2 homeostasis in arabidopsis thaliana · 2/26/2016 · 5 121 contains a hemerythrin domain (hhe)...

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Two bHLH Transcription Factors, bHLH34 and bHLH104, Regulate Iron 1

Homeostasis in Arabidopsis thaliana 2

Xiaoli Li1,2, Huimin Zhang1,2, Qin Ai2,3, Gang Liang2*, Diqiu Yu2* 3 1School of Life Sciences, University of Science and Technology of China, 4

Hefei, Anhui 230027, China 5

2Key Laboratory of Tropical Plant Resources and Sustainable Use, 6

Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan 650223, China 7 3University of Chinese Academy of Sciences, Beijing 100049, China 8

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*Corresponding author: 10

Dr. Gang Liang ( [email protected]) 11

Dr. Diqiu Yu ([email protected]) 12

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AUTHOR CONTRIBUTIONS 14

X.L., G.L, and D.Y. designed the study. X.L., H.Z., Q.A., and G.L. performed 15

research. X.L., H.Z., G.L., and D.Y. analyzed data. G.L. wrote the article. X.L., 16

G.L., H.Z., Q.A. and D.Y. revised the article. All authors read and approved 17

the final manuscript. 18

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FUNDING 20

This work was supported by the Youth Innovation Promotion Association of 21

CAS, Candidates of the Young and Middle Aged Academic Leaders of 22

Yunnan Province [2015HB095] and the program for Innovative Research 23

Team of Yunnan Province [ 2014HC017]. 24

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Short title: 26

bHLH34 and bHLH104 Regulate Iron Homeostasis 27

One sentence summary 28

bHLH34 and bHLH10 positively regulate Fe homeostasis by directly 29

activating the transcription of Ib subgroup bHLH genes, bHLH38/39/100/101. 30

Plant Physiology Preview. Published on February 26, 2016, as DOI:10.1104/pp.15.01827

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ABSTRACT 31

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Regulation of iron (Fe) homeostasis is critical for plant survival. Although the 33

systems responsible for reduction, uptake and translocation of Fe have been 34

described, the molecular mechanism by which plants sense Fe status and 35

coordinate the expression of Fe deficiency responsive genes is largely 36

unknown. Here, we report that two basic helix-loop-helix-type transcription 37

factors, bHLH34 and bHLH104, positively regulate Fe homeostasis. 38

Loss-of-function of bHLH34 and bHLH104 causes the disruption of Fe 39

deficiency response and the reduction of Fe content, whereas their 40

overexpression plants constitutively promote the expression of Fe deficiency 41

responsive genes and Fe accumulation. Further analysis indicates that 42

bHLH34 and bHLH104 directly activate the transcription of Ib subgroup bHLH 43

genes, bHLH38/39/100/101. Moreover, overexpression of bHLH101 partially 44

rescues the Fe deficiency phenotypes of bhlh34bhlh104 double mutants. 45

Further investigation suggests that bHLH34, bHLH104 and bHLH105 (ILR3) 46

function as homodimers or heterodimers to non-redundantly regulate Fe 47

homeostasis. This work reveals that plants have evolved complex molecular 48

mechanisms to regulate Fe deficiency response genes to adapt to Fe 49

deficiency conditions. 50

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INTRODUCTION 61

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Iron (Fe) is of particular importance as a cofactor for a wide variety of proteins 63

in living organisms. In humans, Fe deficiency is one of the major reasons for 64

anemia. A plant diet is a major resource for human; thus it is important to 65

clarify the mechanisms of Fe homeostasis in plants. Fe is an indispensable 66

micronutrient for plant growth and development, and is involved in many 67

cellular functions, including chlorophyll biosynthesis, photosynthesis, and 68

respiration (Hänschand Mendel, 2009). Therefore, a decrease in Fe uptake 69

directly affects crop yield and crop quality. Plants take up Fe from the soil, 70

where Fe availability is limited as its major form is as insoluble ferric 71

hydroxides, especially in calcareous soils which make up one-third of the 72

world’s cultivated areas. 73

To cope with low Fe environments, higher plants have evolved two major 74

strategies for Fe acquisition. Non-graminaceous plants employ a reduction 75

strategy (strategy I), which involves three stages in low Fe conditions. The 76

acidification of soil by protons extruded by H+-ATPases and phenolic 77

compounds makes Fe(III) more soluble (Santi and Schmidt, 2009; Kobayashi 78

and Nishizawa, 2012). The ferric chelate reductase activity of FERRIC 79

REDUCTION OXIDASE 2 (FRO2) is induced by low Fe; this enzyme reduces 80

Fe(III) to Fe(II) (Robinson et al., 1999). The Fe(II) is then transported into 81

roots by the major Fe transporter of plant roots, IRON-REGULATED 82

TRANSPORTER 1 (IRT1) (Eide et al., 1996; Henriques et al., 2002; Varotto et 83

al., 2002; Vert et al., 2002). In contrast, graminaceous plants use a 84

chelation-based strategy (strategy II) (Walker and Connolly, 2008; Morrissey 85

and Guerinot, 2009). These plants release mugineic acid family 86

phytosiderophores, which have high affinity for Fe(III) and efficiently bind 87

Fe(III) in the rhizosphere. The resulting chelate complexes are then 88

transported into plant roots via a specific transport system (Mori, 1999). 89

Transcriptional regulation of genes involved in Fe uptake under Fe-deficient 90



4

conditions is intensively controlled by plants. In the past decade, several 91

bHLH proteins have been identified as regulators of Fe deficiency response. 92

The bHLH protein, tomato FER, was the first characterized to control Fe 93

deficiency responses, and its mutant failed to activate Fe acquisition strategy 94

I (Ling et al., 2002). FE-DEFICIENCY INDUCED TRANSCRIPTION FACTOR 95

(FIT), a homolog of FER in Arabidopsis thaliana, is required for proper 96

regulation of ferric chelate reductase activity and Fe transport into the plant 97

root, and its mutation was lethal when plants were grown in normal soils 98

(Colangelo and Guerinot, 2004; Jakoby et al., 2004; Yuan et al, 2005). 99

However, overexpression of FIT is not sufficient to constitutively induce the 100

expression of its target genes, such as IRT1 and FRO2, in normal growth 101

conditions. In fact, FIT is dually regulated by Fe starvation. At the 102

transcriptional level, FIT is induced by Fe deficiency (Colangelo and Guerinot, 103

2004; Jakoby et al., 2004; Yuan et al., 2005). At the post-transcriptional level, 104

FIT is actively destabilized and degraded by the 26S proteasome during Fe 105

limitation (Meiser et al., 2011; Sivitz et al., 2011). In addition, FIT can form a 106

heterodimer with members of the Ib subgroup of bHLH proteins 107

(bHLH38/39/100/101) to constitutively activate the transcription of IRT1 and 108

FRO2 (Yuan et al., 2008; Wang et al., 2013). Both FIT and 109

bHLH38/39/100/101 function as positive regulators. In contrast, POPEYE 110

(PYE) was identified as playing a negative role in Fe deficiency response in 111

Arabidopsis thaliana (Long et al., 2010). PYE transcript levels were elevated 112

when plants were subjected to low Fe conditions. Chip-on-chip experiments 113

revealed that PYE directly targets several genes involved in metal 114

homeostasis, such as NICOTIANAMINE SYNTHASE 4 (NAS4), FRO3, and 115

ZINC INDUCED FACILITATOR 1 (ZIF1). In a pye-1 mutant, the expression of 116

these target genes is significantly upregulated in Fe-deficient conditions, 117

suggesting that PYE functions as a negative regulator. 118

It is unclear how plants perceive Fe status and transmit signals to 119

downstream pathways. In mammals, FBXL5 functions as an Fe sensor, and 120



5

contains a hemerythrin domain (HHE) for Fe binding and an F-box domain for 121

ubiquitination and degradation of IRP2. The latter governs cellular Fe 122

homeostasis by regulation of translation and stability of mRNAs involved in 123

Fe homeostasis (Salahudeen et al., 2009; Vashisht et al., 2009). Interestingly, 124

two different groups (Kobayashi et al., 2013; Selote et al., 2015) confirmed 125

that Arabidopsis thaliana BRUTUS (BTS) and its rice orthologs, 126

HAEMERYTHRIN MOTIF-CONTAINING REALLY INTERESTING NEW 127

GENE (RING) AND ZINC-FINGER PROTEIN 1 (HRZ1) and HRZ2, possess 128

HHE domains for binding Fe. Although they lack an F-box domain similar to 129

that in FBXL5, BTS and HRZ1/2 have RING domains that can mediate the 130

ubiquitination reaction. Functional analysis revealed that BTS and HRZ1/2 131

negatively regulate Fe deficiency response in plants. Given that BTS and 132

HRZ1/2 are structurally and functionally similar to FBXL5, they are thought to 133

be potential Fe sensors in plants. Although no substrates for HRZ1/2 have 134

been found, it is established that BTS interacts with three PYE-like proteins 135

