running head: mdareb2 regulates soluble sugar …111. supply (khan et al., 2016). 112. as mentioned...

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Running head MdAREB2 regulates soluble sugar accumulation via MdSUT2 4

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Correspondence 8

Yu-Jin Hao (haoyujinsdaueducn) 9

National Key Laboratory of Crop Biology 10

National Research Center for Apple Engineering and Technology 11

College of Horticulture Science and Engineering 12

Shandong Agricultural University Tai-An Shandong 271018 China 13

Tel +86-538-824-6692 14

Fax number +86-538-824-2364 15

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Plant Physiology Preview Published on June 9 2017 as DOI101104pp1700502

Copyright 2017 by the American Society of Plant Biologists

wwwplantphysiolorgon March 4 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

2

Transcription factor AREB2 is involved in soluble sugar accumulation by 24

activating sugar transporter and amylase genes 25

26

Qi-Jun Ma Mei-Hong Sun Jing Lu Ya-Jing Liu Da-Gang Hu Yu-Jin Hao 27

28

29

National Key Laboratory of Crop Biology National Research Center for Apple 30

Engineering and Technology College of Horticulture Science and Engineering 31


33

34

35

The corresponding author Yu-Jin Hao 36

Tel +86-538-824-6692 37

E-mail haoyujinsdaueducn 38

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One Sentence Summary the ABA-responsive transcription factor MdAREB2 directly activates 40

the expression of amylase and sugar transporter genes to promote soluble sugar accumulation 41

42

43

44

45

46

47


3

48

49

Abstract 50

Sugars play important roles in plant growth and development crop yield and 51

quality as well as responses to abiotic stresses ABA is a multi-functional hormone 52

However the exact mechanism by which ABA regulates sugar accumulation is largely 53

unknown in plants Here we tested the expression profile of several sugar transporter 54

and amylase genes in response to ABA treatment MdSUT2 and MdAREB2 were 55

isolated and genetically transformed into apples to investigate their roles in 56

ABA-induced sugar accumulation The MdAREB2 transcription factor was found to 57

bind to the promoters of the sugar transporter and amylase genes and activate their 58

expression Both MdAREB2 and MdSUT2 transgenic plants produced more soluble 59

sugars than controls Furthermore MdAREB2 promoted the accumulation of sucrose 60

and soluble sugars in an MdSUT2-dependent manner Our results demonstrate that the 61

ABA-responsive transcription factor MdAREB2 directly activates the expression of 62

amylase and sugar transporter genes to promote soluble sugar accumulation 63

suggesting a mechanism by which ABA regulates sugar accumulation in plants 64

65

Key words ABA MdAREB2 sugar transporters amylase genes soluble sugar 66

67

Introduction 68

Sugars are major products of carbon and energy during photosynthesis In plants 69

they act not only as an essential source of carbon but also as signaling molecules in 70

response to the integration of information from environmental signals as well as 71

developmental and metabolic cues (Lastdrager et al 2014) They are therefore very 72

important for growth and development In addition when sugars are transported into 73

and accumulate in the vacuole they become crucial players in the development of 74

fruit quality and stress tolerance 75

In higher plants high sugar levels are accumulated in sinks depending on the 76

expression of various genes associated with sugar biosynthesis metabolism and 77

transportation The genes SuSy and AINV encode sucrose synthase and vacuolar acid 78

invertase respectively They play an important role in the regulation of sugar 79

accumulation because they are involved in the regulation of sucrose decomposition 80

through which sucrose is generally broken down into glucose fructose or 81


4

UDP-glucose in fruit (Yamaki 2010) In addition sucrose transporters also play an 82

important physiological role in the regulation of fruit development by stimulating 83

sugar accumulation in fruit and thus improving fruit yield and quality (Lu et al 2000) 84

In grapevines the sucrose transporters VvSuc11 and VvSuc12 regulate soluble sugar 85

accumulation by controlling sucrose transport (Lecourieux et al 2013) The transcript 86

level of CitSUT1 increases with the development and ripening of citrus fruits Its 87

expression pattern is similar to those of VvSuc11 and VvSuc12 and its overexpression 88

increases sucrose accumulation in transgenic citrus lines (Zheng et al 2014 Islam et 89

al 2015) A sucrose uptake transporter (SUT) gene is highly expressed in pollen 90

which leads to the accumulation of sucrose in tobacco (Chung et al 2014) When the 91

SUT1 gene is symplasmically isolated during fruit development soluble sugars 92

accumulate at a high level through an apoplasmic phloem unloading pathway in apple 93

fruit (Zhang et al 2004) In addition ectopic expression of MdSUT2 gene confers 94

enhanced stress tolerance early flowering time and increased plant size in transgenic 95

Arabidopsis (Ma et al 2016) 96

Notably sugar accumulates at a relatively high level in vacuoles and produces 97

high turgor pressure Therefore sugar is also involved in the response to abiotic 98

stresses by affecting osmotic potentials (Gibson 2005) Soluble sugars (SS) respond 99

to environmental stresses including water stress and salinity (Moustakas et al 2011 100

Rasheed et al 2011) In Arabidopsis soluble sugars anthocyanins and proline all 101

increase together under drought stress (Sperdouli and Moustakas 2012) Previous 102

studies have indicated that the sucrose and fructan levels increased together in 103

salt-stressed wheat (Kafi et al 2003) Transgenic cotton plants with a vacuolar 104

H+-PPase gene are more resistant to NaCl than non-transgenic plants which may be 105

attributed to their enhanced sugar levels upon exposure to salt stress (Lv et al 2008 106

Yao et al 2010) The levels of compatible solutes in particular of glucose fructose 107

mannitol and several amino acids were increased in the halophyte Thellungiella upon 108

exposure to high salt levels Abiotic stresses such as salt cold and drought severely 109

impact crop performance and productivity at least in part because they affect the sugar 110

supply (Khan et al 2016) 111

As mentioned above various environmental stimuli such as drought stress 112

promote sugar accumulation which is very important for stress tolerance and fruit 113

quality by inducing the expression of genes that are associated with sugar 114

biosynthesis and transport (Ferrandino and Lovisolo 2014 Medici et al 2014) 115


5

However the exact mechanism through which environment signals induce the 116

expression of those genes is not particularly clear Abscisic acid (ABA) is well-known 117

as a key hormone that acts in response to various abiotic stresses It regulates the 118

accumulation of soluble sugars by affecting the transcript levels of genes that are 119

associated with sugar synthesis and transport in plants (Gibson 2004) The expression 120

of TREHALOSE 6-PHOSPHATE SYNTHASE (TPS1) which plays a key role in 121

modulating trehalose 6-phosphate levels in the vegetative tissues of Arabidopsis is 122

regulated by ABA It is involved in regulating the accumulation of soluble sugars 123

(Goacutemez et al 2010) ABA induces the expression of the cellulose synthase gene 124

AtCesA8IRX1 which plays a role in sugar metabolism AtCesA8IRX1 mutant plants 125

accumulate more abscisic acid (ABA) proline and soluble sugars than the wild type 126

(Chen et al 2005) The transcripts of an ADP-glucose pyrophosphorylase gene called 127

OsAPL3 accumulate significantly in response to increased levels of sucrose and 128

abscisic acid (ABA) in the medium (Akihiro et al 2005) In addition a high 129

concentration of ABA reduces sucrose transport into the grains and lowers the ability 130

of the grains to synthesize starch (Bhatia and Singh 2002) However an appropriate 131

concentration of ABA enhances sucrose synthase (SUS) activity and increases the 132

expression of genes related to sugar metabolism (Akihiro et al 2005 Tang et al 133

2009) 134

Some evidence shows that plant monosaccharide transport proteins such as the 135

hexose transporter (HT) are also regulated by ABA VvHT1 is transcriptionally 136

regulated by VvMSA the expression of which is induced by ABA but only in the 137

presence of sucrose (Rook et al 2006) VvHT5 expression is regulated by ABA 138

during the hexose transition from source to sink in response to infection by biotrophic 139

pathogens (Hayes et al 2010) ABA also plays a vital role in regulating the key 140

enzymes that are involved in fructan and Suc metabolism in wheat (Yang et al 2004) 141

Although genetic analyses have revealed that sugar transporters may be involved in 142

interactions between the sugar signaling pathways and the ABA plant hormone 143

(Gibson 2004 Rolland et al 2006) the exact mechanism by which ABA regulates 144

sugar transport and accumulation is largely unknown 145

In this study the sugar transporters MdTMT1 and MdSUT2 were found to be 146

noticeably induced at the transcriptional level by ABA hormone and they were 147

involved in the accumulation of soluble sugars The transcription factor MdAREB2 148

was directly bound to the ABRE recognition site in the promoter regions of several 149


6

genes encoding sugar transporters and amylase and it positively regulated their 150

expression This discovery shed light on the molecular mechanism by which ABA 151

regulates sugar accumulation in apples 152

Results 153

Sugar transporter genes MdTMT1 and MdSUT2 are crucial for the ABA-induced 154

accumulation of soluble sugars 155

To determine whether ABA affects the soluble sugars content the in vitro shoot 156

cultures of lsquoGalarsquo apples at the same size were treated with 100 μM ABA The starch 157

staining demonstrated that ABA treatment markedly reduced the starch contents of the 158

shoot cultures (Fig 1A-B) Simultaneously the soluble sugar sucrose and glucose 159

contents noticeably increased with ABA treatment However the ABA treatment did 160

not significantly influence the fructose content (Fig 1C-D) 161

Generally tonoplast monosaccharide transporter (TMT) genes are responsible for 162

monosaccharide (such as glucose) transport while sucrose transporters (SUTs) are 163

responsible for sucrose transport (Barker et al 2000 Slewinski 2011) To find out 164

the MdTMT and MdSUT genes in the apple genome a BLAST search against the 165

apple genome database (The Apple Gene Function amp Gene Family DataBase v10) 166

was performed using the Arabidopsis AtTMT1 and AtSUT2 as query respectively As 167

a result there are a total of 5 MdTMT and 4 MdSUT genes ie MdTMT1 168

(MDP0000381084) MdTMT2 (MDP0000212510) MdTMT3 (MDP0000250194) 169

MdTMT4 (MDP0000868028) MdTMT5 (MDP0000250193) MdSUT1 170

(MDP0000426862) MdSUT2 (MDP0000277235) MdSUT3 (MDP0000584979) and 171

MdSUT4 (MDP0000275743) (Fig S1A-B) Their expression in response to ABA 172

treatment was examined by qRT-PCR The result showed that only the transcript 173

levels of the MdTMT1 and MdSUT2 genes were noticeably induced (Fig 1E) In 174

addition the amylase MdAMY and MdBAM genes were also examined BLAST 175

search (httpswwwrosaceaeorgtoolsncbi) was performed using Arabidopsis 176

AtAMY1 and AtBAM1 as query respectively The results showed that the expression 177

of MdAMY1 MdAMY3 MdBAM1 and MdBAM3 were markedly increased with ABA 178

treatment (Fig 1E) In addition it was found that those genes constitutively expressed 179

in different organs such as root stem leaf flower and fruit (Fig S2A-B) 180

To elucidate the functions of sugar transporter genes in apples the sucrose 181

transporter MdSUT2 was selected and cloned by polymerase chain reaction (PCR) 182

According to the phylogenetic tree MdSUT2 (MDP0000277235) was located 183


7

together with Arabidopsis sucrose transporter AtSUT2 supported by a bootstrap value 184

of 100 The MdSUT2 gene was located on apple chromosomes 3 A gene structure 185

analysis showed that there were 14 exons and 13 introns in the MdSUT2 ORF (Fig 186

S3A) Hydrophobicity plots showed that MdSUT2 has an N-terminal domain that is 187

approximately 30 amino acids longer than the apple sucrose transporters MdSUT1 3 188

and 4 AtSUT2 and MdSUT2 were overlaid and the extended central cytoplasmic 189

loop is approximately 50 amino acids longer at amino acid residue position 319 (Fig 190

