1 Short title: Zn uptake transporter in rice2 31 KAKENHI Grant Number 16H06296 to J.F.M.). 32 33...

1

Short title: Zn uptake transporter in rice 1

2

Title 3

The ZIP transporter family member OsZIP9 contributes to root Zn uptake in rice 4

under Zn-limited conditions 5

6

Authors 7

Sheng Huang1, Akimasa Sasaki1, Naoki Yamaji, Haruka Okada, Namiki Mitani-Ueno 8

and Jian Feng Ma† 9

Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, 10

710-0046 Japan 11 1These authors contribute to this work equally 12 †To whom correspondence should be addressed. 13

Jian Feng Ma 14

Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, 15

710-0046 Japan 16

E-mail: [email protected] 17

Tel: +81-86-434-1209, Fax: +81-86-434-1209 18

19

One-sentence summary: The zinc transporter OsZIP9, expressed at the exodermis and 20

endodermis of root mature region, contributes to Zn uptake from soil in rice. 21

22

Author contributions 23

S. H., A. S., and J.F.M. conceived and designed the experiments; S. H., A. S., N. Y., H. 24

O., N.M-U., and J. F. M. performed experiments. S. H., A. S., and J.F.M. analyzed data; 25

S. H., A. S., and J.F.M. wrote the manuscript; all authors discussed the results and 26

commented on the manuscript. 27

28

Funding information 29

This work was supported by Grant-in-Aid for Specially Promoted Research (JSPS 30

Plant Physiology Preview. Published on May 5, 2020, as DOI:10.1104/pp.20.00125

Copyright 2020 by the American Society of Plant Biologists

www.plantphysiol.orgon June 24, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

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2

KAKENHI Grant Number 16H06296 to J.F.M.). 31

32

Abstract 33

Zinc is an important essential micronutrient for plants and humans; however, the exact 34

transporter responsible for root zinc (Zn) uptake from soil has not been identified. Here, 35

we found that OsZIP9, a member of ZIP (ZRT, IRT-like protein) family, is involved in 36

Zn uptake in rice under Zn-limited conditions. OsZIP9 was mainly localized to the 37

plasma membrane and showed transport activity for Zn in yeast. Expression pattern 38

analysis showed that OsZIP9 was mainly expressed in the roots throughout all growth 39

stages and its expression was up-regulated by Zn-deficiency. Furthermore, OsZIP9 was 40

expressed in the exodermis and endodermis of root mature regions. For plants grown in 41

a hydroponic solution with low Zn concentration, knockout of OsZIP9 significantly 42

reduced plant growth, which was accompanied by decreased Zn concentrations in both 43

the root and shoot. However, plant growth and Zn accumulation did not differ between 44

knockout lines and wild-type rice under Zn-sufficient conditions. When grown in soil, 45

Zn concentrations in the shoots and grains of knockout lines were decreased to half of 46

wild-type rice, whereas the concentrations of other mineral nutrients were not altered. 47

A short-term kinetic experiment with stable isotope 67Zn showed that 67Zn uptake in 48

knockout lines was much lower than that in wild-type rice. Combined, these results 49

indicate that OsZIP9 localized at the root exodermis and endodermis functions as an 50

influx transporter of Zn and contributes to Zn uptake under Zn-limited conditions in 51

rice. 52

53

Introduction 54

Zinc (Zn) is an essential micronutrient for plant growth and development (Marschner, 55

2012). Zn plays structural and catalytic roles in large number of proteins. However, Zn 56

deficiency is the most widely occurring micronutrient deficiency in crops worldwide, 57

which has been a limiting factor of crop production on millions of hectares of arable 58

land, especially in alkaline soil (Barker and Pilbeam, 2015). Furthermore, this 59

deficiency also results in Zn deficiency in humans because Zn in edible parts of crops 60



3

is our primary source of Zn intake. Therefore, it is important to understand the 61

molecular mechanisms of Zn transport and regulation in crops for enhancing crop 62

tolerance to Zn deficiency and preserving Zn accumulation in edible plant parts. 63

The predominant form of Zn in soil solution is the divalent cation (Zn2+) in most 64

soils, although it may be present as Zn(OH)+ at high pH. The transport of Zn from soil 65

to different organs and tissues have been proposed to be mediated by different 66

transporters such as members of the Zn-regulated transporter, iron-regulated 67

transporter-like proteins (ZIP; ZRT-IRT-related protein), yellow-stripe1-like (YSL) 68

family, heavy metal ATPases (HMA), cation diffusion facilitator (CDF) (Grotz et al., 69

1998; Guerinot, 2000; Sinclair and Krämer, 2012). Among them, several members of 70

the ZIP family have been implicated in uptake and transport of Zn. ZIP transporters 71

were first identified in yeast (ZRT1) and Arabidopsis (IRT) (Zhao and Eide, 1996; Eide 72

et al., 1996). Homologous ZIP proteins are present in many plant species. For example, 73

there are 15 members in Arabidopsis (Milner et al., 2013), 17 in rice (Oryza sativa) 74

(Chen et al., 2008 ), 14 in wheat (Triticum aestivum) (Evens et al., 2017), 12 in barley 75

(Hordeum vulgare) (Tiong et al., 2014), and 23 in common bean (Phaseolus vulgaris 76

L.) (Astudillo et al., 2013). Most ZIP proteins have 309–470 amino acids and are 77

predicted to have eight trans-membrane domains and a similar membrane topology in 78

which the amino- and carboxy-terminal ends of the proteins are located on the outside 79

surface of the plasma membrane (Guerinot, 2000). Based on transport assays mainly in 80

yeast mutants, ZIP transporters show broad substrate transport activity; in addition to 81

transporting Zn and Fe, they also transport Mn, Cd, and Co, although some members 82

only transport Zn (Korshunova et al., 1999; Waters and Sankaran, 2011; Milner et al., 83