(bHLH104, bHLH105/ILR3, and bHLH115) and facilitates 26S 136

proteasome-mediated degradation of bHLH105/IAA-LEUCINE RESISTANT3 137

(ILR3) and bHLH115 in the absence of Fe in vitro (Selote et al., 2015). 138

Recently, Zhang et al. (2015) revealed that bHLH104 and bHLH105 positively 139

regulate Fe homeostasis by directly activating transcription of 140

bHLH38/39/100/101 and PYE. Further investigation is required to explore the 141

functions of the other PYE-like proteins. 142

Here, through screening regulators upstream of bHLH101, we identified 143

bHLH transcription factor bHLH34, a homolog of bHLH104 in Arabidopsis 144

thaliana. Mutational analysis suggests that bHLH34 and bHLH104 positively 145

regulate Fe deficiency response. Further analysis reveals that both bHLH34 146

and bHLH104 directly activate the transcription of bHLH38/39/100/101. In 147

agreement with this, constitutive expression of bHLH101 partially 148

complements the Fe deficiency symptoms of bhlh34bhlh104 double mutants. 149

Protein interaction assays indicate that both heterodimers and homodimers 150



6

occur between bHLH34, bHLH104 and bHLH105. These data reveal that 151

bHLH34 and bHLH104 play major roles in regulating Fe homeostasis by 152

activating the transcription of bHLH38/39/100/101 under Fe deficiency 153

conditions. 154

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RESULTS 157

Identification of bHLH34 158

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bHLH38/39/100/101 have been characterized as key transcription factors 160

regulating Fe-deficient response in Arabidopsis thaliana and their transcript 161

levels are also up-regulated by Fe deficiency. To identify transcription factors 162

functioning upstream of bHLH38/39/100/101, yeast one-hybrid (Y1H) 163

screening was performed using the promoter of bHLH101 as bait. From the 164

screening, we obtained nine positive colonies, three of which encoded a 165

bHLH protein, bHLH34. As a close homolog of bHLH34 (Supplemental Figure 166

1), bHLH104 has been characterized to bind the promoter of bHLH101 167

(Zhang et al., 2015). Further experiments confirmed that both bHLH34 and 168

both104, but not the other candidates, can activate the promoter of bHLH101 169

(Figure 1), which suggests that both bHLH34 and bHLH104 regulate 170

bHLH101 gene. Given that other Fe homeostasis-associated transcription 171

factors, such as bHLH38/39/100/101, FIT, PYE, MYB10/72, are induced by 172

Fe deficiency, we wanted to know whether bHLH34 and bHLH104 are also 173

responsive to Fe deficiency. Transcript abundance analysis indicated that 174

neither bHLH34 nor bHLH104 is affected by Fe deficiency (Supplemental 175

Figure 2). 176

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bhlh34, bhlh104, and bhlh34bhlh104 Mutant Plants Display Fe 178

Deficiency Symptoms 179

180

To investigate the functions of bHLH34 and bHLH104, two T-DNA insertion 181

alleles for the bHLH34 and bHLH104 genes were obtained from The 182

Arabidopsis Information Resource (TAIR). bhlh34 contains a T-DNA in the 183

second exon and bhlh104 has a T-DNA insertion in the third intron (Figure 2A). 184

The homozygous lines of bhlh34 and bhlh104 were isolated by PCR, and the 185

positions of the insertions were confirmed. The loss of full-length transcripts in 186



8

the T-DNA lines were determined by RT-PCR (Figure 2B), indicating that both 187

T-DNA lines are knockout mutants. Considering their putative function 188

redundancy, we constructed bhlh34bhlh104 double mutants by crossing two 189

single knock-out mutants. When grown on Fe sufficient media, neither single 190

mutants nor double mutants showed any obvious phenotypic differences 191

compared with wild-type plants (Supplemental Figure 3). However, when they 192

were grown on Fe free media, all mutants displayed Fe-deficient symptoms 193

and the double mutants had the most severe phenotypes (Figure 2C ). 194

To further confirm the functions of bHLH34 and bHLH104, we designed an 195

artificial miRNA, amiR-bhlh34/104, which is predicted to target both bHLH34 196

and bHLH104 (Supplemental Figure 4A), and generated amiR-bhlh34/104 197

transgenic plants. qRT-PCR analysis demonstrated that both bHLH34 and 198

bHLH104 were significantly down-regulated in amiR-bhlh34/104 plants 199

(Supplemental Figure 4B). As expected, the amiR-bhlh34/104 transgenic 200

plants grown in soils showed interveinal chlorosis in leaves, which is very 201

similar to that of bhlh34bhlh104 double mutants (Figure 2D). Further 202

phenotype evaluation revealed that the root growth of amiR-bhlh34/104 203

seedlings was strongly inhibited on Fe-deficient media whereas no visible 204



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difference was observed between amiR-bhlh34/104 and wild type plants on 205

normal media (Supplemental Figure 4C, D). These results demonstrate that 206

the amiR-bhlh34/104 plants phenocopy the bhlh34bhlh104 mutants. 207



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Impaired Fe Deficiency Response by Loss-of-function of bHLH34 and 209

bHLH104 210

211

We further determined the Fe-associated phenotypes of plants grown in soils 212

containing various concentration of calcium oxide (Figure 3A), which was 213

used to produce alkaline soil in which the Fe availability was limited because 214

of decreased Fe solubility. In normal soil, no visible phenotypic difference was 215

observed between the bhlh34, bhlh104, and wild-type plants, whereas the 216

bhlh34bhlh104 double mutants had small stature and chlorotic leaves. In soil 217

supplemented with 1‰ (w/w) calcium oxide, the bhlh104 mutants showed 218

slight chlorosis. In contrast, 5‰ calcium oxide caused leaf chlorosis in all 219

three types of mutants, and seedling lethality of the bhlh34bhlh104 double 220

mutants. These data suggest that the loss-of-function of bHLH34 and 221

bHLH104 inhibits the Fe deficiency response of the bhlh34bhlh104 plants. 222

Fe is indispensable for the synthesis of chlorophyll, and Fe deficiency often 223

causes leaf chlorosis with decreased levels of chlorophyll in plants (Terry et 224

al., 1980). The significant chlorosis of the bhlh34bhlh104 double mutant 225

plants implied a decline in chlorophyll content. We measured chlorophyll 226

levels in plants grown on Fe-sufficient and -deficient media. There were no 227

significant differences under Fe-sufficient conditions. However, under 228

Fe-deficient conditions, the mutants showed significantly reduced chlorophyll 229

levels and the bhlh34bhlh104 double mutants had the lowest chlorophyll 230

levels (Supplemental Figure 5). 231

Fe reductase activity is a typical indicator of Fe deficiency. We analyzed Fe 232

reductase activity using the ferrozine assay (Yi and Guerinot, 1996). In 233

Fe-sufficient conditions, no significant difference in Fe reductase activity was 234

observed between wild-type and mutant plants. In response to Fe deficiency, 235

both wild-type and mutant plants increased their Fe reductase activity. 236

However, the Fe reductase activity of the mutant plants was significantly 237



11

lower than that of wild type, and the bhlh34bhlh104 double mutants had the 238

lowest Fe reductase activity (Figure 3B). 239

To determine whether the loss-of-function of bHLH34 and bHLH104 causes 240

altered Fe accumulation, the Fe content of wild-type and mutant 2-week-old 241

seedlings was measured. 11-day-old seedlings grown under Fe-sufficient 242

conditions were transferred to Fe-sufficient or Fe-deficient media for another 243

three days. Roots and shoots were separately harvested and used for Fe 244

content analysis. The shoots of bhlh34bhlh104 plants had 17% less Fe than 245



12

the shoots of wild-type plants under Fe sufficient conditions and 71% less Fe 246

under Fe deficient conditions (Figure 3C). Similar results were also observed 247

in the roots. The roots of bhlh34bhlh104 plants contained 10% less Fe than 248

the roots of wild-type plants under Fe sufficient conditions and 56% less Fe 249

under Fe deficient conditions (Figure 3D). These data suggest that bHLH34 250

and bHLH104 are required for maintaining Fe homeostasis in both 251

Fe-sufficient and -deficient conditions. 252

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Fe Deficiency Responsive Genes are Down-Regulated in the bhlh34, 254

bhlh104, and bhlh34bhlh104 Mutants 255

256

To adapt to Fe-deficient conditions, plants often elevate the expression of 257

Fe-uptake associated genes. Fe deficiency-induced genes FRO2 and IRT1 258

encode two key root membrane proteins for Fe homeostasis; the former 259

converts Fe(III) to Fe (II) and the latter transports ferrous Fe from soil to root 260

