3 Ting Wu, Email: [email protected], Tel.: +86-010-627311187 133 the active Fe contents in M....

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Short title: IRT1 promoter variation alters Fe uptake 1 Corresponding authors: 2 Ting Wu, Email: [email protected], Tel.: +86-010-62731118 3

Zhenhai Han, Email: [email protected], Tel.: +86-010-62732467 4

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Plant Physiology Preview. Published on November 23, 2016, as DOI:10.1104/pp.16.01504

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TATA box insertion provides a selection mechanism underpinning adaptations to 6 Fe deficiency 7

8

Meiling Zhang1†, Yuanda Lv1†, Yi Wang1†, Jocelyn K. C. Rose2, Fei Shen1，9

Zhenyun Han1, Xinzhong Zhang1, Xuefeng Xu1, Ting Wu1*, Zhenhai Han1* 10 11 1Institute for Horticultural Plants, China Agricultural University, Beijing 100193, P. 12

R. China 13 2Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA 14

15

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17 †These authors contributed equally to this work. 18 *To whom correspondence should be addressed. 19

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21 22

One Sentence Summary: TATA-box insertion in the upstream untranslated sequences of 23 genes enhances adaptation to selective pressure. 24 25

26

The author responsible for distribution of materials integral to the findings 27

presented in this article in accordance with the policy described in the Instructions 28

for Authors (www. plant physiol.org) are: Ting Wu ([email protected]); Zhenhai 29

Han ([email protected]). 30

31



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Author contributions: 32 T.W. and Z.H. conceived and designed research. M.Z., Y.L., S.F., Z. H., and X.X. 33

conducted experiments. Y.W. and X.Z. contributed new reagents or analytical tools. 34

M.Z. and T.W. wrote the manuscript. J.R. gave advice and edited the manuscript. All 35

authors read and approved the manuscript. 36

37 Funding information: 38 Financial support was provided by the National Natural Science Foundation of 39

China (No. 31272139 and No. 31401840), Beijing Natural Science Foundation (No. 40

6154028), China Postdoctoral Science Foundation (No. 2016M590157), Special 41

Fund for Agro-scientific Research in the Public Interest (No. 201203075 and 42

No.201303093), Beijing Municipal Education Commission (CEFF-43

PXM2016_014207_000038), and Key Labs Nutrition and Physiology for 44

Horticultural Crops, and Physiology and Molecular Biology of Tree Fruit. JKCR is 45

supported by an affiliation with the Atkinson Center for a Sustainable Future. 46

47 48



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Abstract 49 Intraspecific genetic variation is essential for the responses and adaption of plants to 50

evolutionary challenges, such as changing environmental conditions. The 51

development of the Earth’s aerobic atmosphere has increased the demand for iron (Fe) 52

in organisms, and Fe deficiency has become a limiting environmental factor for plant 53

growth. Here, we demonstrate that apple trees adapt to Fe deficiency through 54

modification of the Iron-Regulated Transporter1 (IRT1) promoter. Specifically, an 55

IRT1 mutant allele with a TATA-box insertion in the promoter region upstream of the 56

coding region exhibited increased IRT1 expression. The altered IRT1 promoter is 57

responsible for enhancing Fe uptake. Increasing the number of synthetic repeat TATA-58

boxes correlates with increased promoter activity. Furthermore, we demonstrate that 59

the insertion of the TATA-box correlates with an increase in transcriptional activation 60

via specific binding of the transcription factor IID (TF IID; MDP0000939369). Taken 61

together, these results indicate that an allelic insertion of a TATA-box in a gene 62

promoter has allowed apple to adapt to the selective pressure posed by Fe deficiency. 63

More broadly, this study reveals a new mechanism for enhancing gene expression to 64

help plants adapt to different environments, providing new insights into molecular 65

genetic divergence in plants. 66

67



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INTRODUCTION 68 A diverse portfolio of crops that can adapt to potentially adverse environmental 69

conditions is essential to meet the demands of increasingly intensive agricultural 70

production (Huang and Han, 2014), particularly in the context of climate change and 71

global population increases. Genetic variants of cultivated species derived from wild 72

progenitors of plants under both natural and human selection provide vital sources of 73

this type of genetic variation. Therefore, there is considerable interest in identifying 74

allelic variants in natural populations and determining whether they have a selective 75

advantage under specific conditions. Some variants account for all of the heritability 76

of gene expression attributable to cis-regulatory elements. Variations in the expression 77

of genes associated with agriculturally important traits due to transposon insertion or 78

differential expansion of microsatellite repeat sequences have been identified 79

(Sureshkumar and Lee, 2009; Wang et al., 2012). 80

A critical factor that can limit crop production is the availability of the 81

micronutrient Fe, which plays important roles in a wide range of cellular processes, 82

including photosynthesis and respiration (Bonner and Varner, 1976; Briat et al., 1995; 83

Le, 2002). During the early period of the Earth’s history, the atmosphere was anoxic, 84

and water-soluble ferrous Fe was the form of Fe used by early organisms. However, 85

the development of an oxygen-rich atmosphere has resulted in the loss of the 86

bioavailable form of Fe, Fe (II), and the accumulation of insoluble Fe (III) (Crichton 87

and Pierre, 2001; Darbani et al., 2013). Therefore, Fe is increasingly becoming a 88

limiting factor for plant growth and development (Abadía et al., 2011). To adapt to 89

these changes, plants have developed two Fe uptake strategies (I and II) (Marschner et 90

al., 1986a; Marschner et al., 1986b; Briat et al., 1995), which are exhibited in non-91

graminaceous and graminaceous plants, respectively. The Strategy I response, also 92

known as the Reduction Strategy, involves the reduction of ferric chelates at the root 93

surface and absorption of the generated ferrous ions across the root plasma 94

membrane. Strategy II relies on the biosynthesis and secretion of Fe(III)-solubilizing 95

molecules, termed mugineic acids (MAs), which chelate Fe; therefore, this strategy is 96

also known as the Chelation Strategy (Marschner et al., 1986a; Marschner et al., 97

1986b; Briat et al., 1995). In Strategy I plants, the transporter IRT1 plays a major role 98

in regulating Fe homeostasis, and its expression is induced by Fe (Henriques et al., 99

2002; Vert, 2002). Some species, such as tomato (Solanum lycopersicum M.), lettuce 100



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(Lactuca sativa L.), and peanut (Arachis hypogaea L.), show different levels of 101

resistance to Fe deficiency, depending on the genotype (Li and Wang, 1995; Chen et 102

al., 2001; Xu et al., 2012; Xia et al., 2013). QTL studies investigating the genetic 103

basis of mineral tolerance in crops have focused on mineral concentrations in seeds or 104

leaves, and most have targeted Strategy II plants, such as rice, wheat, and barley 105

