JOURNAL OF VIROLOGY€¦ · Eckhart, Keith W. C. Peden, Ashok Srinivasan, and James M. Pipas........

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Short title: ERF-WRKY complex involved in anaerobic metabolism 1 2 Corresponding author details: 3

Xue-ren Yin 4

Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, 5

Zhejiang University, Zijingang Campus 6

Hangzhou Zhejiang 7

310058, PR China 8

Tel: +86-571-88982461 9

Fax: +86-571-88982224 10

E-mail: [email protected]

Plant Physiology Preview. Published on March 8, 2019, as DOI:10.1104/pp.18.01552

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High-CO2/hypoxia-responsive transcription factors DkERF24 and 12 DkWRKY1 interact and activate DkPDC2 promoter 13 14 Qing-gang Zhu1, #, Zi-yuan Gong1, #, Jingwen Huang1, Donald Grierson1,3, Kun-song 15 Chen1,2, Xue-ren Yin1,2, * 16 17 1Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, 18

Zhejiang University, Zijingang Campus, Hangzhou 310058, PR China 19 2The State Agriculture Ministry Laboratory of Horticultural Plant Growth, 20

Development and Quality Improvement, Zhejiang University, Zijingang Campus, 21

Hangzhou 310058, PR China 22 3 Plant & Crop Sciences Division, School of Biosciences, University of Nottingham, 23

Sutton Bonington Campus, Loughborough, UK 24

25 # These authors contributed equally to this manuscript. 26

27

One sentence summary: 28

Two high-CO2/hypoxia responsive transcription factors from persimmon fruit, 29

DkERF24 and DkWRKY1, form a complex and synergistically transactivate the 30

promoter of the hypoxia-responsive gene DkPDC2. 31

32

List of author contributions: X.Y. conceived the research plans; X.Y., and K.C. 33

supervised the experiments; Q.Z. and Z.G. performed most of the experiments; J.H. 34

provided technical assistance to Q.Z.; X.Y., Q.Z. and K.C. designed the experiments 35

and analyzed the data; X.Y., Q.Z. and D.G. wrote the article with contributions of all 36

the authors. 37

38

Funding information: This research was supported by the National Key Research and 39

Development Program (2016YFD0400102), the National Natural Science Foundation 40

of China (31672204, 31722042), and the Natural Science Foundation of Zhejiang 41

Province, China (LR16C150001), the Fok Ying Tung Education Foundation, China 42



3

(161028), the Fundamental Research Funds for the Central Universities 43

(2018XZZX002-03) and the 111 Project (B17039). 44

45

*Corresponding author email: [email protected] 46

47

Date of submission: 24 January 2019 48

Tables: 0 49

Figures: 7 50

Color figures in print: Figs.1, 3-7 51

Total word counts: 5268 52

Supporting Information Files: 11 53

Supporting Information Tables: 11 54

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56



4

Abstract 57

Identification and functional characterization of hypoxia-responsive 58

transcription factors is important for understanding plant responses to natural anaerobic 59

environments and during storage and transport of fresh horticultural products. In this 60

study, yeast one-hybrid (Y1H) library screening using the persimmon (Diospyros kaki) 61

pyruvate decarboxylase (DkPDC2) promoter identified three Ethylene Response 62

Factor genes (DkERF23/24/25) and four WRKY transcription factor genes 63

(DkWRKY1/5/6/7) that were differentially expressed in response to high CO2 (95%, 64

with 4% N2 and 1% O2) and high N2 (99% N2 and 1% O2). Y1H assays and 65

electrophoretic mobility shift assays indicated that DkERF23/24/25 and DkWRKY6/7 66

could directly bind to the DkPDC2 promoter. Dual-luciferase assays confirmed that 67

these transcription factors were capable of transactivating the DkPDC2 promoter. 68

DkERF24 and DkWRKY1 in combination synergistically transactivated the DkPDC2 69

promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays 70

confirmed protein–protein interaction between DkERF24 and DkWRKY1. Transient 71

over-expression of DkERF24 and DkWRKY1 separately and in combination in 72

persimmon fruit discs were effective in maintaining insolubilization of tannins, 73

concomitantly with the accumulation of DkPDC2 transcripts. Studies with Arabidopsis 74

thaliana homologs AtERF1 and AtWRKY53 indicated that similar protein–protein 75

interactions and synergistic regulatory effects also occur with the DkPDC2 promoter. 76

We propose that an ERF and WRKY transcription factor complex contributes to 77

responses to hypoxia in both persimmon fruit and Arabidopsis, and the possibility that 78

this is a general plant response requires further investigation. 79

80

Key words: persimmon fruit; Arabidopsis; hypoxia response; ERF; WRKY; 81

protein-protein interaction 82



5

Introduction 83

Transcription factors (TFs) play important roles in plant responses to hypoxia. Our 84

understanding of these responses has been advanced significantly by the 85

characterization of subfamily VII of the ethylene response factors (ERFs). Five ERF 86

genes (HRE1, HRE2, RAP2.2, RAP2.3, RAP2.12) have been reported as the main plant 87

oxygen-sensing regulators and have been shown to control fermentation-related 88

alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) genes in 89

Arabidopsis thaliana ( Hinz et al., 2010; Licausi et al, 2010, Yang et al., 2011; Bui et 90

al., 2015; Papdi et al., 2015). ERFs involved in the regulation of hypoxia responses 91

have also been reported in other plants, such as ERFVII in Rumex acetosa, Rumex 92

palustris, Rorippa sylvestris and Rorippa amphibia (van Veen et al., 2014) and 93

SUBMERGENCE TOLERANCE-RELATED SUBMERGENCE 1 (Sub1) in rice (Oryza 94

sativa) (Xu et al., 2006). Control of the stability of hypoxia responsive ERFs by the 95

N-end rule is thought to be the main mechanism whereby plants sense and respond to 96

low oxygen (Gibbs et al., 2011; Licausi et al., 2011a). 97

However, there may be other important TFs involved in the hypoxia response which 98

may or may not operate via the N-end rule. A few other hypoxia-related TFs have been 99

reported, such as AtMYB2 in Arabidopsis that physically interacts with the AtADH1 100

promoter (Hoeren et al, 1998). Overexpression of AtMYB2 enhanced AtADH1 101

expression (Abe et al., 2003). In another example, the heat shock factor HsfA2 has been 102

shown to be responsive to low-oxygen conditions and transactivate downstream genes 103

(ADH) to enable plants to acquire anoxia tolerance (Banti et al., 2010). Wheat 104

(Triticum aestivum) TaMYB1 is also responsive to low oxygen (Lee et al., 2007). 105

Moreover, omics-based analyses have shown that additional differentially expressed 106

TFs may also be related to the hypoxia response in different Arabidopsis organs 107

(Branco-Price et al., 2005; Liu et al., 2005; Mustroph et al., 2009; Lee et al., 2011; 108

Licausi et al., 2011b). The interactions between different hypoxia responsive TFs and 109

their precise roles are still unclear, however, and relationships between different 110

hypoxia-responsive TFs have rarely been reported. 111



6

Unlike most plants, fruit frequently experience artificial low oxygen environments 112

during postharvest storage, where, for example, controlled atmosphere (CA) storage is 113

widely used (Ali et al., 2016; Bekele et al., 2016; Matityahu et al., 2016). 114

Understanding the fruit response to anaerobic environments could lead to 115

improvements in storage technologies and improve fruit quality, and there have been 116

many physiological and biochemical studies on fruit responses to reduced oxygen 117

environments (Ali et al., 2016). Indeed, it was one such study that led to the discovery 118

of the role of ACC as a precursor in ethylene synthesis (Adams and Yang, 1978). In 119

apple (Malus domestica) fruit, MdRAP2.12 protein has been shown to differentially 120

accumulate in samples held at different oxygen concentrations, indicating that the 121

oxygen sensing mechanisms described in Arabidopsis are also present in apple fruit 122

