERF9-FaMYB98- and 696


Supplemental Table S4 Primers used for reverse transcription quantitative PCR 698

(RT-qPCR) 699

Supplemental Table S5 Primers used to amplify the full-length coding sequences of 700

AP2ERF genes and FaMYB98 in strawberry (Fragaria times ananassa) 701

Supplemental Table S6 Primers used for vector construction 702

703

Acknowledgments 704




708

Figure legends 709

Figure 1 Changes in furanone content and furanone biosynthetic genes during 710

strawberry (Fragaria times ananassa Duch cv Yuexin) fruit development and ripening 711

A Fruits were collected at four ripening stages G (green stage) T (turning stage) IR 712

(intermediate red stage) R (full red stage) B Changes in the contents of furaneol 713

(HDMF) and mesifurane (DMMF) HDMF content was calculated using a standard 714

curve of HDMF while DMMF content was calculated with an internal standard as the 715

reference C Expression levels of FaQR and FaOMT The expression levels of each 716

gene were calculated relative to corresponding values in the basal section at the R 717

stage SEs were calculated from three replicates and LSD values represent the least 718

significant differences at p = 005 719

Figure 2 Regulatory effects of FaAP2ERF on the promoter of FaQR LUCREN 720

values of the empty vector on FaQR promoter were used as the calibrator set as 1 721

and SEs were calculated from five replicates statistical significance was determined 722

by Studentrsquos two-tailed t test ( plt0001) 723

Figure 3 Expression and the subcellular localization of FaERF9 A The expression 724

levels were calculated relative to the levels in the basal section at the R stage B 725


26

Subcellular localization analysis was performed in tobacco leaves The tobacco used 726

in the assay was stably transformed with a specific nucleus localized red fluorescence 727

protein construct and the red fluorescence indicates the nucleus-localized signal 728

(NLS) Scale bar = 25 microm SEs were calculated from three replicates 729

Figure 4 Transient overexpression of FaERF9 in strawberry fruit A Relative 730

expression of FaQR and FaERF9 in FaERF9-OX fruit 7 days after infiltration The 731

expression was calculated relative to the average value in control (SK) fruits B 732

HDMF and DMMF content in FaERF9-OX fruit The content of both furanones 733

were quantified using the peak area of the internal standard (2-octanol) as the 734

reference SEs were calculated from three replicates Statistical significance was 735

determined by Studentrsquos two-tailed t test ( plt005 plt001 plt0001) 736

Figure 5 Scatter plots of 11 strawberry cultivars A Linear regression analysis 737

between FaERF9 expression and FaQR expression B Linear regression analysis 738

between FaERF9 expression and furanone content The relative expression was 739

normalized to that of the housekeeping gene FaRIB413 and furanone content was 740

calculated with internal standard as the reference Significant differences were 741

determined by SPSS Statistics 200 742

Figure 6 Interactions of FaERF9 and FaMYB98 with the FaQR promoter A Yeast 743

one-hybrid analysis of the interaction of FaERF9 and FaQR promoter B Yeast 744

one-hybrid analysis of the interaction of FaMYB98 and FaQR promoter C 745

Regulatory effect of FaMYB98 on the FaQR promoter Auto-activation was tested on 746

SD-Ura (synthetically defined medium without uracil) in presence of Aureobasidin A 747

(AbA) and physical interaction was determined on SD-Leu (synthetically defined 748

medium without leucine) in presence of AbA LUCREN values of the empty vector 749

on FaQR promoter were used as the calibrator set as 1 and error bars were calculated 750

from five replicates Statistical significance was determined by Studentrsquos two-tailed t 751

test ( plt005 plt001 plt0001) 752

Figure 7 Electrophoretic mobility shift assay of FaMYB98 binding to FaQR 753

promoter A The probe sequence used for EMSA and mutated nucleotides are 754

indicated with lowercase letters B Purified DNA-binding domain of FaMYB98 755


27

protein and biotin-labelled DNA probe were mixed and analysed on 6 native 756

polyacrylamide gels and then photographed Presence or absence of specific probes is 757

marked by the symbol + or - 758

Figure 8 The combined activation effects of FaERF9FaMYB98 on the FaQR 759

promoter LUCREN values obtained with the empty vector and FaQR promoter were 760

used as the calibrator set as 1 and error bars were calculated from five replicates 761

Figure 9 Yeast two-hybrid analysis of the protein-protein interactions between 762

FaMYB98 and FaERF9 Positive interactions were determined by the growth of 763

yeast cells on selection plates Bait with pOST acted as positive controls and bait with 764

pPR3 as negative controls DDO synthetically defined medium without tryptophan 765

and leucine QDO synthetically defined medium without tryptophan leucine 766

histidine and adenine QDO+3AT QDO medium supplemented with 1 mM 767

3-aminotriazole 768

Figure 10 BiFC analysis of the protein-protein interactions between FaMYB98 and 769