(Alonso-Blanco et al., 2009). Despite these efforts, the molecular basis of the natural 106

variation in Fe uptake systems and the mechanisms determining Fe uptake affinity 107

remain largely undefined. 108

In the current study, we provide evidence that a TATA-box insertion in the IRT1 109

promoter in the genus Malus increases the expression of IRT1, which accounts for the 110

increase in Fe uptake in various Malus species. Furthermore, our findings suggest that 111

the binding of TFIID to the IRT1 promoter is enhanced by the presence of a TATA-112

box insertion, leading to increased IRT1 transcript levels and enhanced Fe uptake. 113

This represents a novel mechanism for genetic divergence in which a TATA-box 114

insertion promotes environmental adaption. 115

116

RESULTS 117

Microtomography Analysis of Fe Distribution in Roots Reveals Variants with Fe-118 Deficiency Resistance 119

Among members of the genus Malus, M. xiaojinensis and M. baccata have well-120

developed root systems and are valued in China as native apple rootstocks with good 121

grafting compatibility for the development of new apple cultivars (Han et al., 1994; 122

Han et al., 1998). However, while M. xiaojinensis is an Fe-efficient species, M. 123

baccata has much lower Fe uptake efficiency. We confirmed this difference using 124

microtomography analysis of Fe distribution in the roots of plants of both species. 125

Figure 1 shows X-ray fluorescence (XRF) maps of Fe distribution patterns in cross-126

sections of roots under normal (40 μM Fe2+) and low (4 μM Fe2+) Fe conditions. 127

Under both conditions, the Fe intensity in cross-sections of M. xiaojinensis roots was 128

high, whereas reduced levels or the complete absence of Fe was detected in M. 129

baccata (Fig. 1D, E). Fe, the ferrous-iron (Fe2+) form termed “active Fe”, which can 130

be extracted with weak acids and some chelating agents, is closely associated with Fe 131

chlorosis (Köseoğlu and Açikgöz, 1995; Zha et al., 2014). Furthermore, we measured 132



7

the active Fe contents in M. xiaojinensis and M. baccata supplied with 0 μM Fe2+ or 133

40 μM Fe2+ for 0, 3, and 6 days. Under Fe-sufficient conditions, the active Fe contents 134

were significantly lower in the roots of M. baccata than in those of M. xiaojinensis. 135

Similarly, continuous Fe deficiency induced a more rapid decrease in Fe levels in the 136

roots of M. baccata versus M. xiaojinensis. Therefore, the different Fe levels are 137

consistent with different levels of Fe uptake efficiency between M. xiaojinensis and 138

M. baccata (Fig. 2B). 139

Identification of an IRT1 Mutant Allele with a TATA-box Insertion 140

We previously reported that M. xiaojinensis has higher IRT1 mRNA levels than M. 141

baccata (Zha et al., 2014) and that Fe deficiency induces a substantial increase in 142

IRT1 expression in M. xiaojinensis, but not in M. baccata (Fig. 2C), suggesting that a 143

modified allele of IRT1 with enhanced expression might be responsible for the 144

enhanced Fe-efficiency of this species. To elucidate the genetic mechanism 145

underlying this difference, we analyzed the gene sequences of IRT1 from M. 146

xiaojinensis and M. baccata. As shown in Figure 2, M. xiaojinensis has a 147

TTG…ATTATAA insertion in the promoter of the IRT1 coding region that is absent 148

in the equivalent sequence of M. baccata. The insertion, -363 to -357 bp from the 149

ATG start site, is predicted to be a core promoter element TATA-box. Alleles 150

containing or lacking the TATA-box insertion were designated FUE (Fe uptake 151

efficiency) and FUI (Fe uptake inefficiency), respectively (Accession No. KX549782, 152

KX549781) (Fig. 2D, Supplemental Fig. S1). 153

We confirmed the association between the FUE allele and Fe uptake efficiency by 154

analyzing segregating progeny from a cross between M. xiaojinensis (FUE/FUI) and 155

M. baccata (FUI/FUI). We observed a strong negative correlation (R2=0.7536, 156

P

8

IRT1 expression in plants of the same ploidy level and found that the presence of a 166

high copy number of the TATA-box insertion helped prevent Fe deficiency-induced 167

chlorosis in 2x, 3x, and 4x plants (Fig. 3). Furthermore, our results show a positive 168

correlation between the frequency of TATA-box insertion and IRT1 expression in 2x, 169

3x, and 4x plants. However, in 5x plants, no correlation between chlorosis and TATA-170

box insertion was observed that could be explained by gene dosage effects (Fig. 3). 171

Therefore, the results of genetic analysis indicate that the TATA-box insertion 172

conferred Fe-deficiency resistance to most of the progeny. 173

A TATA-box Insertion in the Promoter of IRT1 Alters its Expression 174

Since the TATA-box can play a role in the activation of eukaryotic genes transcribed 175

by RNA polymerase II (Burley and Roeder, 1996), a possible consequence of the 176

TATA-box insertion within the IRT1 promoter is an increase in the basal activity of 177

the promoter. We investigated this possibility using transient expression assays in 178

Arabidopsis thaliana mesophyll protoplasts. We fused the FUE and FUI promoter 179

sequences to the green fluorescence protein (GFP) coding sequence, expressed the 180

fusion construct in protoplasts, and measured GFP expression relative to that of co-181

expressed β-glucuronidase (GUS) driven by the constitutive 35S promoter. The GFP 182

reporter exhibited 1.58-fold greater expression when driven by the FUE promoter 183

compared to the FUI promoter (Fig. 4). We also assessed the activities of the 184

promoters via transient expression in tobacco (Nicotiana benthamiana) leaves 185

harboring each promoter fused to the GUS coding sequence. Again, the FUE promoter 186

had higher activity than the FUI promoter (Supplemental Fig. S3). Taken together, 187

these results suggest that the TATA-box insertion enhances the basal activity of the 188

IRT1 promoter. 189

To further investigate the effects of the TATA-box insertion on IRT1 promoter 190

activity, we produced a series of synthetic constructs to test the effects of the number 191

of TATA-box repeat units in the upstream region of IRT1 on its transcriptional 192

activity. These constructs, which were based on the native promoter sequences, but 193

with the addition of zero (0) to three (3) TATA-boxes fused to the GUS reporter (Fig. 194

4C, Supplemental Data 1), were assayed as above. The results show a strong positive 195

correlation between the number of repeat TATA-box insertions and promoter activity 196

(Fig. 4D). 197

We also found that the TATA-box insertion is tightly linked to a base mutation in 198



9

the coding sequence (CDS) at + 96 bp (G/T), which results in a missense mutation 199

and an amino acid change from E to D (Fig. 2D). To assess the effects of this mutation 200

of FUE on Fe uptake, we performed functional complementation of yeast mutant 201

DEY1453 (fet3fet4). The CDS of both FUE and FUI successfully complemented the 202

high-affinity Fe uptake defect of the mutant. We therefore conclude that the TATA-203

box-linked base mutation does not affect the function of IRT1 (Supplemental Fig. S4). 204

FUE:IRT1-Overexpressing Plants Exhibit Increased Fe Storage 205

To further investigate the effects of the TATA-box insertion on Fe storage, we 206

introduced IRT1 driven by either the FUE or FUI promoter into tobacco. X-ray 207

fluorescence (XRF) analyses showed that more Fe accumulated in transgenic 208

FUE:IRT1 versus FUI:IRT1 plants (Fig. 5A). Moreover, IRT1 transcript levels were 209

significantly higher in FUE:IRT1 roots than in FUI:IRT1 roots. We also found that the 210