S3B) They had a very high similarity in terms of the predicted 3D protein structure 191

(Fig S3C) In addition the position of the MdTMT1 gene was found to be at apple 192

chromosome 6 which has 5 exons and 4 introns in the MdTMT1 ORF (Fig S3D) 193

To examine if MdTMT1 and MdSUT2 genes are involved in regulating 194

ABA-induced soluble sugar accumulation a viral vector-based method was used to 195

alter their expression The TRV vector was used to suppress gene expression The two 196

viral constructs MdTMT1-TRV and MdSUT2-TRV were obtained They were 197

separately or simultaneously transformed into in vitro shoot cultures of lsquoGalarsquo apples 198

while the empty TRV vector was used as the control An expression analysis 199

demonstrated that the empty TRV infection did not influence the expression of 200

MdTMT1 and MdSUT2 genes (Fig S4A-C) The transcript levels of MdTMT1 and 201

MdSUT2 were markedly reduced in MdTMT1-TRV MdSUT2-TRV and 202

MdTMT1-TRV + MdSUT2-TRV-infected shoot cultures (Fig 1F) suggesting that the 203

TRV viral vector-based method worked well in this study 204

The MdTMT1-TRV MdSUT2-TRV or MdTMT1-TRV + MdSUT2-TRV-infected 205

shoot cultures were subsequently used to check if MdTMT1 and MdSUT2 genes are 206

involved in ABA-induced glucose and sucrose accumulation with 100 microM ABA 207

treatments respectively The result indicated that the MdTMT1-TRV-mediated 208

suppression of the MdTMT1 gene successfully counteracted the ABA-induced 209

increase in the glucose content while the MdSUT2-TRV-mediated suppression of the 210

MdSUT2 gene did the same for the sucrose content (Fig 1H) As a result both 211

MdTMT1-TRV and MdSUT2-TRV-infected shoot cultures produced lower amounts 212

of soluble sugars than the empty TRV control (Fig 1G) In addition simultaneous 213

suppression of two genes ie MdTMT1-TRV+MdSUT2-TRV double infection 214

resulted in decreased amounts of glucose sucrose and soluble sugar (Fig 1G and 1H) 215

Therefore ABA-induced glucose and sucrose accumulation required the functions of 216

MdTMT1 and MdSUT2 respectively 217


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MdSUT2 functions as a sucrose transporter 218

The MdSUT2 gene was subsequently chosen for further investigation To 219

characterize the function of MdSUT2 in planta an expression construct called 220

35SMdSUT2 was constructed and genetically transformed into the lsquoGalarsquo apple As 221

a result nine independent transgenic lines of MdSUT2 were obtained Expression 222

analysis demonstrated that the transgenic lines MdSUT2-1 MdSUT2-2 and 223

MdSUT2-5 produced much greater amounts of MdSUT2 transcripts than the WT 224

control (Fig 2A-B Fig S5) And it seems that they grew faster than the WT control 225

(Fig 2B) And the three transgenic lines accumulated more MdSUT2 proteins and 226

exhibited higher sucrose transport activity than the WT control (Fig 2C-D) They 227

accumulated more total soluble sugar and sucrose than the WT control (Fig 2E-F) 228

As a result it seems that they grew faster than the WT control (Fig 2B) 229

ABRE cis-element in the promoter region of the MdSUT2 gene is required for its 230

ABA-induced expression 231

In our previous study the expression of MdSUT2 was found to be induced by 232

ABA (Ma et al 2016) To explain why its expression is induced by ABA the 233

promoter region of the MdSUT2 gene was analyzed The MdSUT2 promoter was 234

found to contain various cis-elements (Table S1) Among them there is an 235

ABA-responsive element (ABRE) which has a CACGTC core sequence (Fig 3A) To 236

examine if the transcription activity of the MdSUT2 promoter is induced by ABA a 237

GUS reporter gene was fused downstream from the promoter The resulting 238

pMdSUT2GUS construct was then genetically transformed into the apple calli GUS 239

staining demonstrated that ABA treatment noticeably increased the GUS activity 240

indicating that the promoter is ABA-responsive (Fig 3B-C) 241

To examine if the ABRE core GCACGTCC sequence is crucial for the ABA 242

response a mutant MdSUT2 promoter that contains CTGGAT but not CACGTC was 243

artificially constructed and used for the GUS analysis In this case the 244

pMdSUT2GUS(m) construct was transformed into the apple calli When the 245

pMdSUT2GUS(m) transgenic calli were treated with ABA it was found that ABA 246

treatment did not influence the GUS activity (Fig 3B-C) At the same time 247

pMdSUT2GUS and pMdSUT2(m)GUS were genetically transformed into 248

Arabidopsis The transgenic Arabidopsis showed the same GUS phenotype as the 249

transgenic apple calli (Fig 3D) In other words the mutation in the core sequence 250

destroyed the responsiveness of the transgenic calli to the ABA treatment These 251


9

results indicated that the ABRE cis-element plays a crucial role in the ABA response 252

ABA-responsive transcription factor MdAREB2 binds to the promoters of the 253

sugar transporter and amylase genes in response to ABA in apples 254

To identify the potential transcription factors that recognize and bind to the 255

ABRE cis-element the oligonucleotide probe 256

TCCATACAGCACGTCCTAGTTTGATTTTTAGCACTCGTGAATTA with the 257

ABRE core sequence underlined was designed on the basis of the MdSUT2 promoter 258

The resulting probe was used for DNA-affinity trapping and EMSA assays When the 259

nuclear protein extracts were incubated with biotin-labeled MdSUT2 promoter 260

oligonucleotides a DNA-protein complex with a slower mobility in EMSA was 261

observed (Fig 4A) Subsequently a mass spectrometry analysis was performed to 262

identify the proteins that bind to the oligonucleotide The result showed that the 263

protein encoded by the MDP0000248567 gene was a candidate from among the 264

LCMS data (Appendix S1) To identify MDP0000248567 a phylogenetic tree was 265

conducted with MEGA 50 software It was found that MDP0000248567 was located 266

together with Arabidopsis transcription factor AtAREB2 supported by bootstrap 267

values of 100 (Fig S6A) Therefore MDP0000248567 was named MdAREB2 268

MdAREB2 gene is localized to chromosome 5 and has 4 exons and 3 introns (Fig 269

S6B) The expression analysis demonstrated that the transcript level of the MdAREB2 270

gene was markedly induced by ABA drought and PEG (Fig S6C) indicating that it 271

is an ABA-responsive gene 272

To determine whether MdAREB2 actually binds to the ABRE recognition site of 273

the MdSUT2 promoter EMSA was performed using MdAREB2-GST fusion proteins 274

A specific DNA-MdAREB2 protein complex was detected when the 275

CACGTC-containing oligonucleotide was used as a labeled probe The formation of 276

these complexes was reduced with increasing the amounts of the unlabeled ABRE 277

competitor probe with the same sequence This competition was not observed when 278

using the mutated version This competition specificity confirmed that the specific 279

binding of MdAREB2 to the MdSUT2 promoter required the ABRE recognition 280

sequence (Fig 4B) 281

To verify the specific in vivo binding of MdAREB2 to MdSUT2 promoter 282

ChIP-PCR assays were conducted using pAREB2MdAREB2-GFP and 283

pAREB2GFP transgenic apple calli treated with 50 μM ABA respectively The 284

ABRE element-containing promoter regions of MdSUT2 but not those of MdSUT1 285


10

and MdSUT4 were enriched by ChIP in the pAREB2MdAREB2-GFP transgenic 286

calli compared to the pAREB2GFP control (Fig 4C Fig S7A-B) Another primer 287

without ABRE core sequence based on the promoter sequence of MdSUT2 gene was 288

designed ChIP-PCR showed that MdAREB2 failed to bind to the sequence (Fig 4C) 289

It showed that MdAREB2 specifically bound to the ABRE cis-acting element on the 290

promoter of MdSUT2 291

In addition ChIP-PCR was performed using in pMdAREB2MdAREB2-GFP 292

pMdAREB2MdAREB2-GFPpMdSUT2(ABRE)GUS and 293

pMdAREB2MdAREB2-GFPpMdSUT2(mABRE)GUS co-expressed apple 294

protoplasts treated with or without ABA Apple isolated protoplasts from 295

pMdAREB2GFP were used as control The result showed that the enrichment of 296

MdSUT2 promoter region increased in the protoplasts treated with ABA compared 297

with those without ABA treatment When the ABRE element of MdSUT2 promoter 298

was mutated however the enrichment of MdSUT2 promoter region did not increase 299

in protoplasts even under ABA treatment These results indicated that ABA increased 300

the specific binding of MdAREB2 to the ABRE element of MdSUT2 promoter (Fig 301

4D) 302

For the yeast one-hybrid (Y1H) assays the MdSUT2 promoter was fused to the 303

pAbAi vector and the MdAREB2 gene was fused to the activation domain AD When 304

fused pMdSUT2-AbAi was co-expressed with MdAREB2-AD in yeast the strain was 305

able to grow on an SD-LeuAbA600 plate No growth was observed in the negative 306

control expression in which the MdSUT2 promoter was mutated (Fig 4E) These 307

results provided in vivo evidence for the specific binding of MdAREB2 to the 308

MdSUT2 promoter 309

GUS assays were conducted to examine if MdAREB2 influences the 310

transcription activity of MdSUT2 promoter in response ABA The 35SMdAREB2 311

construct was genetically transformed into the pMdSUT2GUS transgenic calli The 312

GUS analysis showed that the transgenic calli containing pMdSUT2GUS plus 313

35SMdAREB2 exhibited a much higher GUS activity than those harboring 314

pMdSUT2GUS alone (Fig 4F) indicating that MdAREB2 specifically activated 315

GUS transcription as driven by the MdSUT2 promoters in response to ABA 316

The MdTMT1 promoter was found to have two ABRE cis-acting elements ie 317

ABRE1 and ABRE2 (Fig 4G Table S1) ChIP-PCR demonstrated that MdAREB2 318

specifically binds to ABRE1 but not to ABRE2 (Fig 4H) suggesting that MdAREB2 319


11

may also be involved in the transcriptional regulation of MdTMT1 Furthermore the 320

genes MdTMT3 MdAMY1 MdAMY2 MdAMY3 MdBAM1 MdBAM2 and MdBAM3 321

also have one ABRE cis-acting element (Fig 4G Table S1 Fig S7A) ChIP-PCR 322

assays showed that MdAREB2 could bind to the promoters of α-amylase genes 323

MdAMY1 and MdAMY3 as well as β-amylase genes MdBAM1 and MdBAM3 but not 324

those of MdAMY2 and MdBAM2 which suggested that MdAREB2 is also involved 325

in regulating starch degradation (Fig 4H Fig S7C) In addition we designed another 326

primer without ABRE core sequence based on the promoter sequence of MdTMT1 327

MdAMY1 MdAMY3 MdBAM1 and MdBAM3 genes ChIP-PCR showed that 328

MdAREB2 failed to bind to these sequences (Fig 4H) 329

MdAREB2 promotes the accumulation of sucrose and soluble sugars in response 330

to ABA 331

To characterize the function of MdAREB2 the gene was introduced into the 332

pCXMyc-P plant transformation vector downstream of the CaMV 35S promoter and 333

then transformed into the lsquoGalarsquo apples As a result eight transgenic lines of 334

MdAREB2 were obtained The three independent transgenic lines MdAREB2-1 335

MdAREB2-2 and MdAREB2-4 were used for the functional analysis The RT-PCR 336

analysis showed that the transcript levels of three transgenic lines noticeably 337

increased compared with the lsquoGalarsquo control (Fig 5B Fig S8) A Western blot with an 338

anti-Myc antibody indicated that the MdAREB2-Myc protein was detected in the 339

transgenic plants (Fig 5C) The overexpression of the MdAREB2 gene markedly 340

upregulated the expression levels of the MdTMT1 MdSUT2 MdAMY1 MdAMY3 341

MdBAM1 and MdBAM3 genes in response to ABA in the three transgenic lines 342

compared with the WT control (Fig 5D) The transcript levels of the other MdTMTs 343