(Robinson et al., 1999; Vert et al., 2002). Given the low Fe levels in the 261

bhlh34bhlh104 mutants, we wanted to know whether the expression of these 262

two Fe-uptake associated genes is altered in the mutants. In response to Fe 263

deficiency, both genes were induced in the roots of wild-type and mutant 264

plants. However, their expression levels were low in the mutants compared 265

with wild type under both Fe-sufficient and -deficient conditions (Figure 4). 266

Moreover, their expression levels in the bhlh34bhlh104 under Fe-deficient 267

conditions were close to that in the wild type in Fe-sufficient conditions. These 268

alterations suggest that there is decreased Fe uptake in the mutant plants, 269

which is in agreement with the reduction in both Fe reductase activity and Fe 270

accumulation. These data also demonstrate that bHLH34 and bHLH104 271

directly or indirectly activate the expression of FRO2 and IRT1. 272

Two MYB transcription factors, MYB10 and MYB72, were identified as Fe 273

deficiency responsive genes, which are responsible for the activation of 274

expression of the NAS4 gene in Fe-deficient conditions in Arabidopsis 275



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thaliana (Palmer et al., 2013). Considering the fact that the bhlh34bhlh104 276

mutants display interveinal chlorosis similar to nas4x-1 mutants (Klatte et al., 277

2009), we asked whether these two MYB genes are affected in the 278

bhlh34bhlh104 mutants. As expected, compared with wild type, the mutants 279

show considerably down-regulated transcript abundance of MYB10 and 280

MYB72 regardless of whether Fe is deficient or not (Figure 4), suggesting that 281

bHLH34 and bHLH104 positively regulate the transcription of MYB10 and 282

MYB72. 283

The well-known transcription factors for Fe homeostasis in Arabidopsis 284

thaliana are four Ib subgroup bHLH transcription factors (bHLH38/39/100/101) 285

and another bHLH transcription factor, FIT, all of which are induced by Fe 286

deficiency (Colangelo and Guerinot, 2004; Wang et al., 2007). Each of 287

bHLH38/39/100/101 can interact with FIT to regulate the expression of Fe 288

uptake-associated genes (Yuan et al, 2008; Wang et al., 2013). To determine 289



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the position of bHLH34 and bHLH104 in Fe signaling pathways, it is 290

necessary to investigate their regulatory relationship with these bHLH 291

transcription factors. Gene expression analysis revealed that 292

bHLH38/39/100/101 were strongly down-regulated in the bhlh34bhlh104 293

mutants under both Fe-sufficient and -deficient conditions, whereas FIT was 294

moderately decreased (Figure 4). These data imply that bHLH34 and 295

bHLH104 act upstream of bHLH38/39/100/101 and FIT. 296

In addition, we also determined the expression of genes that are involved in 297

Fe distribution in plants, finding that NAS2, NAS4, ZIF1, FRD3, OPT3 and 298

PYE were down-regulated in the bhlh34bhlh104 mutants in response to Fe 299

deficiency (Supplemental Figure 6). Taken together, our data suggest that 300

bHLH34 and bHLH104 positively regulate Fe deficiency response. 301

302

Overexpression of bHLH34 and bHLH104 Activates Fe Deficiency 303

Response 304

305

Considering the inhibited Fe deficiency response caused by loss-of-function 306

of bHLH34 and bHLH104, we asked whether elevated expression of bHLH34 307

and bHLH104 would enhance the Fe deficiency response. First, we 308

performed complementation assays. ProbHLH34:Myc-bHLH34 and 309

ProbHLH104:Myc-bHLH104 constructs were introduced into the bhlh34 and 310

bhlh104 mutants, respectively. When grown on Fe deficiency media, both 311

ProbHLH34:Myc-bHLH34/bhlh34 and ProbHLH104:Myc-bHLH104/bhlh104 plants 312

were comparable with wild type plants (Supplemental Figure 7), suggesting 313

the both mutants were rescued. Next, we constructed transgenic plants 314

expressing Pro35S:Myc-bHLH34 and Pro35S:Myc-bHLH104. When grown in 315

normal soils, about 10% of Pro35S:Myc-bHLH34 and 33% of 316

Pro35S:Myc-bHLH104 T0 transgenic plants displayed visible phenotypes 317

(small rosettes and leaf necrosis) (Figure 5A) and no visible phenotype was 318

observed for the other transgenic plants. We further assessed the root 319



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phenotypes of T1 plants when grown on Fe deficiency media, finding that the 320

progenies of T0 plants with visible phenotypes produced short roots whereas 321

the root length of the other progenies was similar to that of wild type plants 322



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(Figure 5B). In contrast, no visible phenotypes were observed between wild 323

type and transgenic plants when grown on Fe sufficient media for ten days 324

(Supplemental Figure 8A). The determination of transgene abundance 325

revealed that the transgenic plants with visible phenotypes have significantly 326

higher transgene levels than those without visible phenotypes (Figure 5C; 327

Supplemental Figure 8B) suggesting that the visible phenotypes are closely 328

associated with the transgene levels. For the further investigation, 329

Pro35S:Myc-bHLH34#3 and Pro35S:Myc-bHLH104#5 were chosen as 330

representatives of plants with visible phenotypes and Pro35S:Myc-bHLH34#7 331

and Pro35S:Myc-bHLH104#9 as representatives of plants without visible 332

phenotypes. 333

Given that the bhlh34bhlh104 mutants contain less Fe than wild type, we 334

asked whether overexpression transgenic plants accumulate more Fe than 335

wild type. The leaves of four-week-old plants grown in normal soil were used 336

for Fe concentration determination. As expected, overexpression plants have 337

higher Fe content than wild type, and the Fe concentration is particularly high 338

in Pro35S:Myc-bHLH34#3 and Pro35S:Myc-bHLH104#5 plants (Figure 5D). In 339

contrast, the concentration of Mn, Cu and Zn in overexpression plants is 340

comparable to that in wild type (Supplemental Figure 9). These data imply 341

that the visible phenotypes may be linked with the high transgene abundance 342

and high Fe concentration. 343

Correspondingly, we examined the transcript abundance of Fe deficiency 344

responsive genes. In contrast to the bhlh34bhlh104 mutants, overexpression 345

plants constitutively activate FIT, bHLH38/39/100/101, MYB10/72, IRT1, and 346

FRO2 (Figure 6), as well as NAS2, NAS4, ZIF1, FRD3, OPT3 and PYE 347

(Supplemental figure 10). These data suggest that bHLH34 and bHLH104 348

positively regulate Fe deficiency response. 349

350

bHLH34 and bHLH104 Directly Regulate Transcription of 351

bHLH38/39/100/101 352



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353

Although bHLH38/39/100/101 and FIT are the major regulators of Fe 354

homeostasis, their transcripts are still up-regulated in response to Fe 355

deficiency. This implies that unidentified transcription factors activate their 356

transcription in Fe-deficient conditions. Our yeast one-hybrid assays indicated 357

that bHLH34 and bHLH104 can bind to the promoter of bHLH101. 358

Considering the fact that the transcript abundance of bHLH38/39/100/101 359

and FIT is decreased in the bhlh34bhlh104 mutants, we proposed that 360

bHLH38/39/100/101 and FIT are the direct target genes of bHLH34 and 361

bHLH104. To confirm this hypothesis, we designed a reporter-effector 362

transient expression assay system (Figure 7A; Supplemental figure 11A). The 363

promoter of bHLH101 was fused with the GFP reporter gene containing a 364

nuclear localization sequence (NLS) for construction of the reporter 365



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expression cassette, ProbHLH101:NLS-GFP. For the construction of effectors, 366