FaERF9 The pairs of fusion proteins tested were FaMYB98-YFPN+FaERF9-YFPC 770

FaERF9-YFPN+FaMYB98-YFPC FaMYB98-YFPN+FaMYB98-YFPC and 771

FaERF9-YFPN+FaERF9-YFPC The other combinations were negative controls 772

The tobacco used in the assay was stably transformed with a specific 773

nucleus-localized red fluorescence protein construct and the red fluorescence 774

indicated the nucleus-localized signal (NLS) Fluorescence of the yellow fluorescence 775

protein (YFP) visualized the interaction in vivo Scale bar = 25 microm 776

777

Literature Cited 778

779

Agarwal P Agarwal PK Joshi AJ Sopory SK Reddy MK (2010) Overexpression 780

of PgDREB2A transcription factor enhances abiotic stress tolerance and activates 781

downstream stress-responsive genes Mol Biol Rep 37 1125-1135 782

Araguumlez I Osorio S Hoffmann T Rambla JL Medina-Escobar N Granell A 783

Botella MAacute Schwab W Valpuesta V (2013) Eugenol production in achenes and 784


28

receptacles of strawberry fruits is catalyzed by synthases exhibiting distinct kinetics 785

Plant Physiol 163 946-958 786

Carvalho RF Carvalho SD OrsquoGrady K Folta KM (2016) Agroinfiltration of 787

strawberry fruit mdash a powerful transient expression system for gene validation Curr 788

Plant Biol 6 19-37 789

Chai L Shen YY (2016) FaABI4 is involved in strawberry fruit ripening Sci Hortic 790

(Amsterdam) 210 34-40 791

Chang SJ Puryear J Cairney J (1993) A simple and efficient method for isolating 792

RNA from pine trees Plant Mol Biol Rep 11 113-116 793

Colquhoun TA Kim JY Wedde AE Levin LA Schmitt KC Schuurink RC 794

Clark DG (2011) PhMYB4 fine-tunes the floral volatile signature of 795

Petuniatimeshybrida through PhC4H J Exp Bot 62 1133-1143 796

Dal Cin V Tieman DM Tohge T McQuinn R de Vos RCH Osorio S Schmelz 797

EA Taylor MG Smits-Kroon MT Schuurink RC Haring MA Giovannoni J 798

Fernie AR Klee HJ (2011) Identification of genes in the phenylalanine metabolic 799

pathway by ectopic expression of a MYB transcription factor in tomato fruit Plant 800

Cell 23 2738-2753 801

Fu XM Cheng SH Zhang YQ Du B Feng C Zhou Y Mei X Jiang YM Duan 802

XW Yang ZY (2017) Differential responses of four biosynthetic pathways of 803

aroma compounds in postharvest strawberry ( Fragaria times ananassa Duch) under 804

interaction of light and temperature Food Chem 221 356-364 805

Gao LM Zhang J Hou Y Yao YC Ji QL (2015) RNA-Seq screening of 806

differentially-expressed genes during somatic embryogenesis in Fragaria times 807

ananassa Duch lsquoBenihopprsquo J Hortic Sci Biotechnol 90 671-681 808

Ge H Zhang J Zhang YJ Li X Yin XR Grierson D Chen KS (2017) EjNAC3 809

transcriptionally regulates chilling-induced lignification of loquat fruit via physical 810

interaction with an atypical CAD-like gene J Exp Bot 68 5129-5136 811

Goacutemez-Maldonado J Avila C Torre F Cantildeas R Caacutenovas FM Campbell MM 812

(2004) Functional interactions between a glutamine synthetase promoter and MYB 813

proteins Plant J 39 513-526 814


29

Han Y Dang R Li J Jiang J Zhang N Jia M Wei L Li Z Li B Jia W (2015) 815

Sucrose nonfermenting1-related protein kinase26 an ortholog of open stomata1 is 816

a negative regulator of strawberry fruit development and ripening Plant Physiol 817

167 915-930 818

Hoffmann T Kalinowski G Schwab W (2006) RNAi-induced silencing of gene 819

expression in strawberry fruit (Fragaria times ananassa) by agroinfiltration a rapid 820

assay for gene function analysis Plant J 48 818-826 821

Jetti RR Yang E Kurnianta A Finn C Qian MC (2007) Quantification of 822

selected aroma-active compounds in strawberries by headspace solid-phase 823

microextraction gas chromatography and correlation with sensory descriptive 824

analysis J Food Sci 72 S487-S496 825

Jia HF Chai YM Li CL Lu D Luo JJ Qin L Shen YY (2011) Abscisic acid 826

plays an important role in the regulation of strawberry fruit ripening Plant Physiol 827

157 188-199 828

Jiang YQ Deyholos MK (2006) Comprehensive transcriptional profiling of 829

NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes BMC 830

Plant Biol 6 20 831

Kasahara RD Portereiko MF Sandaklie-Nikolova L Rabiger DS Drews GN 832

(2005) MYB98 is required for pollen tube guidance and synergid cell differentiation 833

in Arabidopsis Plant Cell 17 2981-2992 834

Klein D Fink B Arold B Eisenreich W Schwab W (2007) Functional 835

characterization of enone oxidoreductases from strawberry and tomato fruit J 836

Agric Food Chem 55 6705-6711 837

Koehler G Wilson RC Goodpaster JV Sonsteby A Lai X Witzmann FA You 838

J-S Rohloff J Randall SK Alsheikh M (2012) Proteomic study of 839

low-temperature responses in strawberry cultivars (Fragaria x ananassa) that differ 840

in cold tolerance Plant Physiol 159 1787-1805 841

Krishnaswamy S Verma S Rahman MH Kav NNV (2011) Functional 842

characterization of four APETALA2-family genes (RAP26 RAP26L DREB19 843

and DREB26) in Arabidopsis Plant Mol Biol 75 107-127 844


30

Kulkarni R Chidley H Deshpande A Schmidt A Pujari K Giri A Gershenzon 845

J Gupta V (2013) An oxidoreductase from lsquoAlphonsorsquo mango catalyzing 846

biosynthesis of furaneol and reduction of reactive carbonyls SpringerPlus 2 494 847