MdSUTs and amylase genes barely changed As a result three transgenic lines 344

accumulated less starch but much more glucose sucrose and soluble sugars than the 345

WT control (Fig 5A 5E-G) 346

In addition TRV viral-based gene suppression demonstrated that the suppression 347

of MdAREB2 downregulated the expression of MdSUT2 MdTMT1 MdAMY1 348

MdAMY3 MdBAM1 and MdBAM3 genes (Fig 6B) indicating that MdAREB2 349

positively regulates their expression As a result the MdAREB2-TRV transiently 350

transgenic shoot cultures accumulated more starch but less soluble sugars than the 351

empty vector control under ABA treatment (Fig 6A 6C-D) while no significantly 352

difference for starch and soluble sugar contents was found in these materials without 353


12

ABA treatment (Fig S9) Furthermore HPLC assays showed that the fructose 354

glucose and sucrose contents were noticeably reduced in MdAREB2-TRV shoot 355

tissues (Fig 6E) Therefore MdAREB2 is required for ABA-induced sugar 356

accumulation in apples 357

MdAREB2-promoted sucrose accumulation under ABA treatment partially 358

needs MdSUT2 359

To examine if MdAREB2 regulates sucrose accumulation in response to ABA 360

in an MdSUT2-dependent manner a VIGS (virus-induced gene silencing) method 361

was performed using the in vitro leaves of three MdAREB2 transgenic apple lines 362

The MdSUT2-TRV vector was used for to suppress the MdSUT2 gene It was 363

transiently transformed into the transgenic lines MdAREB2-1 MdAREB2-2 and 364

MdAREB2-4 while the empty TRV vector was used as a control The resulting leaves 365

that were infected with empty MdSUT2-TRV and TRV vectors were then transferred 366

onto plates containing MS medium for 4 days under normal light at room temperature 367

The expression analysis demonstrated that the MdSUT2-TRV infection successfully 368

suppressed the expression level of MdSUT2 in three MdAREB2 transgenic lines (Fig 369

7A) 370

The sucrose and soluble sugar contents were subsequently determined The result 371

showed that the infection with the TRV empty vector barely influenced the function of 372

MdAREB2 in regulating soluble sugar and sucrose accumulation (Fig 7B-C) 373

However the infection with the MdSUT2-TRV vector at least partially if not 374

completely inhibited the MdAREB2 function In addition sugar content inlsquoGalarsquo375

apple leaves infected with empty vector TRV MdAREB2-TRV 376

MdAREB2-TRVMdSUT2-IL60 and MdAREB2-TRVMdSUT2-TRV respectively 377

were detected MdAREB2-TRV infection noticeably decreased sucrose content 378

compared with TRV injection MdAREB2-TRVMdSUT2-TRV double injection 379

further decreased sucrose content while MdAREB2-TRVMdSUT2-IL60 double 380

injection restored sucrose content to the TRV control level (Fig S10) Therefore 381

MdAREB2 regulates sucrose and sugar accumulation in response to ABA at least 382

partially in an MdSUT2-dependent manner 383

MdAREB2 activated the expression of MdSUT2 which affects the soluble sugar 384

contents of apple fruits 385

To examine if ABA regulates starch and sugar accumulation in fruit the fruits of 386

the Red Delicious apple cultivar were treated with 0 10 20 and 50 microM ABA The 387


13

sugar transport genes MdTMT1 MdTMT4 MdSUT1 MdSUT2 and MdSUT4 were 388

increased under ABA treatment In addition MdAMY1 MdAMY3 MdBAM1 and 389

MdBAM3 were significantly induced by ABA (Fig 8B) The result showed that ABA 390

treatment markedly reduced the starch content but it increased the contents of sucrose 391

and soluble sugars (Fig 8A 8C-E) just as it did in the apple shoot cultures and 392

callus 393

To investigate the function of MdAREB2 in regulating MdSUT2 in apple fruits 394

these two genes were connected to the IL60 vector and the TRV vector which were 395

used for transient gene overexpression and transient gene silence respectively The 396

empty vectors were used as controls The qRT-PCR results showed that 397

MdAREB2-IL60 and MdSUT2-IL60 generated higher transcript levels while 398

MdAREB2-TRV and MdSUT2-TRV had a lower transcription level (Fig 8F) The 399

results indicated that MdAREB2-TRV and MdSUT2-TRV reduced the soluble sugar 400

and sucrose contents (Fig 8G-H) MdAREB2-IL60 and MdSUT2-IL60 showed the 401

opposite trend These results showed that MdAREB2 positively activated the 402

expression of MdSUT2 increasing the soluble sugar contents of apple fruits 403

404

Discussion 405

Crops and other plants under natural conditions are routinely affected by a broad 406

range of abiotic and biotic stresses that act simultaneously One of the pivotal events 407

in abiotic stress responses is the rapid accumulation of ABA which regulates the 408

expression of ABA-responsive genes that protect plants from damage (Lee and Luan 409

2012) Simultaneously different environmental stimuli increase sugar accumulation 410

by regulating the expression of sugar metabolism genes In this study the 411

ABA-induced transcription factor known as MdAREB2 was found to regulate sugar 412

accumulation by directly binding to the promoters of several genes which encode 413

sugar transporters and amylases and by activating their expression 414

SUT mediates the stress-induced sugar accumulation in the vacuolar 415

Sugars are synthesized from not only photosynthetic carbon fixation but also 416

starch Starch is the most abundant form in which plants store carbohydrates Starch 417

metabolism is regulated by various abiotic stresses (Valerio et al 2011 Zanella et al 418

2016) and the resulting accumulation of starch metabolites including sugars lowers 419

the water potential of the cell which promotes water retention in the plant (Verslues 420

and Sharma 2011 Krasensky and Jonak 2012) Starch is degraded by a set of 421


14

glucan-hydrolyzing enzymes (including α-amylases and β-amylases) In Arabidopsis 422

α-amylase genes such as AMY3 and BAM1 are involved in stress-induced starch 423

degradation (Thalmann et al 2016) In apple MdAMY1 MdAMY3 MdBAM1 and 424

MdBAM3 are homologous to Arabidopsis AMY3 and BAM1 Their expression is 425

induced by ABA (Fig 1) It is reasonable to speculate these genes may contribute to 426

ABA-induced starch degradation and subsequent sugar accumulation (Fig 5) The 427

results indicated that these genes were expressed in different tissue (Fig S2) Sugar 428

accumulation at least partially comes from starch degradation in response to ABA in 429

all tissue 430

Sugars not only play as osmotic adjustment substances but also help in 431

stabilizing proteins and cell structures under stress conditions (Sami et al 2016) In 432

addition they also reduce the effect of stress-induced ROS accumulation (Hu et al 433

2012) Sugars as osmotic adjustment substances mainly accumulate in the vacuolar 434

The vacuole is involved in the long-term or temporary storage of sugars (Martinoia et 435

al 2007) Various vacuolar sugar transporters are responsible for the transportation of 436

sugars into the vacuole TMTs (tonoplast monosaccharide transporters) are vacuolar 437

carrier proteins that have transport activity for monosaccharides such glucose (Wormit 438

et al 2006) Sucrose transporters (SUTsSUCs) are proton-coupling sucrose uptake 439

transporters which are responsible for the transportation of sucrose into vacuolar 440

(Shitan and Yazaki 2013 Schneider et al 2012) Both TMTs and SUTsSUCs 441

abundantly work together to modulate soluble sugar accumulation in the vacuolar 442

(Reuscher et al 2014) As a result the expression levels of TMT1 genes were 443

upregulated maybe through a feed-back regulatory manner in Arabidopsis mutant 444

suc2 and apple MdSUT2-TRV shoot cultures (Fig S11A Fig 1F) Meanwhile 445

MdSUT2 overexpression decreased the expression level of MdTMT1 gene in 446

transgenic apple plantlets (Fig S11B) Similarly apple MdTMT1-TRV shoot cultures 447

generated more MdSUT2 transcripts than the TRV control (Fig 1F) Therefore 448

MdSUT2-TRV shoot cultures accumulated more glucose and MdTMT1-TRV shoot 449

cultures did more sucrose than the TRV control although both of them accumulated 450

less soluble sugars than the TRV control (Fig 1G) In addition transient 451

overexpression of MdTMT1 gene produced more soluble sugar and glucose in apple 452

leaves than the empty vector control (Fig S12) suggesting that MdTMT1 acted as a 453

functional glucose transporter 454

In addition all of three distinct SUT subfamilies (type SUT1 SUT2 and SUT4) 455


15

have 12 predicted transmembrane domains in Arabidopsis (Sun et al 2008) Type 456

SUT2 also has two additional domains that is the extended N-terminal and 30 to 50 457

amino acid-center loop domains compared to type SUT1 and SUT4 (Stolz et al 1999 458

Fig S3B) In apple MdSUT2 contains those conserved domains and exhibits sucrose 459

transporter activity (Ma et al 2016 Fig S3) 460

Vacuolar sugar transporters are regulated by abiotic stresses In the apoplasm the 461

TMT1 and TMT2 genes are induced by salt drought and cold stresses (Wormit et al 462

2006) Arabidopsis AtSUC1 AtSUC2 and rice OsSUT2 transcripts are up-regulated in 463

response to low temperatures drought and salinity stresses (Lundmark et al 2006 464

Ibraheem et al 2011) The ectopic overexpression of an apple MdSUT2 gene 465

improves tolerance to abiotic stresses in apple calli and Arabidopsis (Ma et al 2016) 466

Therefore vacuolar sugar transporters play crucial roles in the response to various 467

abiotic stresses by promoting sugar accumulation 468

ABA-induced transcription factor MdAREB2 regulates sugar accumulation 469

The endogenous ABA level in plant cells increases with osmotic stresses such as 470

drought and high salinity leading to the expression of stress-responsive genes (Rook 471

et al 2006) The AREB transcription factors play an important role in ABA signaling 472

The AREBABF genes encode basic-domain leucine zipper (bZIP) transcription 473

factors and belong to a group-A subfamily which comprises nine homologs in the 474

Arabidopsis genome and they harbor three N-terminal and one C-terminal conserved 475

domains (Yoshida et al 2015) Among them AREB1ABF2 AREB2ABF4 and 476

ABF3 are induced by dehydration high salinity or ABA treatment in vegetative 477

tissues (Fujita et al 2005 Kim et al 2011) The areb1areb2abf3 triple knockout 478

mutant shows the impaired expression of ABA and osmotic stress-responsive genes 479

resulting in increased sensitivity to drought and decreased sensitivity to ABA in 480

primary root growth (Yoshida et al 2010) Arabidopsis (ABI5 AREB1 and AREB2) 481

rice (TRAB1 and OREB1) and wheat (TaABF) ABF family members have been 482

reported to be crucial for the regulation of ABA-responsive gene expression (Yoshida 483

et al 2010 Kagaya et al 2002 Johnson et al 2002) 484

In addition the AREB transcription factors are also involved in sugar 485

accumulation In Arabidopsis AREB transcription factors are suggested to activate 486

the expression of BAM1 and AMY3 genes through an ABA-dependent SnRK2 487

signaling pathway (Thalmann et al 2016) In ABA-treated mosses the concentration 488

of soluble sugars significantly increased which may promote cytoplasm vitrification 489


16

and protect membranes (Mayaba et al 2001) In carrots a sucrose-upregulated bZIP 490

transcription factor called CAREB1 responds to the ABA and sugar signal (Guan et al 491

2009) ABI5 overexpression confers increased sensitivity to the glucose inhibition of 492

seedling growth indicating that ABI5 mediates the sugar response (Finkelstein et al 493