2013). The ZIP genes also show different expression patterns; some are only expressed 84

in the roots (Bughio et al., 2002; Ishimaru et al., 2006), whereas others are expressed in 85

different tissues (Ishimaru, et al., 2005; Yang et al. 2009; Lee et al., 2010a; Lee et al., 86

2010b; Kavitha et al., 2015; Sasaki et al., 2015). The response of ZIP genes to different 87

Zn concentrations differ between members, but most ZIP genes reported are 88

up-regulated by Zn-deficiency (Ishimaru et al., 2005; Lee et al., 2010a; Lee et al., 89

2010b; Yang et al. 2009; Kavitha et al., 2015). Furthermore, two basic-region 90



4

leucine-zipper (bZIP) transcription factors, bZIP19 and bZIP23, are reported to be 91

involved in regulation of ZIP expression (Assuncao et al., 2010; Inaba et al., 2015). 92

Rice is a staple food for half of the global population, and therefore it provides an 93

important source of dietary Zn intake in rice-eating populations. However, the 94

transport system of Zn in rice has not been well understood. Several ZIP members have 95

been functionally characterized in terms of transport activity, expression patterns, and 96

ectopic expression analysis. OsZIP1, OsZIP3, OsZIP4, OsZIP5, OsZIP7a, and OsZIP8 97

showed influx transport activity for Zn in yeast (Ramesh et al., 2003, Ishimaru et al., 98

2005; Yang et al., 2009; Lee et al., 2010a; Lee et al., 2010b; Tan et al., 2019). However, 99

OsZIP2 in yeast and OsZIP6 in Xenopus oocytes did not show transport activity for Zn 100

(Ramesh et al. 2003; Kavita et al., 2015). Rice ZIP genes also show different 101

expression patterns; OsZIP1, OsZIP4, OsZIP5, OsZIP6, OsZIP7a, and OsZIP8 are 102

expressed in both the roots and shoots (Ramesh et al., 2003; Ishimaru et al., 2005; 103

Kavitha et al., 2015; Yang et al., 2009; Lee et al., 2010a; Lee et al., 2010b), whereas 104

OsZIP3 is mainly expressed in the nodes (Sasaki et al., 2015). Furthermore, the 105

expression of OsZIP4, OsZIP5, OsZIP6, OsZIP7a, and OsZIP8 is up-regulated by 106

Zn-deficiency, whereas OsZIP1 and OsZIP3 are constitutively expressed (Suzuki et al., 107

2012; Sasaki et al., 2015). On the other hand, overexpression of OsZIP4 and OsZIP5 108

causes decreased Zn accumulation in the shoots, but increased Zn accumulation in the 109

roots (Ishimaru et al., 2007; Lee et al., 2010a). Based on these findings, OsZIP1 has 110

been proposed to function in Zn uptake from soil (Ramesh et al. 2003, Bashir et al. 111

2012), whereas OsZIP4, OsZIP5, OsZIP7, and OsZIP8 are involved in Zn 112

translocation/distribution in the shoots (Ishimaru et al., 2005; Lee et al., 2010a; Lee et 113

al., 2010b; Sasaki et al., 2015; Tan et al., 2019). However, aside for OsZIP3, the exact 114

physiological roles of these ZIP genes in planta remain poorly understood. OsZIP3 115

localized in the node is responsible for the preferential distribution of Zn to developing 116

tissues (Sasaki et al. 2015). Here, we report on a previously uncharacterized rice ZIP 117

member, OsZIP9. Through detailed functional analyses, we found that OsZIP9 is a 118

transporter that contributes to Zn uptake in both Zn-limited hydroponic conditions and 119



5

in soil. 120

121 122



6

Results 123

Cloning of OsZIP9 124

According to the database (http://aramemnon.uni-koeln.de/), there are 15 members of 125

the ZIP family in rice (Supplemental Fig. S1A). We amplified the full-length coding 126

region of OsZIP9 (LOC_Os05g39540/Os05g0472400) by PCR from complementary 127

DNA (cDNA) of rice roots (cv Nipponbare). The primers used were designed 128

according to the Rice Annotation Project (http://rice.plantbiology.msu.edu/). OsZIP9 is 129

composed of three exons and two introns (Supplemental Fig. S2) and encodes a protein 130

of 363 amino acids. OsZIP9 shares 23–52% identify with other ZIP members 131

(Supplemental Fig. S1B) and forms a separate clade from other ZIP members 132

(Supplemental Fig. S1A). Similar to other rice ZIP members, OsZIP9 protein was 133

predicted to have eight trans-membrane domains (TMHMM Server v. 2.0; 134

http://www.cbs.dtu.dk/services/TMHMM/) (Supplemental Figs. S1C and S2C). 135

136

Transport activity test of OsZIP9. 137

To examine whether OsZIP9 is able to transport Zn, we expressed it in Zn uptake–138

defective yeast cells (ZHY3) under control of the galactose-inducible promoter. A 139

time-course experiment with stable isotope 67Zn showed that in the presence of glucose 140

(no OsZIP9 expression) there was no difference in Zn accumulation (Δ67Zn) between 141

vector control and yeast expressing OsZIP9 (Fig. 1A). However, when the expression 142

of OsZIP9 was induced by the presence of galactose, yeast expressing OsZIP9 showed 143

much higher Δ67Zn compared with the empty vector control (Fig. 1B). 144

To examine the transport specificity of OsZIP9 for metals, we compared the 145

transport activity for Fe, Cu, and Zn using respective stable isotopes, specifically 67Zn, 146 65Cu, or 57Fe, in wild-type yeast cells (BY4741). In the presence of galactose, OsZIP9 147

transported only Zn and not Fe or Cu (Fig. 1C). 148

149

Expression pattern analysis of OsZIP9. 150

The expression pattern of OsZIP9 was investigated in plants grown in either soil or 151

nutrient solution by reverse transcription quantitative PCR (RT-qPCR). In samples 152