FUE:IRT1 promoter increased the expression of the GFP reporter 1.5-fold in roots 211

relative to the FUI:IRT1 promoter (Fig. 5B). 212

The TATA-box Insertion Enhances IRT1 Expression via the Specific Binding of 213 TF IID 214

RNA polymerase II-mediated transcription by the pre-initiation complex (PIC) begins 215

with the binding of TFIID to the TATA-box. We therefore investigated the binding of 216

TFIID to the TATA-box identified in this study using yeast one-hybrid (Y1H) 217

analysis. Phylogenetic analysis identified 29 genes that are most closely related to 218

TFIID in the apple genome (Supplemental Fig. S5), 11 of which are expressed in the 219

roots of M. xiaojinensis. We therefore investigated the ability of the corresponding 220

proteins to bind to the TATA-box (Supplemental Table S1). One TFIID protein 221

(MDP0000495467) bound to both the FUE and FUI promoters, with no difference in 222

binding activity detected between the two. However, another TFIID protein 223

(MDP0000939369) bound to the FUE promoter, but not to the FUI promoter (Fig. 224

6A). To confirm this finding, we analyzed in vitro binding using electrophoretic 225

mobility shift assays (EMSAs). Recombinant GST-tagged TFIID protein 226

(MDP0000939369) was purified from Escherichia coli and incubated with DNA 227

probes representing the FUE or FUI promoter (Table S2). MDP0000939369 228

specifically bound only to biotin-labeled FUE (Fig. 6B). Finally, we measured TBP 229

(MDP0000939369) expression in M. xiaojinensis and M. baccata supplied with 0 μM 230



10

Fe2+ or 40 μM Fe2+ for 0, 3, or 6 days, finding that Fe deficiency induced a substantial 231

increase in TBP expression in M. xiaojinensis, but not in M. baccata (Fig. 6C). In 232

addition, TBP expression was positively correlated with IRT1 expression (R2=0.9043, 233

P < 0.001) (Fig. 6D). 234

Based on the data presented in this study, we propose a model for the regulation of 235

IRT1 expression by the TATA-box insertion regulatory module. The model suggests 236

that the specific binding of TFIID protein (MDP0000939369) to FUE leads to an 237

increase in IRT1 transcript levels and, consequently, Fe uptake (Fig. 6E). 238

Evolutionary Analysis of the TATA-box Insertion 239

To confirm the role of the FUE allele in the evolution of Malus species, we 240

allelotyped the FUE/FUI loci of 52 accessions belonging to four different Malus 241

species (Phipps et al. 1990; Yao et al. 2015) (Supplemental Table S1): the 52 242

accessions represent the diversity of the four Malus species. We found that the 243

nucleotide diversity (Θ and π values) of the IRT1 allele was significantly lower in M. 244

domestica than in the other species (Table 1). Tajima’s D tests (Tajima, 1989) detected 245

departure from neutrality for the IRT1 genes of M. sieversii and M. domestica, 246

indicating that the IRT1 allele is under selection. 247

248

DISCUSSION 249

Our data suggest that a TATA-box insertion in the upstream regulatory region of IRT1 250

functions in a novel regulatory mechanism. Specifically, we present evidence that the 251

TATA-box insertion enhances IRT1 expression to control Fe uptake efficiency as a 252

result of specific binding of TFIID to the TATA box. 253

Plants exhibit substantial natural variation in mineral use efficiency, root uptake, 254

translocation from roots to shoots, and accumulation (Alonso-Blanco et al., 2009). 255

QTL studies in various crop species have focused on seed or leaf mineral 256

concentrations, primarily in rice, wheat, and barley, which use the Chelation Strategy 257

(Strategy II) to acquire Fe. These studies have resulted in the identification of several 258

natural alleles differing in missense mutations in conserved motifs exhibiting reduced 259

P-Type ATPase activity and copper (Cu) translocation to the shoot in Arabidopsis 260

(Kobayashi et al., 2008), as well as a 1-kb insertion in the promoter that increases the 261

expression of citrate transporter gene HvAACT1 in several Al-tolerant barley cultivars 262



11

(Fujii et al., 2012). In addition, natural variation in the rice Nramp aluminum 263

transporter (NRAT1) gene results in significant differences in Al tolerance (Li et al., 264

2014). Finally, a deletion in the promoter of a mitochondrial molybdenum transporter 265

gene (MOT1) in Arabidopsis is associated with reduced gene expression and low 266

molybdenum (Mo) levels in the shoot, suggesting that this regulatory mutation is the 267

causal nucleotide polymorphism (Baxter et al., 2008). QTL studies of the Strategy I 268

plant Arabidopsis have focused on the accumulation of several minerals, including 269

nitrogen (Loudet et al., 2003; Harada et al., 2004), potassium (Harada and Leigh, 270

2006), Cu (Sasaki et al., 2008), Mo (Baxter et al., 2008), and sodium (Rus et al., 271

2006). Compared to Arabidopsis, a relatively high level of genetic variation has been 272

maintained between or among domesticated crops and their close wild relatives 273

(Huang and Han, 2014). Self-incompatible species, such as apple, have particularly 274

high levels of genetic variation; the use of highly heterozygous Malus genotypes has 275

allowed us to demonstrate that a TATA-box insertion in the promoter of a key gene 276

involved in the regulation of Fe homeostasis contributes to Fe-uptake efficiency. Our 277

results provide compelling evidence that natural allelic differences in promoter 278

sequences alter Fe uptake efficiency in this Strategy I plant. 279

The best-characterized core-promoter elements, which can function independently 280

or synergistically, are TATA elements, located 25 base pairs (bp) upstream of the 281

transcription start site (Burley and Roeder, 1996). The TATA box-binding protein 282

(TBP) was first identified as a component of a class II initiation factor (transcription 283

factor [TF] IID), a universal transcription initiation factor (Nikolov and Burley, 1994). 284

Thus, in addition to identifying a polymorphic gene, our study lays the foundation for 285

elucidating the detailed regulatory mechanism associated with natural variation. 286

Based on the newly identified role of the FUE allele in Fe uptake, we identified a 287

specific transcription factor (TF) IID protein (MDP0000939369) that binds to the 288

TATA box. Our model provides a new framework for future investigations of 289

mechanisms driving molecular genetic divergence in plants. Moreover, our results 290

establish that a synthetic repeat TATA-box insertion in the promoter can enhance gene 291

expression, which might represent a general tool for precisely regulating gene 292

expression. 293

Polyploidy has a major influence on gene flow in species. Multiple ploidy levels 294

exist within many species, and gene flow between ploidy levels is restricted by the 295

low fitness of the progeny produced by crosses between ploidy levels (Martinez-296