(Cukrov et al., 2016). 123

Persimmon (Diospyros kaki) fruit are ideal material for studies on hypoxia response 124

in fruit. Most cultivated persimmons are of the astringent type and are rich in soluble 125

condensed tannins (SCTs) (Akagi et al., 2009). Mature fruit require postharvest 126

treatments to remove the astringency by insolubilization of SCTs (Wang et al., 1997). 127

The mechanism operates by induction of pyruvate decarboxylase (PDC), and to a lesser 128

extent alcohol dehydrogenase (ADH), which leads to acetaldehyde accumulation. This 129

precipitates soluble tannins, removing the astringency (Taira et al., 2001; Salvador et 130

al., 2007; Min et al., 2012). High CO2 treatment is the most effective and widely used 131

method, with CO2 concentrations usually set at 95% and O2 reduced to 1%, elevating 132

ADH and PDC activities and triggering acetaldehyde metabolism (Ikegami et al., 2007; 133

Salvador et al., 2007; Min et al., 2012; Yin et al., 2012). Thus, for fruit, the molecular 134

basis of the hypoxia response has been most extensively studied in persimmon. Some 135

TFs have been identified and shown to activate transcription of hypoxia-responsive 136

genes (Hoeren et al, 1998; Abe et al., 2003; Min et al., 2012, 2014; Fang et al., 2016; 137

Zhu et al., 2018), but there are still gaps in our knowledge. Twenty-two high 138

CO2/hypoxia-responsive DkERF genes have been isolated previously from persimmon 139



7

and DkERF9/10/19/22 were characterized as direct regulators of the alcoholic 140

fermentation genes DkPDC2 and DkADH1 (Min et al., 2012, 2014). However, only 141

DkERF3 and DkERF10 are group VII subfamily members with conserved N-end MC 142

domains (MCGGAII), suggesting they may be involved in a similar hypoxia response 143

mechanism in persimmon to that described in Arabidopsis (Gibbs et al., 2011; Licausi 144

et al., 2011a). Evidence for a cascade involving additional DkERFs 145

(DkERF18/19/21/22) and DkMYB (DkMYB6/10) was found, however, involving 146

transcriptional regulation of DkERF9/10/19 (Zhu et al., 2018). The potential roles of 147

these other TFs in persimmon fruit deastringency and also the Arabidopsis anaerobic 148

response are unclear. 149

In the present study, yeast one-hybrid (Y1H) library screening was conducted to 150

identify additional persimmon TFs that interact with the DkPDC2 promoter, and 151

interactions between TFs and the DkPDC2 promoter were investigated by 152

dual-luciferase and Y1H assays. Further yeast two-hybrid (Y2H) and bimolecular 153

fluorescence complementation (BiFC) experiments identified a synergistic interaction 154

involving an ERF-WRKY complex that transactivated the PDC2 promoter. Parallel 155

experiments confirmed the ability of Arabidopsis ERF-WRKY homologs to participate 156

in this hypoxia response. 157

158

Results 159

Identification and characterization of transcription factors targeting the hypoxia 160

responsive DkPDC2 promoter by Y1H library screening 161

To understand the control of gene expression in response to hypoxia, especially the 162

cross-talk between master regulators, the ideal objective would be to identify all 163

hypoxia-responsive TFs that interact with the same promoter. Here, Y1H-based library 164

screening was used with the DkPDC2 promoter as the bait; 95 colonies were sequenced 165

and 45 sequences obtained (Supplementary Table S1). Among them, three DkERFs 166

(DkERF23/24/25, MH054905-7) and four DkWRKYs (DkWRKY1, KY849608; 167



8

DkWRKY5/6/7, MH054908-10) TF genes were identified and characterized. Individual 168

Y1H assays generated similar results to the library screening, except for DkWRKY1 and 169

DkWRKY5 (Fig. 1A). Electrophoretic mobility shift assays (EMSAs) were performed 170

in order to verify the interactions and locate more precisely the cis-element involved in 171

binding. A GCC box (GCCGCC) for AP2/ERF TFs and a W-box (GGTCAA) 172

(Birkenbihl et al., 2017) for WRKY TFs were found in the DkPDC2 promoter region 173

(Supplemental Fig. S1). Biotinylated probes containing these sequences were able to 174

bind DkERF23/24/25 and DkWRKY6/7 proteins, and the addition of a high 175

concentration of cold probe significantly reduced the binding affinity of the 176

biotinylated probe (Fig 1B-F), whereas DkWRKY1/5 could not bind to the DkPDC2 177

promoter (Supplemental Fig. S2). These results indicated that DkERFs could 178

physically bind to the GCC-box motif, and DkWRKYs targeted the W-box motif of the 179

DkPDC2 promoter. Dual-luciferase assays indicated that DkERF23/24/25 and 180

DkWRKY1/7 had significant activation effects on transcription from the DkPDC2 181

promoter (more than 2-fold increase), while DkWRKY5 and DkWRKY6 had no 182

significant effects on the DkPDC2 promoter (Fig. 1G). The regulatory effects of these 183

TFs on the DkPDC2 promoter were confirmed by histochemical staining of GUS 184

activity in Nicotiana benthamiana leaves, which showed that transient over-expression 185

(TOX) of DkERF23/24/25 and DkWRKY1/7 could significantly up-regulate the 186

DkPDC2 promoter-GUS expression (Supplemental Fig. S3). These TFs had either 187

limited or no effect on promoters of the other deastringency-related persimmon genes, 188

with the exception of DkERF24, which was able to activate the DkERF9 promoter 189

above the 2-fold threshold (Supplemental Fig. S4). 190

191

Expression of TFs in response to high CO2 treatment in various persimmon 192

cultivars 193

In order to analyze the responses of TFs to deastringency treatment, three different 194

cultivars were selected, ‘Mopanshi’, ‘Jingmianshi’, ‘Tonewase’. Our previous data 195



9

indicated that CO2 treatment (95%, 1 d) was effective in insolubilizing the soluble 196

tannins in postharvest fruit of all three cultivars (Wang et al., 2017; Zhu et al., 2018). 197

Expression of the seven TFs was analyzed by reverse transcription quantitative PCR 198

(RT-qPCR), and, with the exception of DkWRKY6, the other six TFs were responsive to 199

high CO2 treatment in all three cultivars (Fig. 2), and were designated as high CO2/low 200

oxygen-responsive. Some genes showed quantitatively different expression patterns 201

between the cultivars, eg. DkERF23 showed higher than 200-fold induction in 202

‘Jingmianshi’ and much less activation in ‘Tonewase’ fruit (about 5-fold) at 1 d; in 203

contrast, DkERF25 showed highest induction by CO2 treatment in ‘Tonewase’ (about 204

40-fold at 1 d) (Fig. 2). 205

In order to clarify potentially different effects of high CO2 and hypoxia, the hypoxia 206

treatment (99% N2, 1% O2) was applied to ‘Gongcheng-shuishi’ (Zhu et al., 2018). The 207

expression analysis indicated that DkERF25 and DkWRKY5 were only up-regulated by 208

high CO2, but not the hypoxia treatment. DkERF23/24 and DkWRKY1 were 209

responsive to both high CO2 and hypoxia. Expression of DkWRKY1 was significantly 210

weaker in response to hypoxia treatment compared to high CO2. In contrast, DkERF23 211

showed greater abundance in response to hypoxia treatment (Supplemental Fig. S5). 212