bHLH34 and bHLH104 were fused with the CaMV 35S promoter to form 367

Pro35S:Myc-bHLH34 and Pro35S:Myc-bHLH104. When the 368



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ProbHLH101:NLS-GFP reporter was separately co-expressed with the empty 369

vector (the backbone vector of Pro35S:Myc-bHLH34 and 370

Pro35S:Myc-bHLH104), only a weak GFP signal was observed (Figure 7B). In 371

contrast, the Pro35S:Myc-bHLH34 or Pro35S:Myc-bHLH104 effectors 372

dramatically enhanced the GFP signal of the ProbHLH101:NLS-GFP reporter. To 373

confirm the activation specificity, we also constructed a Pro35S:Myc-MYC2 374

effector. MYC2 is a bHLH transcription factor with transcription activation 375

activity (Fernández-Calvo et al., 2011). Co-expression assays indicated that 376

use of the Pro35S:Myc-MYC2 resulted in as low a GFP signal as was observed 377

for the empty vector (Figure 7B). We further quantified the GFP transcript 378

levels, which showed that the GFP mRNA abundance was considerably 379

increased when Pro35S:Myc-bHLH34 or Pro35S:Myc-bHLH104 effectors were 380

co-expressed with ProbHLH101:NLS-GFP (Figure 7C). Correspondingly, we 381

also constructed ProbHLH38:NLS-GFP, ProbHLH39:NLS-GFP, 382

ProbHLH100:NLS-GFP, and ProFIT:NLS-GFP reporters, finding that 383

Pro35S:Myc-bHLH34 and Pro35S:Myc-bHLH104 activated ProbHLH38:NLS-GFP, 384

ProbHLH39:NLS-GFP, and ProbHLH100:NLS-GFP, but not ProFIT:NLS-GFP 385

(Supplemental figure 11B). These data suggest that both bHLH34 and 386

bHLH104 specifically activate the transcription of bHLH38/39/100/101. 387

388

Overexpression of bHLH101 Partially Rescues bhlh34bhlh104 Mutants 389

Previous studies have confirmed that the FRO2 and IRT1 genes are 390

regulated directly by bHLH38/39/100/101and their expression is drastically 391

decreased in a bHLH39, bHLH100, and bHLH101 triple knockout mutant 392

(Yuan et al., 2008; Wang et al., 2013). Thus, we speculated that the inability 393

of bhlh34bhlh104 to induce bHLH38/39/100/101 is the primary reason for the 394

Fe deficiency symptoms and the reduced expression of FRO2 and IRT1 in 395

the bhlh34bhlh104 mutants. It was expected that elevated expression of 396

bHLH38/39/100/101 would cure or relieve the Fe deficiency symptoms. 397

Because of the functional redundancy of bHLH38/39/100/101 (Wang et al., 398



20

2013), we selected bHLH101 as a representative. We employed the CaMV 399

35S promoter to drive the expression of bHLH101. The Pro35S:bHLH101 400

cascade was introduced into the bhlh34bhlh104 mutants by Agrobacterium. 401



21

Of the 78 transgenic plants screened, 42 showed significant relief of the leaf 402

chlorosis symptom. Transcript level analysis indicated that bHLH101 was 403

constitutively overexpressed in the bhlh34bhlh104/Pro35S:bHLH101 plants 404

(Supplemental figure 12A). The T2 generation plants from two different 405

transgenic lines, bhlh34bhlh104/Pro35S:bHLH101#1 and 406

bhlh34bhlh104/Pro35S:bHLH101#11, were used for further analyses. When 407

grown for 10 days on Fe-deficient media, these two lines displayed significant 408

growth advantages compared with the bhlh34bhlh104 mutants (Figure 8A), 409

including increased root length and chlorophyll content (Figure 8B,C). We 410

further assessed the expression of IRT1, FRO2, MYB10 and MYB72, finding 411

that IRT1 and FRO2, but not FIT, MYB10 or MYB72, were elevated in the two 412

transgenic lines exposed to Fe-deficient conditions compared with the 413

bhlh34bhlh104 mutants (Figure 8D; Supplemental figure 12B-D). These data 414

reveal that the overexpression of bHLH101 partially rescues the 415

bhlh34bhlh104 mutants. 416

417

bHLH34 Can Physically Interacts with bHLH104 418

419

bHLH34 and bHLH104 belong to the IVc bHLH subgroup (Heim et al., 2003). 420

A typical feature of bHLH proteins is the formation of homodimers or 421

heterodimers. To investigate the potential interaction between bHLH34 and 422

bHLH104, we conducted yeast-two-hybrid assays (Figure 9A). When 423

full-length bHLH34 was fused with the BD domain of GAL4 in vector pGBK-T7, 424

it showed strong autoactivation activity. In contrast, the C-terminal truncated 425

versions containing the bHLH domain (bHLH34C) displayed no autoactivation 426

activity; this form was used in the subsequent yeast-two-hybrid assays. 427

Protein interaction test in yeast indicated that bHLH34C can interact with 428

bHLH104. 429

Next, we employed bimolecular fluorescence complementation (BiFC) 430

assays to confirm their interaction in plant cells. bHLH34 and bHLH104 were 431



22

fused with the N-terminal fragment (nYFP) and C-terminal fragment (cYFP) of 432

YFP, respectively. When bHLH34-nYFP was transiently co-expressed with 433

bHLH104-cYFP, strong YFP fluorescence was visible in the nucleus of 434

epidermal cells in Nicotiana benthamiana leaves whereas no YFP 435

fluorescence was detected in negative controls (bHLH34-nYFP co-expressed 436

with cYFP, or nYFP co-expressed with bHLH104-cYFP) (Figure 9B). 437

To further confirm whether bHLH34 and bHLH104 form a protein complex 438



23

in plant cells, we performed co-immunoprecipitation assays (Figure9C). 439

bHLH34 and bHLH104 were transiently co-expressed in tobacco leaves. The 440

total proteins were incubated with Flag antibody and A/G-agarose beads and 441

then separated on SDS-PAGE for immunoblotting with Myc antibody. In 442

agreement with the results from BiFC, bHLH34 and bHLH104 were present in 443

the same protein complex. Taken together, these data suggest that bHLH34 444

could physically interact with bHLH104. 445

446

Interaction between bHLH34/ bHLH104 and bHLH105 447

448

This work confirms the interaction between bHLH34 and bHLH104. Recent 449

study suggested that bHLH104 interacts with bHLH105 and modulates Fe 450

homeostasis in Arabidopsis thaliana (Zhang et al., 2015). Considering that 451

these three proteins share the same target genes (bHLH38/39/100/101), and 452

their overexpressors up-regulate the same Fe-deficiency responsive genes, 453

we proposed that they function as heterodimers. To investigate our 454

hypothesis, yeast-two-hybrid assays were performed. As shown in Figure 455

10A, each of these three proteins can interact with itself and the other two 456

proteins, implying that they function as homodimers or heterodimers. 457

To further investigate their genetic interactions, we further produced two 458

double mutants, bhlh34bhlh105 and bhlh104bhlh105. We tried to generate 459

bhlh34bhlh104bhlh105 triple mutants but failed. Then, we evaluated the 460

Fe-deficiency tolerance ability of various mutants. We found that under Fe 461

deficiency conditions both bhlh34bhlh105 and bhlh104bhlhl105 double 462

mutants displayed the enhanced Fe-deficiency symptoms (shorter roots) 463

compared with the three single mutants (Figure 10B). The expression of 464

Fe-deficiency responsive genes was lower in the bhlh34bhlh105 and 465

bhlh104bhlhl105 double mutants than in the three single mutants 466

(Supplemental figure 13). All these data suggest that bHLH34, bHLH104 and 467

bHLH105 play non-redundant but additive roles in modulating Fe 468



24

homeostasis. 469

Given their similar molecular functions, we wanted to know whether the 470

expression patterns of these three genes are different. We constructed 471



25

ProbHLH34:GUS, ProbHLH104:GUS and ProbHLH105:GUS transgenic plants. 472

Although different expression strength was observed, bHLH34, bHLH104 and 473

bHLH105 have similar tissue-specific expression patterns in rosette leaves, 474

cauline leaves, stems and siliques, but not in flowers(Supplemental figure 475

14A). When seedlings were further analyzed by microscope, all plants 476

contained GUS staining in the pericycle of root maturation zone and veins of 477

leaves (Figure 11). ProbHLH105:GUS was detected in the elongation zone of 478

root tips and early lateral roots whereas ProbHLH34:GUS and ProbHLH104:GUS 479

were not observed. In contrast, ProbHLH34:GUS and ProbHLH104:GUS were 480

detected in the hypocotyls whereas ProbHLH105:GUS was not. The difference 481

of tissue-specific expression patterns may explain the functional 482

non-redundancy between bHLH34, bHLH104 and bHLH105. In addition, 483

GUS staining of seedlings exposed to Fe-sufficient and Fe-deficient 484

conditions suggests that their promoter activity is stable irrespective of the Fe 485