12

Reyna and Vogel, 2002; Henry and Crawford, 2005). In addition, the physiological 297

and genetic effects of polyploidy may directly result in adaptive differentiation within 298

a species (Otto and Whitton, 2000). However, the role of polyploidy in shaping 299

intraspecific diversity is poorly understood (Levin, 1983; Ramsey and Schemske, 300

2002). Cultivated apple is an example of a plant with secondary polyploidy (Velasco 301

et al., 2010), as the wild Malus species, M. xiaojinensis, is tetraploid (Wang et al., 302

2012). The progeny of M. xiaojinensis × M. baccata comprise hybrids of 2x, 3x, 4x, 303

or 5x ploidy (Wang et al., 2012). The physiological and genetic effects of polyploidy 304

may directly result in adaptive differentiation within a species. Since the complexity 305

of ploidy differences complicates efforts to quantify the contribution of the TATA-box 306

insertion to iron deficiency tolerance in the progeny, we performed separate Pearson 307

correlation analyses with plants of the same ploidy level to better understand the 308

relationship between the TATA-box insertion and the Fe deficiency tolerance 309

phenotype. Taken together, the results of relative genetic analysis between the FUE 310

allele and phenotype suggest that the TATA-box insertion contributes to the Fe 311

deficiency-resistant trait, but this contribution varies with ploidy level. 312

Phylogenetic analysis has suggested that IRT genes from dicotyledonous species A. 313

thaliana and M. xiaojinensis are closely related and belong to a single clade (Victoria 314

et al., 2012). Additionally, the close relationship between dicotyledon and 315

monocotyledon IRT genes suggests incipient divergence in these two groups 316

(Guerinot, 2000; Maser, 2001; Vert, 2002; Gross et al., 2003), which we found 317

evidence for in our analysis of IRT1. M. toringoides species, comprising the 318

hybridization complex M. toringoides, M. kansuensis, and M. transitoria, produce 319

highly phenotypically variable progenies due to their self-incompatibility or long 320

juvenile phase. M. xiaojinensis is a product of the complicated evolutionary history of 321

M. toringoides (Chen et al., 2000), which originated in (and is distributed throughout) 322

Sichuan province. The soil type in this region is brown acidic, with a pH value 323

slightly lower than 7 and high levels of available Fe. Therefore, there is no anticipated 324

selective pressure for Fe homeostasis-related genes in plants in this region. We found 325

that wild relatives of M. xiaojinensis growing in similar soils are homozygous for FUI 326

and therefore lack a TATA-box insertion in IRT1 (Fig. 7A). Consistently, in our 327

evolutionary analysis of TATA-box insertions, we did not detect significant departure 328

from neutrality in M. toringoides species. Although the ecological effects of the 329

genetic insertion, which occurred in a geographic region with high levels of available 330



13

Fe, have not yet been assessed, our findings suggest that the TATA-box insertion may 331

have promoted the migration of M. xiaojinensis from the acidic soils of Southwest 332

China to the alkaline soils in the North. 333

Geographic analyses of genetic variation in several plant species have revealed 334

clear genetic signals of local adaptation caused by differences in selective regimes 335

among locations (Linhart and Grant, 1996). At the beginning of our study, we 336

confirmed that M. baccata is homozygous for a lack of the TATA-box insertion. 337

However, we subsequently found M. baccata plants of different ecotypes containing a 338

TATA insertion from regions with alkaline soil (Fig. 7B). M. baccata and M. 339

xiaojinensis do not share a common ancestor (Chen et al., 2001), suggesting that the 340

TATA insertion in Fe uptake-efficient genus Malus occurred independently. Our 341

results provide evidence that the acquisition of tolerance to Fe deficiency is an 342

adaptive trait of the genus Malus. This adaptation likely facilitates the expansion of 343

apple production areas using genotypes tolerant to low Fe availability. 344

Previous studies have suggested that natural variation in cis elements can underlie 345

phenotypic variation (Sureshkumar and Lee, 2009). We found that this variation can 346

occur in the form of a TATA-box insertion in a gene promoter. This model provides an 347

important framework for future investigations of the role of molecular genetic 348

divergence in environmental adaption. 349

350

MATERIALS AND METHODS 351

Materials and Treatments 352

For X-ray fluorescence microscopy (XRF) analysis, M. xiaojinensis and M. baccata 353

seedlings were propagated on Murashige and Skoog (MS) medium containing 0.5 354

mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L indole-3-butytric acid (IBA) for one 355

month. The seedlings were transferred to MS medium with 1.0 mg/L IBA for rooting. 356

After an additional 1.5 months, the rooted seedlings were transferred to Hoagland’s 357

nutrient solution, pH 6.0 (Han et al., 1994), and cultured at 24°C, 60–70% relative 358

humidity with a photoperiod of 16-h light/8-h dark under a light intensity of 250 359

mmol quanta m-2s-1. The nutrient solution was replaced weekly. After 3 weeks, the 360

plants were placed in Hoagland nutrient solution containing 0 μM (Fe-deficient) or 40 361

μM (Fe-sufficient) FeNaEDTA and grown for 6 days. 362



14

To measure IRT1 expression, M. xiaojinensis, M. baccata, and M. xiaojinensis × M. 363

baccata progeny seedlings were grown as above under Fe-sufficient conditions. Roots 364

were collected for RNA isolation and gene expression analysis. 365

To analyze active Fe levels and the expression of IRT1 and the selected 366

transcription factor genes, M. xiaojinensis and M. baccata seedlings were grown in 367

Hoagland nutrient solution under Fe deficient conditions for 0, 3, and 6 days and their 368

roots analyzed. 369

To sequence the IRT1 coding region and upstream region, leaves were collected 370

from M. xiaojinensis, M. baccata, and M. xiaojinensis × M. baccata progeny plants at 371

the Institute of Forestry and Pomology, Beijing Academy of Agricultural and Forestry 372

Sciences, the Institute of Botany, Chinese Academy of Sciences, and the horticulture 373

research garden in Changping district, Beijing, respectively. 374

To evaluate leaf chlorosis, the in vitro-rooted progeny of M. xiaojinensis × M. 375

baccata were transplanted to the field in May 2013 and cultured in fine quartz sand in 376

a 90×30×30 cm (L×W×H) plastic container. The plants were drip-irrigated with 377

Hoagland nutrient solution containing 4 μM Fe (for Fe-deficient treatment) and 40 378

μM Fe (for Fe-sufficient treatment). 379

To analyze the evolution of the TATA-box insertion in the IRT1 promoter, the 380

leaves of 67 Malus species were collected from different regions of China: M. 381

toringoides, Southwest University in Chongqing; M. baccata, Shanxi, Liaoning, 382

Heilongjiang; M. domestica and M. sieversii, the horticulture research garden, 383