Among the seven TFs, DkWRKY7 was undetectable in ‘Gongcheng-shuishi’. 213

214

Subcellular localization analysis of TFs 215

Subcellular localization assays were performed in N. benthamiana leaves stably 216

transformed with a nuclear marker in order to visualize the subcellular locations of the 217

seven TFs. DkERF23/25 and DkWRKY1/5/6/7 all gave strong signals in the nucleus, 218

while DkERF24 showed signals in both nucleus and the cell membrane (Fig. 3). 219

220

Synergistic effects of DkERF24 and DkWRKY1 on transcription from the 221

DkPDC2 promoter and analysis of protein–protein interactions 222



10

The effects of different combinations of TFs on the DkPDC2 promoter were 223

analyzed by the dual-luciferase assay. A few combinatorial effects were found between 224

various TFs (Supplemental Fig. S6A), the most notable being the interaction between 225

DkERF24 and DkWRKY1. This showed a 13-fold synergistic activation of DkPDC2 226

promoter compared to the less than 4-fold activation of either TF separately (Fig. 4A). 227

The Y2H assay indicated that the interaction between DkERF24 and DkWRKY1 (Fig. 228

4B) was the only direct protein-protein interaction (Supplemental Fig. S6B). The 229

interaction between DkERF24 and DkWRKY1 was also verified by the BiFC assay 230

(Fig. 4C). The BiFC results showed that negative combinations, such as 231

YFPN/DkWRKY1-YFPC, YFPC/DkWRKY1-YFPN, YFPN/DkERF24-YFPC, 232

YFPC/DkERF24-YFPN and YFPN/YFPC did not produce any detectable fluorescence 233

signals, while the positive combination of PHR2-YFPN/SPX4-YFPC, and the 234

co-expression of DkERF24-YFPN/DkWRKY1-YFPC, 235

DkERF24-YFPC/DkWRKY1-YFPN gave strong signals located in the nuclei (Fig. 4C). 236

237

Transient overexpression analysis in persimmon fruit discs 238

Transient overexpression (TOX) analyses were performed with fruit discs to verify the 239

functions of TFs involved in persimmon fruit deastringency. DkERF1, which had no 240

transactivation effect on the DkPDC2 promoter (Min et al., 2012), was chosen as a 241

negative control. The content of soluble tannins in the discs treated with both the empty 242

vector (SK, the 2nd negative control) and TFs declined during incubation (Fig. 5A). 243

With the exception of the DkERF1 negative control, the combination of DkERF24 and 244

DkWRKY1, or the two individual TFs, resulted in significantly lower content of soluble 245

tannins compared with the controls (Fig. 5A). Interactions between TFs and DkPDC2 246

were also analyzed and the results indicated that TOX of these TFs could significantly 247

up-regulate the endogenous DkPDC2 transcript in persimmon fruit discs, supporting 248

the evidence for interactions of hypoxia responsive TFs with the DkPDC2 promoter 249



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(Fig. 5C-5E). In particular, the combination of DkERF24 and DkWRKY1 showed a 250

more obvious up-regulation in DkPDC2 transcripts than either TF alone (Fig. 5C-E). 251

252

Arabidopsis AtERF1 and AtWRKY53 also transactivate the DkPDC2 promoter 253

Based on the phylogenetic trees (Supplemental Fig. S7 and S8), AtERF1, AtWRKY41 254

and AtWRKY53, are genes homologous to DkERF24 and DkWRKY1 in Arabidopsis. 255

AtWRKY53 has been shown to be responsive to hypoxia, while AtERF1 showed a slight 256

negative response (Mustroph et al., 2009). The possibility of a conserved mechanism 257

for the hypoxia response involving ERF and WRKY action in both Arabidopsis and 258

persimmon was investigated. Using dual-luciferase assays, AtERF1 and AtWRKY53 259

had significant transcriptional activations of the DkPDC2 promoter (approximately 260

3.0- and 2.3-fold, respectively) (Fig. 6A), while AtWRKY41 had no effect on the 261

DkPDC2 promoter (Supplemental Fig. S9A). Histochemical GUS staining using the 262

DkPDC2 promoter fused to the ß-glucuronidase reporter gene gave more intensive blue 263

color with AtERF1 and AtWRKY53 than the empty vector (SK). The gus (formerly 264

uidA) transcripts in AtERF1TOX and AtWRKY53TOX were higher than the empty vector 265

(Supplemental Fig. S10). Furthermore, using the Y1H assay, it was found that both 266

AtERF1 and AtWRKY53 could physically bind to the DkPDC2 promoter (Fig. 6B). 267

The combination of AtERF1 and AtWRKY53 also showed higher activation of the 268

DkPDC2 promoter (LUC/REN=6.12) than either of them singly (1.92-fold and 269

1.63-fold) (Fig. 6C), the combination of AtERF1 and AtWRKY41 showing no additive 270

effect on the DkPDC2 promoter (Fig S9B). BiFC analysis showed that the 271

co-expression of AtERF1-YFPN/AtWRKY53-YFPC and 272

AtERF1-YFPC/AtWRKY53-YFPN also gave strong signals in the nucleus (Fig. 6D), 273

confirming protein–protein interaction between AtERF1 and AtWRKY53. 274

275

Discussion 276



12

Multiple TFs involved in anaerobic metabolism/hypoxia response may target the 277

same promoter 278

In our previous studies, at least two DkERFs (DkERF9/19, Min et al., 2012, 2014), 279

and DkMYB6 (Fang et al., 2016) were shown to be direct activators of the DkPDC2 280

promoter. Several types of TFs, in addition to ERFs, have been suggested to be 281

hypoxia-responsive in both persimmon (e.g., TGA, Zhu et al., 2016; MYB, Zhu et al., 282

2018) and Arabidopsis. Omics-based approaches in Arabidopsis, indicated that 283

hundreds of TFs are regulated by low-oxygen environments (e.g., submergence, Liu et 284

al., 2005; Licausi et al., 2011b). It is possible, therefore, that several different TFs may 285

contribute to plant hypoxia tolerance, but there is very little information about such 286

interactions. In the present research, six additional TFs (DkERF23/24/25 and 287

DkWRKY1/6/7) were shown to have either binding affinity and/or trans-activation 288

ability for the DkPDC2 promoter. Y1H-based cDNA library screening and 289

dual-luciferase assay demonstrated that some of theseTFs (ERF and WRKY) could 290

bind to and/or trans-activate the DkPDC2 promoter (Fig. 1). Taken together, there 291

appear to be at least eight TFs that can participate in regulating the DkPDC2 promoter 292

(Fig. 7), providing new candidates for unravelling the regulatory complexities of the 293

response to anaerobic or more particularly, hypoxic conditions. 294

Not all these persimmon ERFs belong to the well characterized Group VII subfamily 295

involved in plant hypoxia responses (Hinz et al., 2010; Licausi et al., 2010, 2011b; 296

Yang et al., 2011; Gasch et al., 2016). In persimmon only DkERF3 and DkERF10 297

belong to the Group VII subfamily, but DkERF3 (Supplemental Fig. S11) and 298

DkERF10 have no effects on the DkPDC2 promoter, and only DkERF10 had a 299

trans-activation effect on the DkADH1 promoter (Min et al., 2012). At least three of the 300

TFs (DkERF9/19/22) involved in persimmon fruit hypoxic responses and the 301

deastringency process (Min et al., 2012, 2014) do not belong to Group VII, but to 302

different subfamilies (DkERF9-Group IV, DkERF19-Group IX and DkERF22-RAV 303

subfamily) (Min et al., 2012, 2014; Zhu et al., 2018). Here, all the newly identified ERF 304



13

genes DkERF23/24/25 belong to Group IX (Supplemental Fig. S7), which indicates 305

that multiple subfamilies of ERFs may contribute to fruit anaerobic metabolism, not 306

just Group VII family members. Moreover, there have been a few studies, both in 307

model plants and fruit, indicating that Group IX ERFs are involved in stress responses, 308

such as Arabidopsis ERF6 for oxidative stress (Wang et al., 2013), ESE1 for salt 309

response (Zhang et al., 2011) and tomato Pti4 for plant defence (Gu et al., 2000). 310

