Post on 12-Jan-2020
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Short title: Dof regulates kiwifruit starch degradation 1
Corresponding author details: 2
Xueren Yin 3
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, 4
Zhejiang University, Zijingang Campus, 310058 Hangzhou, PR China 5
E-mail, xuerenyin@zju.edu.cn 6
7
Transcriptome analysis identifies a zinc finger protein regulating 8
starch degradation in kiwifruit 9
10
Ai-di Zhang1,2, Wen-qiu Wang1,2, Yang Tong1, Ming-jun Li3, Donald Grierson1,4, Ian 11
Ferguson1,5, Kun-song Chen1,2, Xue-ren Yin1,2* 12
13
1 Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, 14
Zhejiang University, Zijingang Campus, Hangzhou, PR China 15
2 The State Agriculture Ministry Laboratory of Horticultural Plant Growth, 16
Development and Quality Improvement, Zhejiang University, Zijingang Campus, 17
Hangzhou, PR China 18
3 State Key Laboratory of Crop Stress Biology in Arid Areas/College of Horticulture, 19
Northwest A&F University, Yangling, PR China 20
4 Plant & Crop Sciences Division, School of Biosciences, University of Nottingham, 21
Sutton Bonington Campus, Loughborough, UK 22
5 New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, 23
Auckland, New Zealand 24
25
One sentence summary: 26
An ethylene responsive C2H2-type zinc finger transcription factor, AdDof3, regulates 27
starch degradation in kiwifruit via trans-activation of the AdBAM3L promoter. 28
Plant Physiology Preview. Published on August 22, 2018, as DOI:10.1104/pp.18.00427
Copyright 2018 by the American Society of Plant Biologists
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29
Author contributions: 30
X.Y. and K.C. designed the study; A.Z., W.W. and Y.T. performed the experiments and 31
analyses, with input from all other authors; M.L. contributed to the stable 32
transformation and analyses; A.Z., D.G., I.F. and X.Y. wrote the manuscript. All 33
authors read and approved the final manuscript. 34
35
Funding: 36
This research was supported by the National Key Research and Development Program 37
(2016YFD0400102), the National Natural Science Foundation of China (31722042), 38
the Natural Science Foundation of Zhejiang Province, China (LR16C150001), the Fok 39
Ying Tung Education Foundation, China (161028, 20170101210004) and the 111 40
Project (B17039). 41
42
Address correspondence to xuerenyin@zju.edu.cn 43
44
Abstract 45
Ripening, including softening, is a critical factor in determining postharvest shelf-life 46
of fruit and is controlled by enzymes involved in cell wall metabolism, starch 47
degradation and hormone metabolism. Here, we used a transcriptomics-based 48
approach to identify transcriptional regulatory components associated with texture, 49
ethylene and starch degradation in ripening kiwifruit (Actinidia deliciosa). Twelve 50
differentially expressed structural genes— including seven involved in cell wall 51
metabolism, four in ethylene biosynthesis and one in starch degradation— and 14 52
transcription factors (TFs) induced by exogenous ethylene treatment and inhibited by 53
the ethylene signalling inhibitor 1-methylcyclopropene were identified as changing in 54
transcript levels during ripening. Moreover, analysis of the regulatory effects of 55
differentially expressed genes (DEGs) identified a zinc finger TF, DNA BINDING 56
WITH ONE FINGER (AdDof3), which showed significant trans-activation on the 57
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AdBAM3L (β-amylase) promoter. AdDof3 physically interacted with the AdBAM3L 58
promoter, and stable overexpression of AdBAM3L resulted in lower starch content in 59
transgenic kiwifruit leaves, suggesting that AdBAM3L is a key gene for starch 60
degradation. Moreover, transient overexpression analysis showed that AdDof3 61
upregulated AdBAM3L expression in kiwifruit. Thus, transcriptomics analysis not 62
only allowed prediction of some ripening regulating genes but also facilitated 63
characterization of a TF, AdDof3, and a key structural gene, AdBAM3L, in starch 64
degradation. 65
Key words: Fruit ripening, cell wall, Dof transcription factor, ethylene, kiwifruit, 66
starch degradation, stable transformation 67
68
Introduction 69
Ripening is a programmed process involving substantial changes in fruit quality 70
properties, such as color, aroma, flavor and texture (Prasanna et al., 2007; Klee and 71
Giovannoni, 2011). The overall process of fruit ripening, however, is that of 72
senescence, accompanied by fruit quality deterioration and postharvest loss (Seymour 73
et al., 2013). Thus, the postharvest control of ripening is critical for the fruit industry. 74
In tomato (Solanum lycopersicum), multiple regulators of fruit ripening have been 75
identified, such as Ripening-inhibitor (Rin, Vrebalov et al., 2002), Colorless 76
non-ripening (CNR, Manning et al., 2006), Never-ripe (Nr, Wilkinson et al., 1995), 77
APETALA2a (AP2a, Karlova et al., 2011), etc. Transgenic studies of such genes in 78
tomato fruit, or use of mutants, has shown the impacts of these genes on fruit ripening. 79
However, such studies have been less frequent in other fruit crops, especially 80
perennial fruit where transgenic studies and the availability of mutants is limited. 81
Moreover, the function of these ripening regulators may differ in various crops, e.g. 82
tomato rin mutant (Vrebalov et al., 2002) and FaMADS9 83
(MCM1/AGAMOUS/DEFICIENS/SRF(MADS)-box) antisense transgenic strawberry 84
(Fragaria x ananassa Duch.) fruit (Seymour et al., 2011) inhibit or retard ripening, 85
while MdMADS8/9-suppressed apples (Malus x domestica) have a phenotype of small 86
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fruit with reduced flesh (Ireland et al., 2013). These findings highlight the need for 87
investigations on ripening regulation in different fruit types and species. 88
Kiwifruit (Actinidia), one of the most recently domesticated fruit crops, now has 89
global distribution (Huang et al., 2001). Its rise as an important economic crop has led 90
to extensive research on regulation of kiwifruit ripening. It is a typical climacteric 91
fruit exhibiting an ethylene burst and also is particularly sensitive to ethylene 92
(McDonald and Harman, 1982). Postharvest ripening indices for kiwifruit are similar 93
to those in other fruit, including starch degradation/soluble solids accumulation, 94
ethylene biosynthesis, cell wall metabolism, and volatile emission (Atkinson et al., 95
2011). A number of structural genes related with these physiological changes have 96
been characterized, but most are associated with multiple gene families involved with 97
regulation of fruit softening (CkPGA/B/C, polygalacturonase, Wang et al., 2000; 98
AdXTH4/5/6/7/8/10/13, xyloglucan endotransglucosylase/hydrolase, Atkinson et al., 99
2009; AdBAM3L/3.1/9, AdAMY1 α-amylase, AdAGL3 α-glucosidase, Hu et al., 2016), 100
and prove difficult to manipulate in relation to genetic improvement. Other genes 101
have been identified with multiple quality traits, e.g. knock-down of AdACO1 102
(1-aminocyclopropane-1-carboxylic acid oxidase) resulted in less ethylene production 103
and firm fruit but lower levels of the quality attributes of aroma and flavor (Atkinson 104
et al., 2011). 105
In terms of regulating fruit ripening, targeting transcription factors (TFs) is an 106
alternative option. In tomato, the above-mentioned CNR and RIN genes encode SBP 107
(SQUAMOSA promoter binding protein) and MADS TFs (Dong et al., 2013). Other 108
TFs have also been shown to be involved in fruit ripening, mostly characterized in 109
tomato, including AP2/Ethylene Response Factor (eg. SlERF6, Lee et al., 2012; 110
SlERF.B3-SRDX, Liu et al., 2014), NAC (eg. SlNAC1/4, Ma et al., 2014; Zhu et al., 111
2014; Meng et al., 2016), and Homeobox (eg. LeHB1, Lin et al., 2008). In other fruit 112
species, potential roles of TFs in fruit ripening have been identified (Xie et al., 2016), 113
but few have full or partial functional characterization. Citrus CitERF13 is a regulator 114
of ethylene-driven fruit postharvest degreening via binding and regulation of the 115
CitPPH (pheophorbide hydrolase) promoter (Yin et al., 2016); a jasmonate (JA) 116
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responsive MdMYC2 transcription factor mediates JA regulation of ethylene 117
biosynthesis and apple fruit ripening (Li et al., 2017). 118
In the present research, we analyzed three major ripening traits in kiwifruit, 119
including texture (firmness and cell wall components), ethylene production, and 120
starch degradation (as well as total soluble solids (TSS)). We performed 121
transcriptomic analysis on ethylene- and 1-methylcyclopropene (1-MCP)-treated fruit 122
with the aim of identifying the most responsive TFs and structural genes during fruit 123
ripening. Using the dual-luciferase assay, multiple TFs showed regulatory effects on 124
different promoters, including trans-activation by AdDof3 on a starch degradation 125
gene (AdBAM3L). Furthermore, electrophoretic mobility shift assays (EMSAs) 126
indicated specific cis-elements of the AdBAM3L promoter for AdDof3 binding. We 127
investigated the functions of AdDof3 and AdBAM3L, as well as their in vivo regulation 128
in fruit, with stable transformation (analysis using leaves) or transient overexpression 129
in kiwifruit (analysis with the core tissues at two sites within a single fruit). This 130
strategy provided insights into the molecular basis of starch degradation during 131
kiwifruit ripening. 132
133
Results 134
Analyses of kiwifruit ripening 135
Ethylene treatment significantly accelerated fruit ripening, with treated fruit 136
reaching the ethylene climacteric peak of 81.4 nl g-1 h-1 at 8 days in storage (DIS), 137
which was higher and occurred earlier than that of control fruit, which peaked at 17 d 138
with 30.2 nl g-1 h-1 production. Ethylene production was inhibited in 1-MCP-treated 139
fruit and was therefore undetectable over the whole experimental period (Fig. 1a). 140
Starch degradation is considered the first sign of kiwifruit postharvest ripening 141
and also contributes to the TSS. Starch content decreased from 61.5 mg/g at 0 DIS to 142
1.5 mg/g at 18 DIS in control fruit. In ethylene-treated fruit, starch content rapidly 143
decreased to 1.9 mg/g by 8 DIS. 1-MCP-treated fruit exhibited a significantly slower 144
rate of decrease with 24.3 mg/g starch remaining at 18 DIS (Fig. 1b). TSS showed the 145
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opposite trends to starch content, increasing from 6.2% at 0 DIS to 15.6% at 18 DIS 146
for the control fruit. In ethylene- and 1-MCP-treated fruit, TSS reached 15.2% at 8 147
DIS and 11.7% at 18 DIS, respectively (Fig. 1b). 148
Changes in fruit firmness were similar to those of starch content. 149
Ethylene-treated fruit reached an ‘eating-ripe’ stage (firmness of 7.9 N) at 6 DIS 150
whereas control fruit reached a similar firmness (7.4 N) at 18 DIS (Fig. 1c). In parallel 151
with fruit softening, extractable cell wall materials (CWM) also decreased, with the 152
decrease accelerated by ethylene and inhibited by 1-MCP (Fig. 1c). With regard to 153
main cell wall components, in general only cellulose and covalent binding pectin 154
(CBP) showed similar patterns to that of fruit firmness. Both cellulose and CBP 155
contents were relatively lower in ethylene-treated and higher in 1-MCP-treated fruit. 156
Water soluble pectin (WSP) content showed an increasing trend during ripening (Fig. 157
1c). Both hemicellulose and ionic soluble pectin (ISP) decreased during fruit ripening, 158
but their decreasing rate and contents were similar among the three treatments (Fig. 159