Changping district, Beijing. The leaves were collected for DNA isolation and gene 384

expression detection. 385

Synchrotron Radiation Microscopic X-ray Fluorescence Analysis (SR-μXRF) 386

The micro-X-ray fluorescence (μ-XRF) microspectroscopy experiment was 387

performed at the 4W1B endstation at the Beijing Synchrotron Radiation Facility, 388

which runs a 2.5 GeV electron beam with a current of 150 mA to 250 mA. The 389

incident X-ray energy was monochromatized with a W/B4C Double-Multilayer-390

Monochromator (DMM) at 15 keV and focused to a 50 μm diameter beam using a 391

polycapillary lens. M. xiaojinensis and M. baccata roots was sliced transversely with 392

a scalpel. To obtain a flat surface, a Cryotome was used to produce ~200 μm thick 393

latitudinal root sections. The samples were placed on Kapton® tape and freeze-dried 394

in a vacuum freeze dryer (LGJ-10B, Beijing Four-Ring Science Instrument Factory). 395



15

Two-dimensional mapping was performed in step-mode: the sample was held on a 396

precision motor-driven stage, scanning 50 μm stepwise. A Si (Li) solid-state detector 397

was used to detect X-ray fluorescence emission lines with a live time of 60 s. Data 398

reduction and processing were performed using PyMCA (Kim et al., 2006; Solé et al., 399

2007). 400

Cloning and Sequencing of the IRT1 Genome Fragment 401

Genomic DNA was extracted from leaves of Malus species and their progeny using 402

the CTAB method (Gasic et al., 2004). The PCR primers listed below were used to 403

amplify the different MdIRT1 DNA fragments with KOD FX DNA polymerase 404

(Toyobo). The amplified product was purified, ligated into the pEASY-T1 vector 405

(TransGen Biotechnology Co., Ltd., Beijing, China), cloned into E. coli strain DH5α, 406

and sequenced. The PCR primers aIRT251-F: 5’-GCAGCTGCCAACCTCAATCC-3’ 407

and aIRT251-R: 5’-CGACCGGCTTGACACCAAAA-3’ were used to amplify the 408

region 5’ 578 bp + IRT1 (3 exons + 2 introns) 1637 bp + 87 bp 3’. The primers 409

IRT721-F: 5’-AAGCCACCGGAGGACATGG-3’ and IRT721-R: 5’-410

GGGCTATGATTTTGAG GGGCA-3’ were used to amplify the region 5’ 621 bp + 411

IRT1 (100 bp cDNA) to detect the sequence mutation. STR (Short tandem repeats) 412

analysis was performed using fluorescently labeled primers IRT721-F: 5’-413

AAGCCACCGGAGGACATGG-3’ and IRT721-R: 5’-414

GGGCTATGATTTTGAGGGGCA-3’ (Beijing Microread Genetics Co., Ltd.). 415

Total RNA Isolation and Quantitative RT-PCR 416

Total RNA was extracted from root tissue using a modified CTAB method (Gasic et 417

al., 2004), followed by DNase I digestion to remove any DNA contamination 418

(TaKaRa Biotechnology Co., Ltd., Dalian, China). To generate cDNA, the RNA 419

samples were reverse-transcribed using oligo-dT primer and reverse transcriptase 420

(TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s 421

instructions. 422

The relative expression levels of IRT1 were measured using an Applied Biosystems 423

7500 real-time PCR system and SYBR Green fluorescent dye (Takara Biotechnology 424

Co., Ltd., Dalian, China). The primers MdIRT1-F: 5’- 425

TGACAAGGGAGAAAACGGAGAC-3’, MdIRT1-R: 5’-426

AACAACTGAATGGACAATGATACCC-3’ were designed using Primer Premier 5 427



16

(Zhang and Gao, 2004), and the efficiency of the primers was confirmed by RT-PCR. 428

β-actin was used as the reference gene. The primers were actin-F: 5’-429

TGGCATATACTCTGGAGGCT-3’ and actin-R: 5’TGGTGAGGCTCTATTCCAAC-430

3’. MdIRT1 relative expression levels were calculated according to the 2-ΔΔCT method 431

(Livak and Schmittgen, 2001). Each reaction was performed with three biological 432

replicates. 433

Measuring Chlorosis in the Leaves in M. xiaojinensis × M. baccata Progeny 434

Leaf chlorosis was evaluated as previously described (Xu et al., 2012b; Zhu et al., 435

2015). 436

Separation Ratio of TATA-box Insertions or Deletions in the IRT1 Promoters of 437 M. xiaojinensis × M. baccata Progeny 438

To investigate the influence of the TATA-box on iron deficiency resistance in the 439

progeny of M. xiaojinensis × M. baccata, progeny with 2x, 3x, 4x, and 5x ploidy were 440

used to measure the separation ratio of TATA-box insertion or deletion. DNA 441

fragments (721 bp) were PCR amplified using primers IRT721-F (5’-442

AAGCCACCGGAGGACATGG-3’) and IRT721-R (5’-443

GGGCTATGATTTTGAGGGGCA-3’), ligated into the pEASY-T1 vector, and cloned 444

into E. coli strain DH5α. The sequences of 20 monoclonal colonies per M. 445

xiaojinensis × M. baccata progeny (grown in ampicillin LB medium) were used as the 446

basis for estimating the proportion of TATA-box insertions. 447

Analysis of Active Fe 448

Active Fe was evaluated by polarized Zeeman atomic absorption 449

spectrophotometry (Z-5000, Hitachi, Tokyo, Japan) as previously described (Zha et 450

al., 2014). 451

Construction of Recombinant Plasmids 452

Fusion products of the promoters with or without the TATA-box and the two 453

versions of the full-length IRT1 open reading frame (ORF) were generated using 454

overlap PCR technology (Lee et al., 2010). The promoters were amplified with the 455

primers: a-IRT600-F (5’-AAGCCACCGGAGGACATGG-3’) and a-IRT600-R (5’-456

GTGGCAGCCATTGACCCTGAT-3’). The full-length IRT1 ORF was amplified 457

with the primers: b-IRT1100-F (5’-ATCAGGGTCAATGGCTGCCAC-3’) and b-458



17

IRT1100-R (5’-AGCCCAC TTTGCCAATA-3’). The recombinant fusion products 459

were renamed FUE and FUI and cloned into the pGWEB404 vector with GFP as a tag 460

using the Gateway method (Curtis and Grossniklaus, 2003). 461

A. thaliana Protoplast Isolation and Transformation 462

Protoplasts were isolated from fully expanded healthy leaves of 3 to 4-week-old A. 463

thaliana using a modified version of a published protocol (Sheen, 2001). Protoplasts 464

were transformed with polyethylene glycol (PEG) essentially as described (He et al., 465

2007; Yoo et al., 2007) using recombinant plasmid DNA including FUE or FUI, with 466

empty vector used as the negative control. The detail protocol is presented in 467

Supplemental Method S2. After 16 h, the protoplasts were harvested and stored at -468

80°C for RNA extraction and GFP expression analysis. 469

Investigating Promoter Activity in IRT1 Promoters with Different Numbers of 470 TATA-box Insertions 471

To investigate the effects of different numbers of TATA-box insertions on IRT1 472

promoter activity, IRT promoters with 0, 1, 2, or 3 copies of the TATA-box were 473

generated by artificial gene synthesis (Beijing Kwinbon Biotechnology Co., Ltd. 474