MdERF2, a Group IX ERF, mediated regulatory effects of jasmonate on ethylene 311

biosynthesis and ripening in apple fruit (Li et al., 2017). Also, our previous results 312

indicated that DkERF19, a Group IX ERF, is involved in persimmon fruit 313

deastringency (Wang et al., 2017). Thus, the multiple Group IX ERFs 314

(DkERF19/23/24/25) shown to be responsive to high CO2/low oxygen are also likely to 315

be involved in high CO2/low oxygen driven persimmon fruit deastringency and 316

ripening. 317

In Arabidopsis, many WRKYs play important roles in plant stress tolerance (Chen et 318

al., 2017), although there is no report on WRKY involvement in plant hypoxia 319

tolerance or persimmon fruit deastringency, and the identification of DkWRKY genes 320

(Fig.1) provides new targets in researching responses to anaerobic treatments. 321

322

Interactions between persimmon DkERF24 and DkWRKY1 regulate expression 323

of the hypoxia responsive DkPDC2 promoter 324

To analyze the relations between different hypoxia responsive TFs from persimmon 325

fruit, experiments with random paired combinations of these TFs were carried out. The 326

combination of DkERF24 and DkWRKY1 showed significantly higher activation 327

(13-fold) of the DkPDC2 promoter than either of them acting individually, and the 328

results of the Y2H and BiFC assays confirmed protein-protein interaction between 329

DkERF24 and DkWRKY1 (Fig. 4). Taken together, these results suggest that high 330

CO2/low oxygen treatment could trigger both DkERF24 and DkWRKY1 accumulation 331

and the induced TFs may form a complex which synergistically stimulates substantially 332



14

higher activation of the DkPDC2 target gene and accelerates persimmon fruit 333

deastringency. Another well-known protein–protein interaction related to the response 334

to aerobic environments occurs between the hypoxia responsive RAP2.12 (an ERF) 335

and acyl-coA binding protein (ACBP), which dissociates under an anaerobic 336

environment whereupon RAP2.12 moves into the nucleus to transcriptionally regulate 337

anaerobic related genes (Licausi et al., 2011a). ACBP is not a TF, however, and the 338

interactions between hypoxia responsive ERF-WRKY TFs described here reveal the 339

role of a different TF complex in the hypoxia response. 340

341

Arabidopsis ERF and WRKY also regulate the DkPDC2 promoter 342

Parallel analysis with Arabidopsis homologs indicated that AtERF1 and 343

AtWRKY53 could interact with each other in Y2H and BiFC assays. The combination 344

of AtERF1 and AtWRKY53 also strongly activated the DkPDC2 promoter (Fig.6). 345

These results suggest that the protein–protein interaction between hypoxia responsive 346

ERFs and WRKY may be conserved in different plants. In Arabidopsis, the 347

homologous gene AtERF1 is a key integrator of jasmonate and ethylene signals in the 348

regulation of ethylene/jasmonate-dependent defense in response to different plant 349

pathogens (Solano et al., 1998; Berrocal-Lobo et al., 2002; Lorenzo et al., 2003). 350

Overexpression of AtWRKY53 in Arabidopsis could accelerate leaf senescence, 351

coordinated by the interaction of salicylic acid and jasmonate signaling pathways 352

(Miao et al., 2004), although their involvement in the regulation of the plant hypoxia 353

response has not been comprehensively analyzed. At the transcriptomic level, at least 354

AtWRKY53 could be significantly induced by low oxygen conditions and is among the 355

most sensitive of the WRKY family members (Mustroph et al., 2009). Based on the 356

present analysis, it could be proposed that hypoxia-responsive AtWRKY53 could 357

regulate alcoholic fermentation-related genes, via interaction with AtERF1. 358

359



15

In vivo interactions of DkERF24 and DkWRKY1 with persimmon fruit DkPDC2 360

measured by insolubilizing soluble tannins 361

Due to the difficulty of stable transformation of perennial fruit, a TOX system was 362

employed to analyze persimmon gene function and the regulation of endogenous genes 363

by their transcriptional regulators. In the persimmon system, the reduction in soluble 364

tannin content can be used as a measurement of the induction of PDC and the anaerobic 365

response. Overexpression of DkMYB4 in kiwifruit calluses could significantly enhance 366

tannin biosynthesis (Akagi et al., 2009), and expression of DkPDC2 and DkADH1 in 367

persimmon leaves decreased soluble tannin content (Min et al., 2012; Mo et al., 2016). 368

Overexpression of DkERF18/19 and DkMYB6/10 caused a rapid decrease in the content 369

of soluble tannins in persimmon fruit discs (Zhu et al., 2018) and up-regulated the 370

expression of the corresponding genes (DkERF9/19). Here, a slight modification was 371

introduced, by adding a negative control, DkERF1, which was shown not to activate the 372

DkPDC2 promoter (Min et al., 2012). This additional control increased the reliability 373

of the TOX system, and the results indicated that DkERF1TOX produced no significant 374

change in expression of DkPDC2 (Fig. 5). DkERF24TOX, DkWRKY1TOX and DkERF24 375

plus DkWRKY1TOX, however, significantly accelerated insolubilization of tannins in 376

persimmon fruit discs, indicating that they participate in persimmon fruit 377

deastringency. The synergistic effects of DkERF24 and DkWRKY1 on trans-activation 378

of the expression of DkPDC2 was also confirmed. These results not only support the 379

conclusions about the regulatory roles and involvement in a transcriptional complex of 380

DkERF24 and DkWRKY1, but also confirm the role of these TFs in persimmon 381

anaerobic response and fruit deastringency. 382

383

In conclusion, Y1H library screening and further investigations with the 384

dual-luciferase assay, EMSA, Y2H and BiFC, identified multiple novel hypoxia 385

responsive TFs. TOX analysis confirmed the role of this complex in persimmon fruit 386

deastringency, which is critical for the persimmon industry. Furthermore, the fact that 387



16

there is a similar synergistic effect of DkERF24 and DkWRKY1 homologs from 388

Arabidopsis on the DkPDC2 promoter suggests this may be a conserved feature of the 389

anaerobic response in plants. 390

391

Materials and Methods 392

Plant material and treatments 393

The persimmon fruit material was from the same batch as those used in our previous 394

studies (Wang et al., 2017; Zhu et al., 2018). In brief, three astringent-type persimmon 395

(Diospyros kaki) fruit, two Chinese cultivars (‘Mopanshi’ and ‘Jingmianshi’) and one 396

Japanese cultivar (‘Tonewase’), were collected from an orchard at mature stage at 397

Qingdao (Shandong, China) in 2014. The fruit were treated with air (as control) or CO2 398

(95%, with 4% N2 and 1% O2) to accelerate insolubilization of soluble tannins 399

(deastringency), for 1 d. The mature persimmon fruit ‘Gongcheng-shuishi’ were 400

harvested from Guilin (Guangxi, China) was treated with air (control), CO2 (95%, with 401

4% N2 and 1% O2) and N2 (99% N2 and 1% O2). The physiological data and sampling 402

information are described in Wang et al., (2017) and Zhu et al., (2018). 403

404

Construction of cDNA library and Yeast one-hybrid library screening 405

Total RNA was extracted from ‘Mopanshi’ persimmon fruit flesh and used for 406

cDNA library construction according to the MatchmakerTM Gold Yeast One-Hybrid 407

Library Screening System Kit user manual (Clontech, USA). The DkPDC2 promoter 408

was constructed in pAbAi vector as in Min et al., (2014). The screening was according 409

to the protocol of the YeastmakerTM Yeast Transformation System 2 User Manual 410