1c). 160
161
Expression of TF and structural genes during ripening 162
RNA-seq provided an overview of genes differentially expressed during ripening 163
and in response to exogenous ethylene and 1-MCP treatments. The transcript 164
abundances of genes were estimated by fragments per kilobase of exon per million 165
fragments mapped (FPKM). The boxplot distribution of the log10FPKM values in 166
Supplemental Fig. S1a showed that the median and quartile values of the expression 167
values across the libraries compared for differential expression were comparable. De 168
novo assembly also predicted a total of 4542 genes which have not appeared in the 169
‘Hong Yang’ genome database annotated by COG, GO, KEGG, KOG, Pfam, eggnog, 170
Swiss-Prot and nr databases (Supplemental Fig. S1b). At 1 and 4 DIS, 6326 and 3994 171
DEGs were found between the control and ethylene treatments, while only 25 and 34 172
DEGs were found between the control (CK) and 1-MCP treatments (Supplemental Fig. 173
S1c, d). KEGG pathway analysis revealed that the DEGs between control and 174
ethylene treatments were mainly enriched in carbon metabolism, biosynthesis of 175
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amino acids and starch and sucrose metabolism at both 1 and 4 DIS (Supplemental 176
Fig. S1e, f). 177
The selective thresholds were set using a false discovery rate (FDR) ≤0.01, 178
estimated absolute log2fold change >2, FPKM ratio of CK/ETH >40 or 179
ETH/1-MCP >100 and the highest FPKM values >9. Thirteen target genes, including 180
four ethylene biosynthesis genes, eight cell wall-related genes, one starch degradation 181
gene (Fig. 2a) and 14 transcription factors (Fig. 2b) emerged from all DEGs 182
(Supplemental Table S1). Expression of the 13 structural genes positively associated 183
with kiwifruit postharvest ripening and softening, as all genes were induced by 184
ethylene treatment and suppressed by 1-MCP (Fig. 2). This was verified by reverse 185
transcription quantitative PCR (RT-qPCR) for all genes (Fig. 3) except AdAFC1 (acid 186
β-fructofuranosidase, Supplemental Fig. S2). However, these structural genes also 187
showed different responses to exogenous and internal ethylene. AdXTH5, AdXTH6, 188
AdACO5 and AdACO7 were responsive to ethylene treatment and peaked at 1 or 2 189
DIS, then declined to similar basal levels of the control and 1-MCP-treated fruit. 190
AdPME1 (pectin methyl esterase), AdPL2 (pectin lyase), AdMAN1 191
(endo-β-mannanase), AdBAM3L and AdACO1 were rapidly up-regulated by ethylene 192
treatment and then showed a significantly higher expression peak at the ethylene 193
climacteric peak (17 DIS for control and 4-8 DIS for ethylene-treated fruit). 194
Expression of AdPG1, AdPL1 and AdACS1 (1-aminocyclopropane-1-carboxylate 195
synthase) followed the pattern of the ethylene climacteric peak (Fig. 3). In contrast to 196
the expression patterns of structural genes, 11 transcription factors were putative 197
activators and were up-regulated by ethylene (Fig. 4a), while AdbZIP1 (basic leucine 198
zipper protein), AdDof3 and AdHB1 were putative repressors down-regulated by 199
ethylene treatment (Fig. 4b). In general, all of these structural genes and transcription 200
factors could be potential candidates involved in programming kiwifruit ripening. It 201
should be noted that we only looked at three key ripening processes in the present 202
study, and other structural genes involved in fruit ripening could also be targets for 203
these transcription factors. 204
205
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In vivo regulation of ripening-associated TFs and structural genes 206
The regulatory effects of ripening-associated TFs on structural genes were tested 207
using the dual-luciferase assay. Although these TFs and structural genes had very 208
obvious associations at the transcript level, limited in vivo interactions were found 209
between them (Fig. 5). AdDof4 acted as a repressor on promoters of AdPL1 and 210
AdACS1 where luciferase activity (LUC/REN value) was reduced to approx. 0.5. 211
AdNAC5 and AdWRKY1 both targeted the AdACS1 promoter, with 3.7- and 2.3-fold 212
induction, respectively. The AdBAM3L promoter was only up-regulated by AdDof3, 213
with an approx. 3-fold induction. Except for these regulatory effects, relations of the 214
other ripening-associated TFs and structural genes remained unclear. 215
216
Investigation of AdDof3 binding elements on the AdBAM3L promoter 217
Based on the dual-luciferase assays (Fig. 5) and RNA-seq results (Supplemental 218
Fig. S1), the mechanism of AdDof3 regulation on the AdBAM3L promoter was 219
selected for further investigation. Firstly, subcellular localization results indicated that 220
AdDof3 located at the nucleus (Fig. 6a), which is similar to most TFs. The core 221
binding sequence for the Dof family is AAAG/CTTT (Yanagisawa et al., 1999). In the 222
region of the AdBAM3L promoter (-1790 to -1 bp), six motifs were found (Fig. 6c). 223
EMSA results indicated that the region (-160 to -200 bp) that contains the other three 224
motifs showed the binding band in the presence of AdDof3 (Fig. 6b and d). 225
In order to determine the exact binding site between -160 to -200 bp, eight 226
different probes were designed with deletion or mutagenesis (Fig. 6b). EMSA showed 227
that AdDof3 could bind to the probes of P-abc (probe with three motifs), P-a, P-bc and 228
P-ab, but not the probe P-c (Fig. 6d). These results suggested that AdDof3 physically 229
binds to the AdBAM3L promoter (sites ‘a’ and ‘b’), which was further confirmed by 230
mutated probes. In the presence of AdDof3, P-Δabc (probe with b and c motifs and 231
mutated a motif) and P-aΔbc showed binding signals with different intensity, while 232
the P-ΔaΔbc failed to generate any visible binding signal (Fig. 6d). Moreover, the 233
shifted band disappeared with the addition of an unlabeled competitor with the same 234
sequence P-abc. 235
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236
Stable transformation suggests AdBAM3L is an important gene for starch 237
degradation in kiwifruit 238
For most perennial fruits, it is difficult to perform stable transformation due to 239
low efficiency and long periods required for growth and development. Here, 240
AdBAM3L was overexpressed in ‘Qinmei’ (Actinidia deliciosa) tissue cultured 241
plantlets. Although the aim of the present research is fruit ripening, only leaves from 5 242
month plantlets on Murashige and Skoog (MS) medium were used for transgenic 243
verification and phenotype analyses (Fig. 7a), due to the very slow growth rate of 244
kiwifruit plantlets (Supplemental Fig. S3). Firstly, the integration of AdBAM3L into 245
the genome was confirmed by conventional PCR analyses (Fig. 7b). Further analysis 246
by RT-qPCR (Fig. 7c) and GUS staining (Fig. 7d) confirmed the overexpression of 247
AdBAM3L in the two transgenic lines. The expression of AdBAM3L in transgenic 248
plants (lines 4 and 6) was more than 10-fold higher than in the WT (Fig. 7c). Starch 249
analyses indicated that both transgenic lines had lower starch contents in leaves than 250
the WT plants (Fig. 7e, f). At the same period (about 5 months), leaves of WT plants 251
contained 60 mg/g fresh weight (FW) starch, while the transgenic lines (line-4 and 252
line-6) only contained 6.20 and 9.98 mg/g FW starch, respectively (Fig. 7f). 253
254
Transient overexpression analyses suggest regulation of AdBAM3L by AdDof3 255
during kiwifruit ripening 256
The results from the dual-luciferase assay, EMSA and stable transformation 257
analyses suggest a regulatory pathway between AdDof3 and AdBAM3L. AdDof3 258
could contribute to kiwifruit ripening via physical binding and activation on the 259
promoter of AdBAM3L, a key gene of starch degradation. Further experiments were 260
designed to test the proposed regulatory model in kiwifruit. Transient overexpression 261
experiments were carried out in ‘Hayward’ fruit core tissue because this tissue has 262
high permeability and provides the ideal single fruit control with two ends (Fig. 8a). 263
RT-qPCR analyses indicated that the expression of AdDof3 and AdBAM3L was similar 264
in the two different fruit ends (Supplemental Fig. S4). Thus, one end of the core tissue 265
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was infiltrated with Agrobacterium tumefaciens strain EHA105 as control, and the 266
other injected with AdDof3-pCAMBIA1301-EHA105 (Fig. 8b). GUS staining showed 267
that the strain with the vector carrying AdDof3 not only infiltrated into the kiwifruit 268
core tissue but was also translated and functioned normally, while the control tissue 269
had no visible GUS staining (Fig. 8b). 270
Fruit at two different stages of maturity were selected for transient expression 271
experiments, including immature fruit (80 days after full bloom, DAFB) and 272
commercially mature fruit (170 DAFB). Both AdDof3 and AdBAM3L expression 273
levels were much higher in the mature fruit core tissue than in immature fruit (Fig. 8c, 274
d). The abundances of AdDof3 and AdBAM3L transcripts increased at 1 d and 2 d after 275
AdDof3 infiltration in both stages. Injecting with AdDof3 resulted in 16.1% (1 d after 276
injection) and 25.2% (2 d after injection) reductions in 80 DAFB samples, and 12.5% 277
(1 d after injection) and 9.0% (2 d after injection) reductions in 170 DAFB samples 278
(Supplemental Fig. S5). However, these reductions were not significant at p<0.05 279
level. 280
281
Discussion 282
Characterization of genes associated with postharvest ripening of kiwifruit by 283
transcriptomics analysis 284
Kiwifruit is an ideal species for studying fruit ripening and softening and 285
contains four distinct softening phases (Atkinson et al., 2011). Here, the measurement 286
of three characteristic indices (ethylene production, firmness, starch content) indicated 287
that ripening and softening of ‘Hayward’ kiwifruit was, as expected, accelerated by 288
ethylene and retarded by 1-MCP, as previously reported in kiwifruit (Koukounaras et 289
al., 2007; Atkinson et al., 2011; Mworia et al., 2012). 290
Transcriptomic analysis indicated there were more than 5000 DEGs between 291
ethylene-treated and control fruit at 1 d, which is consistent with general ideas on the 292
multigenic traits of fruit ripening. Our selective analysis identified 12 structural genes 293
related to ethylene biosynthesis, cell wall and starch degradation. As only three 294
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characteristic traits were selected in this research, it is likely that additional DEGs will 295
exist for the other ripening-related traits, such as aroma, sugar and acid degradation 296
(Nieuwenhuizen et al., 2015; Tang et al., 2016). In addition, 14 TFs reached the 297
selective threshold set for DEGs, with 11 putative activators and 3 putative repressors, 298
based on their sequence similarity with previously published sequences. Unlike the 299
structural genes, these 14 TFs could be potential regulators of any aspect of kiwifruit 300
ripening, including other ripening traits in addition to softening, starch and ethylene. 301
Most of these structural genes have been previously reported from gene expression or 302
activity analyses (AdXTH5/6, Atkinson et al., 2009; AdBAM3L, Hu et al., 2016; 303
AdACO1, Xu et al., 1998) or functional analysis (AdACS1, Atkinson et al., 2011). 304
However, some new genes, such as a PG gene (AdPG1) and two PLs (AdPL1/2), were 305
identified in the present work and may contribute to kiwifruit pectin solubilization and 306
softening. For hemicellulose degradation, isolation of another new gene, AdMAN1, 307