2002) In this study the expression of the MdAREB2 gene was also found to be 494

induced by ABA (Fig S6) and MdAREB2 overexpression increased the soluble sugar 495

contents in transgenic apple plantlets in response to ABA (Fig S5) 496

As is well known AtSUT2 shows a similar structure to those of yeast sugar 497

sensors RGT2 and SNF3 (Oumlzcan et al 1998) MdSUT2 and AtSUT2 exhibited highly 498

similar amino acid sequences and 3D structures to each another (Fig S1A S3C) It 499

may present a hypothesis that both of them may function as sugar sensors and 500

influence expression levels of the related genes This hypothesis is supported by the 501

fact that the transcript levels of AREB2 genes decreases in Arabidopsis mutant suc2 502

and apple MdSUT2-TRV shoot cultures (Fig S13A Fig 7A) but increases in 503

MdSUT2 transgenic apple plantlets (Fig S13B) Therefore MdSUT2 may functions a 504

sugar sensor to positively regulate the expression of AREB2 gene although it is 505

unknown concerning the exact mechanism In addition it was found that the 506

expression level of MdAREB2 gene and the content of sucrose reduced in the 507

MdSUT2-TRV leaves of three MdAREB2 apple transgenic plantlets (Fig 7A 7C) 508

MdSUT2 was ovexpressed in MdAREB2-TRV transgenic plants which could 509

increased sucrose content (Fig S10) It showed that MdAREB2 promotes sugar 510

accumulation at least partially if not all via MdSUT2 which increases SUT2 activity 511

directly by itself and indirectly by enhancing MdAREB2 expression 512

ABA-induced sugars contribute to plant development and fruit quality 513

Soluble sugars (sucrose glucose and fructose) play an important role in 514

maintaining the growth and development of plants The soluble sugars trigger the 515

proliferation of organs and produce larger and thicker leaves increasing the size and 516

number of tubers and adventitious roots For example high sugar concentrations 517

stimulate the formation of adventitious roots in Arabidopsis (Gibson 2005) and they 518

lead to an increase in the number of tubers (Sami et al 2016) The SUT1 mRNA and 519

protein levels can control leaf senescence The antisense SUT1 plants delay plant 520

growth (Lalonde et al 2004) 521

In addition sugar acts as a signaling molecule that is involved in plant growth 522

During germination glucose is known to be a very potent modulator in activating 523


17

genes involved in ABA biosynthesis (Price et al 2003) A higher concentration of 524

sugars triggers the repression of genes associated with photosynthesis (Hammond et 525

al 2011) 526

Moreover sugar molecules also act as nutrients and substrates that contribute to 527

fruit or crop quality characteristics such as sweetness SUT2 has a major impact on the 528

sugar contents of potatoes (Barker et al 2000) Similarly MdSUT2 overexpression 529

increases the soluble sugar and sucrose contents in fruits (Fig 8) In addition sugar 530

also regulates anthocyanin biosynthesis In Arabidopsis sucrose induces the 531

expression of MYB75PAP1 gene which activates the expression of structural genes 532

associated with anthocyanin synthesis (Solfanelli et al 2006) 533

Finally a model for the ABA regulation of soluble sugar accumulation is 534

established It shows that ABA induces the expression of MdAREB2 which 535

subsequently binds to the promoters of sugar transport genes MdSUT2 and MdTMT1 536

as well as amylase genes including MdAMY1 MdAMY3 MdBAM1 and MdBAM3 537

This signal then transcriptionally activates the expression of MdSUT2 (and maybe 538

other MdAREB2-regulated genes) finally resulting in soluble sugar accumulation to 539

influence the abiotic stress tolerance fruit quality plant growth and development (Fig 540

9) In fruit this regulatory pathway sheds light on why ABA and the related stresses 541

such as drought promote fruit quality and thereby provides theoretical guidance for 542

developing new cultivation techniques to improving fruit quality 543

544

Materials and Methods 545

Plant materials growth conditions and ABA treatments 546

The in vitro shoot cultures of apple cultivar lsquoGalarsquo were grown on MS medium 547

supplemented with 10 mgL-1

naphthyl acetate (NAA) and 05 mg L-1

548

6-benzylaminopurine (6-BA) at 25degC under long-day conditions (16 h light8 h dark) 549

They were subcultured at 30-day intervals For rooting in vitro shoot cultures were 550

grown on MS medium supplemented with 05 mg L-1

indole-3-acetic acid (IAA) 551

Finally the rooted apple plantlets were transferred to pots containing a mixture of 552

soilperlite (11) and grown in the greenhouse under a 16 h8 h lightdark and 25degC 553

daynight cycle 554

For ABA treatment apple shoot cultures were cultivated on MS medium 555

supplemented with 05 mg L-1

indole-3-acetic acid (IAA) 15 mg L-1

556

6-benzylaminopurine (6-BA) and 100 μM ABA for two days Apple calli were 557


18

cultivated on MS medium supplemented with 15 mgL-1

6-benzylaminopurine (6-BA) 558

and 05 mgL-1

2 4-Dichlorophenoxyacetic acid and 100 μM ABA for one day 559

Arabidopsis seedlings were cultivated on MS medium containing 50 μM ABA for one 560

day Fruits of apple cultivar lsquoRed deliciousrsquo were sprayed with 0 10 20 50 μM ABA 561

in the dark for 4 days 562

Construction of the expression vectors and genetic transformation into apple 563

To construct MdSUT2 and MdAREB2 overexpression vectors the full-length 564

DNAs of MdSUT2 and MdAREB2 were isolated from lsquoGalarsquo apples using PCR All 565

the DNAs were digested with EcoRIBamHI and cloned into the MYC plant 566

transformation vectors downstream of the CaMV 35S promoters All the primers used 567

here are listed in Table S2 These vectors were genetically introduced into apple 568

plants using Agrobacterium-mediated transformation as described by Zhao et al 569

(2016) 570

RNA extraction RT-PCR and qRT-PCR assays 571

The total fruit RNAs were extracted using the hot borate method as described by 572

Yao et al (2010) The total RNAs from other tissues were extracted using the Trizol 573

Reagent (Invitrogen Life Technologies Carlsbad CA USA) Two micrograms of the 574

total RNAs was used to synthesize first-strand cDNA with a PrimeScript First Strand 575

cDNA Synthesis Kit (TaKaRa Dalian China) For the real-time qRT-PCR analysis 576

the reactions were performed with iQ SYBR Green Supermix in an iCycler iQ5 577

system (Bio-Rad Hercules CA USA) according to the manufacturerrsquos instructions 578

The specific mRNA levels were analyzed by relative quantification using the cycle 579

threshold (Ct) 2-ΔΔCt method For all of the analyses the signal that was obtained for 580

a gene of interest was normalized against the signal that was obtained for the 18s gene 581

All of the samples were tested in three to four biological replicates All of the primers 582

that were used for the qRT-PCR are listed For the semi-quantitative RT-PCR the 583

reactions were performed according to the manufacturerrsquos instructions (TransGen 584

Beijing China) using the following thermal profile pre-incubation at 95 degC for 5 min 585

followed by 30 cycles of 95 degC (30 s) 58 degC (30 s) and 72 degC (30 s) with a final 586

extension at 72 degC for 5 min The primers are listed in Table S2 587

Starch quantification 588

Starch which is composed of glucose residues is a polysaccharide It can be 589

divided into glucose under acidic conditions by heating and hydrolysis The 590

chromogenic reagent known as anthrone was used The 1g (fresh weight) samples 591


19

were transferred into 50 ml volumetric flask add 20 ml hot distilled water placed in a 592

boiling water bath boil 15 min then added 2 ml 92 mol L perchloric acid to extract 593

for 15 min after cooling filtering with filter paper (or centrifuging at 2500r min for 594

10 min) Then set the volume with distilled water This reaction can be subjected to 595

colorimetric determination 596

Soluble sugar contents 597

1g (fresh weight) samples were ground in 5 mL of 95 (vv) ice-cold methanol 598

The methods were described by Hu et al (2016) Three milliliters of supernatant was 599

passed through a 045-μm membrane filter and the filtrate was then analyzed with 600

High performance liquid chromatography (HPLC) 601

Yeast one-hybrid 602

Genomic DNAs extracted from apple tissue culture was used as template to 603

amplify promoter pMdSUT2(ABRE) In addition they were also used to generate 604

mutated MdSUT2 promoter pMdSUT2(mABRE) with a PCR-based point mutagenesis 605

method The first-stage PCR was performed with the mutagenic primer ie 606

pMdSUT2-F1 5-GCCATATCAGAGACGGGAAG-3 and pMdSUT2-R1 607

5-CAAACTAGGACCTACTGTATGGA-3 (the mutant nucleotides were underlined) 608

as well as pMdSUT2-F2 5-TCCATACAGTAGGTCCTAGTTTG-3 (the mutant 609

nucleotides were underlined) and pMdSUT2-R2 610

5-GGCGACTCGGTTTCACTGACTCAGT-3 The resultant PCR products were 611

recovered and purified They were then 11 mixed and used as template for the second 612

round of PCR amplification with primers pMdSUT2-F1 613

5-GCCATATCAGAGACGGGAAG-3 and pMdSUT2-R2 614

5-GGCGACTCGGTTTCACTGACTCAGT-3 The promoter sequence of MdSUT2 615

gene was shown in Table S3 616

The wild-type and mutated promoters pMdSUT2(ABRE) and 617

pMdSUT2(mABRE) were ligated into the one-hybrid vector pAbAi (Clontech Palo 618

Alto USA) respectively The resultant vectors pMdSUT2-pAbAi and 619

pMdSUT2(m)-pAbAi were transferred into yeast one-hybrid strain YM187 And the 620

resulting strain cell was plated on SD-Ura medium plus 600ngml Aureobasidin A a 621

competitive inhibitor for yeast growth The recombinant vector pGADT7-MdAREB2 622

was constructed and transferred into the yeast strain containing pMdSUT2 623

(AREB)-pAbAi and pMdSUT2 (mABRE)-pAbAi The resulting strain cells were 624

grown on SD-LeuAbA600 medium The plaque indicates that MdAREB2 bind to the 625


20

MdSUT2 promoter Otherwise it does not bind 626

Chromatin immunoprecipitation (ChIP) qPCR analysis 627

The transgenic calli pMdAREB2GFP and pMdAREB2MdAREB2-GFP were 628

applied to ChIP analysis Apple protoplasts were isolated from transgenic 629

pMdAREB2MdAREB2-GFP calli (Hu et al 2016) The constructed vectors 630

pMdSUT2 (ABRE)GUS and pMdSUT2 (mABRE)GUS were expressed in the 631

pMdAREB2MdAREB2-GFP transformed protoplasts An anti-GFP antibody 632

(Beyotime Haimen China) was used for ChIP qPCR as described by Hu et al (2016) 633

The immunoprecipitated samples were used as template for qPCR analysis with 634

primers as listed in Table S2 635

Electrophoretic mobility shift assay (EMSA) 636

An EMSA was conducted according to Xie et al (2012) MdAREB2 was cloned 637

into the expression vector pGEX4T-1 The MdAREB2-GST recombinant protein was 638

expressed in Escherichia coli strain BL21 and purified using glutathione sepharose 639

beads (Thermo Shanghai China) An oligonucleotide probe of the MdSUT2 promoter 640

was labeled with an EMSA probe biotin labeling kit (Beyotime Haimen China) 641

according to the manufacturerrsquos instructions The binding reaction was performed for 642

20 min at room temperature The DNA-protein complexes were electrophoresed on 643

65 non-denaturing polyacrylamide gels electrotransferred and detected according 644

to the manufacturerrsquos instructions The binding specificity was also examined by 645

testing its competition with a fold excess of unlabeled oligonucleotides The primers 646

that were used are listed in Table S2 647

GUS assays 648

Transient expression assays were conducted using apple calli The normal and 649

mutant promoters of MdSUT2 were cloned into pBI121-GUS to fuse with the reporter 650

gene GUS The resulting MdSUT2GUS constructs were obtained and genetically 651

transformed into apple calli via an Agrobacterium-mediated method Subsequently 652