26

status (Supplemental figure 14B). 486

487

DISSCUSSION 488

489

Fe is indispensable for plant growth and development. Fe deficiency often 490

causes delayed growth and reduced photosynthesis, and hence decreased 491

crop yields. When suffering from a Fe-deficient environment, plants can 492

sense external Fe status and employ transcription factors and intricate 493

mechanisms to regulate the expression of Fe-uptake-associated genes, 494

which then facilitate Fe influx from soils to meet the internal demands of the 495

plant. However, excess Fe is toxic to plant cells owing to the generation of 496

hydroxyl radicals by the Fenton reaction (Thomine and Vert, 2013). Therefore, 497

it is crucial to maintain Fe homeostasis in plants. Plants have evolved 498

sophisticated regulatory mechanisms to maintain Fe homeostasis. It is well 499

known that bHLH38/39/100/101 and FIT have synergistic effects on the 500

regulation of Fe deficiency responses. Their co-overexpression constitutively 501

activates Fe acquisition strategy I in Arabidopsis thaliana. It is noteworthy that, 502

like other Fe deficiency responsive genes, bHLH38/39/100/101 and FIT are 503

induced by Fe deficiency. Thus, to reveal how bHLH38/39/100/101 and FIT 504

are activated in Fe-deficient conditions would provide insights into the 505

mechanism that plants employ to maintain Fe homeostasis. Here, we 506

characterized that both bHLH34 and bHLH104 activate the transcription of 507

bHLH38/39/100/101 and they non-redundantly regulate Fe deficiency 508

responses in Arabidopsis thaliana. 509

bHLH34 was identified by screening proteins that bound to the promoter of 510

the bHLH101 gene. The bhlh34, bhlh104, and bhlh34bhlh104 mutants display 511

sensitivity to Fe deficiency, including reduced root length, chlorotic leaves, 512

and decreased ferric-chelate reductase activity (Figure 2C; Figure 3 B). 513

Correspondingly, amiR-bhlh34/104 plants show phenotypes similar to those 514

of bhlh34bhlh104 mutants (Figure 2D; Supplemental Figure 3D). These data 515



27

indicate that loss-of-function of bHLH34 and bHLH104 causes sensitivity to 516

Fe deficiency. Fe deficiency sensitivity can be caused by limited Fe uptake or 517

disrupted Fe distribution. For example, irt1, frd1, and fit mutants display 518

severe Fe deficiency sensitivity because of Fe uptake limitation (Robinson et 519

al., 1999; Vert et al., 2002; Colangelo and Guerinot, 2004). In contrast, frd3 520

and opt3 mutation enhances Fe deficiency sensitivity by disrupting Fe 521

translocation (Durrett et al., 2007; Zhai et al., 2014). The determination of Fe 522

concentration confirms that the bhlh34bhlh104 mutants contain less Fe than 523

wild-type plants in both Fe-sufficient and -deficient conditions (Figure 3C, D), 524

suggesting that limited Fe uptake contributes to the Fe deficiency sensitivity 525

of the bhlh34bhlh104 mutants. We also observed that new leaves of 526

bhlh34bhlh104 mutants are yellower than old leaves (Figure 2D), whereas 527

the opposite phenomenon occurs in the bHLH34 and bHLH104 528

overexpression plants (Figure 5A), implying that bHLH34 and bHLH104 may 529

interrupt Fe distribution between leaves. 530

Fe deficiency responsive genes, including IRT1 and FRO2, MYB10/72, FIT 531

and bHLH38/39/100/101, are repressed in the bhlh34bhlh104 mutants 532

(Figure 4), indicating that the loss-of-function of bHLH34 and bHLH104 533

impairs the transduction of Fe deficiency response signaling. Although bhlh34 534

and bhlh104 mutants display reduced activation of Fe deficiency responsive 535

genes, the bhlh34bhlh104 mutants have the strongest inhibitory effect on the 536

Fe deficiency responsive genes. When either ProbHLH34:Myc-bHLH34 or 537

ProbHLH104:Myc-bHLH104 was introduced into bhlh34bhlh104 double mutants, 538

the Fe-deficiency symptoms of bhlh34bhlh104 were partially rescued 539

(Supplemental figure 15). These data suggest that bHLH34 and bHLH104 540

have non-redundant functions under Fe-deficient conditions. In contrary to 541

bhlh34 and bhlh104 mutants, bHLH34 and bHLH104 overexpression plants 542

activate the expression of Fe deficiency responsive genes (Figure 6). In 543

agreement with this, Fe is over accumulated in the overexpression plants 544

(Figure 5D). Taken together, our data suggest that bHLH34 and bHLH104 act 545



28

as positive regulators in Fe-deficiency response signaling. 546

The bhlh34bhlh104 mutants displayed enhanced Fe-deficiency sensitivity. 547

However, unexpectedly, we found that about 10% of Pro35S:Myc-bHLH34 and 548

30% of Pro35S:Myc-bHLH104 plants showed the short roots on Fe-deficient 549

media although they accumulated high Fe content and activated Fe 550

deficiency responsive genes including bHLH38/39/100/101, MYB10/72, 551

FRO2, IRT1 and so on. In fact, similar observations were made for a pye-1 552

mutant which up-regulates Fe deficiency responsive genes and accumulates 553

excessive Fe, but displays short roots under Fe deficiency conditions (Long et 554

al., 2010). NAS4 and ZIF1 are significantly up-regulated in the bHLH34 and 555

bHLH104 overexpression plants and particular high when overexpression 556

plants were subjected to Fe deficiency conditions (Supplemental figure 10). It 557

is noteworthy that NAS4 and ZIF1 are directly negatively regulated by PYE 558

(Long et al., 2010). As a negative regulator in Fe homeostasis, PYE is also a 559

direct target of bHLH104 and bHLH105 (Zhang et al., 2015). Our results 560

suggest that PYE is positively regulated by bHLH34, bHLH104 and bHLH105. 561

The expression analysis of NAS4 and ZIF1 between bhlh34bhlhl104 and 562

pye-1 mutants (Supplemental Figure 16) indicates that NAS4 and ZIF1 are 563

positively and negatively regulated by bHLH34/bHLH104 and PYE, 564

respectively. Plants have evolved this mechanism to fine-tune the Fe 565

homeostasis. 566

Recently, Zhang et al. (2015) revealed that overexpression of bHLH104 567

and bHLH105 activates the Fe deficiency responsive genes and 568

overaccumulation of Fe. However, the Fe deficiency sensitivity of our 569

overexpression plants is completely different from the results from Zhang et al. 570