suggests a potential metabolic pathway for hemicellulose degradation in addition to 308
the role of the XTHs (Cutillasiturralde et al., 1994; Atkinson et al., 2009). It is 309
particularly interesting that AdPG1 could not be found in the current version of the 310
kiwifruit genome database. Of the TFs identified, only AdERF10 and AdERF64 have 311
been previously reported from gene expression analysis (Yin et al., 2010; Zhang et al., 312
2016), and all others were newly identified regulator genes potentially implicated in 313
controlling kiwifruit ripening and quality. 314
315
Characterization of multiple links between TFs and ripening-related genes 316
Ripening-associated TFs have been widely reported in various fruit; however, 317
most (especially from perennial fruit) have only been characterized through 318
correlations between gene transcripts and fruit ripening. Few TFs have reported 319
regulatory effects on target genes, e.g. a number of tomato fruit ripening regulators 320
(Rin, Vrebalov et al., 2002; CNR, Manning et al., 2006; Nr, Wilkinson et al., 1995; 321
AP2a, Karlova et al., 2011), a few banana (Musa acuminata) ripening related genes 322
(MabHLH6, basic Helix-Loop-Helix, Xiao et al., 2017; MaDREB1-MaDREB4, 323
dehydration responsive element binding protein, Kuang et al., 2017; MaERF9, 324
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MaDof23, Feng et al., 2016), apple ERF genes (MdCBF, Tacken et al., 2010; 325
MdERF2, Li et al., 2016), and papaya (Carica papaya) CpERF9 (Fu et al., 2016). 326
Here, among 14 TFs, at least four (AdDof3, AdDof4, AdNAC5 and AdWRKY1) 327
showed significant regulatory activity on different target kiwifruit ripening genes, 328
suggesting potential roles in ripening and softening. AdDof3 was an activator on the 329
AdBAM3L promoter, AdNAC5 and AdWRKY1 were activators for the AdACS1 330
promoter, and AdDof4 was a repressor on both AdPL1 and AdACS1 promoters. In 331
kiwifruit, AdERF9 has been characterized as a regulator of the AdXTH5 promoter 332
(designated AdXET5, Yin et al., 2010). Thus, the roles of these TFs on fruit 333
ripening-related genes may provide new information on kiwifruit ripening regulation. 334
Compared to the model fruit tomato and perennial fruit banana and apple, our 335
results appear to show previously uncharacterized links between TFs and 336
ripening-related genes. In tomato, PL was recently characterized as an important 337
regulator for fruit softening (Yang et al., 2017) and long shelf-life (Uluisik et al., 338
2016), but its transcriptional regulation remains unclear. In tomato, high-resolution 339
mapping showed that a QTL contained an ERF and three PME genes (Chapman et al., 340
2012). In apple, an ERF gene (MdCBF) was also characterized as an activator of the 341
apple MdPG (Tacken et al., 2010). In kiwifruit, the physiology analysis also 342
confirmed that pectin degradation is important for postharvest softening. Thus, the 343
regulation of AdDof4 on AdPL1 suggests a regulatory link between TFs and pectin 344
modification. 345
In general, the interactions between the four TFs (AdDof3, AdDof4, AdNAC5 and 346
AdWRKY1) and ripening-related genes were not only benefit to understand kiwifruit 347
ripening, but also provided new examples for other fruit species. The differences 348
between the present results in kiwifruit and previous findings from other fruit, while 349
probably reflecting species differences, could also be due to the selective threshold 350
setting. Thus, homologs of the ripening-related TFs from other fruit may also exist in 351
kiwifruit. Moreover, the structural genes were only limited to ethylene response and 352
cell wall and starch degradation, thus the 10 other TFs still retain the potential for fruit 353
ripening regulation via other ripening-related genes. 354
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355
Functional verification of the role of AdBAM3L as a key gene in starch 356
degradation 357
The two AdBAM3L overexpressed transgenic lines of kiwifruit showed obvious 358
phenotypes. The transgenic lines had low gene expression levels and lower starch 359
contents. In addition, sucrose content was higher in transgenic lines, but fructose and 360
glucose contents were similar (Supplemental Fig. S6). Thus, the transformation and 361
expression analysis (both postharvest ripening and on-tree development) suggests that 362
AdBAM3L is a key gene for kiwifruit starch degradation (Fig. 3; Supplemental Fig. 363
S7). For perennial fruit, stable transformation is extremely difficult and has only been 364
used in a few species (e.g. papaya, apple). Thus, most fruit-related genes (except for 365
tomato) have been characterized with in vitro molecular biology platforms. Among 366
the three structural genes (AdPL1, AdBAM3L and AdACS1) that could be regulated by 367
differentially expressed TFs, AdBAM3L was one of the predicted starch degradation 368
related genes from our previous research, using various postharvest treatments and 369
RT-qPCR (Hu et al., 2016). Thus, the functional verification of AdBAM3L with 370
overexpression analysis in kiwifruit is an extension of previous results, with the 371
approach providing a shortcut to overcoming long-term transformation in kiwifruit 372
(Supplemental Fig. S3) and also the basis for selections of AdDof3-AdBAM3L 373
interactions for further analysis. The associations between expression of BAM, as well 374
as some other structural genes, and ripening have been previously reported in fruit, 375
such as kiwifruit (Tang et al., 2016; Hu et al., 2016), banana (Xiao et al., 2017), and 376
Poncirus trifoliate (Peng et al., 2014). However, none of these genes had been 377
functionally characterized with stable transformation. 378
379
In vitro and in vivo regulation of AdBAM3L by AdDof3 380
The dual-luciferase assay showed that AdDof3 trans-activated the promoter of 381
AdBAM3L, which is further supported by the associations between AdDof3 and 382
AdBAM3L expression in both developing kiwifruit (Supplemental Fig. S7) and fruit 383
undergoing postharvest ripening (Fig. 2). EMSA analyses further indicated that 384
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AdDof3 physically bound to AAAG/CTTT elements within the AdBAM3L promoter, 385
which is similar to Dof homologs from other plants. For instance, a maize (Zea mays) 386
Dof class protein, PBF (prolamin-box binding factor), can trans-activate the γ-zein 387
gene promoter by binding to the AAAG motif (Marzabal et al., 2008). Moreover, 388
ZmDof3 recognized and bound to the AAAG core sequence in promoters of the starch 389
biosynthesis genes Du1 and Su2 in maize and functioned as a positive regulator (Qi et 390
al., 2017). Similar to these plants, AdDof3 is a direct activator on the AdBAM3L 391
promoter and may contribute to starch degradation in kiwifruit. Banana MabHLH6 is 392
also an activator of starch degradation genes via physical binding to target promoters 393
(Xiao et al., 2017). Here, AdDof3 provided another type of regulator on fruit starch 394
degradation. 395
Moreover, while MabHLH6 showed regulatory function on starch degradation 396
genes based on EMSA and dual-luciferase assays, its regulatory function in banana 397
fruit remains unclear. Transient expression experiments have been widely used for 398
gene function analysis in various fruit species (Akagi et al., 2009; Li et al., 2017), but 399
the precision of such analyses is largely influenced by bias that is generated between 400
different fruit tissues (discs or slices) and also different fruits. Our results indicate that 401
kiwifruit core tissue had very high water permeability and the two ends of the fruit 402
had similar gene expression levels (Fig. 8), thus providing an ideal system for 403
transient overexpression analysis. With the benefit of this system, as well as GUS 404
staining (Fig. 8), AdDof3 was shown to regulate AdBAM3L in vivo in ‘Hayward’ 405
kiwifruit, in both immature (80 DAFB) and mature fruit (170 DAFB). However, 406
analysis of the starch contents in transient overexpressed tissues indicated that 407
AdDof3 could reduce starch contents compared to the empty control (SK), which is 408
consistent with its regulation of starch degradation genes. However, the reduction was 409
not statistically significant (Supplemental Fig. S5). To find out whether such an effect 410
is significant requires more extended transient overexpression experiments or stable 411
transformation. 412
413
Conclusions 414
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Transcriptomic analysis predicted 12 structural genes (for ethylene biosynthesis, 415
cell wall degradation, and starch degradation) and 14 TFs that showed high potential 416
for fruit ripening regulation. Moreover, analysis of relationships between these TFs 417
and structural genes indicated previously uncharacterized novel potential 418
transcriptional regulation links, including four TFs (AdDof3, AdDof4, AdNAC5 and 419
AdWRKY1) and three structural genes (AdBAM3L, AdACS1 and AdPL1). Most 420
significantly, using stable transformation, EMSA, and transient analysis, AdBAM3L 421
was confirmed as a key regulator of kiwifruit starch degradation, which could be 422
trans-activated by AdDof3 via binding on AAAG/CTTT elements. Thus, the present 423
findings advance our understanding of the regulation of fruit starch degradation, a 424
critical step for both fruit initial ripening and the ultimate fruit flavor contributed by 425
soluble sugars. 426
427
Materials and Methods 428
Plant material and treatments 429
Mature kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. 430
deliciosa cv. Hayward) were harvested from a commercial orchard, Shanxi, China in 431
2015, with mean TSS of 6.19%. Fruits of uniform size free from visible defects were 432
selected and divided into three batches for three treatments. Each treatment contained 433
three biological replicates of approximately 200 fruit. The fruit were treated with 434
ethylene (100 μl l-1, 24 h, 20 oC), 1-MCP (1 μl l-1, 24 h, 20 oC) or air as the control (24 435
h, 20 oC) in 20 L air-tight containers, respectively. After treatment, the fruit were 436
transferred to normal air at 20 oC. At each sampling point, 3 replicates of 4 fruit were 437
collected from each batch. The outer pericarp (without skin or seeds) of the fruit was 438
cut into small pieces and rapidly frozen in liquid nitrogen and then stored at -80 oC for 439
further experiments. 440
441
Fruit physiological properties 442
A number of kiwifruit postharvest quality properties were measured, including 443
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ethylene production, TSS and starch content, firmness, and CWM. Ethylene was 444
measured by gas chromatography (Agilent Technologies 7890A GC System) and 445
firmness using a TA-XT2i texture analyzer (Stable Micro systems, UK), with the 446
same parameters as described in our previous report (Zhang et al., 2016). TSS were 447
measured using an Atago digital hand-held refractometer (Tokyo, Japan). Two ends of 448
each fruit were sliced and then three drops of juice squeezed from each slice onto the 449
refractometer. Ethylene was measured with three replicates (4 fruit in each replicate) 450
for each treatment. Firmness and TSS were measured with ten single fruit replicates. 451
Total starch was measured on three replicates of 0.1 g frozen samples using a 452
total starch assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland), 453
following a method described previously (Stevenson et al., 2006; Hu et al., 2016). 454