(Clontech, USA), performed on SD/-Leu+AbA250 plates in a 30 oC incubator for 4 d. 411

Single colonies were selected, amplified by PCR, the DNA sequences determined and 412

those encoding TFs used as candidates for further investigations. 413

414

Gene isolation and sequence analysis 415



17

After BLAST analysis, the partial coding sequences (CDS) were amplified with the 416

primers (Supplementary Table S2) and a SMART RACE cDNA amplification Kit 417

(Clontech, USA) to obtain the complete CDS. The sequences of full-length TF 418

candidates were confirmed and amplified with primers spanning the start and stop 419

codons (Supplementary Table S3) and translated with the ExPASy software 420

(http://web.expasy.org/translate). These newly isolated TFs were named according to 421

their homologs in Genbank and the previously reported TFs in persimmon. 422

423

Yeast one-hybrid assay (Y1H) 424

The Y1H assay, to identify individual interactions between a single TF and the target 425

gene promoter, was used to verify the interactions indicated by library screening, using 426

the MatchmakerTM Gold Yeast One-Hybrid Library Screening System (Clontech, 427

USA). The full-length TF candidate sequences were subcloned into the pGADT7 AD 428

vector (primers are listed in Supplementary Table S4) and the interaction analyses were 429

conducted according to the manufacturer’s protocol. 430

431

Dual-luciferase assay and GUS (β-glucuronidase) histochemical staining 432

Dual-luciferase assays have been widely used for investigations on the 433

trans-regulation of target promoters by TFs (Zeng et al., 2015; Wang et al., 2017). 434

Full-length TFs were cloned into the pGreen II 002962-SK vector (SK) (primers are 435

listed in Supplementary Table S5) and tested with the promoters of DkADH1, 436

DkPDC2, DkERF9, DkERF10, and DkERF19, originally constructed in the pGreen II 437

0800-LUC vector (LUC) by Min et al., (2012, 2014). All constructs were 438

electroporated into Agrobacterium tumefaciens GV1301. The constructed SK and LUC 439

plasmids were transiently expressed in Nicotiana benthamiana leaves as described by 440

Min et al., (2012). The Agrobacterium were suspended in infiltration buffer (10 mM 441

MES, 10 mM MgCl2, 150 mM acetosyringone, pH 5.6) to an OD600 of approximately 442

0.75. TFs and promoter were combined in a v/v ratio of 10:1 and infiltrated into N. 443


http://web.expasy.org/translate


18

benthamiana leaves by needleless syringes. A dual-luciferase assay kit (Promega) was 444

used to analyze the transient expression in N. benthamiana leaves after 3 d of 445

infiltration. Absolute LUC and REN were measured in a GLOMAXTM 96 Microplate 446

Luminometer (Promega). 447

GUS histochemical assays were performed according to Xiao et al., (2018), but 448

with some minor revisions and the GUS gene was amplified by PCR using forward 449

primer: 5’-CGCCCATGGTACGTCCTGTAGAAACCC-3’; and reverse primer: 450

5’-GATTCTAGATCATTGTTTGCCTCCCTGCTG-3’). The PCR product was 451

inserted into the pGreen II 0800-LUC vector by replacing the LUC region to generate 452

the construct containing the GUS-coding region under the control of the DkPDC2 453

promoter. The constructs were electroporated into Agrobacterium tumefaciens 454

GV1301 and then the cultured Agrobacterium were suspended in infiltration buffer to 455

an OD600 of approximately 0.75. TFs and promoter were combined in a v/v ratio of 5:1 456

and infiltrated into N. benthamiana leaves by needleless syringes. The photos were 457

taken after 5 d of infiltration. The staining buffer was 0.1 M sodium phosphate buffer 458

(pH 7.0), 10 mM EDTA, 1 mM ferricyanide, 1 mM ferrocyanide, 0.5% Triton X-100, 459

and 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). The infiltrated 460

leaves of N. benthamiana were immersed in the staining buffer under vacuum for 30 461

min and then incubated at 37°C for 24 h. The leaves were decolorized in 75% ethanol 462

for 2 h for proper visualization. The gus (uidA) transcripts were detected by RT-qPCR 463

using N. benthamiana leaves collected at 1-day intervals after injections. The RT-qPCR 464

primers for gus are forward primer: 5’- CGGGTGAAGGTTATCTCTAT-3’; and 465

reverse primer: 5’-TTCGGTCATTTCATCTTGCC -3’. RT-qPCR was carried out on a 466

Bio-Rad CFX96 Real-Time PCR System using the SsoFastTM EvaGreen Supermix 467

(Bio-Rad) following the manufacturer’s instructions. The housekeeping gene NtACT 468

(GenBank No. JQ256516) (Zhang et al., 2017) was chosen as the internal control and 469

the 2-ΔCt method was used to calculate the relative expression (Livak and Schmittgen, 470

2001). 471



19

472

Electrophoretic mobility shift assay (EMSA) 473

The intact open reading frames of DkERF23/24/25 and DkWRKY1/5/6/7 were 474

inserted into pET-32a (Clontech) (primers are listed in Supplementary Table S6) to 475

generate a TF-His fusion protein. The reconstructed plasmids were transformed into 476

Escherichia coli strain BL21. The transformed cells were treated with 0.5 mM 477

isopropyl β-D-1-thiogalactopyranoside (IPTG) followed by incubation at 16°C for 20 478

h. HisTALONTM Gravity Columns (Clontech) were used to purify proteins. 479

EMSA experiments were performed according to the Lightshift Chemiluminescent 480

EMSA kit (Thermo) manufacturer’s instructions. Oligonucleotide probes were 481

synthesized and labeled with biotin (HuaGen Biotech, Shanghai, China). The 3’ biotin 482

end-labeled double-stranded DNA probes were prepared by annealing complementary 483

oligonucleotides, heated at 95°C for 5 min and the temperature gradually decreased to 484

25°C at the rate of 0.1°C s-1. The probes used for EMSA are listed in Supplementary 485

Table S6. EMSA was performed as previously described in Ge et al., (2017). 486

487

RNA extraction and cDNA synthesis 488

Total RNA was extracted from frozen persimmon fruit flesh (2.0 g for each), using 489

the method described by Chang et al., (1993). Contaminating genomic DNA in total 490

RNA was removed by TURBO DNA-free kit (Ambion). After quantification by 491

Nanophotometer Pearl (Implen), 1.0 μg DNA-free RNA was initiated for cDNA 492

synthesis with iScriptTM cDNA Synthesis Kit (Bio-Rad). For each sampling point, three 493

biological replicates were used for RNA extraction and the subsequent cDNA 494

synthesis. 495

496

Reverse transcription quantitative PCR (RT-qPCR) analysis 497

For RT-qPCR, gene specific oligonucleotide primers were designed and are 498

described in Supplementary Table S7. The quality and specificity of each pair of 499



20

primers were checked by melting curves and product resequencing. RT-qPCR was 500

carried out on a Bio-Rad CFX96 Real-Time PCR System using the SsoFastTM 501

EvaGreen Supermix (Bio-Rad) following the manufacturer’s instructions. The 502

housekeeping gene DkACT (GenBank No. AB473616) (Akagi et al., 2009) was chosen 503

as the internal control and the 2-ΔΔCt method was used to calculate the relative 504

expression (Livak and Schmittgen, 2001). 505

506

Subcellular localization analysis 507

Full-length TFs were cloned into pCAMBIA1300-sGFP (35S-TFs-GFP, Lv et al., 508

2014), using the primers described in Supplementary Table S8. All constructs were 509

electroporated into Agrobacterium tumefaciens GV1301. 35S-TFs-GFP were 510

transiently expressed in transgenic N. benthamiana leaves (stably transformed with 511

nuclear location signal, NLS-mCherry) (Li et al., 2017) using needleless syringes with 512

the infiltration buffer. Fluorescence from green fluorescent protein transiently 513

expressed in N. benthamiana leaves was imaged 2 d after infiltration using a Zeiss 514