(2015), who confirmed that bHLH104 overexpression plants produced longer 571

roots than wild type on Fe deficient media. They used Pro35S:bHLH104-GFP 572

as an overexpression construct whereas we used Pro35S:Myc-bHLH104. To 573

investigate whether the C-terminal fused GFP affects the function of bHLH34 574

or bHLH104, we constructed Pro35S:Myc-bHLH34-GFP and 575



29

Pro35S:Myc-bHLH104-GFP transgenic plants. No visible phenotype was found 576

in all Pro35S:Myc-bHLH34-GFP and Pro35S:Myc-bHLH104-GFP transgenic 577

plants compared with wild type when plants were grown in normal soils. In 578

agreement with the results from Zhang et al., about 70% of these GFP fusion 579

transgenic plants produced significantly longer roots than wild type on Fe 580

deficient media (Supplemental Figure 17A). The expression of Fe-deficiency 581

responsive genes in GFP fusion transgenic plants with longer roots is 582

significantly lower than that in Myc fusion transgenic plants with shorter roots, 583

but comparable with the Myc fusion transgenic plants with normal roots 584

(Supplemental Figure 17B). It is noteworthy that the overexpression plants 585

with visible phenotypes have extremely high transgene levels compared with 586

overexpressors with invisible phenotypes (Figure 5C; Supplemental Figure 587

8B). Therefore, it is likely that the extremely high expression of transgene 588

caused abnormal regulation of Fe-uptake-associated genes and Fe overload. 589

In any case, it is an efficient approach to generate Fe deficiency tolerant 590

plants by constitutively expressing bHLH34 or bHLH104 with a C-terminal 591

fused with a GFP gene. 592

Under both Fe-sufficient and -deficient conditions, the transcript levels of 593

bHLH38/39/100/101 are always lower in the bhlh34, bhlh104, and 594

bhlh34bhlh104 mutants than in wild-type plants (Figure 4). Transient 595

expression assays reveal that both bHLH34 and bHLH104 activate the 596

promoters of bHLH38/39/100/101 (Figure 7B, C; Supplemental figure 11). 597

These data suggest that bHLH34 and bHLH104 directly and positively 598

regulate the transcription of bHLH38/39/100/101. In agreement with our 599

results, Zhang et al. (2015) showed the direct regulation of 600

bHLH38/39/100/101 by bHLH104. bHLH38/39/100/101 are strongly induced 601

when wild-type plants are exposed to Fe-deficient conditions. However, it is 602

worth noting that, in some mutants where Fe homeostasis is disrupted, such 603

as irt1, frd1, frd3, and fit, bHLH38/39/100/101 transcript abundance is 604

increased even under Fe-sufficient conditions compared with wild-type plants 605



30

(Wang et al., 2007). The fact that Fe deficiency responsive genes are 606

regulated by internal Fe demand via shoot-to-root signaling was previously 607

demonstrated (Grusak and Pezehgi, 1996; Vert et al., 2003). Interestingly, 608

although the bhlh34bhlh104 mutants have lower Fe content than wild-type 609

plants, the induction of bHLH38/39/100/101 by Fe deficiency is strongly 610

inhibited in the bhlh34bhlh104 mutants, suggesting that the transcriptional 611

activation of bHLH38/39/100/101 by internal Fe demand is largely dependent 612

on bHLH34 and bHLH104. 613

It has been confirmed that bHLH38/39/100/101 have redundant functions in 614

Fe deficiency response (Yuan et al., 2008; Wang et al., 2013). Our 615

experiments indicate that constitutive expression of bHLH101 partially 616

rescued the severe Fe deficiency symptoms of bhlh34bhlh104 mutants 617

(Figure 8), implying that regulation of bHLH38/39/100/101 by bHLH34 and 618

bHLH104 is required for Fe homeostasis. Because of the imperfect 619

complementation by overexpression of bHLH101, it is possible that the 620

ectopic expression of bHLH101 caused ectopic expression of its target genes, 621

which then led to the abnormal Fe distribution, or that bHLH34 and bHLH104 622

also regulate other genes that are involved in Fe homeostasis but are not 623

regulated by bHLH101. In fact, we found that the induction of FIT, MYB10, 624

and MYB72 by Fe deficiency is not rescued in 625

bhlh34bhlh104/Pro35S:bHLH101#1 and bhlh34bhlh104/Pro35S:bHLH101#11 626

plants (Supplemental figure 12). It has been reported that MYB10 and MYB72 627

are required for growth in Fe-limiting conditions by directly regulating the 628

expression of NAS4 (Palmer et al., 2013). NAS genes are responsible for the 629

synthesis of nicotianamine, which plays a role in intercellular and intracellular 630

distribution of Fe (Takahashi et al., 2003; Klatte et al., 2009). Therefore, the 631

elevated expression of bHLH101 may only rescue the Fe uptake phenotype, 632

but not the Fe distribution phenotype, in the bhlh34bhlh104 mutants. The 633

transcript abundance of bHLH34 and bHLH104 is not induced by Fe 634

deficiency (Supplemental Figure 2), which implies that they may be regulated 635



31

at the post-transcription level because their target genes are up-regulated by 636

Fe deficiency. To further analyze their protein stability and screen their 637

interaction partners will provide insights into their regulation mechanisms 638

under Fe deficiency conditions. The stronger Fe-deficiency symptoms of 639

double mutants than single mutants suggest that bHLH34, bHLH104 and 640

bHLH105 play non-redundant roles in regulating Fe homeostasis. We also 641

observed differential expression patterns among ProbHLH34:GUS, 642

ProbHLH104:GUS and ProbHLH105:GUS, which might explain the non-redundant 643

roles. 644

A putative Fe deficiency responsive signaling pathway was shown in Figure 645

12. BTS is considered a potential Fe sensor because its structure, its ability to 646

bind Fe, and its capacity to catalyze ubiquitination are similar to the 647

mammalian Fe sensor FBXL5 (Kobayashi et al., 2013; Selote et al., 2015). 648

BTS is induced and its product is stabilized by Fe deficiency; however, it has 649

been confirmed to negatively regulate Fe homeostasis (Long et al, 2010; 650

Selote et al., 2015; Zhang et al., 2015). Long et al. (2010) confirmed that 651

bHLH104 and bHLH105, but not bHLH34, interacts with BTS in yeast. 652

Although its interaction with BTS, bHLH104 protein is not affected by BTS 653

(Selote et al., 2015). bHLH34, bHLH104 and bHLH105 function as 654

homodimers or heterodimers. It is likely that the interaction of BTS with 655

bHLH104 competitively interferes with the formation of dimers, which then 656

affects regulation of downstream genes, such as bHLH38/39/100/101 and 657

PYE. FIT interacts with bHLH38/39/100/101 to activate the expression of 658

IRT1 and FRO2. PYE functions as the negative regulator of ZIF1 and NAS4. 659

MYB10 and MYB72 function redundantly to regulate Fe homeostasis by the 660

direct activation of NAS4. The induction of MYB10/72 by Fe deficiency is 661

partially dependent of FIT; and bHLH34/104/105 are required for the 662

induction of FIT and MYB10/72. It is worth mentioning that the model has no 663

spatial dimension; it reflects interactions globally but is not applicable in any 664

particular cell type because the expression patterns of the different players do 665



32

not always overlap. Future research should aim at identifying the distinct 666

modules that are active in the root cortex and epidermis, in the root stele and 667

in various leaf cell types. 668

669

670



33

METHODS 671

Plant Materials and Growth Conditions 672

The Arabidopsis thaliana ecotype Columbia-0 was used as the wild type in 673

this study. The T-DNA insertion lines for bHLH34 (CS411089), bHLH104 674

(Salk_099496C) and bHLH105 (Salk_043690C) were confirmed using PCR 675

with a T-DNA primer and gene-specific primers (Supplemental Table 1). pye-1 676

was described previously (Long et al., 2010). Plants were grown in long 677

photoperiods (16 hour light/8 hour dark) or in short photoperiods (8 hour 678

light/16 hour dark) at 22°C. Surface sterilized seeds were stratified at 4°C for 679

2 to 4 d before being planted on media. Fe-sufficient (+Fe) media is half 680

Murashige and Skoog (MS) media with 1%sucrose, 0.8% agar, and 0.1 mM 681

FeEDTA. Fe deficient (–Fe) media is the same without FeEDTA. 682

683

Plasmid Construction 684

Standard molecular biology techniques were used for the cloning procedures. 685

Genomic DNA from genomic was used as the template for amplification of the 686

upstream regulatory promoter sequence for ProbHLH34:GUS, ProbHLH104:GUS , 687

ProbHLH105:GUS, ProFIT:NLS-GFP, ProbHLH38:NLS-GFP, ProbHLH39:NLS-GFP, 688

ProbHLH100:NLS-GFP and ProbHLH101:NLS-GFP. The NLS sequence was 689

amplified from the pGADT7 plasmid. For Pro35S:Flag-bHLH34, 690

Pro35S:Myc-bHLH34, Pro35S:Myc-bHLH104, Pro35S:bHLH101, 691

Pro35S:Myc-GUS and Pro35S:Myc-MYC2, the corresponding cds sequences 692

were inserted between the CaMV35S promoter and polyA of the binary vector 693

pOCA30. The amiR-bhlh34/104 was integrated into the MIR319a backbone 694

and the construction strategy was described previously (Liang et al., 2012). 695

Primers used for these constructs were listed in Supplemental Table 1. 696

Arabidopsis thaliana transformation was conducted by the floral dip method 697

(Clough and Bent, 1998). Transgenic plants were selected with the use of 50 698

μg/mL kanamycin. 699

700



34

Histochemical GUS staining 701

The whole seedlings were immersed immediately in 1.5 ml staining solution 702

containing 0.5 mg/mL 5-bromo-4-chloro-3-indoyl-b-D-glucuronide (X-gluc, 703

Sigma) in 0.1 M sodium phosphate buffer (pH 7.3) in a microfuge tube. The 704

reaction was performed in the dark at 37°C until a blue indigo color appeared. 705