Larsen M Poll L (1992) Odour thresholds of some important aroma compounds in 848

strawberries Z Lebensm-Unters Forsch 195 120-123 849

Li L Luo ZS Huang XH Zhang L Zhao PY Ma HY Li XH Ban ZJ Liu X 850

(2015) Label-free quantitative proteomics to investigate strawberry fruit proteome 851

changes under controlled atmosphere and low temperature storage J Proteomics 852

120 44-57 853

Li SJ Yin XR Wang WL Liu XF Zhang B Chen KS (2017) Citrus CitNAC62 854

cooperates with CitWRKY1 to participate in citric acid degradation via 855

up-regulation of CitAco3 J Exp Bot 68 3419-3426 856

Li X Xu YY Shen SL Yin XR Klee HJ Zhang B Chen KS (2017) Transcription 857

factor CitERF71 activates the terpene synthase gene CitTPS16 involved in the 858

synthesis of E-geraniol in sweet orange fruit J Exp Bot 68 4929-4938 859

Licausi F Giorgi FM Zenoni S Osti F Pezzotti M Perata P (2010) Genomic and 860

transcriptomic analysis of the AP2ERF superfamily in Vitis vinifera BMC 861

Genomics 11 719 862

Licausi F Ohme-Takagi M Perata P (2013) APETALA2Ethylene Responsive 863

Factor (AP2ERF) transcription factors mediators of stress responses and 864

developmental programs New Phytol 199 639-649 865

Liu F Xiao ZN Yang L Chen Q Shao L Liu JX Yu YX (2017) PhERF6 866

interacting with EOBI negatively regulates fragrance biosynthesis in petunia 867

flowers New Phytol 215 1490-1502 868

Liu XF Yin XR Allan AC Lin Wang K Shi YN Huang YJ Ferguson IB Xu 869

CJ Chen KS (2013) The role of MrbHLH1 and MrMYB1 in regulating 870

anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) 871

during anthocyanin biosynthesis Plant Cell Tissue Organ Cult 115 285-298 872


31

Lv QD Zhong YJ Wang YG Wang ZY Zhang L Shi J Wu ZC Liu Y Mao 873

CZ Yi KK Wu P (2014) SPX4 negatively regulates phosphate signaling and 874

homeostasis through its interaction with PHR2 in rice Plant Cell 26 1586-1597 875

Medina-Puche L Molina-Hidalgo FJ Boersma M Schuurink RC 876

Loacutepez-Vidriero I Solano R Franco-Zorrilla J-M Caballero JL 877

Blanco-Portales R Muntildeoz-Blanco J (2015) An R2R3-MYB transcription factor 878

regulates eugenol production in ripe strawberry fruit receptacles Plant Physiol 168 879

598-614 880

Meacutenager I Jost M Aubert C (2004) Changes in physicochemical characteristics 881

and volatile constituents of strawberry (cv Cigaline) during maturation J Agric 882

Food Chem 52 1248-1254 883

Min T Yin XR Shi YN Luo ZR Yao Y Grierson D Ferguson IB Chen KS 884

(2012) Ethylene-responsive transcription factors interact with promoters of ADH 885

and PDC involved in persimmon (Diospyros kaki) fruit de-astringency J Exp Bot 886

63 6393-6405 887

Molina-Hidalgo FJ Medina-Puche L Cantildeete-Goacutemez C Franco-Zorrilla JM 888

Loacutepez-Vidriero I Solano R Caballero JL Rodriacuteguez-Franco A 889

Blanco-Portales R Muntildeoz-Blanco J Moyano E (2017) The fruit-specific 890

transcription factor FaDOF2 regulates the production of eugenol in ripe fruit 891

receptacles J Exp Bot 68 4529-4543 892

Mu Q Wang BJ Leng XP Sun X Shangguan LF Jia HF Fang JG (2016) 893

Comparison and verification of the genes involved in ethylene biosynthesis and 894

signaling in apple grape peach pear and strawberry Acta Physiol Plant 38 44 895

Nakano T Suzuki K Fujimura T Shinshi H (2006) Genome-wide analysis of the 896

ERF gene family in Arabidopsis and rice Plant Physiol 140 411-432 897

Owens CL Thomashow MF Hancock JF Iezzoni AF (2002) CBF1 orthologs in 898

sour cherry and strawberry and the heterologous expression of CBF1 in strawberry 899

J Am Soc Hortic Sci 127 489-494 900

Pirrello J Prasad BN Zhang WS Chen KS Mila I Zouine M Latcheacute A Pech 901

JC Ohme-Takagi M Regad F Bouzayen M (2012) Functional analysis and 902


32

binding affinity of tomato ethylene response factors provide insight on the 903

molecular bases of plant differential responses to ethylene BMC Plant Biol 12 190 904