7

derived from rice grown in the field, OsZIP9 was found to be mainly expressed in the 153

roots at all growth stages (Fig. 2A). In samples from hydroponically cultivated rice, the 154

expression of OsZIP9 in the roots was strongly induced by Zn-deficiency, but not by 155

Cu- or Mn-deficiency (Fig. 2B). OsZIP9 expression was also induced by Fe-deficiency, 156

but to a lesser extent. Time-dependent expression analysis showed that OsZIP9 157

expression was significantly up-regulated following 1 day and further increased 158

following 3 days of Zn deficiency (Supplemental Fig. S3A). However, 1 day of Fe 159

deficiency did not induce OsZIP9 expression, although expression induction was 160

observed following 3 days of Fe deficiency (Supplemental Fig. S3B). 161

We also investigated the spatial expression pattern of OsZIP9 in different root 162

regions. The expression of OsZIP9 was very low in the root tip region (0–0.5 cm from 163

the root tip) (Fig. 2C). However, higher expression was detected in root mature regions 164

(>1.0 cm). 165

166

Tissue specificity of OsZIP9 expression 167

To investigate the tissue specificity of OsZIP9 expression, we generated transgenic 168

lines carrying the promoter of OsZIP9 fused with GFP. Immunostaining using GFP 169

antibody showed that the signal was very weak in both the root tip (0.2 cm from the 170

root tip) and mature region (1.5 cm from the root tip) of plants supplied with Zn (Fig. 3, 171

A and D). However, in Zn-deficient roots, ZIP9 was strongly expressed at the 172

exodermis and endodermis of the root mature region (Fig. 3, E, G and H). The signal in 173

the root tip of Zn-deficient plants was also weak, which is consistent with the spatial 174

expression pattern of OsZIP9 (Fig. 2C). No signal was detected in wild-type plants 175

(Fig. 3, C and F), indicating the specificity of the antibody. 176

177

Subcellular localization of OsZIP9 178

Subcellular localization of OsZIP9 was investigated by transiently expressing a 179

GFP-OsZIP9 fusion in rice protoplasts and onion epidermal cells. In rice protoplasts 180

expressing GFP alone, the GFP signal was detected in the cytoplasm and nuclei 181

(Supplemental Fig. S4, A-D). However, in protoplasts expressing GFP-OsZIP9, the 182



8

GFP signal was mainly localized to the peripheral membrane of the cells, although 183

some signal was also detected in the endomembrane (Supplemental Fig. S4, E-H). 184

Similar results were obtained in onion epidermal cells (Supplemental Fig. S4, I-L). 185

To further confirm OsZIP9 subcellular localization, we performed double staining 186

using DAPI and an OsZIP9 antibody. In the roots of plants exposed to -Zn conditions 187

for 4 days, OsZIP9 was localized to the periphery of the cells, outside of the nuclei 188

stained by DAPI (Supplemental Fig. S4, M-P). No signal was detected in the knockout 189

line (Supplemental Fig. S4Q). Taken together, these results indicate that OsZIP9 is 190

most likely localized to the plasma membrane. 191

192

Phenotypic analysis of OsZIP9 knockout lines in hydroponic and soil culture 193

To investigate the role of OsZIP9 in Zn transport, we generated OsZIP9 knockout lines 194

by the CRISPR/Cas9 technique. We obtained two independent knockout lines with 195

different target positions (oszip9-1 and oszip9-2): one (oszip9-1) with a 1-bp deletion at 196

the first exon, and the other (oszip9-2) with a 1-bp insertion at the second exon 197

(Supplemental Fig. S2B). 198

We first grew the wild-type rice and two independent knockout lines in a nutrient 199

solution containing different Zn concentrations (0.02, 0.2, or 2 µM). At 0.02 µM Zn, 200

growth of the two knockout lines was obviously inhibited compared with wild-type 201

rice (Fig. 4A). New leaves showed typical Zn-deficiency symptoms in the knockout 202

lines, but not in the wild-type rice (Fig. 4D). The shoot fresh weight of the knockout 203

lines was 65% of the wild-type rice (Fig. 4E), although the root fresh weight did not 204

differ between different lines (Fig. 4F). However, at 0.2 and 2 µM Zn, growth was 205

similar between wild-type rice and the knockout lines (Fig. 4, B, C, E and F). 206

We then compared mineral element profiles in the roots and shoots of wild-type rice 207

and the knockout lines exposed to different Zn concentrations. At 0.02 µM Zn, both the 208

concentration and content of Zn in the roots and shoots were significantly lower in the 209

knockout lines than in wild-type rice (Fig. 5, A-D). At 0.2 µM Zn, shoot Zn 210

concentration and content were lower in the knockout lines than in wild-type rice, but 211

root Zn concentration and content were similar between different lines. However, Zn 212



9

concentration and content in both the roots and shoots of the different lines were 213

comparable at 2 µM Zn (Fig. 5, A-D). 214

There was no difference in the concentrations of Ca, Mg, K, P, Fe, Cu, and Mn in the 215

roots of wild-type rice and the knockout lines (Supplemental Figs. S5, A-D and S6, 216

A-C); however, the knockout mutants showed higher concentrations of Ca, Mg, Fe, Cu, 217

and Mn in the shoots at 0.02 µM Zn, but not at 0.2 and 2.0 µM Zn (Supplemental Figs. 218