LSM780NLO confocal laser scanning microscope. 515

516

Yeast two-hybrid assay (Y2H) 517

Protein-protein interactions were investigated in yeast with the DUAL hunter system 518

(Dual-systems Biotech, Switzerland). Full-length coding sequences of DkWRKY1 were 519

cloned into the pDHB1 vector as bait, and the full-length DkERF24 was cloned into 520

pPR3-N vector as prey. The primers used for vector construction are described in 521

Supplementary Table S9. All constructs were transformed into the yeast strain NMY51. 522

The assays were performed with different media: (1) DDO (SD medium lacking Trp 523

and Leu); (2) QDO (SD medium lacking Trp, Leu, His, and Ade; and (3) QDO+3AT 524

(QDO with 5 mM 3-amino-1,2,4-triazole). Auto-activations were tested with empty 525

pPR3-N vectors and target genes with pDHB1, which were co-transformed into 526

NMY51 and plated on QDO. Auto-activations were indicated by the presence of 527



21

colonies. Protein-protein interaction assays were performed with co-transformation of 528

DkWRKY1 in pDHB1 and DkERF24 in pPR3-N. The presence of colonies in QDO and 529

QDO+3AT indicated protein-protein interaction. 530

531

Bimolecular fluorescence complementation (BiFC) assay 532

BiFC was used to confirm the results of Y2H analysis. Full-length DkERF24 and 533

DkWRKY1 were cloned into both C-terminal and N-terminal fragments of yellow 534

fluorescent protein (YFP) vectors, and BiFC assays were performed according to the 535

protocols of Sainsbury et al., (2009). Primers used are listed in Supplementary Table 536

S10. All constructs were transiently expressed in N. benthamiana leaves by 537

Agrobacterium-mediated infiltration (GV3101) based on previous reports (Li et al., 538

2017). The YFP fluorescence of N. benthamiana leaves was imaged 2 d after 539

infiltration using a Zeiss LSM780NLO confocal laser scanning microscope. 540

541

Transient overexpression in persimmon fruit discs 542

In order to verify the potential roles of these TFs in the persimmon fruit 543

anaerobic/deastringency response, transient overexpression (TOX) was performed with 544

persimmon fruit (‘Gongcheng-shuishi’) discs, using the same protocol as in our 545

previous report (Zhu et al., 2018). Discs of 1 cm diameter and 0.3 cm thickness were 546

prepared and divided into eight batches. The discs were incubated with Agrobacterium 547

carrying constructs in the same buffer used for the dual-luciferase assay for 1 h. The 548

discs were then transferred to filter papers (wetted by Murashige and Skoog medium) in 549

tissue-culture dishes, and placed in an incubator at 25°C for 3 d. All of the experiments 550

were performed with three biological replicates. Each day, samples of the discs were 551

dried on filter papers, and then frozen in liquid nitrogen and stored at -80°C for further 552

use. 553

554

Soluble condensed tannins 555



22

Soluble condensed tannins are the main source of astringency for persimmon fruit. 556

The decreasing in soluble condensed tannins in high CO2 treatment is one of the main 557

responses of persimmon fruit to anaerobic environments. The content of soluble 558

condensed tannins in persimmon fruit discs was measured from 1 g frozen samples with 559

Folin-Ciocalteu reagent, with three biological replicates, according to the method 560

described by Yin et al., (2012). The results were calculated using a standard curve of 561

tannic acid equivalents. 562

563

Arabidopsis gene isolation and analysis 564

Based on the phylogenetic trees (Fig S7 and S8), AtERF1 (AT3G23240), AtWRKY41 565

(AT4G11070), and AtWRKY53 (AT4G23810) are the potential homologs of DkERF24 566

and DkWRKY1 in Arabidopsis. The three Arabidopsis genes were cloned into the 567

vectors pGreen II 002962-SK and AtERF1 and AtWRKY53 were also cloned into 568

pGADT7 AD and N-terminal and C-terminal fragments of yellow fluorescent protein 569

(YFP). The primers used for gene isolation and vector construction are listed in 570

Supplementary Table S11. The protocol for dual-luciferase assay, the Y1H assay, the 571

GUS histochemical staining, the genes expression and BiFC were as described above. 572

573

Statistical analysis 574

The statistical significance of differences was determined using Student’s t-test (A 575

t-test was performed if two values were compared which displayed in the paper as 576

asterisks) or an ANOVA test for significance analysis and least significant difference 577

(LSD) for multiple comparisons by DPS 2.05 (Zhejiang University, Hangzhou, China). 578

Figures were drawn using Origin 8.0 (Microcal Software Inc. Northampton, MA). 579

580

Accession Numbers 581

All sequences reads are available at GenBank MH054905 to MH054910 and 582

KY849608 583



23

584

SUPPLEMENTAL DATA 585

Additional Supporting Information may be found in the online version of this article: 586

Supplemental Figure S1. Promoter of DkPDC2 from persimmon with marked motifs. 587

Supplemental Figure S2. Electrophoretic mobility shift assay (EMSA) of 588

DkWRKY1/5/6 binding to the DkDPC2 promoter. 589

Supplemental Figure S3. GUS histochemical assays of effects of TFs on the DkPDC2 590

promoter. Error bars indicate S.E.s from three biological replicates.(*p<0.05, 591

**p<0.01, ***p<0.001) 592

Supplemental Figure S4. Regulatory effects of DkERF23/24/25 and DkWRKY1/5/6/7 593

on the promoters of DkADH1 and DkERF9/10/19. Error bars indicate S.E.s from five 594

biological replicates (***p<0.001). 595

Supplemental Figure S5. Expression of transcription factors responsive to CO2 and N2 596

treatments in ‘Gongcheng-shuishi’ persimmon fruit. 597

Supplemental Figure S6. The combinatorial effects of various TFs on activation of the 598

DkPDC2 promoter. 599

Supplemental Figure S7. Phylogenetic tree analysis of DkERFs in persimmon and 600

AtERFs in Arabidopsis. 601

Supplemental Figure S8. Phylogenetic tree of DkWRKYs in persimmon and AtWRKYs 602

in Arabidopsis. 603

Supplemental Figure S9. Regulatory effect of AtWRKY41 on the DkPDC2 promoter. 604

Error bars indicate S.E.s from five biological replicates. Letters above the columns 605

represent no differences (p<0.05) between different constructs. 606

Supplemental Figure S10. GUS histochemical assays of effects of AtERF1 and 607

AtWRKY53 on the DkPDC2 promoter. Error bars indicate S.E.s from three biological 608

replicates.(*p<0.05, **p<0.01, ***p<0.001) 609

Supplemental Figure S11. Regulatory effect of DkERF3 on the DkPDC2 promoter. 610

Supplemental Table S1. Y1H library screening with DkDPC2 promoter. 611



24

Supplemental Table S2. Primer sequences used for RACE. 612

Supplemental Table S3. Primer sequences used for full-length amplification. 613

Supplemental Table S4. Primer sequences used for yeast one-hybrid assay. 614

Supplemental Table S5. Primer sequences for gene vector construction used for 615

dual-luciferase assays. 616

Supplemental Table S6. Primer sequences for EMSA experiments. 617

Supplemental Table S7. Primer sequences for RT-qPCR. 618

Supplemental Table S8. Primer sequences for subcellular localization. 619

Supplemental Table S9. Primer sequences for yeast two-hybrid assay. 620

Supplemental Table S10. Primer sequences for BiFC. 621

Supplemental Table S11. Primer sequences for SK, AD and BiFC of genes from 622

Arabidopsis. 623

624

ACKNOWLEDGEMENTS 625

The authors would like to thank Dr. Ian Ferguson (Plant & Food Research, NZ) for 626

critical reading of the manuscript. 627

628

FIGURE LEGENDS 629

Fig. 1 Action of DkERFs and DkWRKYs on the promoter of DkPDC2. (A) Physical 630

interactions between DkERFs, DkWRKYs, and DkPDC2 promoter, using yeast 631

one-hybrid analysis. Auto-activation of promoters were tested on SD medium lacking 632