Starch contents were measured from frozen fruit flesh with three biological replicates. 455
CWM extraction and isolation was performed as described previously with slight 456
modifications (Vicente et al., 2013; Minas et al., 2014). The CWM extractions were 457
performed on three biological replicates. For each extraction, approximately 3.0 g of 458
frozen fruit flesh was placed in 20 mL of 80% (V/V) ethanol and boiled for 20 min. 459
After centrifuging at room temperature, the low molecular weight solutes and 460
insoluble materials were separated. The supernatant was discarded and sediments 461
were sequentially washed with 80% (V/V) ethanol, chloroform: methanol (1:1) and 462
acetone and dried at 40 oC for 24 h. The dried residue was collected and weighed. 463
Approximately 50 mg CWM from each sample was used to determine the contents of 464
different cell wall components. Firstly, they were suspended in 6 mL of 50 mM acetic 465
acid-sodium acetic buffer (pH 6.5) and stirred at 20 oC for 6 h, then centrifuged and 466
vacuum filtered. The filtrate were designated as WSP. Secondly, the residue was 467
suspended in 6 mL of 50 mM acetic acid-sodium acetic buffer (pH 6.5) with 50 mM 468
ethylene diamine tetraacetic acid (EDTA), for 6 h continuous shaking. After 469
centrifugation, the supernatant was filtered and measured as ISP. Thirdly, the 470
EDTA-insoluble pellet was extracted with 6 mL of 50 mM Na2CO3 at 4 oC for 18 h, 471
then turned to 20 oC shaking for 2 h. The slurry was centrifuged and the supernatant 472
was filtered as CBP. Subsequently, the residue was extracted with 3 mL of 4 M KOH 473
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at 20 oC for 5 h continuous shaking, and the supernatant was designated as 474
hemicellulose content. Finally, the insoluble residue was washed with 3 mL of 0.3 M 475
acetic acid and 6 mL of 80% (V/V) ethanol and then centrifuged. The pellet was dried 476
at 40 oC and weighted as α-cellulose. Pectin and hemicellulose were measured 477
according to reported protocols (Blumenkrantz and Asboe-Hansen, 1973; Yemm and 478
Wills, 1954). For pectin, 3 mL H2SO4 was added to 0.5 mL extracting solution and 479
boiled for 20 minutes. The mixture was cooled down to room temperature and 0.2 mL 480
carbazole-anhydrous ethanol (1.5 g/L) added. After 30 min standing, absorbance was 481
measured at 530 nm. Hemicellulose content was determined by the anthrone-sulfuric 482
acid method. The hemicellulose extracting solution comprised 5 mL anthrone reagent 483
(2 g anthrone dissolved in 80% H2SO4, and diluted with 80% H2SO4 to 1000 mL). The 484
mixture was heated in a 100 oC water bath for 10 min. After cooling to room 485
temperature, absorbance measurements were made at 625 nm. The results were 486
expressed as mg GalA (galacturonic acid) /g FW and mg Glu (glucose) /g FW, 487
respectively. 488
489
RNA extraction and RNA-seq 490
Total RNA was extracted from frozen kiwifruit flesh following our previous 491
protocol (Yin et al., 2008). For RNA-seq, at least 1.0 µg RNA from each sampling 492
point (1 d and 4 d) and each treatment (control, 1-MCP, ethylene) were sent for 493
sequencing. Three replicates were used for RNA-seq. 494
RNA-seq and bioinformatics analyses were conducted by Biomarker (Beijing, 495
China). The library constructions were carried out following the manufacturer’s 496
instruction of NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530) and 497
NEBNext Multiplex Oligos for Illumina (NEB, E7500), and were sequenced with 498
Illumina HiSeqTM 4000 sequencing platform. Transcriptome analysis used reference 499
genome-based reads mapping. The clean reads filtered from raw data were mapped to 500
the Hong Yang (Actinidia chinensis) genome database using Tophat2 software (Kim et 501
al., 2013). Low quality reads were removed by perl script (unknown nucleotides >5%, 502
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or low Q-value≤20%). FPKM were used to estimate gene expression levels by the 503
Cufflinks software (Trapnell et al., 2010). FDR was used to identify the p-value 504
threshold in multiple texts. Sequences were compared against various protein 505
databases, including NCBI (the National Center for Biotechnology Information), Nr 506
(non-redundant protein) and Swiss-Prot (a manually annotated and reviewed protein 507
sequence database) by a cut-off E-value of 10-5. Gene function was annotated with 508
GO (gene ontology), KO (KEGG ortholog database), Swiss-Prot and Nr annotation. 509
GO enrichment analysis of the DEGs was carried out using Wallenius non-central 510
hyper-geometric distribution based on GOseq R packages (Young et al., 2010), which 511
can adjust for gene length bias in DEGs. KEGG is a database resource for 512
understanding high-level functions and utilities of the biological system (Kanehisa et 513
al., 2008). KOBAS software was used to test the statistical enrichment of DEGs in 514
KEGG pathways (Mao et al., 2005). 515
516
cDNA synthesis and RT-qPCR 517
For cDNA synthesis, TURBO Dnase (Ambion) was used for removing 518
contaminating genomic DNA. Reverse Transcription System (Promega) was used as 519
cDNA synthesis. Three biological replicates with three independent RNA extractions 520
and cDNA synthesis were performed for each sampling point and each treatment. 521
RT-qPCR was carried out using a LightCycler® 480 instrument (Roche), with 522
LightCycler® 480 SYBR Green I Master (Roche). The specificity of primers was 523
double checked by melting curves and product resequencing (Yin et al., 2010). 524
Primers for RT-qPCR analysis are listed in Supplemental Table S2. Kiwifruit actin 525
(Genbank no. EF063572) was employed as the housekeeping gene (Zhang et al., 526
2006). 527
528
Genomic DNA extraction 529
Kiwifruit genomic DNA was extracted from leaves. Approximately 0.1 g of 530
tissue was put into 900 μL TPS buffer (100 mM Tris-HCL, 10 mM EDTA and 1 M 531
KCL) for 1 h in a water bath at 75 oC, then centrifuged at 12000 rpm for 10 min and 532
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added with equal volume of isopropyl alcohol to the supernatant to subside genomic 533
DNA. After 5 min, the tube was centrifuged again and supernatant was discarded. The 534
pellet (genomic DNA) was dissolved in sterile water. Three individual plants (five 535
months old) from each line were used as three replicates. The PCR template using a 536
plasmid containing the target sequence acted as the positive control and water was the 537
negative control. 538
539
Gene isolation, promoter cloning and promoter motif analysis 540
Based on the RNA-seq results, differentially expressed sequences (DES) 541
associated with ethylene biosynthesis, starch degradation and cell wall metabolism 542
were selected. Genes induced by ethylene and repressed by 1-MCP relative to the 543
control were assigned as candidates, and the threshold of DES was set as 50-fold 544
based on FPKM values between control, ethylene and 1-MCP treatments on 1 d and 4 545
d, separately. The full-length coding sequences for these DES were obtained from the 546
kiwifruit genome database (Huang et al., 2013) and the sequences were verified by 547
PCR using gene specific primers (Supplemental Table S3), with cDNA from the 548
‘Hayward’ cultivar. 549
Promoters of postharvest ripening related genes (the structural genes) were 550
isolated according to the genome database, except for AdPG1 (not present in the 551
genome database). With the promoter sequences from the genome database, the 552
forward primers (FP) were located in promoter regions and reverse primers (RP) were 553
from coding sequences. For AdPG1, genome walking was carried out to obtain its 554
promoter, with the GenomeWalker kit (Clontech), using primary RP 555
(5’-TGCATGGCCCGCTAAACATAGTC-3’) and secondary RP 556
(5’-GCCGCAAGCTGAATCCCATGCG-3’). The analysis of cis-elements within 557
promoter regions was conducted using the online website http://jaspar.genereg.net. All 558
promoter sequences used in the following experiments are listed in Supplemental 559
Table S4. 560
561
Dual luciferase assays 562
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The dual luciferase assay was used to investigate the regulatory roles of different 563
transcription factors on target promoters, according to our previous protocols (Min et 564
al., 2012). Full length sequences of eleven transcription factors were inserted into 565
pGreen II 0029 62-SK vector (SK), while the promoters of eight softening-related 566
genes were recombined to the pGreen II 0800-LUC vector. Primers used for vector 567
constructions are listed in Supplemental Table S5. All the constructs were transferred 568
into Agrobacterium tumefaciens (GV3101), and the cultures were adjusted to an 569
OD600 of 0.75 with infiltration buffer (150 mM acetosyringone, 10 mM MES, 10mM 570
MgCl2, pH 5.6). The ratio of A. tumefaciens mixtures of transcription factors and 571
promoters was 10:1, which were then infiltrated into the leaves of Nicotiana 572
benthamiana. The N. benthamiana plants were cultivated in a glasshouse for 3 d. 573
Firefly luciferase and Renilla luciferase were assayed with the Dual-Luciferase 574
Reporter Assay System (Promega). For each TF-promoter interaction, triplicate 575
transient assay measurements were performed. Those with significant regulatory 576
effects were confirmed by at least three independent experiments. 577
578
Recombinant protein and EMSA analysis 579
According to the results of the dual luciferase assay, the regulatory action of 580
AdDof3 on the AdBAM3L promoter was further analyzed by an electrophoretic 581
mobility shift assay (EMSA). 582
In order to obtain the recombinant protein, the full-length AdDof3 was amplified 583
with FP (5’-GCTGATATCGGATCCGAATTCATGCCTCCGGAAACTTCCG-3’) and 584
RP (5’-GCAAGCTTGTCGACGGAGCTCGACTTGAGACCTTTGCCTG-3’) and 585
inserted into the pET-32a (Novagen) vector with double digestions of EcoRI and SacI. 586
The construct was purified and transformed into Escherichia coli strain BL21 (DE3). 587
The recombinant protein was induced by 0.5 mM isopropyl 588
β-D-1-thiogalactopyranoside (IPTG) at 20 oC for 18 h and purified as follows. The E. 589
coli cells were lysed with the buffer (20 mM Tris-HCl pH=8.0, 0.5 M NaCl, 10 mM 590
β-mercaptoethanol and 10% glycerol) and then subjected to sonication on ice at 200W 591
with 3s/2s on/off cycle for 30 minutes and centrifuged at 9000g for 20 minutes at 4 oC. 592
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Following this, the Ni-NTA resin (TRAN) was added to the supernatant to combine 593
His-tagged proteins and the His-tagged proteins were eluted with gradient imidazole 594
containing buffers (75, 100, 125, 150, 175, 250 and 500 mM). The portions eluted by 595
500 mM imidazole were further used in the following EMSA experiment 596
(Supplemental Fig. S8). 597
The probes were 3’biotin end-labeled by HuaGene (Shanghai, China) and 598
converted to double-stranded DNA probes by annealing complementary 599
oligonucleotides. EMSA was performed using the LightShift Chemiluminescent 600
EMSA kit (Thermo Fisher Scientific, 20148). The binding specificity was examined 601
by mutant probes and competition probe (1000 folds unlabeled oligonucleotides). 602
603
Stable transformation and analysis in kiwifruit 604
To obtain transgenic kiwifruit, the 1641-bp AdBAM3L coding sequence was 605
amplified with primers (FP, 5’- 606
AGAGAACACGCCCGGGGATCCATGGCTTTAACATTACATTG -3’; RP, 5’- 607
CTTGCATGCCTGCAGGTCGACTTACACAAAAGCAGCCTCCT -3’) and inserted 608
downstream of the CaMV 35S promoter into the modified pCAMBIA1301 vector via 609
the BamHI/SalI restriction sites. The pCAMBIA1301 vector also contained a GUS 610