S5, E-F and S6, D-F). Moreover, the contents of these elements except Fe were similar 219

between the different lines at all Zn concentrations tested (Supplemental Figs. S7, E-F 220

and S8, D-F), indicating that the higher concentrations observed at 0.02 µM Zn were 221

caused by decreased growth. The shoot concentration and content of K were slightly 222

decreased in the knockout lines, whereas those of P were not altered compared with 223

wild-type rice (Supplemental Figs. S5, G-H and S7, G-H). 224

When grown in soil until maturity, the knockout lines accumulated less than half the 225

amount of Zn in wild-type rice in straw and brown rice grain (Fig. 6). However, the 226

concentrations of other elements, including Cu, Fe, and Mn, in straw and brown rice 227

were comparable between wild-type rice and the knockout lines, except that the 228

concentration of Mn in straw was slightly increased in the knockout lines compared 229

with wild-type rice (Fig. 6). We also compared accumulation of Cd and As in straw and 230

brown rice. No difference in the accumulation of these two toxic elements was found 231

in either straw or brown rice between wild-type rice and the OsZIP9 knockout lines 232

(Supplemental Fig. S9). Combined, these results indicate that OsZIP9 is a specific 233

transporter for Zn in rice roots. 234

235

Short-term uptake experiments with stable isotope 67Zn 236

To confirm whether Zn uptake was altered in the knockout lines, we performed a short 237

term (24 h) labeling experiment with stable isotope 67Zn. Following the exposure of 238

Zn-deficient plants to 0.4 µM 67Zn for 24 h, the OsZIP9 knockout lines accumulated 239

much less 67Zn (as ∆67Zn) in both the roots and shoots compared with wild-type rice 240

(Fig. 7A). The ∆67Zn uptake in the knockout lines was 41% of wild-type rice (Fig. 7B); 241

however, there was no difference in the root-to-shoot translocation of ∆67Zn between 242



10

the different lines (Fig. 7C). To confirm these results, we also used an OsZIP9 RNAi 243

line, which showed about 80% reduction in OsZIP9 expression compared wild-type 244

rice (Supplemental Fig. S10A). Similar to the knockout lines, the ∆67Zn concentration 245

in both the roots and shoots was lower in the RNAi line than in wild-type rice 246

(Supplemental Fig. S10B). The ∆67Zn uptake in the RNAi line was 66% of that in 247

wild-type rice (Supplemental Fig. S10C), whereas the root-to-shoot translocation was 248

similar between the RNAi line and wild-type rice (Supplemental Fig. S10D). 249

Furthermore, we performed a kinetic uptake experiment with 67Zn in Zn-deficient 250

plants at 4°C and 25°C. At 4°C, there was no difference in ∆67Zn uptake (30 min) 251

between wild-type rice and the knockout lines (Fig. 7D). However, at 25°C, the ∆67Zn 252

uptake was higher in wild-type rice than in the knockout lines, although the uptake 253

increased with increasing 67Zn concentrations in the nutrient solution in all lines (Fig. 254

7D). The net uptake of ∆67Zn calculated was significantly higher in wild-type rice than 255

in the knockout lines (Fig. 7E). Knockdown of OsZIP9 also significantly reduced the 256

net uptake of ∆67Zn (Supplemental Fig. S10E). Together, these results support that 257

OsZIP9 contributes to Zn uptake in rice roots. 258

259 260



11

Discussion 261

Based on analyses of expression pattern, transport activity in heterologous systems, 262

and ectopic expression, a number of transporters have been suggested to function in 263

root Zn uptake, such as AtZIP2, AtIRT1, and AtIRT3 in Arabidopsis (Vert et al., 2002; 264

Lin et al., 2009; Palmer and Guerinot, 2009; Milner et al., 2013); OsZIP1 and OsZIP3 265

in rice (Ramesh et al., 2003); and HvZIP7 in barley (Tiong et al., 2014). However, the 266

exact transporter for Zn uptake in roots has not been identified in plants (Olsen and 267

Palmgren, 2014). In the present study, we functionally characterized OsZIP9 in rice in 268

terms of growth stage- and organ-dependent expression pattern, subcellular 269

localization, transport activity in both yeast and knockout/knockdown lines, 270

tissue-specificity of localization, and detailed phenotypic analysis of knockout lines 271

growing in both nutrient solution and soil. We revealed that OsZIP9 contributes to Zn 272

uptake under Zn-limited conditions, especially in soil. This conclusion is supported by 273

several lines of evidence: 1) OsZIP9 is localized to the plasma membrane 274

(Supplemental Fig. S4); 2) OsZIP9 shows transport activity for Zn (Fig. 1); 3) OsZIP9 275

is mainly expressed in the roots through the whole growth period (Fig. 2A); 4) OsZIP9 276

expression is induced by Zn-deficiency (Fig. 2B); 5) OsZIP9 is expressed at the 277

exodermis and endodermis of mature root region (Figs. 2C and 3); 6) Knockout or 278

knockdown of OsZIP9 results in remarkably decreased Zn uptake at low Zn 279

concentration in nutrient solution, but not at high Zn concentrations (Figs. 5 and 7, 280

Supplemental Fig. S10); and 7) Knockout of OsZIP9 decreases Zn uptake from soil 281

(Fig. 6). 282

OsZIP9 showed transport activity for Zn in yeast, but not for Fe and Cu (Fig. 1). 283

Transport activity for Mn was not tested in yeast because a stable isotope of Mn was 284

not available. However, no difference in Mn accumulation was found between 285

wild-type rice and knockout lines of OsZIP9 (Supplemental Fig. S8). Furthermore, 286

OsNramp5 is reported to mediate Mn uptake in rice (Sasaki et al., 2012). Therefore, it 287

is unlikely that OsZIP9 contributes to Mn uptake. 288

Rice roots are characterized by two Casparian strips at both the exodermis and 289

endodermis (Enstone et al. 2002). Furthermore, mature roots have a highly developed 290



12

aerenchyma in which almost all of the cortex cells between the exodermis and 291

endodermis are destroyed. Therefore, rice has developed an efficient uptake system for 292

mineral elements, which is mediated by the cooperation of influx and efflux 293

transporters expressed at both the exodermis and endodermis of the root mature 294

regions (Yamaji and Ma, 2007; Sasaki et al. 2016, Mitani et al., 2018). Such uptake 295

systems for Si and Mn have been elucidated in rice roots (Ma et al., 2006; Ma, et al., 296