Pisarnitskii AF Demechenko AG Egorov IA Gvelesiani RK (1992) 905

Methylpentoses are possible precursors of furanones in fruits Prikl Biokhim 906

Mikrobiol 28 123-127 907

Punwani JA Rabiger DS Drews GN (2007) MYB98 positively regulates a battery 908

of synergid-expressed genes encoding filiform apparatus localized proteins Plant 909

Cell 19 2557-2568 910

Pyysalo T Honkanen E Hirvi T (1979) Volatiles of wild strawberries Fragaria 911

vesca L compared to those of cultivated berries Fragariatimes ananassa cv Senga 912

Sengana J Agric Food Chem 27 19-22 913

Raab T Loacutepez-Raacuteez JA Klein D Caballero JL Moyano E Schwab W 914

Muntildeoz-Blanco J (2006) FaQR required for the biosynthesis of the strawberry 915

flavor compound 4-hydroxy-25-dimethyl-3(2H)-furanone encodes an enone 916

oxidoreductase Plant Cell 18 1023-1037 917

Riechmann JL Heard J Martin G Reuber L Jiang CZ Keddie J Adam L 918

Pineda O Ratcliffe OJ Samaha RR Creelman R Pilgrim M Broun P Zhang 919

JZ Ghandehari D Sherman BK Yu GL (2000) Arabidopsis transcription 920

factors genome-wide comparative analysis among eukaryotes Science 290 921

2105-2110 922

Roscher R Bringmann G Schreier P Schwab W (1998) Radiotracer studies on the 923

formation of 25-dimethyl-4-hydroxy-3(2H)-furanone in detached ripening 924

strawberry fruits J Agric Food Chem 46 1488-1493 925

Roscher R Herderich M Steffen JP Schreier P Schwab W (1996) 926

25-dimethyl-4-hydroxy-3[2H]-furanone 6o-malonyl-β-d-glucopyranoside in 927

strawberry fruits Phytochemistry 43 155-159 928

Schwab W (1998) Application of stable isotope ratio analysis explaining the 929

bioformation of 25-dimethyl-4-hydroxy-3(2H)-furanone in plants by a biological 930

maillard reaction J Agric Food Chem 46 2266ndash2269 931


33

Schwab W (2013) Natural 4-hydroxy-25-dimethyl-3(2H)-furanone (Furaneolreg) 932

Molecules 18 6936-6951 933

Schwab W Roscher R (1997) 4-Hydroxy-3(2H)-furanones natural and maillard 934

products Recent Res Dev Phytochem 1 643-673 935

Schwieterman ML Colquhoun TA Jaworski EA Bartoshuk LM Gilbert JL 936

Tieman DM Odabasi AZ Moskowitz HR Folta KM Klee HJ Sims CA 937

Whitaker VM Clark DG (2014) Strawberry flavor diverse chemical 938

compositions a seasonal influence and effects on sensory perception PLoS One 9 939

e88446 940

Sharma MK Kumar R Solanke AU Sharma R Tyagi AK Sharma AK (2010) 941

Identification phylogeny and transcript profiling of ERF family genes during 942

development and abiotic stress treatments in tomato Mol Genet Genomics 284 943

455-475 944

Shen SL Yin XR Zhang B Xie XL Jiang Q Grierson D Chen KS (2016) 945

CitAP210 activation of the terpene synthase CsTPS1 is associated with the 946

synthesis of (+)-valencene in lsquoNewhallrsquo orange J Exp Bot 67 4105-4115 947

Shigyo M Ito M (2004) Analysis of gymnosperm two-AP2-domain-containing genes 948

Dev Genes Evol 214 105-114 949

Song C Hong X Zhao S Liu J Schulenburg K Huang F-C Franz-Oberdorf K 950

Schwab W (2016) Glucosylation of 4-hydroxy-25-dimethyl-3(2H)-furanone the 951

key strawberry flavor compound in strawberry fruit Plant Physiol 171 139-151 952

Spitzer-Rimon B Farhi M Albo B Cnarsquoani A Ben Zvi MM Masci T Edelbaum 953

O Yu YX Shklarman E Ovadis M Vainstein A (2012) The R2R3-MYB-like 954

regulatory factor EOBI acting downstream of EOBII regulates scent production 955

by activating ODO1 and structural scent-related genes in petunia Plant Cell 24 956

5089-5105 957

Tieman D (2017) Transcriptional control of strawberry ripening ndash two to tango J Exp 958

Bot 68 4407-4409 959

Vallarino JG Osorio S Bombarely A Casantildeal A Cruz-Rus E Saacutenchez-Sevilla 960

JF Amaya I Giavalisco P Fernie AR Botella MA Valpuesta V (2015) Central 961


34

role of FaGAMYB in the transition of the strawberry receptacle from development 962

to ripening New Phytol 208 482-496 963

Verdonk JC Haring MA van Tunen AJ Schuurink RC (2005) ODORANT1 964

regulates fragrance biosynthesis in petunia flowers Plant Cell 17 1612-1624 965

Wang SF Song MY Guo JX Huang Y Zhang F Xu C Xiao YH Zhang LS 966

(2018) The potassium channel FaTPK1 plays a critical role in fruit quality 967

formation in strawberry (Fragaria times ananassa) Plant Biotechnol J 16 737-748 968