reporter gene following one CaMV 35S promoter (Fig. 7b). The construct was then 611
introduced into A. tumefaciens strain EHA105. 612
Leaves of plantlet ‘Qinmei’ (Actinidia deliciosa) in tissue culture were used for 613
transformation. Firstly, the leaves were cut into pieces (1 cm * 1 cm) and cultured in 614
co-culture medium for 24 h in dark. They were then co-incubated for 10 min with A. 615
tumefaciens cultures containing 1 mL/L acetosyringone solution (AS, 100 mM), and 616
put back into co-culture medium with sterilized filter paper for 48 h in the dark. Then 617
the explants were transferred and screened on shoot induction medium under long day 618
conditions (16 h light/8 h dark) until the regenerated shoots reached ~2 cm and then 619
were transferred to rooting medium. All the processes were performed at 25 oC and 620
the representative status of transformed plant at different stages are shown in 621
Supplemental Fig. S3. The co-culture medium components were MS+1 mL/L Nitsch 622
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& Nitsch vitamin solution (NV) +4 mg/L 6-benzylaminopurine (6-BA) +1 mg/L 623
Naphthaleneacetic acid solution (NAA); the shoot induction medium was MS+1 mL/L 624
NV+4 mg/L 6-BA+1 mg/L NAA+5 mg/L hygromycin+50 mg/L meropenem; the 625
rooting medium contained MS+1 mL/L NV+5 mg/L hygromycin+50 mg/L 626
meropenem. 627
Transgenic plants overexpressing AdBAM3L were identified by DNA detection, 628
RT-qPCR, GUS staining and starch content analysis. Verification of DNA level used 629
genomic DNA from the transgene plants as template with primers across the 35S 630
promoter region (-424 bp) and CDS region (Supplemental Table S6). The methods of 631
RT-qPCR and starch content have been described above, with reduced amounts of 632
materials (0.25 g for RNA and 0.05 for starch). Three biological replicates were 633
carried out for each line. 634
635
GUS (β-glucuronidase) Staining 636
Histochemical staining was conducted to confirm the expression of the GUS 637
reporter co-transformed with AdBAM3L. The staining buffer was 0.1 M sodium 638
phosphate buffer (pH 7.0), 10 mM EDTA, 1 mM ferricyanide, 1 mM ferrocyanide, 0.5% 639
Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucur-onide (X-Gluc). The 640
leaves of transgenic kiwifruit plantlets were immersed in the staining buffer under 641
vacuum for 30 mins and then incubated overnight at 37 oC. 75% ethanol was used for 642
degradation of the chlorophyll of stained leaves. 643
644
Subcellular localization of AdDof3 645
The AdDof3 full-length CDS without the stop codon was amplified using 646
specific primers (FP: 647
5’-GGACGAGCTCGGTACCATGCCTCCGGAAACTTCCG-3’; RP: 648
5’-TGCTCACCATGTCGACCTTGAGACCTTTGCCTGGAG-3’) and then was 649
fused to the pCAMBIA1300-sGFP vector (KpnI/SalI). The construct 650
(35S-AdDof3-GFP) was transformed into A. tumefaciens strain (GV3101) and then 651
transiently expressed in transgenic N. benthamiana (expressed with nucleus located 652
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mCherry) leaves. The green fluorescent protein (GFP) and red fluorescent protein 653
(RFP) fluorescence was imaged by fluorescence microscope (Zeiss). 654
655
Transient overexpression in ‘Hayward’ fruit 656
The in vivo regulation of AdDof3 on AdBAM3L was investigated by transient 657
overexpression in ‘Hayward’ fruit with core tissue. In order to eliminate the variation 658
across different fruit, the controls (empty SK vector) and AdDof3 (same as dual 659
luciferase assays) were separately infiltrated into two different ends of core tissue 660
within intact fruit (Fig. 8a). The infiltrated materials analyzed by GUS staining were 661
injected EHA105 strain as control and EHA105 strain with the pCAMBIA1301 vector 662
with AdDof3 and the GUS reporter gene (Fig. 8b). The primers for AdDof3 for GUS 663
staining were forward primer 664
5’-AGAGAACACGCCCGGGGATCCATGCCTCCGGAAACTTCCG-3’ and reverse 665
primer 5’-ACGACGGCCAGTGCCAAGCTTCTACTTGAGACCTTTGCCTG-3’. 666
The fruit at 80 and 170 days after full bloom (DAFB) were harvested from 667
Shaanxi in 2017. The buffers together with A. tumefaciens carrying constructs were 668
the same as for the dual luciferase assay. Either 0.2 ml of AdDof3 or empty vector 669
were infiltrated into the core from the two ends, and then fruits were stored in an 670
incubator at 25 oC for 2 d. The material was collected at 1 d and 2 d, with three 671
biological replicates. 672
673
Statistical analysis 674
Least significant difference (LSD) analysis and Student’s t test were conducted 675
by DPS7.05 (Zhejiang University, Hangzhou, China). Figures were drawn with Origin 676
8.0 (Microcal Software Inc., Northampton, MA, USA). The heatmaps were conducted 677
with the log10FPKM values using online software 678
(https://console.biocloud.net/static/index.html#/drawtools/intoDrawTool). 679
680
Accession numbers 681
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All sequence reads are available at GenBank SRR6885590-SRR6885601. 682
683
Acknowledgments 684
The authors would like to thank Prof. Zhenghe Li (Zhejiang University) for providing 685
the transgenic N. benthamiana that expressed with nucleus located mCherry. 686
687
Supplemental Data 688
Supplemental Figure S1. RNA-seq analysis of differentially-expressed genes 689
between control, ethylene-treated, and 1-MCP-treated kiwifruit. 690
Supplemental Figure S2. AdAFC1 gene expression. 691
Supplemental Figure S3. Transgenic plants overexpressing AdBAM3L regenerated 692
and cultured on MS medium for up to 22 weeks. 693
Supplemental Figure S4. RT-qPCR analyses of AdDof3 and AdBAM3L expression at 694
apical and basal ends of kiwifruit core tissue. 695
Supplemental Figure S5. Starch contents in transient overexpressed core tissue with 696
AdDof3 or empty vector (SK). 697
Supplemental Figure S6. Sugar content in transgenic AdBAM3L kiwifruit. 698
Supplemental Figure S7. Expression of AdDof3 and AdBAM3L during the 699
development of ‘Hayward’ kiwifruit. 700
Supplemental Figure S8. AdDof3 protein purification. 701
Supplemental Table S1. Ethylene-responsive structural genes and transcription 702
factors. 703
Supplemental Table S2. Primers for RT-qPCR. 704
Supplemental Table S3. Primers for full-length amplification. 705
Supplemental Table S4. Sequences (5’ to 3’) for promoter isolation. 706
Supplemental Table S5. Primers for vector construction in dual-luciferase assays. 707
Supplemental Table S6. PCR primers for DNA detection in transgenic kiwifruit 708
plants. 709
710
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Figure legends 711
Figure 1. Effects of ethylene and 1-MCP treatment on kiwifruit ripening and 712
softening. Fruits were treated with either 100 μL/L ethylene (ETH), 1 μL/L 1-MCP, or 713
air (control; CK) for 24 h at 20oC. (a) Ethylene production of ‘Hayward’ kiwifruit 714
during storage. Error bars represent ±SE from three replicates. (b) Total soluble solids 715
(TSS) and starch content in ‘Hayward’ fruit. For TSS and starch content, error bars 716
represent ±SE from ten and three replicates, respectively. (c) Firmness, cell wall 717
material (CWM) content, cellulose content, hemicellulose content and pectin content 718
(covalent binding pectin (CBP), water soluble pectin (WSP) and ionic soluble pectin 719
(ISP)) of fruit in storage. Error bars of firmness represent ±SE based on twelve 720
replicates. All others were from three replicates. FW, fresh weight. LSDs represent 721
least significant difference at p=0.05. 722
Figure 2. Comparison of differentially-expressed genes (DEGs) between control, 723
ethylene-treated and 1-MCP treated kiwifruit. Fruits were treated with either 100 μL/L 724
ethylene (ETH), 1 μL/L 1-MCP, or air (control; CK) for 24 h at 20oC, and 725
comparisons were made at 1 and 4 days. (a) DEGs of 13 structural genes with 726
putative function in kiwifruit ethylene biosynthesis, cell wall modification and starch 727
degradation. (b) DEGs of 14 transcriptional factors. There were three replicates at 728
each point. Transcript abundance is indicated by color. The names in black represent 729
new genes, which were not included in ‘Hongyang’ genome database, those in blue 730
and red are published structural and transcription factor genes, respectively. 731
Figure 3. Expression of structural genes in response to ethylene or 1-MCP treatment 732
during kiwifruit ripening. Fruits were treated with either 100 μL/L ethylene (ETH), 1 733
μL/L 1-MCP, or air (control; CK) for 24 h at 20oC. Gene expression was analyzed by 734
RT-qPCR. ACO, 1-aminocyclopropane-1-carboxylate oxidase; ACS, 735
1-aminocyclopropane-1-carboxylate synthase; BAM, β-amylase; MAN, 736
endo-β-mannanase; PG, polygalacturonase; PL, pectin lyase; PME, pectin methyl 737
esterase; XTH, xyloglucan endotransglucosylase/hydrolase. Error bars represent ±SE 738
based on three replications. LSDs represent least significant difference at p=0.05. 739
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Figure 4. Expression of transcription factors in response to ethylene or 1-MCP 740
treatment during kiwifruit ripening. Fruits were treated with either 100 μL/L ethylene 741
(ETH), 1 μL/L 1-MCP, or air (control; CK) for 24 h at 20oC. Gene expression was 742
analyzed by RT-qPCR. (a) Expression of putative activators. (b) Expression of 743
putative repressors. BEE, brassinosteroid enhanced expression; bHLH, basic 744
helix-loop-helix protein; bZIP: basic leucine zipper protein; CBF, cold binding factor; 745
Dof, Dof zinc finger protein; ERF, ethylene response factor; GT, Trihelix transcription 746
factor; HB, Homeobox-leucine zipper protein. Error bars represent ±SE based on 747
three replications. LSDs represent least significant difference at p=0.05. 748
Figure 5. Regulatory effects of TFs on promoters of ethylene biosynthesis, cell wall 749
modifying and starch degradation genes as determined by dual-luciferase assays. The 750
ratio of LUC/REN of the empty vector plus promoter was set as 1. SK represents the 751
empty pGreen II 0029 62-SK vector. Error bars indicate ±SE from three replicates 752
(**P<0.01 and *** P<0.001). 753
Figure 6. Subcellular localization of AdDof3 and electrophoretic mobility shift assays 754
(EMSA). (a) Subcellular localization of AdDof3-GFP in transgenic Nicotiana 755
benthamiana leaves (expressed with nucleus located mCherry). AdDof3 was inserted 756
into the pCAMBIA1300-sGFP vector. GFP fluorescence of AdDof3-GFP is indicated. 757
Bars=25 μm. (b) Oligonucleotides used for the EMSA with the Dof core sequences in 758
red. The mutated bases are indicated in green. (c) The core sequences (AAAG/CTTT) 759
of Dof protein binding sites in the AdBAM3L promoter. (d) EMSA of 3’-biotin-labeled 760
double-stranded DNA probes with the AdDof3 DNA binding domain proteins. 761
Recombinant AdDof3 was purified from E. coli cells and used for DNA binding 762
assays with P-abc, P-a, P-bc, P-c, P-ab, and mutated P-ΔaΔbc, P-Δabc, P-aΔbc 763
together with cold unlabeled competitor as the probes. Water was added in place of 764
AdDof3 protein as control. 765
Figure 7. Overexpression of AdBAM3L in kiwifruit plants. (a) Five-month-old plants 766
on MS medium. (b) Schematic map of the AdBAM3L-pCAMBIA1301 construct and 767
PCR analysis of wild type (WT) and two independently regenerated transgenic lines. 768
The positive control used a plasmid containing the AdBAM3L-pCAMBIA1301 769
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 27 -
construct as a template. (c) Expression of AdBAM3L in WT and transgenic lines. (d) 770
GUS staining of WT and AdBAM3L transgenic plants. (e) Starch content reflected by 771
quinoneimine dye. The color intensity represents starch concentration. Positive 772