2007; Sasaki et al., 2012; Ueno et al., 2015). Si uptake is mediated by Lsi1 and Lsi2 297

(Ma et al., 2006; Ma et al., 2007), whereas Mn uptake is mediated by OsNramp5 and 298

OsMTP9 (Sasaki et al., 2012; Ueno et al., 2015), which are polarly localized at the 299

exodermis and endodermis of the roots. Expression of OsZIP9 at the exodermis and 300

endodermis in the root mature region supports its importance in Zn uptake (Figs. 2C 301

and 3). Since OsZIP9 likely functions as an influx transporter based on yeast transport 302

assay results (Fig. 1), an efflux transporter for cooperative Zn transport with OsZIP9 is 303

required for efficient Zn uptake, which remains to be identified in the future. 304

Knockout or knockdown of OsZIP9 resulted in decreased Zn uptake only under 305

Zn-limited conditions, but not under Zn-sufficient conditions in nutrient solution (Fig. 306

5, Supplemental Fig. S10), suggesting that OsZIP9 functions as a high-affinity 307

transporter for Zn. This is contrast to AtZIP9 and AtZIP12 in Arabidopsis, whereby 308

knockout of AtZIP9 and AtZIP12 only affects Zn uptake at high Zn concentrations and 309

not at low Zn concentrations (Inaba et a., 2015). In paddy soil, the Zn concentration in 310

soil solution is very low (Wang et al., 2019). In fact, OsZIP9 plays an important role in 311

Zn uptake from soil because knockout mutants of OsZIP9 exhibited significant 312

decreases in Zn accumulation in both the straw and brown rice under flooded 313

conditions (Fig. 6). This is also supported by higher expression of OsZIP9 under 314

flooded conditions compared to upland conditions (Wang et al., 2019). Since knockout 315

of OsZIP9 did not completely abolish Zn uptake even under Zn-limited conditions (Fig. 316

5), other unidentified transporters may also be involved in Zn uptake in rice. One 317

candidate is OsZIP1 because it is also highly expressed in the roots (Ramesh et al., 318

2003), although its exact role in Zn uptake remains to be examined. Furthermore, 319

transporters functioning at high Zn concentrations also require characterization in the 320



13

future. 321

The expression of OsZIP9 was also induced by Fe-deficiency to some extent, 322

although the extent of expression induction was not as high as that caused by Zn 323

deficiency (Fig. 2B). However, expression induction by Zn deficiency occurred earlier 324

than by Fe deficiency (Supplemental Fig. S3), suggesting that induction by Fe 325

deficiency was caused by indirect effects, although the exact mechanism is unknown. 326

In OsZIP9 knockout mutants, higher Fe accumulation in the shoots was observed at 327

low Zn supply in nutrient solution (Fig. 6). Since the mutant plants suffered from Zn 328

deficiency at low Zn concentrations (Figs. 4 and 5), some genes related to Fe uptake in 329

the roots may have been induced. However, in soil culture, knockout of OsZIP9 did not 330

affect Fe accumulation in the shoots (Fig. 6), due to high Fe concentration in soil 331

solution of paddy soil (Wang et al., 2019). 332

Identification of OsZIP9 in the present study provides further understanding of the 333

Zn transport system in rice. Zn in soil is first taken up by OsZIP9 localized at the 334

exodermis and endodermis of the roots and other uncharacterized transporters (Fig. 3). 335

Zn is partially sequestered by OsHMA3 localized at the tonoplast in root cells (Cai et 336

al., 2019) and the remaining Zn is translocated to the shoot by OsHMA2 localized at 337

the pericycle cells (Yamaji et al., 2013). OsZIP7 was also implicated in Zn xylem 338

loading although its exact role remains to be examined (Tan et al., 2019). At the node, 339

Zn is preferentially delivered to developing organs such as new leaves and grains by 340

OsZIP3 and OsHMA2. OsZIP3 is localized to xylem transfer cells in enlarged vascular 341

bundles (EVBs) of the nodes and responsible for unloading of Zn from the xylem of 342

EVB (Sasaki et al. 2015), whereas OsHMA2 is localized at the phloem region of both 343

EVBs and diffuse vascular bundles (DVBs) and is responsible for loading Zn to the 344

phloem of DVBs and EVBs (Yamaji et al. 2013). However, some missing transporters, 345

such as Zn efflux transporter(s) in root and node, remain to be identified in future 346

investigations to gain a holistic understanding of the Zn transport system in rice. This 347

will contribute to breeding rice cultivars with high tolerance to Zn deficiency that 348

exhibit high Zn accumulation in the grain. 349

In conclusion, OsZIP9 identified in this study is a transporter for Zn and it 350



14

contributes to root Zn uptake in soil. 351

352

Materials and methods 353

Plant materials and growth conditions 354

Seeds of the wild-type rice (cv. Nipponbare), two independent CRISPR/Cas9 OsZIP9 355

knockout lines (T2), one RNAi line, and transgenic lines (T2) carrying the promoter of 356

ZIP9 fused with GFP were soaked in water in dark at 30°C. After 2 days, the 357

germinated seeds were placed on a plastic net floating on a 0.5 mM CaCl2 solution in a 358

1.2-L plastic pot. The seedlings (7-d-old) were transferred to a 3.5-L plastic pot 359

containing 1/2 Kimura B solution (0.4 µM Zn, pH 5.6) (Ma et al., 2002). The nutrient 360

solution was exchanged every 2 days. All plants were grown in a controlled 361

greenhouse at 25–30°C, under natural light. 362

363

Cloning of full-length cDNA of OsZIP9 364

The full-length ORF of OsZIP9 was amplified by PCR using primers listed in 365

Supplemental Table S1, which were designed based on a putative cDNA clone 366

(Os05g0472400) in the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/) 367

with a putative translational start and stop site. Total RNA was extracted from rice 368

roots (cv. Nipponbare) using a RNeasy Plant Mini Kit (Qiagen, 369

http://www.qiagen.com) and then converted to cDNA using the protocol supplied by 370

the manufacturer of ReverTra Ace qPCR RT Master Mix with gDNA remover 371

(TOYOBO). The amplified cDNA was cloned into pGEM®-T vector (Promega, 372

https://www.promega.com/) and the sequence was confirmed by a sequence analyzer 373