35SMdAREB2 was co-transformed into the above-mentioned transgenic calli 653

Finally histochemical staining was performed to detect the GUS activity in the 654

transgenic calli It was determined using the method described by Zhao et al (2016) 655

Construction of the viral vectors and transient expression in apple fruits 656

To construct the antisense expression viral vectors the conserved MdAREB2 and 657

MdSUT2 cDNA fragments were amplified respectively with RT-PCR using apple 658

fruit cDNA as the template The resulting products were cloned into a tobacco rattle 659


21

virus (TRV) vector in the antisense orientation under the control of the dual 35S 660

promoter The resulting vectors were named MdAREB2-TRV and MdSUT2-TRV 661

respectively To generate the viral overexpression vectors full-length cDNAs of 662

MdAREB2 and MdSUT2 were inserted into the IL-60 vector under the control of the 663

35S promoter The resulting vectors were named MdAREB2-IL60 and MdSUT2-IL60 664

All of the vectors were transformed into Agrobacterium tumefaciens strain GV3101 665

for inoculation Apple fruits were collected from a 6-year-old tree of lsquoRed Deliciousrsquo 666

cultivar The fruits were bagged at 35 days after flowering and harvested at 140 Days 667

They were debagged before injection Fruit infiltrations were performed as described 668

Li et al (2012) 669

DNA-affinity trapping of DNA-binding proteins 670

DNA promoter fragments of the MdSUT2-containing ABRE cis-elements were 671

used to isolate the proteins that were bound to the cis-elements in these promoter 672

fragments Biotinylated DNA promoter fragments were generated by PCR Nuclear 673

protein extracts for EMSA were prepared from the wild-type apple plants that were 674

grown under natural conditions The methods were described by Hu et al (2016) 675

676

Acknowledgements 677

This work was supported by grants from NSFC (31325024 and 31430074) 678

Ministry of Education of China (IRT15R42) and Shandong Province (SDAIT-06-03) 679

680

Author contributions 681

Yu-Jin Hao and Qi-Jun Ma conceived and designed the experiment Qi-Jun Ma 682

Mei-Hong Sun Jing Lu Ya-Jing Liu and Da-Gang Hu preformed the experiments 683

Qi-Jun Ma and Yu-Jin Hao analyzed the data and wrote the paper 684

685

Figure legends 686

Figure 1 ABA promotes the accumulation of soluble sugars and increases the 687

expression of genes encoding sugar transporters and amylases 688

(A) The starch accumulation effect of 100 microm exogenous ABA on the in vitro shoot 689

cultures of lsquoGalarsquo apples (B-D) starch (B) soluble sugar (C) fructose glucose and 690

sucrose (D) contents of lsquoGalarsquo apples without or with ABA (E) ABA effect on the 691

gene expression of MdTMTs MdSUTs MdAMYs and MdBAMs (F) The gene 692

expression of MdTMT1 and MdSUT2 in the in vitro shoot cultures of lsquoGalarsquo apples 693


22

that were transiently infected by the TRV system MdTMT1 and MdSUT2 were 694

cloned into the TRV vector and used for suppression The empty vectors were used as 695

controls (G-H) The contents of soluble sugar (G) fructose glucose and sucrose (H) 696

In the MdTMT1-TRV or MdSUT2-TRV-infected shoot cultures with 100 microM ABA 697

treatment ns indicates a non-significant difference (Pgt001) indicates a 698

statistically significant difference (Plt001 for ) (Plt0001 for ) The values are the 699

means plusmn SD (n =3 independent experiments) 700

701

Figure 2 MdSUT2 functions as a sucrose transporter 702

(A) qRT-PCR was used to examine the transcription levels of the three overexpression 703

lines MdSUT2-1 2 and 5 compared to lsquoGalarsquo (B) Two month-old lsquoGalarsquo plants and 704

three MdSUT2-overexpression lines (MdSUT2-1 2 and 5) (C) Western blot 705

examined the MdSUT2 protein abundance in 35SMdSUT2-Myc transgenic plants 706

(D) The sucrose activity in the roots of transgenic plants (E-F) The soluble sugar (E) 707

and sucrose content (F) indicates a statistically significant difference (Plt001 for ) 708

(Plt0001 for ) The values are the means plusmn SD (n = 3 independent experiments) 709

710

Figure 3 The expression of MdSUT2 was induced by ABA 711

(A) A schematic diagram showing the cis element in the MdSUT2 promoter as 712

analyzed by PlantCARE (httpbioinformaticspsbugentbewebto lsplantcarehtml) 713

Red boxes indicate ABRE (the abscisic acid responsiveness) cis element in the 714

MdSUT2 promoter ABRE(m) indicates the site mutants in which the ABRE core 715

sequence CTGCAC was changed to TAGGTC Blue box represented sequence 716

without ABRE core 717

(B) GUS-stained pMdSUT2-GUS and pMdSUT2-GUS(m) transgenic apple calli in 718

treatments with or without ABA 719

(C) Quantitative statistics of GUS activity in apple calli 720

(D) Quantitative statistics of GUS activity in pMdSUT2-GUS and pMdSUT2-GUS(m) 721

Arabidopsis seeding ns indicates a non-significant difference (Pgt001) indicates a 722

statistically significant difference (Plt0001 for ) The values are the means plusmn SD (n 723

= 3 independent experiments) 724

725

Figure 4 The transcription factor MdAREB2 binds to the cis element of the sugar 726

metabolism promoter 727


23

(A) Identification of the MdAREB2 TF proteins that bind to the cis element of the 728

MdSUT2 promoter with EMSA lsquo+rsquo and lsquo-rsquo represent samples with or without the 729

addition of total protein extracted from the apple plants respectively 730

(B) Interaction of MdAREB2 protein with labeled DNA probes for the cis elements of 731

the MdSUT2 promoter in the EMSA pMdSUT2 is used as a negative control 732

without the MdAREB2 protein 733

(C) The relative enrichment of the MdSUT2 promoter fragments The 734

MdAREB2-DNA complex was co-immunoprecipitated from MdAREB2-GFP 735

transgenic apple calli using an anti-GFP antibody GFP was used as a negative control 736

(D) ChIP analysis of MdAREB2 binding to pMdSUT2(ABRE) and 737

pMdSUT2(mABRE) wiith or without ABA 738

(E) The promoter of MdSUT2 was fused to the pAbAi vector and the MdAREB2 739

gene was fused to activation domain AD in yeast for Y1H assays 740

(F) GUS activity in the transgenic apple calli as labeled 741

(G) The schematic diagram showing the cis element in MdTMT1 742

MdAMY1(MDP0000210170) MdAMY3(MDP0000281786) 743

MdBAM1(MDP0000193684) and MdBAM3(MDP0000397284) promoters as analyzed 744

by PlantCARE (httpbioinformaticspsbugentbewebto lsplantcarehtml) Red 745

boxes indicate the ABRE (the abscisic acid responsiveness) cis element in the 746

promoter of each gene Blue box represented sequence without ABRE core 747

(H) ChIP-PCR showed that MdABRE2 specifically binds to the ABRE cis elements 748

of the MdTMT1 MdAMY1 MdAMY3 MdBAM1 and MdBAM3 promoters in vivo ns 749

indicates non-significant differences (Pgt001) indicates statistically significant 750

differences (Plt001 for ) (Plt0001 for ) The values are the means plusmn SD (n = 3 751

independent experiments) 752

753

Figure 5 MdAREB2-overexpression plants show increased accumulations of sucrose 754

and soluble sugars 755

(A) The starch staining of MdAREB2-overexpression plants (B) qRT-PCR was used 756

to examine the transcription level of the three transgenic lines MdAREB2-1 2 and 4 757

(C) Western Blot to check the MdAREB2 protein content (D) qRT-PCR was used to 758

examine the transcription level of MdTMTs MdSUT2 MdAMYs and MdBAMs in the 759

three transgenic lines MdAREB2-1 2 and 4 (E-G) The contents of starch (E) 760

soluble sugar (F) and sucrose (G) indicates a statistically significant difference 761


24


763

Figure 6 The transient gene-silencing of MdAREB2 through viral vector-based 764

transformation alters the contents of starch and soluble sugar in apple vitro shoot 765

cultures 766

(A) Starch staining of MdAREB2-TRV transiently infected rsquoGalarsquo apple in vitro shoot 767

cultures under ABA treatment The empty vectors were used as controls (B) The gene 768

expression of MdTMTs MdSUT2 MdAMYs and MdBAMs was examined (C-E) The 769

starch (C) soluble sugar (D) fructose glucose and sucrose (E) as treated in (a) plants 770

indicates a statistically significant difference (Plt001 for ) The values are the 771

means plusmn SD (n = 3 independent experiments) 772

773

Figure 7 MdAREB2 promotes the accumulation of sucrose and soluble sugars by 774

MdSUT2 775

(A) qRT-PCR analyses of MdAREB2 and MdSUT2 transcripts in the transgenic plants 776

A VIGS (virus-induced gene silencing) method was performed using the in vitro 777

leaves of three MdAREB2 transgenic apple lines The MdSUT2-TRV vector was used 778

for MdSUT2 gene suppression and it was transiently transformed into the transgenic 779

lines MdAREB2-1 MdAREB2-2 and MdAREB2-4 The TRV empty vector was used 780

as a control (B) (C) The soluble sugar and sucrose contents as treated in (A) plants 781

indicates statistically significant differences (Plt001 for ) The values are the means 782

plusmn SD (n = 3 independent experiments) 783

784


expression of sugar transporters genes The apple fruits of the lsquoRed Deliciousrsquo cultivar 786

was treated with 0 10 20 and 50 microM ABA 787

(A) The starch accumulation effect of exogenous ABA on apple fruits (B) The 788

expression analysis of MdTMT MdSUT2 MdAMYs and MdBAMs genes (C-E) The 789

starch (C) soluble sugar (D) and sucrose (E) (F-H) The transient expression of 790

MdAREB2 and MdSUT2 via viral vector-based transformation alters the soluble 791

sugars and sucrose contents of apple fruits The MdSUT2-IL60 and MdAREB2-IL60 792

vectors were used for the overexpression of MdSUT2 and MdAREB2 genes while 793

the MdSUT2-TRV and MdAREB2-TRV vectors were used for their suppression (F) 794

The expression analysis of MdAREB2 and MdSUT2 genes in each transient expression 795


25

fruit while (G) and (H) show the soluble sugar (g) and sucrose (h) contents 796

respectively indicates a statistically significant difference (Plt001 for ) The 797

values are the means plusmn SD (n = 3 independent experiments) 798

799

Figure 9 A model for the ABA regulation of soluble sugar accumulation 800

801

Supplemental Data 802

Figure S1 Phylogenetic tree analysis of sugar transporters genes 803

Figure S2 qRT-PCR assay that the expression of gene in root stem leaf flower 804

fruit 805

Figure S3 The genome structure analysis of MdSUT2 and MdTMT1 806

Figure S4 The empty TRV infection did not influence the expression of MdTMT1 807

and MdSUT2 genes 808

Figure S5 qRT-PCR examined the transcription level of the six transgenetic lines 809

MdSUT2-3 4 6789 810

Figure S6 The genome structure analysis of MdAREB2 811

Figure S7 The cis element ABRE in the promoters of these genes 812

Figure S8qRT-PCR examined the transcription level of the five transgenetic lines 813

MdAREB2-3 5 678 814

Figure S9 The phenotype of transient gene-silencing of MdAREB2 plants 815

Figure S10 Sugar content inlsquoGalarsquo apple leaves after infection with empty vector 816