control: D-glucose standard (Megazyme International Ireland Ltd., Wicklow, Ireland); 773
negative control: water. (f) Starch content in WT and transgenic plant leaves. Error 774
bars in (c) and (f) indicate ±SE from three replicates (*P<0.05, **P<0.01 and *** 775
P<0.001). 776
Figure 8. Transient overexpression of AdDof3 and its upregulation of AdBAM3L in 777
the core tissue of ‘Hayward’ fruit. (a) Schematic diagram for injection with 778
differential color inks. The arrows show injection sites. (b) GUS staining of kiwifruit 779
core tissue segments injected with AdDof3-pCAMBIA1301-EHA105 or EHA105 at 1 780
day after injection. The segments were photographed separately. Bars = 100 μm. (c) 781
Gene expression of endogenous AdDof3 and AdBAM3L in immature kiwifruit at 80 782
days after full bloom. Injection of Agrobacterium tumefaciens strain (GV3101) with 783
the empty SK vector was the control and the AdDof3 recombined SK vector was the 784
treatment. (d) Gene expression of endogenous AdDof3 and AdBAM3L in mature 785
kiwifruit harvested 170 days after full bloom. Error bars in (c) and (d) indicate ±SE 786
from three replicates (*P<0.05, **P<0.01). 787
788
Literature Cited 789
Akagi T, Ikegami A, Tsujimoto T, Kobayashi S, Sato A, Kono A, Yonemori K 790
(2009) DkMyb4 is a Myb transcription factor involved in proanthocyanidin 791
biosynthesis in persimmon fruit. Plant Physiol 151: 2028-2045 792
Atkinson RG, Johnston SL, Yauk YK, Sharma NN, Schröder R (2009) Analysis of 793
xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and 794
apple. Postharvest Biol Tec 51: 149-157 795
Atkinson RG, Gunaseelan K, Wang MY, Luo L, Wang TC, Norling CL, Johnston 796
SL, Maddumage R, Schröder R, Schaffer RJ (2011) Dissecting the role of 797
climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using 798
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 28 -
a1-aminocyclopropane-1-carboxylic acid oxidase knockdown line. J Exp Bot 62: 799
3821-3835 800
Blumenkrantz N, Asboehansen G (1973) New method for quantitative 801
determination of uronic acids. Anal Biochem 54: 484-489 802
Chapman NH, Bonnet J, Grivet L, Lynn J, Graham N, Smith R, Sun G P, Walley 803
PG, Poole M, Causse M, King GJ, Baxter C, Seymour GB (2012) 804
High-resolution mapping of a fruit firmness-related quantitative trait locus in 805
tomato reveals epistatic interactions associated with a complex combinatorial 806
locus. Plant Physiol 159: 1644-1657 807
Cutillasiturralde A, Zarra I, Fry SC, Lorences EP (1994) Implication of 808
persimmon fruit hemicellulose metabolism in the softening process-importance of 809
xyloglucan endotransglycosylase. Physiol Plantarum 91: 169-176 810
Dong TT, Hu ZL, Deng L, Wang Y, Zhu MK, Zhang JL, Chen GP (2013) A 811
Tomato MADS-Box transcription factor, SlMADS1, acts as a negative regulator 812
of fruit ripening. Plant Physiol 163: 1026-1036 813
Feng BH, Han YC, Xiao YY, Kuang JF, Fan ZQ, Chen JY, Lu WJ (2016) The 814
banana fruit Dof transcription factor MaDof23 acts as a repressor and interacts 815
with MaERF9 in regulating ripening-related genes. J Exp Bot 67: 2263-2275 816
Fu CC, Han YC, Qi XY, Qi XY, Shan W, Chen JY, Lu WJ, Kuang JF (2016) 817
Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes 818
CpPME1/2, and CpPG5, involved in fruit ripening. Plant Cell Rep 35: 2341-2352 819
Hu X, Kuang S, Zhang AD, Zhang WS, Chen MJ, Yin XR, Chen KS (2016) 820
Characterization of starch degradation related genes in postharvest kiwifruit. Int J 821
Mol Sci 17: 2112 822
Huang H, Ferguson AR (2001) Kiwifruit in China. New Zeal J Crop Hort 29: 1-14. 823
Huang SX, Ding J, Deng DJ, Tang W, Sun HH, Liu DY, Zhang L, Niu XL, Meng 824
M, Yu JD, Liu J, Han Y, Shi W, Zhang DF, Cao SQ, Wei ZJ, Cui YL, Xia YH, 825
Zeng HP, Bao K, Lin L, Min Y, Zhang H, Miao M, Tang XF, Zhu YY, Sui Y, 826
Li GW, Sun HJ, Yue JY, Sun JQ, Liu FF, Zhou LQ, Lei L, Zheng XQ, Liu M, 827
Huang L, Song J, Xu CH, Li JW, Ye KY, Zhong SL, Lu BR, He GH, Xiao FM, 828
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 29 -
Wang HL, Zheng HK, Fei ZJ, Liu YS (2013) Draft genome of the kiwifruit 829
Actinida chinensis. Nat Commun 4: 2640 830
Ireland HS, Yao JL, Tomes S, Sutherland PW, Nieuwenhuizen N, Gunaseelan K, 831
Winz RA, David KM, Schaffer RJ (2013) Apple SEPALLATA1/2-like genes 832
control fruit flesh development and ripening. Plant J 73: 1044-1056 833
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, 834
Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking 835
genomes to life and the environment. Nucleic Acids Res 36: 480-484 836
Karlova R, Rosin FM, Busscher-Lange J, Parapunova V, Do PT, Fernie AR, 837
Fraser PD, Baxter C, Angenent GC, De Maagd RA (2011) Transcriptome and 838
metabolite profiling show that APETALA2a is a major regulator of tomato fruit 839
ripening. Plant Cell 23: 923-941 840
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: 841
accurate alignment of transcriptomes in the presence of insertion, deletions and 842
gene fusions. Genome Biol 14: R36 843
Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and 844
quality attributes. Annu Rev Genet 45: 41-59 845
Koukounaras A, Sfakiotakis E (2007) Effect of 1-MCP prestorage treatment on 846
ethylene and CO2 production and quality of ‘Hayward’ kiwifruit during shelf-life 847
after short, medium and long term cold storage. Postharvest Biol Tec 46: 174-180 848
Kuang JF, Chen JY, Liu XC, Han YC, Xiao YY, Shan W, Yang T, Wu KQ, He JX, 849
Lu WJ (2017) The transcriptional regulatory network mediated by banana (Musa 850
acuminata) dehydration-responsive element binding (MaDREB) transcription 851
factors in fruit ripening. New Phytol 214: 762-781 852
Lee JM, Joung JG, McQuinn R, Chung MY, Fei ZJ, Tieman D, Klee H, 853
Giovannoni J (2012) Combined transcriptome, genetic diversity and metabolite 854
profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an 855
important role in ripening and carotenoid accumulation. Plant J 70: 191-204 856
Li T, Jiang ZY, Zhang LC, Tan DM, Wei Y, Yuan H, Li TL, Wang AD (2016) 857
Apple (Malus Domestica) MdERF2 negatively affects ethylene biosynthesis 858
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 30 -
during fruit ripening by suppressing MdACS1 transcription. Plant J 88: 735-748 859
Li T, Xu YX, Zhang LC, Ji YL, Tan DM, Yuan H, Wang AD (2017) The 860
jasmonate-activated transcription factor MdMYC2 regulates ETHYLENE 861
RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene 862
biosynthesis during apple fruit ripening. Plant Cell 29: 1316-1334 863
Lin ZF, Hong YG, Yin MG, Li CY, Zhang K, Grierson D (2008) A tomato HD-Zip 864
homeobox protein, LeHB-1, plays an important role in floral organogenesis and 865
ripening. Plant J 55: 301-310 866
Liu MC, Diretto G, Pirrello J, Roustan JP, Li ZG, Giuliano G, Regad F, 867
Bouzayen M (2014) The chimeric repressor version of an Ethylene Response 868
Factor (ERF) family member, Sl-ERF. B3, shows contrasting effects on tomato 869
fruit ripening. New Phytol 203: 206-218 870
Lv QD, Zhong YJ, Wang YG, Wang ZY, Zhang L, Shi J, Wu ZC, Liu Y, Mao CZ, 871
Yi KK, Wu P (2014) SPX4 Negatively Regulates Phosphate Signaling and 872
Homeostasis through Its Interaction with PHR2 in Rice. Plant Cell 26: 1586-1597 873
Ma NN, Feng HL, Meng X, Li D, Yang DY, Wu CG, Meng QW (2014) 874
Overexpression of tomato SlNAC1 transcription factor alters fruit pigmentation 875
and softening. BMC Plant Biol 14: 351 876
Manning K, Tör M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, 877
Seymour GB (2006) A naturally occurring epigenetic mutation in a gene encoding 878
an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38: 879
948-952 880
Mao XZ, Cai T, Olyarchuk JG, Wei LP (2005) Automated genome annotation and 881
pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. 882
Bioinformatics 21: 3787-3793 883
Marzabal P, Gas E, Fontanet P, Vicente-Carbajosa J, Torrent M, Ludevid MD 884
(2008) The maize Dof protein PBF activates transcription of γ-zein during 885
maize seed development. Plant Mol Biol 67: 441-454 886
Mcdonald B, Harman JE (1982) Controlled-atmosphere storage of kiwifruit. 1. 887
Effect on fruit firmness and storage life. Sci Hortic 17: 113-123 888
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 31 -
Meng C, Yang DY, Ma XC, Zhao WY, Liang XQ, Ma NN, Meng QW (2016) 889
Suppression of tomato SlNAC1 transcription factor delays fruit ripening. J Plant 890
Physiol 193: 88-96 891
Min T, Yin XR, Shi YN, Luo ZR, Yao YC, Grierson D, Ferguson IB, Chen KS 892
(2012) Ethylene-responsive transcription factors interact with promoters of ADH 893
and PDC involved in persimmon (Diospyros kaki) fruit de-astringency. J Exp Bot 894
63: 6393-6405 895
Minas IS, Vicente AR, Dhanapal AP, Manganaris GA, Goulas V, Vasilakakis M, 896
Crisosto CH, Molassiotis A (2014) Ozone-induced kiwifruit ripening delay is 897
mediated by ethylene biosynthesis inhibition and cell wall dismantling regulation. 898
Plant Sci 229: 76-85 899
Mworia EG, Yoshikawa T, Salikon N, Oda C, Asiche WO, Yokotani N, Abe D, 900
Ushijima K, Nakano R, Kubo Y (2012) Low-temperature-modulated fruit 901
ripening is independent of ethylene in ‘Sanuki Gold’ kiwifruit. J Exp Bot 63: 902
963-971 903
Nardozza S, Boldingh HL, Osorio S, Höhne M, Wohlers M, Gleave AP, MacRae 904
EA, Richardson AC, Atkinson RG, Sulpice R, Fernie AR, Clearwater MJ 905
(2013) Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme 906
genotypes reveals hallmarks for fruit starch metabolism. J Exp Bot 64: 5049-5063 907
Nieuwenhuizen NJ, Chen X, Wang MY, Matich AJ, Perez RL, Allan AC, Green 908
SA, Atkinson RG (2015) Natural variation in monoterpene synthesis in kiwifruit: 909
transcriptional regulation of terpene synthases by NAC and 910
ETHYLENE-INSENSITIVE3-like transcription factors. Plant Physiol 167: 911
1243-1258 912
Peng T, Zhu XF, Duan N, Liu JH (2014) PtrBAM1, a β-amylase-coding gene of 913
Poncirus trifoliata, is a CBF regulon member with function in cold tolerance by 914
modulating soluble sugar levels. Plant Cell Environ 37: 2754-2767 915
Prasanna V, Prabha TN, Tharanathan RN (2007) Fruit ripening phenomena-an 916
overview. Crit Rev Food Sci 47: 1-19 917
Qi X, Li SX, Zhu YX, Zhao Q, Zhu DY, Yu JJ (2017) ZmDof3, a maize 918
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 32 -
endosperm-specific Dof protein gene, regulates starch accumulation and aleurone 919
development in maize endosperm. Plant Mol Biol 93: 7-20 920
Seymour GB, Ryder CD, Cevik V, Hammond JP, Popovich A, King GJ, Vrebalov 921
J, Giovannoni JJ, Manning K (2011) A SEPALLATA gene is involved in the 922
development and ripening of strawberry (Fragaria×ananassa Duch.) fruit, a 923
non-climacteric tissue. J Exp Bot 62: 1179-1188 924
Seymour GB, Chapman NH, Chew BL, Rose JKC (2013) Regulation of ripening 925
and opportunities for control in tomato and other fruits. Plant Biotechnol J 11: 926
269-278 927
Stevenson DG, Johnson SR, Jane J, Inglett GE (2010) Chemical and Physical 928
Properties of Kiwifruit (Actinidia deliciosa) Starch. Starch-Stärke 58: 323-329 929
Tacken E, Ireland H, Gunaseelan K, Karunairetnam S, Wang D, Schultz K, 930
Bowen J, Atkinson RG, Johnston JW, Putterill J, Hellens RP, Schaffer RJ 931
(2010) The role of ethylene and cold temperature in the regulation of the apple 932
POLYGALACTURONASE1 gene and fruit softening. Plant Physiol 153: 294-305 933
Tang W, Zheng Y, Dong J, Yu J, Yue JY, Liu FF, Guo XH, Huang SX, Wisniewski 934
M, Sun JQ, Niu XL, Ding J, Liu J, Fei ZJ, Liu YS (2016) Comprehensive 935
transcriptome profiling reveals long noncoding RNA expression and alternative 936
splicing regulation during fruit development and ripening in kiwifruit (Actinidia 937