(ABI Prism 3130; Applied Biosystems, http://www.appliedbiosystems.com/). 374

375

Phylogenetic analysis 376

The alignment was performed with ClustalW using default settings 377

(http://clustalw.ddbj.nig.ac.jp/), and the phylogenetic tree was constructed using the 378

neighbor-joining algorithm with MEGA version 6.0 (Tamura et al., 2013). Bootstrap 379

support was calculated (1000 replications). 380



15

381

Transport activity assay of OsZIP9 382

The OsZIP9-pGEM constructed as above was introduced into pYES2 vector 383

(Invitrogen) through restriction sites of BamHI and XhoI under the control of 384

galactose-inducible promoter, followed by introducing into a wild-type yeast strain 385

(BY4741; MATa his2Δ0 met15Δ0 ura3Δ0) or the Zn uptake defective double mutant 386

(ZHY3; MATa ade6 can1 his3 leu2 trp1 ura3 zrt1∷LEU2 zrt2∷HIS3). In a time-course 387

experiment, ZHY3 expressing OsZIP9 or empty vector were grown in the Sc(-Uracil) 388

medium containing 0.67% (w/v) yeast nitrogen base without amino acids (Difco), 2% 389

(w/v) glucose, 0.2% (w/v) appropriate amino acid, and 2% (w/v) agar at pH 6.0 for 390

selection. The yeast cells were first incubated in Sc(-Uracil) liquid medium with 50 391

mM MES containing 2% (w/v) galactose or 2% (w/v) glucose (as a negative control) 392

for two hours, followed by washing three times with the sterilized milli-Q water. The 393

yeast cells were then exposed to a solution containing 5 µM of stable isotope 67ZnCl2 394

(97% enrichment, Taiyo Nippon Sanso, Tokyo, Japan). At 0, 20, 40, 60, and 120 395

minutes of incubation with shaking at 30°C, the yeast cells were harvested by 396

centrifugation (2300 g, 5 min). Yeast pellet was washed three times with 5 mM CaCl2 397

solution and then digested by 2 N HCl for the determination of metals as described 398

below. 399

To examine the transport activity for Zn, Fe, and Cu, the wild-type yeast cells 400

(BY4741) expressing OsZIP9 or empty vector were prepared as above and then 401

cultured for 4 hours in the presence of 2% (w/v) galactose for gene induction, followed 402

by exposure to a solution containing 5 µM of each stable isotope including 67ZnCl2 403

(97% enrichment), 65CuCl2 (99.7% enrichment), or 57FeCl2 (96.1% 57Fe). 57FeCl2 was 404

prepared from 57FeCl3 by reduction with ascorbic acid. These stable isotopes were 405

purchased from Taiyo Nippon Sanso (Tokyo, Japan). After incubation with shaking for 406

2 hours at 30°C, the yeast cells were harvested by centrifugation (2300 g, 5 min) and 407

subjected to determination of metals as described below. 408

409



16

Expression analysis of OsZIP9 410

To investigate the expression pattern of OsZIP9 in different organs at different growth 411

stage, we used the same cDNA samples collected in the field as described in Sasaki et 412

al. (2015). 413

To investigate the response of OsZIP9 expression in roots to metal deficiency, 414

20-d-old seedlings (cv. Nipponbare) were grown in the 1/2 Kimura B solution with or 415

without Mn, Fe, Cu, or Zn for three days. To further examine the time-dependent 416

response, seedlings (20-d-old) were exposed to -Fe or -Zn for 1 and 3 days and root 417

samples were taken for expression analysis as described below. 418

For spatial expression analysis, different root segments (0–0.5, 0.5–1.0, 1.0–1.5, 1.5–419

2.0, 2.0–2.5, and 2.5–3.0 cm from the root tip) were excised from the roots of 5-d-old 420

seedlings. 421

Samples taken were immediately frozen in liquid nitrogen and then subjected to total 422

RNA extraction using an RNeasy Plant Mini Kit (Qiagen). cDNA was synthesized by 423

ReverTra Ace qPCR RT Kit (TOYOBO) or SuperScript II (Invitrogen) according to the 424

manufacturer’s instruction. The expression analysis of OsZIP9 was determined with 425

SsoFast EvaGreen Supermix (Bio Rad) or KOD SYBR qPCR Mix (TOYOBO) on a 426

real-time PCR machine (CFX384 or CFX96 (Bio-Rad)). Histone H3 and Actin were 427

used as internal controls. Relative gene expression was calculated by the ΔΔCt method. 428

The primer sequences used were listed in Supplemental Table S1. 429

430

Generation of transgenic rice lines 431

For generation of the transgenic lines carrying the promoter of ZIP9 fused with GFP, 432

the promoter region of OsZIP9 (3001 bp) was first amplified with PCR using the 433

primers shown in Supplemental Table S1. The amplified region was introduced into 434

the pGEM®-T easy vector. After confirmation of the sequence, the plasmid was 435

introduced into pPZP2H-lac vector including GFP by KpnI and BamHI, followed by 436

vector transfer to calluses (cv Nipponbare) via A. tumefaciens-mediated transformation 437

(Hiei et al., 1994). 438



17

OsZIP9 knockout lines were generated by using CRISPR/Cas9 using the plant 439

expression vector of Cas9 (pU6gRNA) and single guide RNA expression vector 440

(pZDgRNA_Cas9ver.2_HPT) as described before (Che et al., 2019). Twenty bases 441

upstream of the PAM motif were selected as candidate target sequences (Supplemental 442