TRV MdAREB2-TRV MdAREB2-TRVMdSUT2-IL60 and MdAREB2-TRV 817

MdSUT2-TRV TRV vector was used for transient gene suppression IL60 vector was 818

used for transient gene overexpression 819

Figure S11 The expression of TMT1 820

Figure S12 Sugar content in lsquoGalarsquo apple leaves which contained MdTMT1 transient 821

overexpression vector 35SMdTMT1-IL60 and empty vector IL60 respectively Two 822

independent injections MdTMT1-IL60-1 and MdTMT1-IL60-2 were used 823

Figure S13 The expression of AREB2 824

Table S1 Some important cis-acting regulatory elements in the upstream regulatory 825

sequences of MdSUT2 MdTMT1 MdAMY1 MdAMY3 MdBAM1 and MdBAM3 826

genes 827

Table S2 Primers used in this study 828

Table S3 The promoter of MdSUT2 829


26

830

Literature Cited 831

Akihiro T Mizuno K Fujimura T (2005) Gene expression of ADP-glucose 832

pyrophosphorylase and starch contents in rice cultured cells are cooperatively 833

regulated by sucrose and ABA Plant Cell Physiol 46(6) 937-946 834

Barker L Kuumlhn C Weise A Schulz A Gebhardt C Hirner B Hellmann H 835

Schulze W Ward JM Frommer WB (2000) SUT2 a putative sucrose sensor in 836

sieve elements Plant Cell 12(7) 1153-1164 837

Bhatia S and Singh R (2002) Phytohormone-mediated transformation of sugars to 838

starch in relation to the activities of amylases sucrose-metabolising enzymes in 839

sorghum grain Plant Growth Regul 36 97-104 840

Chen Z Hong X Zhang H Wang Y Li X Zhu JK Gong Z (2005) Disruption of 841

the cellulose synthase gene AtCesA8IRX1 enhances drought and osmotic stress 842

tolerance in Arabidopsis Plant J 43(2) 273-283 843

Chung P Hsiao HH Chen HJ Chang CW Wang SJ (2014) Influence of 844

temperature on the expression of the rice sucrose transporter 4 gene OsSUT4 in 845

germinating embryos and maturing pollen Acta Physiol Plant 36(1) 217-229 846

Ferrandino A and Lovisolo C (2014) Abiotic stress effects on grapevine (Vitis 847

vinifera L) focus on abscisicacid-mediated consequences on secondary 848

metabolism and berry quality Environ Exp Bot 103(1) 138-147 849

Finkelstein R Gibson SI (2002) ABA and sugar interactions regulating development 850

ldquocross-talkrdquo or ldquovoices in a crowdrdquo Curr Opin Plant Biol 5 26-32 851

Fujita Y Fujita M Satoh R Maruyama K Parvez M Seki M Hiratsu K 852

Ohme-Takagi M Shinozaki K Yamaguchi-Shinozaki K (2005) AREB1 is a 853

transcription activator of novel ABRE-dependent ABA signaling that enhances 854

drought stress tolerance in Arabidopsis Plant Cell 17(12) 3470-3488 855

Gibson SI (2004) Sugar and phytohormone response pathways navigating a 856

signalling network J Exp Bot 55(395) 253-264 857

Gibson SI (2005) Control of plant development and gene expression by sugar 858

signaling Curr Opin Plant Biol 8(1) 93-102 859

Goacutemez LD Gilday A Feil R Lunn JE Graham IA (2010) AtTPS1‐mediated 860

trehalose 6‐phosphate synthesis is essential for embryogenic and vegetative 861

growth and responsiveness to ABA in germinating seeds and stomatal guard cells 862

Plant J 64(1) 1-13 863


27

Guan Y Ren H Xie H Ma Z Chen F (2009) Identification and characterization of 864

bZIP‐type transcription factors involved in carrot (Daucus carota L) somatic 865

embryogenesis Plant J 60(2) 207-217 866

Hammond J Broadley M Bowen H Spracklen W Hayden R White P (2011) 867

Gene expression changes in phosphorus deficient potato (Solanum tuberosum L) 868

leaves and the potential for diagnostic gene expression markers PloS One 6 9 869

Hayes MA Feechan A Dry IB (2010) Involvement of abscisic acid in the 870

coordinated regulation of a stress-inducible hexose transporter (VvHT5) and a 871

cell wall invertase in grapevine in response to biotrophic fungal infection Plant 872

Physiol 153(1) 211-221 873

Hu DG Sun CH Ma QJ You CX Cheng L Hao YJ (2016) MdMYB1 regulates 874

anthocyanin and malate accumulation by directly facilitating their transport into 875

vacuoles in apples Plant Physiol 170(3) 1315-1330 876

Hu M Shi Z Zhang Z Zhang Y Li H (2012) Effects of exogenous glucose on seed 877

germination and antioxidant capacity in wheat seedlings under salt stress Plant 878

Growth Regul 68 177e188 879

Ibraheem O Dealtry G Roux S Bradley G (2011) The effect of drought and 880

salinity on the expressional levels of sucrose transporters in rice (Oryza sativa 881

Nipponbare) cultivar plants Plant Omics 4(2) 68 882

Islam MZ Jin LF Shi CY Liu YZ Peng SA (2015) Citrus sucrose transporter 883

genes genome-wide identification and transcript analysis in ripening and 884

ABA-injected fruits Tree Genet Genomes 11(5) 1-9 885

Johnson R R Wagner R L Verhey S D amp Walker-Simmons M K (2002) The 886

abscisic acid-responsive kinase pkaba1 interacts with a seed-specific abscisic 887

acid response element-binding factor taabf and phosphorylates taabf peptide 888

sequences Plant Physiol 130(2) 837 889

Kafi M Stewart WS Borland AM (2003) Carbohydrate and proline contents in 890

leaves roots and apices of salt-tolerant and salt-sensitive wheat cultivars 1 Russ 891

J Plant Physiol 50(2) 155-162 892

Kagaya Y Hobo T Murata M Ban A amp Hattori T (2002) Abscisic acidndashinduced 893

transcription is mediated by phosphorylation of an abscisic acid response 894

element binding factor trab1 Plant Cell 14(12) 3177-89 895

Khan HA Siddique KH Colmer TD (2016) Vegetative and reproductive growth of 896

salt-stressed chickpea are carbon-limited sucrose infusion at the reproductive 897


28

stage improves salt tolerance J Exp Bot erw177 898

Kim JS Mizoi J Yoshida T Fujita Y Nakajima J Ohori T Todaka D 899

Nakashima K Hirayama T Shinozaki K Yamaguchi-Shinozaki K (2011) An 900

ABRE promoter sequence is involved in osmotic stress-responsive expression of 901

the DREB2A gene which encodes a transcription factor regulating 902

drought-inducible genes in Arabidopsis Plant Cell Physiol 52(12) 2136-2146 903

Krasensky J and Jonak C (2012) Drought salt and temperature stress-induced 904

metabolic rearrangements and regulatory networks J Exp Bot 63 1593-1608 905

Lalonde S Wipf D Frommer WB (2004) Transport mechanisms for organic forms 906

of carbon and nitrogen between source and sink Annu Rev Plant Biol 55 907

341-372 908

Lastdrager J Hanson J Smeekens S (2014) Sugar signals and the control of plant 909

growth and development J Exp Bot 65(3) 799-807 910

Lecourieux F Kappel C Lecourieux D Serrano A Torres E Arce-Johnson P 911

Delrot S (2013) An update on sugar transport and signalling in grapevine J Exp 912

Bot ert394 913

Lee SC and Luan S (2012) Aba signal transduction at the crossroad of biotic and 914

abiotic stress responses Plant Cell amp Environ 35(1) 53 915

Li YY Mao K Zhao C Zhao XY Zhang HL Shu HR Hao YJ (2012) MdCOP1 916

ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced 917

anthocyanin biosynthesis and red fruit coloration in apple Plant Physiol 160(2) 918

1011-1022 919

Lu R Guyer DE Beaudry RM (2000) Determination of firmness and sugar content 920

of apples using near-infrared diffuse reflectance1 J Texture Stud 31(6) 615-630 921

Lundmark M Cavaco AM Trevanion S Hurry V (2006) Carbon partitioning and 922

export in transgenic Arabidopsis thaliana with altered capacity for sucrose 923

synthesis grown at low temperature a role for metabolite transporters Plant Cell 924

Environ 29(9) 1703-1714 925

Lv S Zhang K Gao Q Lian L Song Y Zhang J (2008) Overexpression of an 926

H+-PPase gene from Thellungiella halophila in cotton enhances salt tolerance 927

and improves growth and photosynthetic performance Plant Cell Physiol 49(8) 928

1150-1164 929

Ma QJ Sun MH Liu YJ Lu J Hu DG Hao YJ (2016) Molecular cloning and 930

functional characterization of the apple sucrose transporter gene MdSUT2 Plant 931


29

Physiol Bioch 109 442-451 932

Martinoia E Maeshima M Neuhaus HE (2007) Vacuolar transporters and their 933

essential role in plant metabolism J Exp Bot 58(1) 83-102 934

Mayaba N Beckett RP Csintalan Z Tuba Z (2001) ABA increases the desiccation 935

tolerance of photosynthesis in the afromontane understorey moss Atrichum 936

androgynum Ann Bot London 88(6) 1093-11 937

Medici A Laloi M Atanassova R (2014) Profiling of sugar transporter genes in 938

grapevine coping with water deficit FEBS Lett 588(21) 3989-3997 939

Moustakas M Sperdouli I Kouna T Antonopoulou CI Therios I (2011) 940

Exogenous proline induces soluble sugar accumulation and alleviates drought 941

stress effects on photosystem II functioning of Arabidopsis thaliana leaves Plant 942

Growth Regul 65(2) 315-325 943

Oumlzcan S Dover J and Johnston M (1998) Glucose sensing and signaling by two 944

glucose receptors in the yeast Saccharomyces cerevisiae EMBO J 17(9) 945

2566-2573 946

Price J Li TC Kang SG Na JK amp Jang JC (2003) Mechanisms of glucose 947

signaling during germination of Arabidopsis Plant Physiol 132(3) 1424 948

Rasheed R Wahid A Farooq M Hussain I Basra SM (2011) Role of proline and 949

glycinebetaine pretreatments in improving heat tolerance of sprouting sugarcane 950

(Saccharum sp) buds Plant Growth Regul 65(1) 35-45 951

Reuscher S Akiyama M Yasuda T Makino H Aoki K Shibata D amp Shiratake 952

K (2014) The sugar transporter inventory of tomato genome-wide identification 953

and expression analysis Plant Cell Physiol 55(6) 1123-1141 954

Rolland F Baena-Gonzalez E Sheen J (2006) Sugar sensing and signaling in plants 955

conserved and novel mechanisms Annu Rev Plant Biol 57 675-709 956

Rook F Hadingham SA Li Y Bevan MW (2006) Sugar and ABA response 957

pathways and the control of gene expression Plant Cell Environ 29(3) 426-434 958

Sami F Yusuf M Faizan M Faraz A Hayat S (2016) Role of sugars under abiotic 959

stress Plant Physiol Bioch 109 54-61 960

Schneider S Hulpke S Schulz A Yaron I Houmlll J Imlau A Schmitt B Batz S 961

Wolf S Hedrich R Sauer N (2012) Vacuoles release sucrose via 962

tonoplast-localised SUC4-type transporters Plant Biology 14(2) 325-336 963

Shitan N and Yazaki K (2013) New insights into the transport mechanisms in plant 964

vacuoles Int Rev of Cell Mol Bio 305 383-433 965


30

Slewinski TL (2011) Diverse functional roles of monosaccharide transporters and 966

their homologs in vascular plants a physiological perspective Mol Plant 4(4) 967

641-662 968

Solfanelli C Poggi A Loreti E Alpi A Perata P (2006) Sucrose-specific induction 969

of the anthocyanin biosynthetic pathway in Arabidopsis Plant Physiol 140(2) 970

637-646 971

Sperdouli I and Moustakas M (2012) Interaction of proline sugars and 972

anthocyanins during photosynthetic acclimation of Arabidopsis thaliana to 973

drought stress J Plant Physiol 169(6) 577-585 974

Stolz J Ludwig A Stadler R Biesgen C Hagemann K Sauer N (1999) Structural 975

analysis of a plant sucrose carrier using monoclonal antibodies and 976

bacteriophage lambda surface display FEBS Lett 453(3) 375-379 977

Sun AJ Xu HL Gong WK Zhai HL Meng K Wang YQ Wei XL Xiao GF Zhu 978

Z (2008) Cloning and expression analysis of rice sucrose transporter genes 979

OsSUT2M and OsSUT5Z Journal Integr Plant Biol 50(1) 62-75 980

Tang T Xie H Wang YX Lu B Liang JS (2009) The effect of sucrose and abscisic 981

acid interaction on sucrose synthase and its relationship to grain filling of rice 982