Wein M Lavid N Lunkenbein S Lewinsohn E Schwab W Kaldenhoff R (2002) 969

Isolation cloning and expression of a multifunctional O-methyltransferase capable 970

of forming 25-dimethyl-4-methoxy-3(2H)-furanone one of the key aroma 971

compounds in strawberry fruits Plant J 31 755-765 972

Weirauch MT Hughes TR (2011) A catalogue of eukaryotic transcription factor 973

types their evolutionary origin and species distribution In A handbook of 974

transcription factors (Hughes TR Ed) Springer Dordrecht Subcellular 975

Biochemistry 52 25-73 976

Wuumlst M (2017) Biosynthesis of plant-derived odorants In Springer handbook of 977

odor (Buettner A Ed) Springer Cham 9-10 978

Xie XL Shen SL Yin XR Xu Q Sun CD Grierson D Ferguson I Chen KS 979

(2014) Isolation classification and transcription profiles of the AP2ERF 980

transcription factor superfamily in citrus Mol Biol Rep 41 4261-4271 981

Xie XL Yin XR Chen KS (2016) Roles of APETALA2ethylene-response factors in 982

regulation of fruit quality Crit Rev Plant Sci 35 120-130 983

Yao GF Ming ML Allan AC Gu C Li LT Wu X Wang RZ Chang YJ Qi KJ 984

Zhang SL Wu J (2017) Map-based cloning of the pear gene MYB114 identifies an 985

interaction with other transcription factors to coordinately regulate fruit 986

anthocyanin biosynthesis Plant J 92 437-451 987

Yin XR Allan AC Chen KS Ferguson IB (2010) Kiwifruit EIL and ERF genes 988

involved in regulating fruit ripening Plant Physiol 153 1280-1292 989


35

Yin XR Shi YN Min T Luo ZR Yao YC Xu Q Ferguson I Chen KS (2012) 990

Expression of ethylene response genes during persimmon fruit astringency removal 991

Planta 235 895-906 992

Zabetakis I Gramshaw JW Robinson DS (1999) 993

25-dimethyl-4-hydroxy-2H-furan-3-one and its derivatives analysis synthesis and 994

biosynthesis-a review Food Chem 65 139-151 995

Zabetakis I Holden MA (1997) Strawberry flavour analysis and biosynthesis J Sci 996

Food Agric 74 421-434 997

Zeng JK Li X Xu Q Chen JY Yin XR Ferguson IB Chen KS (2015) EjAP2-1 998

an AP2ERF gene is a novel regulator of fruit lignification induced by chilling 999

injury via interaction with EjMYB transcription factors Plant Biotechnol J 13 1000

1325-1334 1001

Zhang AD Hu X Kuang S Ge H Yin XR Chen KS (2016) Isolation 1002

classification and transcription profiles of the Ethylene Response Factors (ERFs) in 1003

ripening kiwifruit Sci Hortic (Amsterdam) 199 209-215 1004

Zhang CH Shangguan LF Ma RJ Sun X Tao R Guo L Korir NK Yu ML 1005

(2012) Genome-wide analysis of the AP2ERF superfamily in peach (Prunus 1006

persica) Genet Mol Res 11 4788-4809 1007

Zhang Y Tang HR Luo Y Wang XR Chen Q Liu ZJ (2014) Isolation and 1008

expression analysis of a CRTDRE-binding factor gene FaCBF1 from Fragaria times 1009

ananassa Acta Hortic 41 240-248 1010

Zhu Q Zhang JT Gao XS Tong JH Xiao LT Li WB Zhang HX (2010) The 1011

Arabidopsis AP2ERF transcription factor RAP26 participates in ABA salt and 1012

osmotic stress responses Gene 457 1-12 1013

Zhuang J Peng RH Cheng ZM (Max) Zhang J Cai B Zhang Z Gao F Zhu B 1014

Fu XY Jin XF Chen JM Qiao YS Xiong AS Yao QH (2009) Genome-wide 1015

analysis of the putative AP2ERF family genes in Vitis vinifera Sci Hortic 1016

(Amsterdam) 123 73-81 1017

Zorrilla-Fontanesi Y Rambla JL Cabeza A Medina JJ Sanchez-Sevilla JF 1018

Valpuesta V Botella MA Granell A Amaya I (2012) Genetic analysis of 1019


36

strawberry fruit aroma and identification of O-methyltransferase FaOMT as the 1020

locus controlling natural variation in mesifurane content Plant Physiol 159 1021

851-870 1022












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Licausi F Giorgi FM Zenoni S Osti F Pezzotti M Perata P (2010) Genomic and transcriptomic analysis of the AP2ERF superfamily inVitis vinifera BMC Genomics 11 719


Licausi F Ohme-Takagi M Perata P (2013) APETALA2Ethylene Responsive Factor (AP2ERF) transcription factors mediators of stressresponses and developmental programs New Phytol 199 639-649


Liu F Xiao ZN Yang L Chen Q Shao L Liu JX Yu YX (2017) PhERF6 interacting with EOBI negatively regulates fragrancebiosynthesis in petunia flowers New Phytol 215 1490-1502