Fig. S2A). Two targets of OsZIP9 were selected. The primers for target sequences in 443

the ORF region of OsZIP9 are listed in Supplemental Table S1. The derived constructs 444

were transformed into calluses as described above. 445

To genotype the resultant mutants, genomic DNA was extracted from leaves of 446

transgenic rice plants. PCR amplifications were carried out using primer pairs flanking 447

the designed target sites as listed in Supplemental Table S1. The PCR products (about 448

500 bp) were sequenced directly using internal specific primers, of which the binding 449

positions are desirably at about 200 bp upstream of the target sites. Two homologous 450

knockout lines without Cas9 were selected and the T2 generation was used in the 451

following phenotypic analysis. 452

An RNAi line was generated according to Miki and Shimamoto (2004) using the 453

primers listed in Supplemental Table S1. The expression level of OsZIP9 in the RNAi 454

line was investigated as described above. 455

Immunostaining analysis for transgenic lines carrying OsZIP9 promoter-GFP 456

To investigate the tissue-specificity of OsZIP9 expression, immunostaining was 457

performed in the transgenic lines (T2) carrying OsZIP9 promoter-GFP by using an 458

antibody against GFP (Thermo Fisher Scientific). Two-week-old plants grown in 1/2 459

Kimura B solution were exposed to a solution containing 0.4 µM Zn or not for 5 days. 460

Cross sections from the root tip (0.2 cm from the tip) and mature region (1.5 cm from 461

the root tip) were prepared and the method for immunostaining was the same as 462

described previously (Yamaji et al., 2007). The signal of fluorescence was observed 463

with a confocal laser scanning microscopy (TCS SP8x, Leica Microsystems). 464

465

Subcellular localization of OsZIP9 466



18

Subcellular localization of OsZIP9 was investigated by transiently expressing 467

GFP-OsZIP9 fusion into rice protoplasts and onion epidermal cells. The ORF of 468

OsZIP9 was amplified by PCR from rice (cv Nipponbare) root cDNA using primers 469

with the BsrGI and NotI site (Supplemental Table S1). The ORF was fused with a 470

linker (SSGSGG) and then inserted into the cauliflower mosaic virus 35S GFP vector 471

at the N terminus according to Sasaki et al. (2012). Rice protoplast transformation was 472

performed by the polyethylene glycol method as described previously (Chen et al., 473

2006). The same plasmid with DsRed was transformed into onion epidermal cells as 474

per the method described previously (Yokosho et al., 2016). The GFP signal was 475

observed with a confocal laser scanning microscope (TCS SP8x, Leica Microsystems). 476

We also performed double staining by using 4’,6-diamidino-2-phenylindole (DAPI) 477

as a nuclei marker and an OsZIP9 antibody for further confirmation of the subcellular 478

localization. The synthetic peptide (DASSSHDHERGN) was used to immunize rabbits 479

to obtain antibodies against OsZIP9. The antiserum was purified through a peptide 480

affinity column. The roots of WT and the knockout line exposed to -Zn for 4 days were 481

used for the immunostaining. The method for immunostaining and secondary antibody 482

incubation were the same as described previously (Yamaji and Ma, 2007). The 483

fluorescence signal was observed through confocal laser scanning microscopy (TCS 484

SP8x, Leica Microsystems). 485

486

Phenotypic analysis of OsZIP9 knockout lines 487

The wild-type rice and two independent OsZIP9 knockout lines (T2, oszip9-1, oszip9-2) 488

generated by CRISPR/Cas9 were used for phenotypic analysis. In a hydroponic 489

solution, seedlings (19-d-old) grown in a 3.5-L plastic pot were transferred to a 1.2-L 490

plastic pot (one plant for each line) with a nutrient solution containing different Zn 491

concentrations; 0.02, 0.2, and 2 µM. The treatment solution was renewed every two 492

days. After 17 days, the plants were photographed. The roots were washed with 5 mM 493

CaCl2 three times and separated from the shoots. The fresh weight of the roots and 494

shoots were recorded. The concentrations of mineral elements in the roots and shoots 495

were determined as described below. 496



19

For soil culture, both wild-type rice and two independent knockout lines were grown 497

in a pot containing 3.5 kg soil collected from a field of the Institute of Plant Science 498

and Resources, Okayama University, under flooded conditions. Tap water was supplied 499

daily and a 2-cm water layer was maintained on the top soil. Plants were grown in a 500

temperature-controlled glasshouse (around 22–30°C) under natural light. At the 501

ripening stage, the plant was harvested and separated into straw and brown rice. The 502

concentrations of mineral elements were determined as described below. 503

504

Short-term uptake experiment with stable isotope 67Zn 505

Seedlings (WT, knockout lines, RNAi line) grown in 0.02 µM Zn for 17 days were 506

exposed to a solution containing 0.4 µM 67Zn. After 24 hours, the roots were washed 507

and separated from the shoots as described above. 508

A kinetic study of Zn uptake was performed by exposing the seedlings (WT, 509

knockout lines, RNAi line) grown in -Zn solution for 7 days to different 67Zn 510

concentrations in the range of 0–2 µM at 25°C and 4°C. After 30 min, the roots were 511

washed three times with 5 mM CaCl2 and harvested for element determination as 512

described below. 513

514

Determination of metals in plant and yeast samples 515

The roots and shoots were dried at 70°C for at least three days before being digested by 516