chinensis). Front Plant Sci 7: 335 938
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, 939
Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification 940
by RNA-Seq reveals unannotated transcripts and isoform switching during cell 941
differentiation. Nature Biotechnology 28: 511-515 942
Uluisik S, Chapan NH, Smith R, Poole M, Adams G, Gillis RB, Besong TMD, 943
Sheldon J, Stiegelmeyer S, Perez L, Samsulrizal N, Wang D, Fisk ID, Yang N, 944
Baxter C, Rickett D, Fray R, Blanco-Ulate B, Powell ALT, Harding SE, 945
Craigon J, Rose JKC, Fich EA, Sun L, Domozych DS, Fraser PD, Tucker GA, 946
Grierson D, Seymour GB (2016) Genetic improvement of tomato by targeted 947
control of fruit softening. Nat Biotechnol 39: 950-952 948
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 33 -
Vicente AR, Manganaris GA, Minas IS,Goulas V, Lafuente M (2013) Cell wall 949
modifications and ethylene-induced tolerance to non-chilling peel pitting in citrus 950
fruit. Plant Sci 210: 46-52 951
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, 952
Schuch W, Giovannoni J (2002) A MADS-box gene necessary for fruit ripening at 953
the tomato ripening-inhibitor (Rin) locus. Science 296: 343-346 954
Wang ZY, MacRae EA, Wright MA, Bolitho KM, Ross GS, Atkinson RG (2000) 955
Polygalacturonase gene expression in kiwifruit: relationship to fruit softening and 956
ethylene production. Plant Mol Biol 42: 317-328 957
Wilkinson JQ, Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ (1995) An 958
ethylene-inducible component of signal transduction encoded by Never-ripe. 959
Science 270: 1807-1809 960
Xiao YY, Kuang JF, Qi XN, Ye YJ, Wu ZX, Chen JY, Lu WJ (2018) A 961
comprehensive investigation of starch degradation process and identification of a 962
transcriptional activator MabHLH6 during banana fruit ripening. Plant Biotechnol 963
J 16: 151-164 964
Xie XL, Yin XR, Chen KS (2016) Roles of APETALA2/Ethylene Responsive 965
Factors in regulation of fruit quality. Crit Rev Plant Sci 35: 120-130 966
Xu ZC, Hyodo H, Ikoma Y, Yano M, Ogawa K (1998) Biochemical characterization 967
and expression of recombinant ACC oxidase in Escherichia coli, and endogenous 968
ACC oxidase from kiwifruit. Postharvest Biol Tec 14: 41-50 969
Yanagisawa S, Schmidt RJ (1999) Diversity and similarity among recognition 970
sequence of Dof transcription factors. Plant J 17: 209-214 971
Yang L, Huang W, Xiong FJ, Xian ZQ, Su DD, Ren MZ, Li ZG (2017) Silencing 972
of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, 973
prolonged shelf-life, and reduced susceptibility to gray mold. Plant Biotechnol J 974
15: 1544-1555 975
Yemm EW, Willis AJ (1954) The estimation of carbohydrates in plant extracts by 976
anthrone. Biochem J 57: 508-514 977
Yin XR, Chen KS, Allan AC, Wu RM, Zhang B, Lallu N, Ferguson I (2008) 978
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
- 34 -
Ethylene-induced modulation of genes associated with the ethylene signaling 979
pathway in ripening kiwifruit. J Exp Bot 59: 2097-2108 980
Yin XR, Allan AC, Chen KS, Ferguson I (2010) Kiwifruit EIL and ERF genes 981
involved in regulating fruit ripening. Plant Physiol 153: 1280-1292 982
Yin XR, Xie XL, Xia XJ, Yu JQ, Ferguson IB, Giovannoni JJ, Chen KS (2016) 983
Involvement of an ethylene response factor in chlorophyll degradation during 984
citrus fruit degreening. Plant J 86: 403-412 985
Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis 986
for RNAseq: accounting for selection bias. Genome Biol 11: 14 987
Zhang AD, Hu X, Kuang S, Ge H, Yin XR, Chen KS (2016) Isolation, 988
classification and transcription profiles of the Ethylene Response Factors (ERFs) 989
in ripening kiwifruit. Sci Hortic 199: 209-215 990
Zhang B, Chen KS, Bowen J, Allan A, Espley R, Karunairetnam S, Ferguson I 991
(2006) Differential expression within the LOX gene family in ripening kiwifruit. J 992
Exp Bot 57: 3825-3836 993
Zhu MK, Chen GP, Zhou S, Tu Y, Wang Y, Dong TT, Hu ZL (2014) A new tomato 994
NAC (NAM/ATAF1/2/CUC2) transcription factor, SlNAC4, function as a positive 995
regulator of fruit ripening and carotenoid accumulation. Plant Cell Physiol 55: 996
119-135 997
998
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
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Parsed CitationsAkagi T, Ikegami A, Tsujimoto T, Kobayashi S, Sato A, Kono A, Yonemori K (2009) DkMyb4 is a Myb transcription factor involved inproanthocyanidin biosynthesis in persimmon fruit. Plant Physiol 151: 2028-2045
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Atkinson RG, Johnston SL, Yauk YK, Sharma NN, Schröder R (2009) Analysis of xyloglucan endotransglucosylase/hydrolase (XTH)gene families in kiwifruit and apple. Postharvest Biol Tec 51: 149-157
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Atkinson RG, Gunaseelan K, Wang MY, Luo L, Wang TC, Norling CL, Johnston SL, Maddumage R, Schröder R, Schaffer RJ (2011)Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a1-aminocyclopropane-1-carboxylic acidoxidase knockdown line. J Exp Bot 62: 3821-3835
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Blumenkrantz N, Asboehansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54: 484-489Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Chapman NH, Bonnet J, Grivet L, Lynn J, Graham N, Smith R, Sun G P, Walley PG, Poole M, Causse M, King GJ, Baxter C, Seymour GB(2012) High-resolution mapping of a fruit firmness-related quantitative trait locus in tomato reveals epistatic interactions associatedwith a complex combinatorial locus. Plant Physiol 159: 1644-1657
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cutillasiturralde A, Zarra I, Fry SC, Lorences EP (1994) Implication of persimmon fruit hemicellulose metabolism in the softeningprocess-importance of xyloglucan endotransglycosylase. Physiol Plantarum 91: 169-176
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dong TT, Hu ZL, Deng L, Wang Y, Zhu MK, Zhang JL, Chen GP (2013) A Tomato MADS-Box transcription factor, SlMADS1, acts as anegative regulator of fruit ripening. Plant Physiol 163: 1026-1036
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Feng BH, Han YC, Xiao YY, Kuang JF, Fan ZQ, Chen JY, Lu WJ (2016) The banana fruit Dof transcription factor MaDof23 acts as arepressor and interacts with MaERF9 in regulating ripening-related genes. J Exp Bot 67: 2263-2275
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Fu CC, Han YC, Qi XY, Qi XY, Shan W, Chen JY, Lu WJ, Kuang JF (2016) Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2, and CpPG5, involved in fruit ripening. Plant Cell Rep 35: 2341-2352
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hu X, Kuang S, Zhang AD, Zhang WS, Chen MJ, Yin XR, Chen KS (2016) Characterization of starch degradation related genes inpostharvest kiwifruit. Int J Mol Sci 17: 2112
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huang H, Ferguson AR (2001) Kiwifruit in China. New Zeal J Crop Hort 29: 1-14.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huang SX, Ding J, Deng DJ, Tang W, Sun HH, Liu DY, Zhang L, Niu XL, Meng M, Yu JD, Liu J, Han Y, Shi W, Zhang DF, Cao SQ, Wei ZJ,Cui YL, Xia YH, Zeng HP, Bao K, Lin L, Min Y, Zhang H, Miao M, Tang XF, Zhu YY, Sui Y, Li GW, Sun HJ, Yue JY, Sun JQ, Liu FF, ZhouLQ, Lei L, Zheng XQ, Liu M, Huang L, Song J, Xu CH, Li JW, Ye KY, Zhong SL, Lu BR, He GH, Xiao FM, Wang HL, Zheng HK, Fei ZJ, LiuYS (2013) Draft genome of the kiwifruit Actinida chinensis. Nat Commun 4: 2640
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ireland HS, Yao JL, Tomes S, Sutherland PW, Nieuwenhuizen N, Gunaseelan K, Winz RA, David KM, Schaffer RJ (2013) AppleSEPALLATA1/2-like genes control fruit flesh development and ripening. Plant J 73: 1044-1056
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008)KEGG for linking genomes to life and the environment. Nucleic Acids Res 36: 480-484
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Karlova R, Rosin FM, Busscher-Lange J, Parapunova V, Do PT, Fernie AR, Fraser PD, Baxter C, Angenent GC, De Maagd RA (2011)Transcriptome and metabolite profiling show that APETALA2a is a major regulator of tomato fruit ripening. Plant Cell 23: 923-941
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presenceof insertion, deletions and gene fusions. Genome Biol 14: R36
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet 45: 41-59Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Koukounaras A, Sfakiotakis E (2007) Effect of 1-MCP prestorage treatment on ethylene and CO2 production and quality of 'Hayward'kiwifruit during shelf-life after short, medium and long term cold storage. Postharvest Biol Tec 46: 174-180
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kuang JF, Chen JY, Liu XC, Han YC, Xiao YY, Shan W, Yang T, Wu KQ, He JX, Lu WJ (2017) The transcriptional regulatory networkmediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening. NewPhytol 214: 762-781
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lee JM, Joung JG, McQuinn R, Chung MY, Fei ZJ, Tieman D, Klee H, Giovannoni J (2012) Combined transcriptome, genetic diversityand metabolite profiling in tomato fruit reveals that the ethylene response factor SlERF6 plays an important role in ripening andcarotenoid accumulation. Plant J 70: 191-204
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li T, Jiang ZY, Zhang LC, Tan DM, Wei Y, Yuan H, Li TL, Wang AD (2016) Apple (Malus Domestica) MdERF2 negatively affects ethylenebiosynthesis during fruit ripening by suppressing MdACS1 transcription. Plant J 88: 735-748
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li T, Xu YX, Zhang LC, Ji YL, Tan DM, Yuan H, Wang AD (2017) The jasmonate-activated transcription factor MdMYC2 regulatesETHYLENE RESPONSE FACTOR and ethylene biosynthetic genes to promote ethylene biosynthesis during apple fruit ripening. PlantCell 29: 1316-1334
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lin ZF, Hong YG, Yin MG, Li CY, Zhang K, Grierson D (2008) A tomato HD-Zip homeobox protein, LeHB-1, plays an important role infloral organogenesis and ripening. Plant J 55: 301-310
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liu MC, Diretto G, Pirrello J, Roustan JP, Li ZG, Giuliano G, Regad F, Bouzayen M (2014) The chimeric repressor version of anEthylene Response Factor (ERF) family member, Sl-ERF. B3, shows contrasting effects on tomato fruit ripening. New Phytol 203: 206-218
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Lv QD, Zhong YJ, Wang YG, Wang ZY, Zhang L, Shi J, Wu ZC, Liu Y, Mao CZ, Yi KK, Wu P (2014) SPX4 Negatively Regulates PhosphateSignaling and Homeostasis through Its Interaction with PHR2 in Rice. Plant Cell 26: 1586-1597
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Ma NN, Feng HL, Meng X, Li D, Yang DY, Wu CG, Meng QW (2014) Overexpression of tomato SlNAC1 transcription factor alters fruitpigmentation and softening. BMC Plant Biol 14: 351
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Manning K, Tör M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, Seymour GB (2006) A naturally occurring epigeneticmutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38: 948-952
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mao XZ, Cai T, Olyarchuk JG, Wei LP (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO)as a controlled vocabulary. Bioinformatics 21: 3787-3793
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Marzabal P, Gas E, Fontanet P, Vicente-Carbajosa J, Torrent M, Ludevid MD (2008) The maize Dof protein PBF activates transcription www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from
Copyright © 2018 American Society of Plant Biologists. All rights reserved.
