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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
Copyright 2019 by the American Society of Plant Biologists
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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
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(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
55
56
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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