China) and renamed as P0, P1 (-358), P2 (-410, -358), and P3 (-415, -410, -358), 475

respectively. The promoters were inserted into the vector pCAMBIA1301 (containing 476

the GUS reporter gene) between the NcoI and KpnI sites. The recombinant plasmids 477

were transformed into A. thaliana protoplast as described above, with empty vector 478

containing the 35S promoter used as the control. Protoplasts were harvested and 479

stored at -80°C for RNA extraction and GUS expression analysis. 480

Generation of Transgenic Tobacco 481

The constructed plasmids containing FUE or FUI were introduced into 482

Agrobacterium tumefaciens (strain EHA105) cells, which were used to transform 483

tobacco (Nicotiana benthamiana) using the leaf disc method (Horsch et al., 1989), 484

with empty vector used as the negative control. Transgenic plants were selected based 485

on kanamycin resistance and transferred to MS liquid medium after rooting. After two 486

weeks, transgenic plants were used to detect the expression of GFP in roots, while the 487

leaves were used for SR-μXRF analysis of Fe content. 488

Identifying TATA-box-binding Proteins via Yeast One-Hybrid Analysis 489



18

The TBP domains were identified using the search term “TATA-box binding protein 490

(TBP)” in the Arabidopsis Information Resource website 491

(http://www.arabidopsis.org/). The domains in the Genome Database for Rosaceae 492

website (https://www.rosaceae.org/) were used queries to search for homologous 493

transcription factors in the apple genome, revealing 29 genes. A phylogenetic analysis 494

of these genes (generated using MEGA5.2) is shown in Supplemental Fig. S3 495

(Tamura et al., 2011). Gene-specific PCR primers were used to amplify the sequences 496

(Supplemental Table S1). Total RNA was extracted from the roots of M. xiaojinensis 497

plants grown under normal and iron-deficient conditions using the modified CTAB 498

method described above and treated with DNase I (TaKaRa Biotechnology Co., Ltd., 499

Dalian, China); cDNA generation and qRT-PCR analysis were performed as described 500

above. According to the qRT-PCR results, only 11 of the 29 transcription factors were 501

expressed under iron-deficient conditions; the corresponding PCR products were 502

purified and cloned into the pEASY Blunt Simple T1 vector (TransGen, Beijing, 503

China) according to the manufacturer’s instructions, followed by sequencing 504

(GENEWIZ, Suzhou, China). 505

Yeast One-Hybrid Analysis 506

Yeast one-hybrid analysis was performed to investigate the interactions of the 11 507

transcription factors with the two IRT1 promoters (with and without the TATA-box). 508

Using EcoRI and XhoI digestion and T4 ligation reactions, the transcription factor 509

genes were inserted into the AD vector (pJG4-5) (Clontech Matchmaker One-Hybrid 510

System User Manual, Dalian, China), and the two promoters (with or without the 511

TATA-box) were inserted into the BD vector (pLacZi). Vector construction and 512

screening were performed according to the manufacturer’s instructions, with minor 513

modifications (Supplemental Method S2). 514

IRT1 Nucleotide Polymorphism Analyses in Various Malus Genotypes 515

To investigate the DNA sequence diversity in the IRT1 promoter, DNA fragments 516

(721 bp) were PCR amplified using primers IRT721-F (5’-517

AAGCCACCGGAGGACATGG-3’) and IRT721-R (5’-518

GGGCTATGATTTTGAGGGGCA-3’). Leaves from 67 Malus species from different 519

regions of China were collected and used for DNA amplification. TransTaq DNA 520

Polymerase High Fidelity (HiFi) (TransGen, Beijing, China) was used for PCR to 521



19

minimize DNA synthesis errors. The PCR products were purified and sequenced 522

(GENEWIZ, Suzhou, China), and the sequences were analyzed using MEGA5.2 523

(Tamura et al., 2011). 524

525 Table 1. Nucleotide polymorphisms in IRT1 in various Malus species. 526

m Ps Θ π DNeutral mutation range

(Tajima’s D value)

M. toringoides 5 0.065693 0.031533 0.032263 0.174412 -1.255–1.737

M. baccata 21 0.030797 0.00856 0.00842 -0.06069 -1.585–1.709

M. sieversii 11 0.030581 0.010441 0.005783 -2.02136 -1.572–1.710

M. domestica 15 0.016514 0.005079 0.002412 -1.96923 -1.584–1.780

527

528

Supplemental data 529 Supplemental Figure S1. Sequences of the 1500 bp FUE and FUI promoters. 530 Supplemental Figure S2. Correlation analysis between chlorosis ratios and relative 531 expression levels of IRT1 in the progeny of M. xiaojinensis and M. baccata under Fe-532

deficiency treatment. 533

Supplemental Figure S3. Transient GUS expression under the control of the FUE 534 and FUI promoters in tobacco. 535

Supplemental Figure S4. Complementation test of IRT1 Fe2+ uptake in the fet3fet4 536 yeast mutant grown on Fe-deficient medium supplemented with 55 μM BPDS 537 (bathophenanthrolinedisulfonic acid disodium salt), pH 5.8. 538

Supplemental Figure S5. Phylogenetic analysis of homologous transcription factors 539 in the apple and A. thaliana genomes. 540

Supplemental Table S1. Primers for the transcription factor genes used in the yeast 541 one-hybrid assay. 542

Supplemental Table S2. Probe sequences used for EMSA. 543 Supplemental Data 1. Sequences of IRT1 promoters with different numbers of the 544 TATA-box insertion. 545



20

Supplemental Method S1. A. thaliana protoplast isolation and PEG-mediated gene 546 transformation. 547

Supplemental Method S2. Modified yeast one-hybrid analysis protocol. 548 Supplemental Method S3. Functional complementation in yeast. 549

550

Acknowledgments 551

The μ-XRF beam time was granted by the 4W1B endstation of the Beijing 552

Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy 553

of Sciences. The staff members of 4W1B are acknowledged for their help with 554

measurements and data analysis. Dr. Ronald F. Korcak is acknowledged for his 555

comments on the experiments and technical editing of the manuscript. 556

557

Figure Legends 558 Figure 1. X-ray fluorescence microtomography showing that the Fe content is higher 559 in M. xiaojinensis roots than in M. baccata roots. Qualitative spatial distribution and 560 concentration gradients of Fe in latitudinal sections of (A and D) M. baccata. 561

(Sufficient Fe supply, 40 μM Fe2+), (B and E) M. xiaojinensis (Sufficient Fe supply, 40 562