of γ-zein during maize seed development. Plant Mol Biol 67: 441-454Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mcdonald B, Harman JE (1982) Controlled-atmosphere storage of kiwifruit. 1. Effect on fruit firmness and storage life. Sci Hortic 17:113-123
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Meng C, Yang DY, Ma XC, Zhao WY, Liang XQ, Ma NN, Meng QW (2016) Suppression of tomato SlNAC1 transcription factor delays fruitripening. J Plant Physiol 193: 88-96
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Min T, Yin XR, Shi YN, Luo ZR, Yao YC, Grierson D, Ferguson IB, Chen KS (2012) Ethylene-responsive transcription factors interactwith promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency. J Exp Bot 63: 6393-6405
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Minas IS, Vicente AR, Dhanapal AP, Manganaris GA, Goulas V, Vasilakakis M, Crisosto CH, Molassiotis A (2014) Ozone-induced kiwifruitripening delay is mediated by ethylene biosynthesis inhibition and cell wall dismantling regulation. Plant Sci 229: 76-85
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mworia EG, Yoshikawa T, Salikon N, Oda C, Asiche WO, Yokotani N, Abe D, Ushijima K, Nakano R, Kubo Y (2012) Low-temperature-modulated fruit ripening is independent of ethylene in 'Sanuki Gold' kiwifruit. J Exp Bot 63: 963-971
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nardozza S, Boldingh HL, Osorio S, Höhne M, Wohlers M, Gleave AP, MacRae EA, Richardson AC, Atkinson RG, Sulpice R, Fernie AR,Clearwater MJ (2013) Metabolic analysis of kiwifruit (Actinidia deliciosa) berries from extreme genotypes reveals hallmarks for fruitstarch metabolism. J Exp Bot 64: 5049-5063
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Nieuwenhuizen NJ, Chen X, Wang MY, Matich AJ, Perez RL, Allan AC, Green SA, Atkinson RG (2015) Natural variation in monoterpenesynthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.Plant Physiol 167: 1243-1258
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Peng T, Zhu XF, Duan N, Liu JH (2014) PtrBAM1, a β-amylase-coding gene of Poncirus trifoliata, is a CBF regulon member with functionin cold tolerance by modulating soluble sugar levels. Plant Cell Environ 37: 2754-2767
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Prasanna V, Prabha TN, Tharanathan RN (2007) Fruit ripening phenomena-an overview. Crit Rev Food Sci 47: 1-19Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Qi X, Li SX, Zhu YX, Zhao Q, Zhu DY, Yu JJ (2017) ZmDof3, a maize endosperm-specific Dof protein gene, regulates starchaccumulation and aleurone development in maize endosperm. Plant Mol Biol 93: 7-20
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Seymour GB, Ryder CD, Cevik V, Hammond JP, Popovich A, King GJ, Vrebalov J, Giovannoni JJ, Manning K (2011) A SEPALLATA geneis involved in the development and ripening of strawberry (Fragaria×ananassa Duch.) fruit, a non-climacteric tissue. J Exp Bot 62:1179-1188
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Seymour GB, Chapman NH, Chew BL, Rose JKC (2013) Regulation of ripening and opportunities for control in tomato and other fruits.Plant Biotechnol J 11: 269-278
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Stevenson DG, Johnson SR, Jane J, Inglett GE (2010) Chemical and Physical Properties of Kiwifruit (Actinidia deliciosa) Starch. Starch-Stärke 58: 323-329
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tacken E, Ireland H, Gunaseelan K, Karunairetnam S, Wang D, Schultz K, Bowen J, Atkinson RG, Johnston JW, Putterill J, Hellens RP,Schaffer RJ (2010) The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruitsoftening. Plant Physiol 153: 294-305
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Tang W, Zheng Y, Dong J, Yu J, Yue JY, Liu FF, Guo XH, Huang SX, Wisniewski M, Sun JQ, Niu XL, Ding J, Liu J, Fei ZJ, Liu YS (2016)Comprehensive transcriptome profiling reveals long noncoding RNA expression and alternative splicing regulation during fruitdevelopment and ripening in kiwifruit (Actinidia chinensis). Front Plant Sci 7: 335
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assemblyand quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology28: 511-515
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Uluisik S, Chapan NH, Smith R, Poole M, Adams G, Gillis RB, Besong TMD, Sheldon J, Stiegelmeyer S, Perez L, Samsulrizal N, Wang D,Fisk ID, Yang N, Baxter C, Rickett D, Fray R, Blanco-Ulate B, Powell ALT, Harding SE, Craigon J, Rose JKC, Fich EA, Sun L, DomozychDS, Fraser PD, Tucker GA, Grierson D, Seymour GB (2016) Genetic improvement of tomato by targeted control of fruit softening. NatBiotechnol 39: 950-952
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vicente AR, Manganaris GA, Minas IS,Goulas V, Lafuente M (2013) Cell wall modifications and ethylene-induced tolerance to non-chilling peel pitting in citrus fruit. Plant Sci 210: 46-52
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, Schuch W, Giovannoni J (2002) A MADS-box gene necessaryfor fruit ripening at the tomato ripening-inhibitor (Rin) locus. Science 296: 343-346
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wang ZY, MacRae EA, Wright MA, Bolitho KM, Ross GS, Atkinson RG (2000) Polygalacturonase gene expression in kiwifruit:relationship to fruit softening and ethylene production. Plant Mol Biol 42: 317-328
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wilkinson JQ, Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ (1995) An ethylene-inducible component of signal transduction encodedby Never-ripe. Science 270: 1807-1809
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xiao YY, Kuang JF, Qi XN, Ye YJ, Wu ZX, Chen JY, Lu WJ (2018) A comprehensive investigation of starch degradation process andidentification of a transcriptional activator MabHLH6 during banana fruit ripening. Plant Biotechnol J 16: 151-164
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xie XL, Yin XR, Chen KS (2016) Roles of APETALA2/Ethylene Responsive Factors in regulation of fruit quality. Crit Rev Plant Sci 35:120-130
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu ZC, Hyodo H, Ikoma Y, Yano M, Ogawa K (1998) Biochemical characterization and expression of recombinant ACC oxidase inEscherichia coli, and endogenous ACC oxidase from kiwifruit. Postharvest Biol Tec 14: 41-50
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yanagisawa S, Schmidt RJ (1999) Diversity and similarity among recognition sequence of Dof transcription factors. Plant J 17: 209-214Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yang L, Huang W, Xiong FJ, Xian ZQ, Su DD, Ren MZ, Li ZG (2017) Silencing of SlPL, which encodes a pectate lyase in tomato, confersenhanced fruit firmness, prolonged shelf-life, and reduced susceptibility to gray mold. Plant Biotechnol J 15: 1544-1555
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yemm EW, Willis AJ (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57: 508-514Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yin XR, Chen KS, Allan AC, Wu RM, Zhang B, Lallu N, Ferguson I (2008) Ethylene-induced modulation of genes associated with theethylene signaling pathway in ripening kiwifruit. J Exp Bot 59: 2097-2108
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.
Yin XR, Allan AC, Chen KS, Ferguson I (2010) Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiol 153: 1280-1292
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yin XR, Xie XL, Xia XJ, Yu JQ, Ferguson IB, Giovannoni JJ, Chen KS (2016) Involvement of an ethylene response factor in chlorophylldegradation during citrus fruit degreening. Plant J 86: 403-412
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNAseq: accounting for selection bias. Genome Biol11: 14
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang AD, Hu X, Kuang S, Ge H, Yin XR, Chen KS (2016) Isolation, classification and transcription profiles of the Ethylene ResponseFactors (ERFs) in ripening kiwifruit. Sci Hortic 199: 209-215
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhang B, Chen KS, Bowen J, Allan A, Espley R, Karunairetnam S, Ferguson I (2006) Differential expression within the LOX gene familyin ripening kiwifruit. J Exp Bot 57: 3825-3836
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Zhu MK, Chen GP, Zhou S, Tu Y, Wang Y, Dong TT, Hu ZL (2014) A new tomato NAC (NAM/ATAF1/2/CUC2) transcription factor, SlNAC4,function as a positive regulator of fruit ripening and carotenoid accumulation. Plant Cell Physiol 55: 119-135
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
www.plantphysiol.orgon January 30, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.