Ura in presence of AbA (SD/-Ura + AbA). Interaction was determined on SD medium 633

lacking Leu in presence of AbA (SD/-Leu + AbA). (B-F) Electrophoretic mobility shift 634

assay (EMSA) of DkERF23/24/25 and DkWRKY6/7 binding to the DkPDC2 635

promoter. Purified transcription factor (TF) proteins and biotin-labeled DNA probe 636

were mixed and analyzed on 6% native polyacrylamide gels. The presence (+) or 637

absence (-) of specific probes is indicated. The concentration of the cold probe is 638

shown; the biotinylated probe concentration was 1 nM. (G) Regulatory effects of 639



25

DkERFs and DkWRKYs on the DkPDC2 promoter. The ratio of LUC/REN of the empty 640

vector plus promoter was used as calibrator (set as 1). Error bars indicate SEs from five 641

biological replicates (the regulatory effects of various TFs on DkPDC2 promoter were 642

all compared to the value of the control (empty vector, SK), *** p<0.001). 643

Fig. 2 Expression of DkERFs and DkWRKYs in response to high CO2 treatment. 644

Persimmon fruit cultivars ‘Mopanshi’, ‘Jingmianshi’, and ‘Tonewase’ were incubated 645

in 95% CO2, 4% N2 and 1% O2 for 1 d and then in air at 20°C. For determination of 646

relative mRNA abundance, the values at day 0 were set as 1. Values are means (+SE) 647

from three biological replicates (gene expression was compared between high CO2 648

treated fruit and control fruit at each sampling point, *p<0.05, **p<0.01, *** p<0.001). 649

Fig. 3 Subcellular localization of DkERFs-GFP and DkWRKYs-GFP in N. 650

benthamiana leaves stably transformed with a red nuclear localization marker and 651

agroinfiltrated with GFP DkERFs-GFP and DkWRKYs-GFP. The fluorescence was 652

measured at 488 nm with a LSM780 microscope and photographed. Bars =25 μm. 653

Fig. 4 Effects of DkERF24 and DkWRKY1 separately and in combination on 654

transcription from the DkPDC2 promoter and analysis of their protein-protein 655

interactions. (A) Effect of the combination of DkERF24 and DkWRKY1 on the 656

DkPDC2 promoter. The ratio of LUC/REN of the empty vector (SK) plus promoter was 657

used as calibrator (set as 1). Error bars indicate SEs from five biological replicates. 658

Different letters above the columns represent significant differences (the combination 659

effects were compared to two individual effects, p<0.05). (B) Interaction between 660

DkERF24 and DkWRKY1 in yeast two-hybrid assays. Liquid cultures of double 661

transformants were plated at OD600=0.01 dilutions on synthetic dropout selective 662

media: (1) SD medium lacking Trp and Leu (DDO); (2) SD medium lacking Trp, Leu, 663

His and Ade (QDO); and (3) SD medium lacking Trp, Leu, His, and Ade, and 664

supplemented with 5 mM 3-amino-1,2,4-triazole (QDO+3AT). Protein-protein 665

interactions were determined on QDO and QDO + 3AT. pOst1-NubI, positive control; 666

pPR3-N, negative control. (C) In vivo interaction between DkERF24 and DkWRKY1, 667



26

determined using BiFC N- and C-terminal fragments of YFP (indicated on the figure as 668

YFPN and YFPC) were fused to the C- and N-terminus of DkERF24 and DkWRKY1, 669

respectively. Combinations of YFPN and YFPC with the corresponding DkERF24 and 670

DkWRKY1 constructs were used as negative controls. Fluorescence of YFP represents 671

protein-protein interaction. Bars=50 μm. 672

Fig. 5 Transient over-expression of transcription factors in persimmon fruit discs. The 673

transient over-expression experiments were conducted with two negative controls 674

(empty vector pGreen II 002962-SK (SK) and DkERF1) and target transcription factors 675

(DkERF24, DkWRKY1 and DkERF24 plus DkWRKY1) using persimmon fruit discs. 676

Tissues from each of the infiltrated discs were taken to measure the soluble tannins 677

content (A) and relative gene expression levels of related downstream genes compared 678

with the SK (B-E) at daily intervals during the 3 d incubation. Soluble tannins content 679

was measured with Folin-Ciocalteu reagent and quantitated by reference to standard 680

tannin acid. Error bars indicate SEs from three biological replicates. The soluble 681

tannins content and gene expression were compared between genetox with the control 682

(empty vector) at each sampling point, **p<0.01, ***p<0.001. 683

Fig. 6 Regulatory roles of AtERF1 and AtWRKY53 on the DkPDC2 promoter. (A) 684

Regulatory effects of AtERF1 and AtWRKY53 on the promoter of DkPDC2. The ratio 685

of LUC/REN of the empty vector plus promoter was used as calibrator (set as 1). Error 686

bars indicate S.E.s from five biological replicates (the regulatory effects of various 687

transcription factors on DkPDC2 promoter were all compared to the value of the 688

control (empty vector, SK), **p<0.01, ***p<0.001). (B) Physical interactions between 689

AtERF1, AtWRKY53 and DkPDC2 promoter, using yeast one-hybrid analysis. 690

Auto-activation of promoters was tested on SD medium lacking Ura in presence of 691

AbA (SD/-Ura + AbA). Interaction was determined on SD medium lacking Leu in 692

presence of AbA (SD/-Leu + AbA). (C) Effect of the combination of AtERF1 and 693

AtWRKY53 on the DkPDC2 promoter. The ratio of LUC/REN of the empty vector (SK) 694

plus promoter was used as calibrator (set as 1). Error bars indicate SEs from five 695



27

biological replicates. Different letters above the columns represent significant 696

differences (the combination effects were compared to two individual effects, p <0.05). 697

(D) In vivo interaction between AtERF1 and AtWRKY53, determined using BiFC. 698

AtERF1 and AtWRKY53 proteins were fused to the N- and C-terminus of YFP (YFPN 699

and YFPC), respectively. PHR2-YFPN and SPX4-YFPC were used as positive controls, 700

while combinations of YFPN and YFPC with the corresponding AtERF1 and 701

AtWRKY53 constructs were used as negative controls. Fluorescence of YFP represents 702

protein-protein interaction. Bars= 50 μm. 703

Fig. 7 Regulatory roles of transcription factors involved in the regulation of the 704

DkPDC2 promoter as part of the hypoxia response/deastringency process in persimmon 705

fruit. DkERF9 and DkERF19 are two direct activators of the DkPDC2 promoter (Min 706

et al., 2012, 2014). In addition, two MYB transcription factors (TFs), DkMYB6 and 707

DkMYB10, were characterized as the upstream activators via binding to DkERF 708

promoters (Zhu et al., 2018). In this study, an ERF/WRKY TF complex responsive to 709

hypoxia and which trans-activated the PDC2 promoter was found with both persimmon 710

fruit (DkERF24 and DkWRKY1) and Arabidopsis (AtERF1 and AtWRKY53) 711

homologs. Meanwhile three other DkWRKY/DkERF also directly recognized and 712

activated the PDC2 promoter, respectively. Solid arrows indicate direct interactions. 713