μM Fe2+), and (C and F) M. xiaojinensis roots (Deficient Fe supply, 0 μM Fe2+). Bar 563

color (blue to red) reflects Fe contents (low to high). The SR-μXRF signals for the 564

map were collected in 50 μm steps. The areas mapped for (A, D), (B, E), and (C, F) 565 were 350 μm × 400 μm, 800 μm × 800 μm, and 650 μm × 650 μm, respectively. Scale 566

bars in A, B, and C indicate 50 µm, 100 µm, and 100 µm, respectively. 567 568 Figure 2. Fe deficiency tolerance, root Fe content, and IRT1 expression in M. 569 xiaojinensis and M. baccata. A, Fe-deficiency symptoms in M. xiaojinensis and M. 570

baccata grown under Fe-deficient conditions and STR (Short tandem repeats) analysis 571

of the IRT1 promoter. The peak at 716 corresponds to the FUE promoter sequence 572

with the TATA-box, and the peak at 727 corresponds to the FUI promoter sequence 573

without the TATA-box. B, Root Fe content in M. xiaojinensis and M. baccata supplied 574

with 0 μM Fe2+ or 40 μM Fe2+ for 0, 3, and 6 days. C, Relative expression of IRT1 in 575

M. xiaojinensis and M. baccata supplied with 0 μM Fe2+ or 40 μM Fe2+ for 0, 3, and 6 576

days. D. Variations in the IRT1 promoter fragment between the FUE and FUI alleles. 577

(See full sequences in Supplemental Fig. S1.) 578



21

579 Figure 3. Analysis of segregating progeny from a cross between M. xiaojinensis and 580 M. baccata. A, Correlation analysis between relative expression levels of IRT1 in the 581

progeny and the TATA-box insertion percentage. B, Fe-deficient chlorosis index in 582

young leaves of the hybrid combinations (M. xiaojinensis and M. baccata) under Fe-583

deficient treatment. C, Varied phenotypic symptoms of Fe deficiency in the 584

segregating progeny. 585 586 Figure 4. Functional analysis of TATA-box insertion in A. thaliana protoplasts. A, 587

Diagrams of the reporter constructs used for transient expression analysis. B, 588

Transient expression analysis of FUE and FUI in A. thaliana protoplasts. A. thaliana 589

protoplasts were transfected with FUE and FUI promoter-GFP fusions, and GFP 590

expression was measured relative to that of GUS driven by the constitutive 35S 591

promoter. Error bars in B indicate s.d. of three biological replicates. C, Schematic of 592

promoters with different numbers of TATA-boxes ranging from zero (P0) to three 593

(P3). D, Transient expression analysis of P0–P3 in A. thaliana protoplasts. A. thaliana 594

protoplasts were transfected with P0-GUS, P1-GUS, P2-GUS, and P3-GUS fusions 595

and GUS expression was measured relative to that of GFP driven by the constitutive 596

35S promoter. Error bars in D indicate s.d. of six biological replicates. 597

598

Figure 5. The TATA-box insertion in FUE increases Fe accumulation. A, X-ray 599 fluorescence microtomography of tobacco leaves transformed with FUE:IRT1:GFP 600

and FUI:IRT1:GFP. Bar color (blue to red) reflects the Fe contents (low to high). The 601

SR-μXRF signals for the map were collected in 100 μm steps. The areas mapped for 602

FUE:IRT transgenic lines 1, 3, and 5 were 600 μm × 600 μm, 600 μm × 600 μm, and 603 1200 μm × 1200 μm, respectively. The areas mapped for FUI:IRT transgenic lines 1, 604 5, and 8 were 1000 μm × 1000 μm, 600 μm × 600, μm and 600 μm × 600 μm, 605

respectively. B, Relative expression of GFP in transgenic tobacco. Error bars in B 606 indicate s.d. of three biological replicates. 607

608

Figure 6. Presence of the TATA-box correlates with an increase in transcriptional 609 activation by specific transcription factor (TF) IID protein binding. A, Yeast one-610 hybrid assay showing the activity of LacZ reporters driven by the FUE and FUI 611

promoters and activated by the activation domain (AD) fused with MDP0000495467 612



22

or MDP0000939369. Empty vectors pJG4-5 and pLacZi were used as negative 613

controls. B, Gel-shift analysis of MDP0000939369 binding to the promoters of FUE. 614

EMSAs of GST-labeled probe fused with TFIID protein (MDP0000939369); DNA 615

probes represent the FUE or FUI (probe sequences are shown in Supplemental Table 616

S2). Protein/DNA complexes are indicated by arrows. C, Relative expression of 617

MDP0000939369 in M. xiaojinensis and M. baccata supplied with 0 μM Fe2+ or 40 618

μM Fe2+ for 0, 3, and 6 days. D, Correlation analysis between relative expression 619

levels of IRT1 and MDP0000939369. E, Schematic model of the regulation of IRT1 620

expression by the TATA-box insertion regulatory module. 621

622

Figure 7. Evolution of the TATA-box in the IRT1 promoter in the genus Malus. A, 623 Phylogenetic analysis of the TATA-box in the IRT1 promoters of M. toringoides, M. 624

toringoides × M. kansuensis progeny, M. maerkangensis, and M. xiaojinensis. B, 625

TATA-box insertion in the IRT1 promoter was induced by geographic ecological 626

selective pressure. The data about available Fe in soil were obtained from published 627

sources (Chen et al., 2000; Cai et al., 2002; Li et al., 2009). 628

629 630 631

632

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Parsed CitationsAbadìa J, Vìzquez SìL, Rellìn-lvarez RìN, El-Jendoubi H, Abadìa AìN, lvarez-Fernìndez A, Lìpez-Millìn AF (2011) Towards aknowledge-based correction of iron chlorosis. Plant Physiol and Biochem 49: 471-482

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

Alonso-Blanco C, Aarts MG, Bentsink L, Keurentjes JJ, Reymond M, Vreugdenhil D, Koornneef M (2009) What has natural variationtaught us about plant development, physiology, and adaptation? Plant Cell 21: 1877-1896


Baxter I, Muthukumar B, Park HC, Buchner P, Lahner B, Danku J, Zhao K, Lee J, Hawkesford MJ, Guerinot ML (2008) Variation inmolybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenumtransporter (MOT1). Plos Genet 4: e1000004


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Gasic K, Hernandez A, Korban SS (2004) RNA extraction from different apple tissues rich in polyphenols and polysaccharides forcDNA library construction. Plant Mol Biol Rep 22: 437-438 www.plantphysiol.orgon August 24, 2020 - Published by Downloaded from