(Oryza sativa L) J Exp Bot 60 2641-2652 983

Thalmann MR Pazmino D Seung D Horrer D Nigro A Meier T Koumllling K 984

Pfeifhofer HW Zeeman SC Santelia D (2016) Regulation of leaf starch 985

degradation by abscisic acid is important for osmotic stress tolerance in plants 986

Plant Cell tpc-00143 987

Valerio C Costa A Marri L Issakidis-Bourguet E Pupillo P Trost P Sparla F 988

(2011) Thioredoxin-regulated beta-amylase (BAM1) triggers diurnal starch 989

degradation in guard cells and in mesophyll cells under osmotic stress J Exp 990

Bot 62 545-555 991

Verslues PE and Sharma S (2011) Proline metabolism and its implications for 992

plant-environment interaction Arabidopsis Book 8 e0140 993

doi101199tab0140 994

Wormit A Trentmann O Feifer I Lohr C Tjaden J Meyer S Schmidt U 995

Martinoia E Neuhaus HE (2006) Molecular identification and physiological 996

characterization of a novel monosaccharide transporter from Arabidopsis 997

involved in vacuolar sugar transport Plant Cell 18 3476ndash3490 998

Xie XB Li S Zhang RF Zhao J Chen YC Zhao Q Yao YX You CX Zhang XS 999


31

Hao YJ (2012) The bHLH transcription factor MdbHLH3 promotes anthocyanin 1000

accumulation and fruit colouration in response to low temperature in apples 1001

Plant Cell Envir 35(11) 1884-1897 1002

Xu F Tan X Wang Z (2010) Effects of sucrose on germination and seedling 1003

development of Brassica Napus Inter J Biol 2 150e154 1004

Yamaki S (2010) Metabolism and accumulation of sugars translocated to fruit and 1005

their regulation J Jpn Soc Hortic Sci 79(1) 1-15 1006

Yang J Zhang J Wang Z Xu G Zhu Q (2004) Activities of key enzymes in 1007

sucrose-to-starch conversion in wheat grains subjected to water deficit during 1008

grain filling Plant Physiol 135(3) 1621-1629 1009

Yao YX Li M You CX Li Z Wang DM Hao YJ (2010) Relationship between 1010

malic acid metabolism-related key enzymes and accumulation of malic acid as 1011

well as the soluble sugars in apple fruit Acta Horticulturae Sinica 37(1) 1-8 1012

Yoshida T Fujita Y Maruyama K Mogami J Todaka D Shinozaki K 1013

Yamaguchi-shinozaki K (2015) Four Arabidopsis AREBABF transcription 1014

factors function predominantly in gene expression downstream of SnRK2 1015

kinases in abscisic acid signalling in response to osmotic stress Plant Cell Envir 1016

38(1) 35-49 1017

Yoshida T Fujita Y Sayama H Kidokoro S Maruyama K Mizoi J Shinozaki K 1018

Yamaguchi-Shinozaki K (2010) AREB1 AREB2 and ABF3 are master 1019

transcription factors that cooperatively regulate ABRE-dependent ABA signaling 1020

involved in drought stress tolerance and require ABA for full activation Plant J 1021

61 672-685 1022

Zanella M Borghi GL Pirone C Thalmann M Pazmino D Costa A Santelia D 1023

Trost P and Sparla F (2016) b-Amylase1 (BAM1) degrades transitory starch to 1024

sustain proline biosynthesis during drought stress J Exp Bot 67 1819-1826 1025

Zhang LY Peng YB Pelleschi-Travier S Fan Y Lu YF Lu YM Gao XP Shen 1026

YY Delrot S Zhang DP (2004) Evidence for apoplasmic phloem unloading in 1027

developing apple fruit Plant Physiol 135(1) 574-586 1028

Zhao Q Ren YR Wang QJ Wang XF You CX and Hao YJ (2016) 1029

Ubiquitination-related MdBT scaffold Proteins Target a bHLH transcription 1030

factor for Iron Homeostasis Plant Physiol 172(3) 1973-1988 1031

Zheng QM Tang Z Xu Q Deng XX (2014) Isolation phylogenetic relationship and 1032

expression profiling of sugar transporter genes in sweet orange (Citrus sinensis) 1033


32

Plant Cell Tiss Org 119(3) 609-624 1034

1035

1036











Parsed CitationsAkihiro T Mizuno K Fujimura T (2005) Gene expression of ADP-glucose pyrophosphorylase and starch contents in rice culturedcells are cooperatively regulated by sucrose and ABA Plant Cell Physiol 46(6) 937-946

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

Barker L Kuumlhn C Weise A Schulz A Gebhardt C Hirner B Hellmann H Schulze W Ward JM Frommer WB (2000) SUT2 aputative sucrose sensor in sieve elements Plant Cell 12(7) 1153-1164


Bhatia S and Singh R (2002) Phytohormone-mediated transformation of sugars to starch in relation to the activities of amylasessucrose-metabolising enzymes in sorghum grain Plant Growth Regul 36 97-104


Chen Z Hong X Zhang H Wang Y Li X Zhu JK Gong Z (2005) Disruption of the cellulose synthase gene AtCesA8IRX1 enhancesdrought and osmotic stress tolerance in Arabidopsis Plant J 43(2) 273-283


Chung P Hsiao HH Chen HJ Chang CW Wang SJ (2014) Influence of temperature on the expression of the rice sucrosetransporter 4 gene OsSUT4 in germinating embryos and maturing pollen Acta Physiol Plant 36(1) 217-229


Ferrandino A and Lovisolo C (2014) Abiotic stress effects on grapevine (Vitis vinifera L) focus on abscisicacid-mediatedconsequences on secondary metabolism and berry quality Environ Exp Bot 103(1) 138-147


Finkelstein R Gibson SI (2002) ABA and sugar interactions regulating development cross-talk or voices in a crowd Curr OpinPlant Biol 5 26-32


Fujita Y Fujita M Satoh R Maruyama K Parvez M Seki M Hiratsu K Ohme-Takagi M Shinozaki K Yamaguchi-Shinozaki K (2005)AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in ArabidopsisPlant Cell 17(12) 3470-3488


Gibson SI (2004) Sugar and phytohormone response pathways navigating a signalling network J Exp Bot 55(395) 253-264Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Gibson SI (2005) Control of plant development and gene expression by sugar signaling Curr Opin Plant Biol 8(1) 93-102Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Goacutemez LD Gilday A Feil R Lunn JE Graham IA (2010) AtTPS1-mediated trehalose 6-phosphate synthesis is essential forembryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells Plant J 64(1) 1-13


Guan Y Ren H Xie H Ma Z Chen F (2009) Identification and characterization of bZIP-type transcription factors involved in carrot(Daucus carota L) somatic embryogenesis Plant J 60(2) 207-217


Hammond J Broadley M Bowen H Spracklen W Hayden R White P (2011) Gene expression changes in phosphorus deficientpotato (Solanum tuberosum L) leaves and the potential for diagnostic gene expression markers PloS One 6 9


Hayes MA Feechan A Dry IB (2010) Involvement of abscisic acid in the coordinated regulation of a stress-inducible hexose wwwplantphysiolorgon March 4 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

transporter (VvHT5) and a cell wall invertase in grapevine in response to biotrophic fungal infection Plant Physiol 153(1) 211-221Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Hu DG Sun CH Ma QJ You CX Cheng L Hao YJ (2016) MdMYB1 regulates anthocyanin and malate accumulation by directlyfacilitating their transport into vacuoles in apples Plant Physiol 170(3) 1315-1330


Hu M Shi Z Zhang Z Zhang Y Li H (2012) Effects of exogenous glucose on seed germination and antioxidant capacity in wheatseedlings under salt stress Plant Growth Regul 68 177e188


Ibraheem O Dealtry G Roux S Bradley G (2011) The effect of drought and salinity on the expressional levels of sucrosetransporters in rice (Oryza sativa Nipponbare) cultivar plants Plant Omics 4(2) 68


Islam MZ Jin LF Shi CY Liu YZ Peng SA (2015) Citrus sucrose transporter genes genome-wide identification and transcriptanalysis in ripening and ABA-injected fruits Tree Genet Genomes 11(5) 1-9


Johnson R R Wagner R L Verhey S D amp Walker-Simmons M K (2002) The abscisic acid-responsive kinase pkaba1 interacts with aseed-specific abscisic acid response element-binding factor taabf and phosphorylates taabf peptide sequences Plant Physiol130(2) 837


Kafi M Stewart WS Borland AM (2003) Carbohydrate and proline contents in leaves roots and apices of salt-tolerant and salt-sensitive wheat cultivars 1 Russ J Plant Physiol 50(2) 155-162


Kagaya Y Hobo T Murata M Ban A amp Hattori T (2002) Abscisic acid-induced transcription is mediated by phosphorylation of anabscisic acid response element binding factor trab1 Plant Cell 14(12) 3177-89


Khan HA Siddique KH Colmer TD (2016) Vegetative and reproductive growth of salt-stressed chickpea are carbon-limitedsucrose infusion at the reproductive stage improves salt tolerance J Exp Bot erw177


Kim JS Mizoi J Yoshida T Fujita Y Nakajima J Ohori T Todaka D Nakashima K Hirayama T Shinozaki K Yamaguchi-Shinozaki K(2011) An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene which encodes atranscription factor regulating drought-inducible genes in Arabidopsis Plant Cell Physiol 52(12) 2136-2146


Krasensky J and Jonak C (2012) Drought salt and temperature stress-induced metabolic rearrangements and regulatorynetworks J Exp Bot 63 1593-1608


Lalonde S Wipf D Frommer WB (2004) Transport mechanisms for organic forms of carbon and nitrogen between source and sinkAnnu Rev Plant Biol 55 341-372


Lastdrager J Hanson J Smeekens S (2014) Sugar signals and the control of plant growth and development J Exp Bot 65(3) 799-807


Lecourieux F Kappel C Lecourieux D Serrano A Torres E Arce-Johnson P Delrot S (2013) An update on sugar transport and wwwplantphysiolorgon March 4 2020 - Published by Downloaded from

Copyright copy 2017 American Society of Plant Biologists All rights reserved

signalling in grapevine J Exp Bot ert394Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lee SC and Luan S (2012) Aba signal transduction at the crossroad of biotic and abiotic stress responses Plant Cell amp Environ35(1) 53


Li YY Mao K Zhao C Zhao XY Zhang HL Shu HR Hao YJ (2012) MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulatelight-induced anthocyanin biosynthesis and red fruit coloration in apple Plant Physiol 160(2) 1011-1022


Lu R Guyer DE Beaudry RM (2000) Determination of firmness and sugar content of apples using near-infrared diffusereflectance1 J Texture Stud 31(6) 615-630


Lundmark M Cavaco AM Trevanion S Hurry V (2006) Carbon partitioning and export in transgenic Arabidopsis thaliana withaltered capacity for sucrose synthesis grown at low temperature a role for metabolite transporters Plant Cell Environ 29(9) 1703-1714