The different red stars indicated the trans-activation effects (One star, 2-5-fold; two 714

stars, higher than 10-fold). 715

716

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https://scholar.google.com/scholar?as_q=Identification of genes involved in proanthocyanidin biosynthesis of persimmon (Diospyros kaki) fruit&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ikegami&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Li T%2C Xu YX%2C Zhang LC%2C Ji YL%2C Tan DM%2C Yuan H%2C Wang AD %282017%29 The jasmonate%2Dactivated transcription factor MdMYC2 regulates ETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=The jasmonate-activated transcription factor MdMYC2 regulates ETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Licausi F%2C Kosmacz M%2C Weits DA%2C Giuntoli B%2C Giorgi FM%2C Voesenek LA%2C Perata P%2C van Dongen JT %282011a%29 Oxygen sensing in plants is mediated by an N%2Dend rule pathway for protein destabilization%2E Nature&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Licausi F%2C van Dongen JT%2C Giuntoli B%2C Novi G%2C Santaniello A%2C Geigenberger P%2C Perata P %282010%29 HRE1 and HRE2%2C two hypoxia%2Dinducible ethylene response factors%2C affect anaerobic responses in Arabidopsis thaliana%2E Plant J&dopt=abstract


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https://www.ncbi.nlm.nih.gov/pubmed?term=Liu F%2C Vantoai T%2C Moy LP%2C Bock G%2C Linford LD%2C Quackenbush J %282005%29 Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis%2E Plant Physiol&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Livak KJ%2C Schmittgen TD %282001%29 Analysis of relative gene expression data using real%2Dtime quantitative PCR and the 2%2D�?��?�CT method%2E Methods&dopt=abstract

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https://scholar.google.com/scholar?as_q=Analysis of relative gene expression data using real-time quantitative PCR and the 2-â�?³â�?³CT method&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Livak&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lorenzo O%2C Piqueras R%2C S�nchez%2DSerrano JJ%2C Solano R %282003%29 ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lorenzo&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lv QD%2C Zhong YJ%2C Wang YG%2C Wang ZY%2C Zhang L%2C Shi J%2C Wu ZC%2C Liu Y%2C Mao CZ%2C Yi KK%2C Wu P %282014%29 SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SPX4 negatively regulates phosphate signaling and homeostasis through its interaction with PHR2 in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lv&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Differential effects of regular and controlled atmosphere storage on the quality of three cultivars of pomegranate (Punica granatum L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Differential effects of regular and controlled atmosphere storage on the quality of three cultivars of pomegranate (Punica granatum L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Matityahu&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Miao&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Min T%2C Yin XR%2C Shi YN%2C Luo ZR%2C Yao YC%2C Grierson D%2C Ferguson IB%2C Chen KS %282012%29 Ethylene%2Dresponsive transcription factors interact with promoters of ADH and PDC involved in persimmon %28Diospyros kaki%29 fruit de%2Dastringency%2E J Exp Bot&dopt=abstract

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https://scholar.google.com/scholar?as_q=Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Min&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Min T%2C Fang F%2C Ge H%2C Shi YN%2C Luo ZR%2C Yao YC%2C Grierson D%2C Yin XR%2C Chen KS %282014%29 Two novel anoxia%2Dinduced ethylene response factors that interact with promoters of deastringency%2Drelated genes from persimmon%2E PLoS ONE&dopt=abstract

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https://scholar.google.com/scholar?as_q=Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Two novel anoxia-induced ethylene response factors that interact with promoters of deastringency-related genes from persimmon.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Min&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Mo RL%2C Yang SC%2C Huang YM%2C Chen WX%2C Zhang QL%2C Luo ZR %282016%29 ADH and PDC genes involved in tannins coagulation leading to natural de%2Dastringency in Chinese pollination constant and non%2Dastringency persimmon %28Diospyros kaki Thunb%2E%29%2E Tree Genet Genomes&dopt=abstract

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https://scholar.google.com/scholar?as_q=ADH and PDC genes involved in tannins coagulation leading to natural de-astringency in Chinese pollination constant and non-astringency persimmon (Diospyros kaki Thunb.).&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=ADH and PDC genes involved in tannins coagulation leading to natural de-astringency in Chinese pollination constant and non-astringency persimmon (Diospyros kaki Thunb.).&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mo&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Mustroph A%2C Zanetti ME%2C Jang CJH%2C Holtan HE%2C Repetti PP%2C Galbraith DW%2C Girke T%2C Bailey%2DSerres J %282009%29 Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis%2E P Nat Acad Sci USA&dopt=abstract

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https://scholar.google.com/scholar?as_q=Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mustroph&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Papdi C%2C P�rez%2DSalam� I%2C Joseph MP%2C Giuntoli B%2C B�gre L%2C Koncz C%2C Szabados L %282015%29 The low oxygen%2C oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2%2E12%2C RAP2%2E2 and RAP2%2E3%2E Plant J&dopt=abstract

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https://scholar.google.com/scholar?as_q=The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP2&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Papdi&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sainsbury&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Physiological and structural changes during ripening and deastringency treatment of persimmon fruit cv&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Salvador&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Wang MM%2C Zhu QG%2C Deng CL%2C Luo ZR%2C Sun NN%2C Grierson D%2C Yin XR%2C Chen KS %282017%29 Hypoxia%2Dresponsive ERFs involved in postdeastringency softening of persimmon fruit%2E Plant Biotechnol J&dopt=abstract

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https://www.ncbi.nlm.nih.gov/pubmed?term=Wang PC%2C Du YY%2C Zhao XL%2C Miao YC%2C Song CP %282013%29 The MPK6%2DERF6%2DROS%2Dresponsive cis%2Dacting element7%2FGCC box complex modulates oxidative gene transcription and the oxidative response in Arabidopsis%2E Plant Physiol&dopt=abstract


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https://scholar.google.com/scholar?as_q=The MPK6-ERF6-ROS-responsive cis-acting element7/GCC box complex modulates oxidative gene transcription and the oxidative response in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang RZ%2C Yang Y%2C Li GC %281997%29 Chinese persimmon germplasm resources%2E Acta Hortic&dopt=abstract


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https://www.ncbi.nlm.nih.gov/pubmed?term=Xiao YY%2C Kuang JF%2C Qi XN%2C Ye YJ%2C Wu ZX%2C Chen JY%2C Lu WJ %282018%29 A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening%2E Plant Biotechno J&dopt=abstract

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https://scholar.google.com/scholar?as_q=A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A comprehensive investigation of starch degradation process and identification of a transcriptional activator MabHLH6 during banana fruit ripening&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xiao&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xu K%2C Xu X%2C Fukao T%2C Canlas P%2C Maghirang%2DRodriguez R%2C Heuer S%2C Ismail AM%2C Bailey%2DSerres J%2C Ronald PC%2C Mackill DJ %282006%29 Sub1A is an ethylene%2Dresponse%2Dfactor%2Dlike gene that confers submergence tolerance to rice%2E Nature&dopt=abstract

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https://scholar.google.com/scholar?as_q=Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yang CY%2C Hsu FC%2C Li JP%2C Wang NN%2C Shih MC %282011%29 The AP2%2FERF transcription factor AtERF73%2FHRE1 modulates ethylene responses during hypoxia in Arabidopsis%2E Plant Physiol&dopt=abstract

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https://scholar.google.com/scholar?as_q=The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yang&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yin XR%2C Shi YN%2C Min T%2C Luo ZR%2C Yao YC%2C Xu Q%2C Ferguson IB%2C Chen KS %282012%29 Expression of ethylene response genes during persimmon fruit astringency removal%2E Planta&dopt=abstract

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JOURNAL OF VIROLOGY€¦ · Eckhart, Keith W. C. Peden, Ashok Srinivasan, and James M. Pipas........

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