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http://www.ncbi.nlm.nih.gov/pubmed?term=Abad�a J%2C V�zquez S�L%2C Rell�n%2Dlvarez R�N%2C El%2DJendoubi H%2C Abad�a A�N%2C lvarez%2DFern�ndez A%2C L�pez%2DMill�n AF %282011%29 Towards a knowledge%2Dbased correction of iron chlorosis%2E Plant Physiol and Biochem 49%3A 471%2D482&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Abad�a J, V�zquez S�L, Rell�n-lvarez R�N, El-Jendoubi H, Abad�a A�N, lvarez-Fern�ndez A, L�pez-Mill�n AF (2011) Towards a knowledge-based correction of iron chlorosis. Plant Physiol and Biochem 49: 471-482http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Abad�a&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Towards a knowledge-based correction of iron chlorosis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Towards a knowledge-based correction of iron chlorosis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Abad�a&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Alonso%2DBlanco C%2C Aarts MG%2C Bentsink L%2C Keurentjes JJ%2C Reymond M%2C Vreugdenhil D%2C Koornneef M %282009%29 What has natural variation taught us about plant development%2C physiology%2C and adaptation%3F Plant Cell 21%3A 1877%2D1896&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Alonso-Blanco C, Aarts MG, Bentsink L, Keurentjes JJ, Reymond M, Vreugdenhil D, Koornneef M (2009) What has natural variation taught us about plant development, physiology, and adaptation? Plant Cell 21: 1877-1896http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Alonso-Blanco&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=What has natural variation taught us about plant development, physiology, and adaptation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=What has natural variation taught us about plant development, physiology, and adaptation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Alonso-Blanco&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Baxter I%2C Muthukumar B%2C Park HC%2C Buchner P%2C Lahner B%2C Danku J%2C Zhao K%2C Lee J%2C Hawkesford MJ%2C Guerinot ML %282008%29 Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter %28MOT1%29%2E Plos Genet 4%3A e1000004&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Baxter I, Muthukumar B, Park HC, Buchner P, Lahner B, Danku J, Zhao K, Lee J, Hawkesford MJ, Guerinot ML (2008) Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1). Plos Genet 4: e1000004http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baxter&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1).&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Variation in molybdenum content across broadly distributed populations of Arabidopsis thaliana is controlled by a mitochondrial molybdenum transporter (MOT1).&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baxter&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Bonner J%2C Varner F %281976%29 Plant biochemistry%2E Eds%2E %28Academic%2C New York%29%2E&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Bonner J, Varner F (1976) Plant biochemistry. Eds. (Academic, New York).http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bonner&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Plant biochemistry.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Plant biochemistry.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bonner&as_ylo=1976&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Briat JO%2C Fobis%2DLoisy I%2C Grignon N%2C Lobr aux SP%2C Pascal N%2C Savino G%2C Thoiron SV%2C Wir n N%2C Wuytswinkel O %281995%29 Cellular and molecular aspects of iron metabolism in plants%2E Biol Cell 84%3A 69%2D81&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Briat JO, Fobis-Loisy I, Grignon N, Lobr aux SP, Pascal N, Savino G, Thoiron SV, Wir n N, Wuytswinkel O (1995) Cellular and molecular aspects of iron metabolism in plants. Biol Cell 84: 69-81http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Briat&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Cellular and molecular aspects of iron metabolism in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Cellular and molecular aspects of iron metabolism in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Briat&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Burley SK%2C Roeder RG %281996%29 Biochemistry and structural biology of transcription factor IID %28TFIID%29%2E Annu Rev Biochem 65%3A 769%2D799&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Burley SK, Roeder RG (1996) Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65: 769-799http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Burley&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Biochemistry and structural biology of transcription factor IID (TFIID)&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Biochemistry and structural biology of transcription factor IID (TFIID)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Burley&as_ylo=1996&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Cai J%2C Cai SL%2C Lu JL %282002%29 Vertical variation of element contents in the main soil types of Heilongjiang province%2E Word Geology 21%3A 364%2D367 Chinese&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Cai J, Cai SL, Lu JL (2002) Vertical variation of element contents in the main soil types of Heilongjiang province. Word Geology 21: 364-367 Chinesehttp://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cai&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Vertical variation of element contents in the main soil types of Heilongjiang province&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Vertical variation of element contents in the main soil types of Heilongjiang province&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cai&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Chen GL%2C Li SJ%2C Zhang FS%2C Li CJ %282001%29 The adaptive mechanism of Fe deficiency to susceptibility of Fe stress of lettuce cultivars%2E Plant Nutr and Fert Sci 7%3A 117%2D120&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Chen GL, Li SJ, Zhang FS, Li CJ (2001) The adaptive mechanism of Fe deficiency to susceptibility of Fe stress of lettuce cultivars. Plant Nutr and Fert Sci 7: 117-120http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=The adaptive mechanism of Fe deficiency to susceptibility of Fe stress of lettuce cultivars&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=The adaptive mechanism of Fe deficiency to susceptibility of Fe stress of lettuce cultivars&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Chen MH%2C Li XL%2C Zhang YG %282000%29 Malus xiaojinensis cheng et jiang%2Da promising stock for apple trees%2E J Southwest Agr Univ 22%3A 383%2D386 Chinese&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Chen MH, Li XL, Zhang YG (2000) Malus xiaojinensis cheng et jiang-a promising stock for apple trees. J Southwest Agr Univ 22: 383-386 Chinesehttp://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chen&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Malus xiaojinensis cheng et jiang-a promising stock for apple trees&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Malus xiaojinensis cheng et jiang-a promising stock for apple trees&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chen&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Crichton RR%2C Pierre JL %282001%29 Old iron%2C young copper%3A From Mars to Venus%2E Biometals 14%3A 99%2D112&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Crichton RR, Pierre JL (2001) Old iron, young copper: From Mars to Venus. Biometals 14: 99-112http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Crichton&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Old iron, young copper: From Mars to Venus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Old iron, young copper: From Mars to Venus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Crichton&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Curtis MD%2C Grossniklaus%2C U %282003%29 A gateway cloning vector set for high%2Dthroughput functional analysis of genes in planta%2E Plant Physiol 133%3A 462%2D469&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Curtis MD, Grossniklaus, U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133: 462-469http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Curtis&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=A gateway cloning vector set for high-throughput functional analysis of genes in planta&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=A gateway cloning vector set for high-throughput functional analysis of genes in planta&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Curtis&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Darbani B%2C Briat JO%2C Holm PB%2C Husted SR%2C Noeparvar S%2C Borg SR %282013%29 Dissecting plant iron homeostasis under short and long%2Dterm iron fluctuations%2E Biotechnol Adv 31%3A 1292%2D1307&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Darbani B, Briat JO, Holm PB, Husted SR, Noeparvar S, Borg SR (2013) Dissecting plant iron homeostasis under short and long-term iron fluctuations. Biotechnol Adv 31: 1292-1307http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Darbani&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Dissecting plant iron homeostasis under short and long-term iron fluctuations&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Dissecting plant iron homeostasis under short and long-term iron fluctuations&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Darbani&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1http://www.ncbi.nlm.nih.gov/pubmed?term=Fujii M%2C Yokosho K%2C Yamaji N%2C Saisho D%2C Yamane M%2C Takahashi H%2C Sato K%2C Nakazono M%2C Ma JF %282012%29 Acquisition of aluminium tolerance by modification of a single gene in barley%2E Nat Commun 3%3A 713&dopt=abstracthttp://search.crossref.org/?page=1&rows=2&q=Fujii M, Yokosho K, Yamaji N, Saisho D, Yamane M, Takahashi H, Sato K, Nakazono M, Ma JF (2012) Acquisition of aluminium tolerance by modification of a single gene in barley. Nat Commun 3: 713http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Fujii&hl=en&c2coff=1http://scholar.google.com/scholar?as_q=Acquisition of aluminium tolerance by modification of a single gene in barley.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1http://scholar.google.com/scholar?as_q=Acquisition of aluminium tolerance by modification of a single gene in barley.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Fujii&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1http://www.plantphysiol.org


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