Lv S Zhang K Gao Q Lian L Song Y Zhang J (2008) Overexpression of an H+-PPase gene from Thellungiella halophila in cottonenhances salt tolerance and improves growth and photosynthetic performance Plant Cell Physiol 49(8) 1150-1164


Ma QJ Sun MH Liu YJ Lu J Hu DG Hao YJ (2016) Molecular cloning and functional characterization of the apple sucrosetransporter gene MdSUT2 Plant Physiol Bioch 109 442-451


Martinoia E Maeshima M Neuhaus HE (2007) Vacuolar transporters and their essential role in plant metabolism J Exp Bot 58(1)83-102


Mayaba N Beckett RP Csintalan Z Tuba Z (2001) ABA increases the desiccation tolerance of photosynthesis in the afromontaneunderstorey moss Atrichum androgynum Ann Bot London 88(6) 1093-11


Medici A Laloi M Atanassova R (2014) Profiling of sugar transporter genes in grapevine coping with water deficit FEBS Lett588(21) 3989-3997


Moustakas M Sperdouli I Kouna T Antonopoulou CI Therios I (2011) Exogenous proline induces soluble sugar accumulation andalleviates drought stress effects on photosystem II functioning of Arabidopsis thaliana leaves Plant Growth Regul 65(2) 315-325


Oumlzcan S Dover J and Johnston M (1998) Glucose sensing and signaling by two glucose receptors in the yeast Saccharomycescerevisiae EMBO J 17(9) 2566-2573


Price J Li TC Kang SG Na JK amp Jang JC (2003) Mechanisms of glucose signaling during germination of Arabidopsis PlantPhysiol 132(3) 1424


Rasheed R Wahid A Farooq M Hussain I Basra SM (2011) Role of proline and glycinebetaine pretreatments in improving heattolerance of sprouting sugarcane (Saccharum sp) buds Plant Growth Regul 65(1) 35-45



Reuscher S Akiyama M Yasuda T Makino H Aoki K Shibata D amp Shiratake K (2014) The sugar transporter inventory of tomatogenome-wide identification and expression analysis Plant Cell Physiol 55(6) 1123-1141


Rolland F Baena-Gonzalez E Sheen J (2006) Sugar sensing and signaling in plants conserved and novel mechanisms Annu RevPlant Biol 57 675-709


Rook F Hadingham SA Li Y Bevan MW (2006) Sugar and ABA response pathways and the control of gene expression Plant CellEnviron 29(3) 426-434


Sami F Yusuf M Faizan M Faraz A Hayat S (2016) Role of sugars under abiotic stress Plant Physiol Bioch 109 54-61Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Schneider S Hulpke S Schulz A Yaron I Houmlll J Imlau A Schmitt B Batz S Wolf S Hedrich R Sauer N (2012) Vacuoles releasesucrose via tonoplast-localised SUC4-type transporters Plant Biology 14(2) 325-336


Shitan N and Yazaki K (2013) New insights into the transport mechanisms in plant vacuoles Int Rev of Cell Mol Bio 305 383-433Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Slewinski TL (2011) Diverse functional roles of monosaccharide transporters and their homologs in vascular plants aphysiological perspective Mol Plant 4(4) 641-662


Solfanelli C Poggi A Loreti E Alpi A Perata P (2006) Sucrose-specific induction of the anthocyanin biosynthetic pathway inArabidopsis Plant Physiol 140(2) 637-646


Sperdouli I and Moustakas M (2012) Interaction of proline sugars and anthocyanins during photosynthetic acclimation ofArabidopsis thaliana to drought stress J Plant Physiol 169(6) 577-585


Stolz J Ludwig A Stadler R Biesgen C Hagemann K Sauer N (1999) Structural analysis of a plant sucrose carrier usingmonoclonal antibodies and bacteriophage lambda surface display FEBS Lett 453(3) 375-379


Sun AJ Xu HL Gong WK Zhai HL Meng K Wang YQ Wei XL Xiao GF Zhu Z (2008) Cloning and expression analysis of ricesucrose transporter genes OsSUT2M and OsSUT5Z Journal Integr Plant Biol 50(1) 62-75


Tang T Xie H Wang YX Lu B Liang JS (2009) The effect of sucrose and abscisic acid interaction on sucrose synthase and itsrelationship to grain filling of rice (Oryza sativa L) J Exp Bot 60 2641-2652


Thalmann MR Pazmino D Seung D Horrer D Nigro A Meier T Koumllling K Pfeifhofer HW Zeeman SC Santelia D (2016) Regulationof leaf starch degradation by abscisic acid is important for osmotic stress tolerance in plants Plant Cell tpc-00143


Valerio C Costa A Marri L Issakidis-Bourguet E Pupillo P Trost P Sparla F (2011) Thioredoxin-regulated beta-amylase (BAM1) wwwplantphysiolorgon March 4 2020 - Published by Downloaded from Copyright copy 2017 American Society of Plant Biologists All rights reserved

triggers diurnal starch degradation in guard cells and in mesophyll cells under osmotic stress J Exp Bot 62 545-555Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Verslues PE and Sharma S (2011) Proline metabolism and its implications for plant-environment interaction Arabidopsis Book 8e0140 doi101199tab0140


Wormit A Trentmann O Feifer I Lohr C Tjaden J Meyer S Schmidt U Martinoia E Neuhaus HE (2006) Molecular identificationand physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transportPlant Cell 18 3476-3490


Xie XB Li S Zhang RF Zhao J Chen YC Zhao Q Yao YX You CX Zhang XS Hao YJ (2012) The bHLH transcription factorMdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples Plant Cell Envir35(11) 1884-1897


Xu F Tan X Wang Z (2010) Effects of sucrose on germination and seedling development of Brassica Napus Inter J Biol 2150e154


Yamaki S (2010) Metabolism and accumulation of sugars translocated to fruit and their regulation J Jpn Soc Hortic Sci 79(1) 1-15Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Yang J Zhang J Wang Z Xu G Zhu Q (2004) Activities of key enzymes in sucrose-to-starch conversion in wheat grains subjectedto water deficit during grain filling Plant Physiol 135(3) 1621-1629


Yao YX Li M You CX Li Z Wang DM Hao YJ (2010) Relationship between malic acid metabolism-related key enzymes andaccumulation of malic acid as well as the soluble sugars in apple fruit Acta Horticulturae Sinica 37(1) 1-8


Yoshida T Fujita Y Maruyama K Mogami J Todaka D Shinozaki K Yamaguchi-shinozaki K (2015) Four Arabidopsis AREBABFtranscription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling inresponse to osmotic stress Plant Cell Envir 38(1) 35-49


Yoshida T Fujita Y Sayama H Kidokoro S Maruyama K Mizoi J Shinozaki K Yamaguchi-Shinozaki K (2010) AREB1 AREB2 andABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stresstolerance and require ABA for full activation Plant J 61 672-685


Zanella M Borghi GL Pirone C Thalmann M Pazmino D Costa A Santelia D Trost P and Sparla F (2016) b-Amylase1 (BAM1)degrades transitory starch to sustain proline biosynthesis during drought stress J Exp Bot 67 1819-1826


Zhang LY Peng YB Pelleschi-Travier S Fan Y Lu YF Lu YM Gao XP Shen YY Delrot S Zhang DP (2004) Evidence forapoplasmic phloem unloading in developing apple fruit Plant Physiol 135(1) 574-586


Zhao Q Ren YR Wang QJ Wang XF You CX and Hao YJ (2016) Ubiquitination-related MdBT scaffold Proteins Target a bHLHtranscription factor for Iron Homeostasis Plant Physiol 172(3) 1973-1988



Zheng QM Tang Z Xu Q Deng XX (2014) Isolation phylogenetic relationship and expression profiling of sugar transporter genesin sweet orange (Citrus sinensis) Plant Cell Tiss Org 119(3) 609-624



Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

2

Transcription factor AREB2 is involved in soluble sugar accumulation by 24

activating sugar transporter and amylase genes 25

26

Qi-Jun Ma Mei-Hong Sun Jing Lu Ya-Jing Liu Da-Gang Hu Yu-Jin Hao 27

28

29

National Key Laboratory of Crop Biology National Research Center for Apple 30

Engineering and Technology College of Horticulture Science and Engineering 31


33

34

35

The corresponding author Yu-Jin Hao 36

Tel +86-538-824-6692 37

E-mail haoyujinsdaueducn 38

39

One Sentence Summary the ABA-responsive transcription factor MdAREB2 directly activates 40

the expression of amylase and sugar transporter genes to promote soluble sugar accumulation 41

42

43

44

45

46

47


3

48

49

Abstract 50















65


67

Introduction 68















4




































5



















2009) 134

















6




Results 153
































7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

3

48

49

Abstract 50















65


67

Introduction 68















4




































5



















2009) 134

















6




Results 153
































7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

4




































5



















2009) 134

















6




Results 153
































7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

5



















2009) 134

















6




Results 153
































7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

6




Results 153
































7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

7




































8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

8




































9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

9




































10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

10

















4D) 302







MdSUT2 promoter 309












11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

11












to ABA 331
























12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

12






needs MdSUT2 359











7A) 370



















13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

13






callus 393











404

Discussion 405


















14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

14









all tissue 430



























15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







31



Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

1036
















































































































































Parsed Citations

Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Parsed Citations

15




































16




































17



al 2011) 526


















544






548







daynight cycle 554




556



18



and 05 mgL-1












(2016) 570























19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































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29












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2566-2573 946





















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Bot 62 545-555 991



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544






548







daynight cycle 554




556



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and 05 mgL-1












(2016) 570























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GUS assays 648













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Li et al (2012) 669







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680





685

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701









710















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753










24


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26

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Plant J 64(1) 1-13 863


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341-372 908





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Growth Regul 65(2) 315-325 943



2566-2573 946





















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doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















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61 672-685 1022













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544






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Li et al (2012) 669







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680





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Figure legends 686









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701









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753










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763



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784













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799


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26

830

































Plant J 64(1) 1-13 863


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Physiol 153(1) 211-221 873



















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341-372 908





Bot ert394 913






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Plant Cell Envir 35(11) 1884-1897 1002















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(2016) 570























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20























GUS assays 648













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Li et al (2012) 669







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680





685

Figure legends 686









22








701









710















725




23


























753










24


763



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MdSUT2 775









784













25




799


801




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MdSUT2-3 4 6789 810




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genes 827




26

830

































Plant J 64(1) 1-13 863


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Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







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341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




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2566-2573 946





















30



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Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















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61 672-685 1022













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19




































20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















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61 672-685 1022













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20























GUS assays 648













21










Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















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61 672-685 1022













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Li et al (2012) 669







676




680





685

Figure legends 686









22








701









710















725




23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













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701









710















725




23


























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24


763



cultures 766







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MdSUT2 775









784













25




799


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MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

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Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













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23


























753










24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

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Figure 1

Figure 2

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24


763



cultures 766







773


MdSUT2 775









784













25




799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



doi101199tab0140 994







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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

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799


801




fruit 805





MdSUT2-3 4 6789 810




MdAREB2-3 5 678 814













genes 827




26

830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

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830

































Plant J 64(1) 1-13 863


27










Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













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1035

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Physiol 153(1) 211-221 873



















J Plant Physiol 50(2) 155-162 892







28











341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



641-662 968



637-646 971




















Bot 62 545-555 991



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Plant Cell Envir 35(11) 1884-1897 1002















38(1) 35-49 1017





61 672-685 1022













32


1035

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341-372 908





Bot ert394 913






1011-1022 919






Environ 29(9) 1703-1714 925




1150-1164 929




29












Growth Regul 65(2) 315-325 943



2566-2573 946





















30



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Plant Cell Envir 35(11) 1884-1897 1002















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61 672-685 1022













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running head: mdareb2 regulates soluble sugar …111. supply (khan et al., 2016). 112. as mentioned...

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