After the reaction, seedlings were rinsed in 0.1 M sodium phosphate buffer 706

(pH 7.3). The samples were then rinsed twice in 70% ethanol to remove 707

chlorophylls. 708

709

Fe Concentration Measurement 710

To determine Fe content, 11-day-old seedlings grown on +Fe media were 711

transferred to +Fe or –Fe media for 3 days. The shoots and roots were 712

separately harvested and used for Fe measurement. Leaves of 4-week-old 713

plants grown in normal soils were used for measurement of Fe content. About 714

100mg dry weight for roots or shoots was used as one sample and three 715

samples were used in each independent experiment. Fe content analysis was 716

performed using inductively coupled plasma spectroscopy. 717

718

Ferric Chelate Reductase Assays 719

Ferric chelate reductase assays were performed as previously described (Yi 720

and Guerinot, 1996). Briefly, ten intact plants for each genotype were 721

pretreated for 30 min in plastic vessels with 4 mL half-strength MS solution 722

without micronutrients (pH 5.5) and then soaked into 4 mL Fe(III) reduction 723

assay solution [half-strength MS without micronutrients, 0.1 mM Fe(III)-EDTA, 724

and 0.3 mM ferrozine, pH adjusted to 5.0 with KOH] for 30 min in darkness. 725

An identical assay solution containing no plants was used as a blank. The 726

purple-colored Fe(II)-ferrozine complex was quantified at 562nm . 727

728

Chlorophyll Measurement 729

Chlorophyll content was measured in 2-week-old plants grown on 730



35

Fe-sufficiency and Fe deficiency media. All leaves are collected and ground 731

to powder in liquid nitrogen. The powder was resuspended in 80% acetone 732

on ice and centrifuged at 10,000g at 4°C, for 5 min. Chlorophyll 733

concentrations were calculated from spectroscopy absorbance 734

measurements at 663.2, 646.8, and 470 nm (Lichtenthaler, 1987). 735

736

Gene Expression Analysis 737

One micrograms total RNA extracted using the Trizol reagent (Invitrogen) was 738

used for oligo(dT)18 primed cDNA synthesis according to the reverse 739

transcription protocol (Fermentas). The resulting cDNA was subjected to 740

relative quantitative PCR using a SYBR Premix Ex TaqTM kit (TaKaRa) on a 741

Roche LightCycler 480 real-time PCR machine, according to the 742

manufacturer’s instructions. ACTIN2 (ACT2) was amplified as an internal 743

control, and gene copy number was normalized to that of ACT2. For the 744

quantification of each gene, at least three biological repeats were used. Each 745

biological repeat contained three technical replicates. One representative 746

result from one biological repeat was shown. The statistic significance 747

analysis was performed by student’s t test. The qRT-PCR primers were listed 748

in Supplemental Table 1. 749

750

Yeast Assays 751

For Y1H assay, the Matchmaker Gold Yeast One-Hybrid Library Screening 752

System (Clontech) was used. The pAbAi vector harboring 1000bp sequence 753

upstream bHLH101 gene was integrated into the genome of yeast strain 754

Y1HGold followed by selection on synthetic dextrose media agar plates 755

without uracil (SD/-Ura). We screened an Arabidopsis thaliana equalized 756

full-length cDNA library on synthetic dextrose media without Leu 757

supplemented with 100 ng/mL Aureobasidin A (AbA) (SD/-Leu/AbA). The AbA 758

resistance is activated by prey proteins that specifically interact with the bait 759

sequence. Candidate cDNA-harboring vectors were amplified and isolated 760



36

via Escherichia coli. The sequence of each candidate was analyzed with the 761

help of the TAIR database. 762

For Y2H assay, the C-terminal truncated bHLH34C and bHLH104C 763

containing the bHLH domain and full-length bHLH105 cDNAs were cloned 764

into pGBKT7, and the full-length cDNAs of bHLH34, bHLH104 and bHLH105 765

were cloned into pGADT7. Growth was determined as described in the Yeast 766

Two-Hybrid System User Manual (Clontech). Primers used for the vector 767

construction were listed in Supplemental Table 1. Experiments were repeated 768

three times. 769

770

Transient Expression Assays 771

Plasmids were transformed into Agrobacterium tumefaciens strain EHA105. 772

Agrobacterial cells were infiltrated into leaves of Nicotiana benthamiana by 773

the infiltration buffer (0.2 mM acetosyringone, 10 mM MgCl2, and 10 mM MES, 774

PH5.6). For BiFC assay, equal volumes of an Agrobacterium culture were 775

mixed before infiltration into N. benthamiana leaves. After infiltration, YFP and 776

DAPI fluorescence were observed under a confocal laser scanning 777

microscope (Olympus, Tokyo, Japan). For transcription activation assay, the 778

final OD600 value was, 0.1 (an internal control; Pro35S:Myc-GUS), 0.5 779

(reporter) and 0.5 (effector). After infiltration, plants were placed in dark at 780