Liu XF Yin XR Allan AC Lin Wang K Shi YN Huang YJ Ferguson IB Xu CJ Chen KS (2013) The role of MrbHLH1 and MrMYB1 inregulating anthocyanin biosynthetic genes in tobacco and Chinese bayberry (Myrica rubra) during anthocyanin biosynthesis Plant CellTissue Organ Cult 115 285-298

Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title wwwplantphysiolorgon February 25 2020 - Published by Downloaded from

Copyright copy 2018 American Society of Plant Biologists All rights reserved

Google Scholar Author Only Title Only Author and Title

Lv QD Zhong YJ Wang YG Wang ZY Zhang L Shi J Wu ZC Liu Y Mao CZ Yi KK Wu P (2014) SPX4 negatively regulates phosphatesignaling and homeostasis through its interaction with PHR2 in rice Plant Cell 26 1586-1597


Medina-Puche L Molina-Hidalgo FJ Boersma M Schuurink RC Loacutepez-Vidriero I Solano R Franco-Zorrilla J-M Caballero JL Blanco-Portales R Muntildeoz-Blanco J (2015) An R2R3-MYB transcription factor regulates eugenol production in ripe strawberry fruitreceptacles Plant Physiol 168 598-614


Meacutenager I Jost M Aubert C (2004) Changes in physicochemical characteristics and volatile constituents of strawberry (cv Cigaline)during maturation J Agric Food Chem 52 1248-1254


Min T Yin XR Shi YN Luo ZR Yao Y Grierson D Ferguson IB Chen KS (2012) Ethylene-responsive transcription factors interact withpromoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency J Exp Bot 63 6393-6405


Molina-Hidalgo FJ Medina-Puche L Cantildeete-Goacutemez C Franco-Zorrilla JM Loacutepez-Vidriero I Solano R Caballero JL Rodriacuteguez-FrancoA Blanco-Portales R Muntildeoz-Blanco J Moyano E (2017) The fruit-specific transcription factor FaDOF2 regulates the production ofeugenol in ripe fruit receptacles J Exp Bot 68 4529-4543


Mu Q Wang BJ Leng XP Sun X Shangguan LF Jia HF Fang JG (2016) Comparison and verification of the genes involved in ethylenebiosynthesis and signaling in apple grape peach pear and strawberry Acta Physiol Plant 38 44


Nakano T Suzuki K Fujimura T Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice Plant Physiol140 411-432


Owens CL Thomashow MF Hancock JF Iezzoni AF (2002) CBF1 orthologs in sour cherry and strawberry and the heterologousexpression of CBF1 in strawberry J Am Soc Hortic Sci 127 489-494


Pirrello J Prasad BN Zhang WS Chen KS Mila I Zouine M Latcheacute A Pech JC Ohme-Takagi M Regad F Bouzayen M (2012)Functional analysis and binding affinity of tomato ethylene response factors provide insight on the molecular bases of plant differentialresponses to ethylene BMC Plant Biol 12 190


Pisarnitskii AF Demechenko AG Egorov IA Gvelesiani RK (1992) Methylpentoses are possible precursors of furanones in fruits PriklBiokhim Mikrobiol 28 123-127


Punwani JA Rabiger DS Drews GN (2007) MYB98 positively regulates a battery of synergid-expressed genes encoding filiformapparatus localized proteins Plant Cell 19 2557-2568


Pyysalo T Honkanen E Hirvi T (1979) Volatiles of wild strawberries Fragaria vesca L compared to those of cultivated berriesFragariatimes ananassa cv Senga Sengana J Agric Food Chem 27 19-22


Raab T Loacutepez-Raacuteez JA Klein D Caballero JL Moyano E Schwab W Muntildeoz-Blanco J (2006) FaQR required for the biosynthesis of thestrawberry flavor compound 4-hydroxy-25-dimethyl-3(2H)-furanone encodes an enone oxidoreductase Plant Cell 18 1023-1037


Riechmann JL Heard J Martin G Reuber L Jiang CZ Keddie J Adam L Pineda O Ratcliffe OJ Samaha RR Creelman R Pilgrim MBroun P Zhang JZ Ghandehari D Sherman BK Yu GL (2000) Arabidopsis transcription factors genome-wide comparative analysisamong eukaryotes Science 290 2105-2110



Roscher R Bringmann G Schreier P Schwab W (1998) Radiotracer studies on the formation of 25-dimethyl-4-hydroxy-3(2H)-furanonein detached ripening strawberry fruits J Agric Food Chem 46 1488-1493


Roscher R Herderich M Steffen JP Schreier P Schwab W (1996) 25-dimethyl-4-hydroxy-3[2H]-furanone 6o-malonyl-β-d-glucopyranoside in strawberry fruits Phytochemistry 43 155-159


Schwab W (1998) Application of stable isotope ratio analysis explaining the bioformation of 25-dimethyl-4-hydroxy-3(2H)-furanone inplants by a biological maillard reaction J Agric Food Chem 46 2266ndash2269


Schwab W (2013) Natural 4-hydroxy-25-dimethyl-3(2H)-furanone (Furaneolreg) Molecules 18 6936-6951Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Schwab W Roscher R (1997) 4-Hydroxy-3(2H)-furanones natural and maillard products Recent Res Dev Phytochem 1 643-673Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Schwieterman ML Colquhoun TA Jaworski EA Bartoshuk LM Gilbert JL Tieman DM Odabasi AZ Moskowitz HR Folta KM Klee HJSims CA Whitaker VM Clark DG (2014) Strawberry flavor diverse chemical compositions a seasonal influence and effects on sensoryperception PLoS One 9 e88446


Sharma MK Kumar R Solanke AU Sharma R Tyagi AK Sharma AK (2010) Identification phylogeny and transcript profiling of ERFfamily genes during development and abiotic stress treatments in tomato Mol Genet Genomics 284 455-475


Shen SL Yin XR Zhang B Xie XL Jiang Q Grierson D Chen KS (2016) CitAP210 activation of the terpene synthase CsTPS1 isassociated with the synthesis of (+)-valencene in Newhall orange J Exp Bot 67 4105-4115


Shigyo M Ito M (2004) Analysis of gymnosperm two-AP2-domain-containing genes Dev Genes Evol 214 105-114Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Song C Hong X Zhao S Liu J Schulenburg K Huang F-C Franz-Oberdorf K Schwab W (2016) Glucosylation of 4-hydroxy-25-dimethyl-3(2H)-furanone the key strawberry flavor compound in strawberry fruit Plant Physiol 171 139-151


Spitzer-Rimon B Farhi M Albo B Cnaani A Ben Zvi MM Masci T Edelbaum O Yu YX Shklarman E Ovadis M Vainstein A (2012) TheR2R3-MYB-like regulatory factor EOBI acting downstream of EOBII regulates scent production by activating ODO1 and structuralscent-related genes in petunia Plant Cell 24 5089-5105


Tieman D (2017) Transcriptional control of strawberry ripening ndash two to tango J Exp Bot 68 4407-4409Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Vallarino JG Osorio S Bombarely A Casantildeal A Cruz-Rus E Saacutenchez-Sevilla JF Amaya I Giavalisco P Fernie AR Botella MAValpuesta V (2015) Central role of FaGAMYB in the transition of the strawberry receptacle from development to ripening New Phytol208 482-496


Verdonk JC Haring MA van Tunen AJ Schuurink RC (2005) ODORANT1 regulates fragrance biosynthesis in petunia flowers PlantCell 17 1612-1624


Wang SF Song MY Guo JX Huang Y Zhang F Xu C Xiao YH Zhang LS (2018) The potassium channel FaTPK1 plays a critical role infruit quality formation in strawberry (Fragaria times ananassa) Plant Biotechnol J 16 737-748


Wein M Lavid N Lunkenbein S Lewinsohn E Schwab W Kaldenhoff R (2002) Isolation cloning and expression of a multifunctional O- wwwplantphysiolorgon February 25 2020 - Published by Downloaded from Copyright copy 2018 American Society of Plant Biologists All rights reserved

methyltransferase capable of forming 25-dimethyl-4-methoxy-3(2H)-furanone one of the key aroma compounds in strawberry fruitsPlant J 31 755-765


Weirauch MT Hughes TR (2011) A catalogue of eukaryotic transcription factor types their evolutionary origin and speciesdistribution In A handbook of transcription factors (Hughes TR Ed) Springer Dordrecht Subcellular Biochemistry 52 25-73


Wuumlst M (2017) Biosynthesis of plant-derived odorants In Springer handbook of odor (Buettner A Ed) Springer Cham 9-10Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Xie XL Shen SL Yin XR Xu Q Sun CD Grierson D Ferguson I Chen KS (2014) Isolation classification and transcription profiles ofthe AP2ERF transcription factor superfamily in citrus Mol Biol Rep 41 4261-4271


Xie XL Yin XR Chen KS (2016) Roles of APETALA2ethylene-response factors in regulation of fruit quality Crit Rev Plant Sci 35 120-130


Yao GF Ming ML Allan AC Gu C Li LT Wu X Wang RZ Chang YJ Qi KJ Zhang SL Wu J (2017) Map-based cloning of the pear geneMYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis Plant J 92437-451


Yin XR Allan AC Chen KS Ferguson IB (2010) Kiwifruit EIL and ERF genes involved in regulating fruit ripening Plant Physiol 1531280-1292


Yin XR Shi YN Min T Luo ZR Yao YC Xu Q Ferguson I Chen KS (2012) Expression of ethylene response genes during persimmonfruit astringency removal Planta 235 895-906


Zabetakis I Gramshaw JW Robinson DS (1999) 25-dimethyl-4-hydroxy-2H-furan-3-one and its derivatives analysis synthesis andbiosynthesis-a review Food Chem 65 139-151


Zabetakis I Holden MA (1997) Strawberry flavour analysis and biosynthesis J Sci Food Agric 74 421-434Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Zeng JK Li X Xu Q Chen JY Yin XR Ferguson IB Chen KS (2015) EjAP2-1 an AP2ERF gene is a novel regulator of fruit lignificationinduced by chilling injury via interaction with EjMYB transcription factors Plant Biotechnol J 13 1325-1334


Zhang AD Hu X Kuang S Ge H Yin XR Chen KS (2016) Isolation classification and transcription profiles of the Ethylene ResponseFactors (ERFs) in ripening kiwifruit Sci Hortic (Amsterdam) 199 209-215