24°C for 48 h before RNA extraction. The transcript abundance of GFP was 781

normalized to GUS. 782

783

CoIP Assay 784

Flag-bHLH34 and Myc-bHLH104 (or Myc-GUS) were transiently 785

co-expressed in N. benthamiana leaves. Infected leaves were harvested 48 h 786

after infiltration and used for protein extraction. Flag-fused bHLH34 was 787

immunoprecipitated using Flag antibody and the coimmunoprecipitated 788

proteins were then detected using Myc antibody. 789

790



37

Accession Numbers 791

Sequence data from this article can be found in the Arabidopsis Genome 792

Initiative or GenBank/EMBL databases under the following accession 793

numbers: bHLH34 (At3g23210), bHLH104 (At4g14410), bHLH105 794

(At5g54680), bHLH38(At3g56970), bHLH39 (At3g56980), bHLH100 795

(At2g41240), bHLH101(At5g04150), FIT (At2g28160), MYB10 (At3g12820), 796

MYB72 (At1g56160), FRO2 (At1g01580), IRT1 (At4g19690), NAS2 797

(At5g56080), NAS4 (At1g56430), ZIF1 (At5g13740), FRD3 (At3g08040), 798

OPT3 (At5g59040) , PYE (At3g47640) and ACT2 (At3g18780). 799

800

Supplemental Data 801

Supplemental Figure 1. Phylogenetic tree of bHLH proteins involved in Fe 802

homeostasis. 803

Supplemental Figure 2. Expression of bHLH34 and bHLH104 in response to 804

Fe deficiency. 805

Supplemental Figure 3. Growth status of wild-type and mutant plants. 806

Supplemental Figure 4. Analysis of amiR-bhlh34/104 and double mutant 807

plants. 808

Supplemental Figure 5.Chlorophyll content of mutant seedlings on +Fe or –809

Fe media. 810

Supplemental Figure 6. NAS2, NAS4, ZIF1, FRD3, OPT3 and PYE 811

transcript levels in various mutant plants. 812

Supplemental Figure 7. Complementation of bhlh34 and bhlh104 mutants. 813

Supplemental Figure 8. Analysis of bHLH34 and bHLH104 overexpression 814

plants. 815

Supplemental Figure 9. Concentration of other metals in overexpression 816

plants. 817

Supplemental figure 10. NAS2, NAS4, ZIF1, FRD3, OPT3 and PYE 818

transcript levels in overexpression plants. 819

Supplemental figure 11. bHLH34 and bHLH104 activate the promoter of 820



38

bHLH38/39/100. 821

Supplemental figure 12. Analysis of bhlh34bhlh104/Pro35S:bHLH101 Plants. 822

Supplemental figure 13. Expression of Fe-deficient responsive genes in 823

various single and double mutants. 824

Supplemental figure 14. GUS staining of ProbHLH34:GUS, ProbHLH104:GUS 825

and ProbHLH105:GUS plants. 826

Supplemental figure 15. Partially complementation of bhlh34blhlh104 827

double mutants by ProbHLH34:Myc-bHLH34 and ProbHLH104:Myc-bHLH104. 828

Supplemental figure 16. Expression of NAS4 and ZIF1 in bhlh34blhlh104 829

and pye-1 mutants. 830

Supplemental Figure 17. Phenotypes of Pro35S:Myc-bHLH34-GFP and 831

Pro35S:Myc-bHLH104-GFP plants. 832

Supplemental Table 1. Primers used in this paper. 833

834

Figure Legends 835

Figure 1. Identification of bHLH34 and bHLH104 by yeast-one-hybrid assay. 836

The promoter of bHLH101 was used as bait and bHLH34/104 as prey. 837

Representative growth status of yeast cell was shown on SD/-Leu agar media 838

with or without AbA from triplicate independent trails. 839

840

Figure 2. Characterization of various mutant plants. 841

(A) T-DNA insertion position in the corresponding gene. Closed boxes 842

indicate the coding sequence. Open boxes indicate untranslated regions. 843

Triangles indicate the position of T-DNA. 844

(B) The corresponding full-length cDNAs were amplified by RT-PCR to 845

confirm the mutants. 846

(C) 10-day-old seedlings germinated directly on -Fe media. 847

(D) Phenotypes of amiR-bhlh34/104 and bhlh34bhlh104 plants in soils. 848

4-week-old plants grown in the normal soil were shown. 849

850



39

Figure 3. Fe deficiency response of various mutants. 851

(A) 3-week-old plants germinated in the soil with various concentration of 852

calcium oxide. 853

(B) Iron reductase activity of plants germinated and grown on +Fe media for 854

10 days and then shifted to –Fe media for 3 days. The ferrozine assay was 855

performed, in triplicate, on 10 pooled plant roots. Significant differences from 856

the wild type are indicated by *(P < 0.05). 857

(C) and (D) Fe content of shoots and roots. Seedlings were germinated and 858

grown on +Fe media for 11 days and then shifted to +Fe or –Fe media for 3 859

days. Significant differences from the wild type are indicated by *(P < 0.05). 860

861

Figure 4. Expression of Fe deficiency responsive genes in various mutants. 862

Wild-type and mutant plants were grown on +Fe media for 10 days, then 863

transferred to +Fe or –Fe media for 3 days. RNA was prepared from root 864

tissues. The data represent means (±SD) of three technical repeats from one 865

representative experiment. Significant differences from the corresponding 866

wild type are indicated by * (P < 0.05). 867

868

Figure 5. Phenotypes of bHLH34 and bHLH104 overexpression plants. 869

(A) 4-week-old plants grown in the normal soil. 870

(B) 10-day-old seedlings grown on –Fe media. The number indicates the 871

frequency of transgenic plants with the corresponding phenotypes. 872

(C) Relative transcript levels of bHLH34 (left) and bHLH104 (right) in 873

overexpression plants. Significant differences from the wild type are indicated 874

by * (P < 0.05). 875

(D) Fe content of leaves in 4-week-old plants grown in normal soils. 876

Significant differences from the wild type are indicated by * (P < 0.05). 877

878

Figure 6. Expression of Fe deficiency responsive genes in various transgenic 879

plants. Wild-type and transgenic plants were grown on +Fe media for 10 days, 880



40

then transferred to +Fe or –Fe media for 3 days. RNA was prepared from root 881

tissues. The data represent means (±SD) of three technical repeats from one 882

representative experiment. Significant differences from the corresponding 883

wild type are indicated by * (P < 0.05). 884

885

Figure 7. bHLH34 and bHLH104 activate the promoter of bHLH101. 886

(A) Schematic representation of the constructs used for transient expression 887

assays. The reporter construct consists of bHLH101 promoter, NLS sequence 888

fused with GFP coding sequence, and polyA terminator. Effector constructs 889

express Myc-bHLH34, Myc-bHLH104, and Myc-MYC2 under the control of 890

the CaMV 35S promoter. 891

(B) bHLH34 and bHLH104 activate the promoter of bHLH101 in transient 892

expression assays. The results are one representative of three biological 893

repeats. 894

(C) GFP transcript abundance. In the transient assays, Pro35S:Myc-GUS was 895

expressed as a control. GFP transcript abundance was normalized to GUS 896

transcript. The value with the empty vector as an effector was set to 1. The 897

results are means (±SD) of three technical repeats from one of three 898

biological repeats. Significant differences from the empty vector are indicated 899

by * (P < 0.05). 900

901

Figure 8. Overexpression of bHLH101 partially rescues bhlh34bhlh104. 902

(A) 10-day-old seedlings grown on +Fe or –Fe media. 903

(B) Root length of seedlings on +Fe or –Fe media .Values are means (±SD) 904

of 10 plants for each genotype. 905

(C) Chlorophyll content of seedlings on +Fe or –Fe media. Significant 906

differences from the wild type are indicated by * (P < 0.05). 907

(D) Expression of IRT1 and FRO2. Plants were grown on +Fe media for 10 908

days, then transferred to +Fe or –Fe media for 3 days. RNA was prepared 909



41

from root tissues. Significant differences from the corresponding wild type are 910

indicated by * (P < 0.05). 911

912

Figure 9. Interaction between bHLH34 and bHLH104. 913

(A) Yeast two hybrid assays. Representative growth status of yeast cells was 914

shown on SD/-LTHA agar media. 915

(B) BiFC assays. N. benthamiana leaves were infiltrated with different 916

combinations of the constructs. 917

(C) CoIP assays. Total protein was immunoprecipitated using Flag antibody, 918

and co-immunoprecipitated protein was then detected using Myc antibody. 919

920

Figure 10. Interactions between bHLH34, bHLH104 and bHLH105. 921

(A). Yeast two-hybrid assays. Interaction was indicated by the ability of cells 922

to grow on synthetic dropout medium lacking Leu/Trp/His/Ade. C-terminal 923

truncated bHLH34 and bHLH104 and full-length bHLH105 were cloned into 924

pGBKT7, and full-length cDNAs of bHLH34, bHLH104 and bHLH105 were 925

cloned into pGADT7, respectively. 926

(B). Fe-deficiency symptoms of various single and double mutants. 927

10-day-old seedlings germinated directly on –Fe or +Fe media. 928

929

Figure 11. GUS staining of ProbHLH34:GUS, ProbHLH104:GUS and 930

ProbHLH105:GUS seedlings. Two-week-old seedlings were used for GUS 931

staining. GUS staining of the root maturation regions, early lateral roots, root 932

tips, leaves, hypocotyls, and whole seedlings was shown. ep, epidermis; c, 933

cortex; e, endodermis; p, pericycle; el, elongation region of root tip; m, 934

meristem zone of root tip. 935

936

Figure 12. Fe deficiency responsive signaling pathway. 937

Fe deficiency stabilizes the BTS protein that interacts with bHLH104 and 938

bHLH105. bHLH34, bHLH104 and bHLH105 can form homodimers or 939



42

heterodimers to activate the transcription of bHLH38/39/100/101 and PYE. 940

FIT interacts with bHLH38/39/100/101 to activate the transcription of FRO2 941

and IRT1. PYE regulates the expression of ZIF1 and NAS4 negatively. 942

MYB10 and MYB72 regulate the expression of NAS4 positively. The induction 943

of MYB10/72 by Fe deficiency is partially dependent of FIT; and 944

bHLH34/104/105 are required for the induction of FIT and MYB10/72. 945

946

947

ACKNOWLEDGMENTS 948

We thank TAIR (The Arabidopsis Information Resource) at the Ohio State 949

University for the T-DNA insertion mutants. 950

951

952

953



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Zhai Z, Gayomba SR, Jung HI, Vimalakumari NK, Piñeros M, Craft E, Rutzke MA, Danku J, Lahner B, Punshon T, et al (2014) OPT3 isa phloem-specific iron transporter that is essential for systemic iron signaling and redistribution of iron and cadmium inArabidopsis. Plant Cell 26: 2249-2264



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2 homeostasis in arabidopsis thaliana · 2/26/2016 · 5 121 contains a hemerythrin domain (hhe)...

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