Zhang CH Shangguan LF Ma RJ Sun X Tao R Guo L Korir NK Yu ML (2012) Genome-wide analysis of the AP2ERF superfamily inpeach (Prunus persica) Genet Mol Res 11 4788-4809


Zhang Y Tang HR Luo Y Wang XR Chen Q Liu ZJ (2014) Isolation and expression analysis of a CRTDRE-binding factor gene FaCBF1from Fragaria times ananassa Acta Hortic 41 240-248


Zhu Q Zhang JT Gao XS Tong JH Xiao LT Li WB Zhang HX (2010) The Arabidopsis AP2ERF transcription factor RAP26 participatesin ABA salt and osmotic stress responses Gene 457 1-12


Zhuang J Peng RH Cheng ZM (Max) Zhang J Cai B Zhang Z Gao F Zhu B Fu XY Jin XF Chen JM Qiao YS Xiong AS Yao QH (2009) wwwplantphysiolorgon February 25 2020 - Published by Downloaded from Copyright copy 2018 American Society of Plant Biologists All rights reserved

Genome-wide analysis of the putative AP2ERF family genes in Vitis vinifera Sci Hortic (Amsterdam) 123 73-81Pubmed Author and TitleGoogle Scholar Author Only Title Only Author and Title

Zorrilla-Fontanesi Y Rambla JL Cabeza A Medina JJ Sanchez-Sevilla JF Valpuesta V Botella MA Granell A Amaya I (2012) Geneticanalysis of strawberry fruit aroma and identification of O-methyltransferase FaOMT as the locus controlling natural variation inmesifurane content Plant Physiol 159 851-870



Article File

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Parsed Citations

2

Article title 28

29

An ETHYLENE RESPONSE FACTOR-MYB Transcription Complex Regulates 30

Furaneol Biosynthesis by Activating QUINONE OXIDOREDUCTASE Expression 31

in Strawberry 32

33

34

Yuanyuan Zhang1 Xueren Yin123 Yuwei Xiao1 Zuying Zhang1 Shaojia Li1 35

Xiaofen Liu1 Bo Zhang123 Xiaofang Yang4 Donald Grierson15 Guihua Jiang4 36

Harry J Klee16 Kunsong Chen123 37

1 College of Agriculture amp Biotechnology Zhejiang University Zijingang Campus 38


2 Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology 40


3 The State Agriculture Ministry Laboratory of Horticultural Plant Growth 42



4 Institute of Horticulture Zhejiang Academy of Agricultural Sciences Hangzhou 45


5 Division of Plant and Crop Sciences School of Biosciences University of 47

Nottingham Sutton Bonington Campus Loughborough LE12 5RD UK 48

6 Horticultural Sciences Plant Innovation Center Genetics Institute University of 49


These authors contributed equally to this manuscript 51

52

One-sentence summary 53

Fragaria times ananassa quinone oxidoreductase which catalyses the final step in 54

furaneol biosynthesis in strawberry is transcriptionally upregulated by a complex 55

consisting of an ethylene response factor and MYB during ripening 56

57


3

Footnotes 58








66





71







4

Abstract 77


















95


5

Introduction 96






















118









6














139














153




7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

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Figure 10

Parsed Citations

3

Footnotes 58








66





71







4

Abstract 77


















95


5

Introduction 96






















118









6














139














153




7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

4

Abstract 77


















95


5

Introduction 96






















118









6














139














153




7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

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Figure 8

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Figure 10

Parsed Citations

5

Introduction 96






















118









6














139














153




7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Figure 7

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Parsed Citations

6














139














153




7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

7









164








172

Results 173














8

186









195












Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

8

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Table S1) 207

208









9







222





















243




10













258









267










11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

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Figure 5

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9







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243




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11


277





















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12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

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Parsed Citations

10













258









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11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

11


277





















298









12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

12






311

Discussion 312






















334



13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

13












347




















14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

14


volatiles 367

368















383


biosynthesis 385












15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

15














409


















16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

16





430

Conclusions 431








439


441
















17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

17


457




461





466





















18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

18




489























512





19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

19

















532













545


20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

20




549



searched 552

553





558



















21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

21

576









585













598









22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

22






611

















628







635


23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

23

636


















654









663

Statistics 664



24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

24








673






679



AP2ERF proteins 682















25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

25




(RT-qPCR) 699




703

Acknowledgments 704




708

Figure legends 709


















26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

26



























test ( plt005 plt001 plt0001) 752





27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

27













3-aminotriazole 768









777


779







28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

28







(Amsterdam) 210 34-40 791










Cell 23 2738-2753 801















29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

29




167 915-930 818










157 188-199 828



Plant Biol 6 20 831















30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

30









120 44-57 853









Genomics 11 719 862












31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

31








598-614 880



Food Chem 52 1248-1254 883




63 6393-6405 887

















32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































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32





Mikrobiol 28 123-127 907



Cell 19 2557-2568 910












2105-2110 922











33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































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33


Molecules 18 6936-6951 933







e88446 940




455-475 944













5089-5105 957


Bot 68 4407-4409 959




34






























35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































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35



Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































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Planta 235 895-906 992





Food Agric 74 421-434 997




1325-1334 1001
















(Amsterdam) 123 73-81 1017




36



851-870 1022































































































































































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short title regulation of furaneol biosynthesis in …...faqr is mainly expressed in 143 the late...

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