HNO3 (60%[w/v]) as described previously (Sasaki et al., 2012). The concentrations of 517

mineral elements in digestion solutions derived from plants and yeast were determined 518

by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS, 7700X; Agilent 519

Technologies). The concentrations of 67Zn, 65Cu, and 57Fe were determined with 520

isotope mode. ΔZn, ΔFe, and ΔCu (net Zn, Fe, or Cu increase) were calculated 521

according to Yamaji et al. (2013). 522

523

Statistical analyses 524

Statistical comparison by using SPSS 19 was performed by ANOVA followed by 525

Tukey-Kramer’s test. 526



20

527

Accession numbers 528

Accession number of OsZIP9 is registered as LC521921 in the GenBank/EMBL 529

databases. 530

531

Supplemental Data 532

Supplemental Figure S1. Sequence analysis of OsZIPs. 533

Supplemental Figure S2. Mutated sequences of OsZIP9 gene in CRISPR/Cas9 534

mutants. 535

Supplemental Figure S3. Time-dependent response of OsZIP9 to Zn- and 536

Fe-deficiency in the roots. 537

Supplemental Figure S4. Subcellular localization of OsZIP9. 538

Supplemental Figure S5. The concentrations of macro-elements in the roots and 539

shoots. 540

Supplemental Figure S6. Concentrations of Fe, Mn, and Cu in the roots and shoots. 541

Supplemental Figure S7. Contents of macro-elements in the roots and shoots. 542

Supplemental Figure S8. Contents of Fe, Mn, and Cu in the roots and shoots. 543

Supplemental Figure S9. Concentrations of Cd and As in straw and brown rice. 544

Supplemental Figure S10. Effect of knockdown of OsZIP9 on Zn uptake and 545

accumulation. 546

Supplemental Table S1. List of primers used in this study 547

548

Acknowledgements 549

We thank Akemi Morita and Sanae Rikiishi for their technical assistance. We also thank 550

Dr. Masaki Endo for providing pU6gRNA and pZDgRNA_Cas9ver.2_HPT for 551

generation of CRISPR/Cas9 lines. 552

553

Figure legends 554

555

Figure 1. Transport activity of OsZIP9 for metals in yeast cells. 556



21

(A-B) Time-dependent uptake of OsZIP9 for 67Zn in the presence of glucose (A) and 557

galactose (B). Zn uptake defective yeast cells (ZHY3) expressing OsZIP9 or empty 558

vector (VC) were exposed to a solution containing 5 µM 67Zn for different time periods. 559

(C) Transport activity for different metals. Wild-type yeast cells (BY4741) expressing 560

OsZIP9 or empty vector (VC) were exposed to a solution containing 5 µM of 67Zn, 57Fe, 561

or 65Cu for two hours in the presence of galactose. The concentration of stable metal 562

isotopes was determined by isotope mode of ICP-MS. ΔMetal was calculated by 563

subtracting the natural abundance of each metal isotope. Data are means ±SD of three 564

biological replicates. The asterisks indicate significant differences (*p<0.05 or**p<0.01 565

by T-test). All data were compared with VC in each part. 566

567

Figure 2. Expression pattern of OsZIP9. 568

(A) Growth stage- and organ-dependent expression of OsZIP9. Samples of various 569

organs were taken from rice grown in the field at different growth stages. (B) Response 570

of OsZIP9 expression to metal deficiency. Rice seedlings were grown in the 1/2 Kimura 571

B solution with or without Cu, Zn, Fe, or Mn for three days. (C) Spatial expression 572

pattern of OsZIP9 in roots. Different root segments (0–0.5, 0.5–1.0, 1.0–1.5, 1.5–2.0, 573

2.0–2.5, and 2.5–3.0 cm from the root tip) were collected from roots of 5-d-old 574

seedlings. The expression level of OsZIP9 was determined by RT-qPCR. Histone H3 (A, 575

B) and Actin (C) were used as internal controls. The expression relative to root at 6 576

weeks (A), control condition (B), and the root segment of 2.5–3.0 cm (C) are shown. 577

Data are means ±SD of three biological replicates. Statistical comparison was 578

performed by ANOVA followed by Tukey-Kramer’s test. Different letters indicate 579

significant difference (p<0.01). 580

581

Figure 3. Tissue specificity of OsZIP9 expression. 582

Two-week-old plants of transgenic lines carrying the OsZIP9 promoter fused with 583

GFP were exposed to a solution containing Zn (A, D) or not (B, E) for 5 days. The 584

root cross sections from the root tip (0.2 cm from the tip) (A-C) and mature region (1.5 585

cm from the tip) (D-F) were prepared and used for immunostaining with an anti-GFP 586



22

antibody. (G, H) Magnified image of orange box area in (E). (C, F) Wild-type rice 587

roots as a negative control. Red color shows signal from the anti-GFP antibody and 588

blue color from auto fluorescence of cell wall. ex, exodermis; en, endodermis. Scale 589

bar, 25 µm. 590

591

Figure 4. Phenotypic analysis of OsZIP9 knockout lines in hydroponic solution. 592

(A-C) Phenotype of the wild-type rice and two OsZIP9 knockout lines (oszip9-1 and 593

oszip9-2). Scale bar, 10 cm. (D) Zn-deficiency symptom of new leaf. Scale bar, 2.5 cm. 594

(E-F) Fresh weight of shoots (E) and roots (F). The plants were grown in a nutrient 595

solution containing 0.02 (A, D), 0.2 µM (B) and 2 µM (C) Zn for 17 days. Data in E 596

and F are means ±SD of three biological replicates. Statistical comparison was 597


significant difference (p<0.01). 599

600

Figure 5. Zn concentrations and contents in wild-type rice and OsZIP9 knockout lines. 601

(A-B) Zn concentrations in root (A) and shoot (B) of wild-type rice and knockout lines. 602

(C-D) Zn contents in the roots and shoots. The plants were grown in a nutrient solution 603

containing 0.02, 0.2, or 2 μM Zn for 17 days. Data are means ±SD of three biological 604

replicates. Statistical comparison was performed by ANOVA followed by 605

Tukey-Kramer’s test. Different letters indicate significant difference (p<0.01). All data 606

were compared with the wild-type rice in each treatment. 607

608

Figure 6. Comparison of metal accumulation between wild-type rice and two 609

independent OsZIP9 knockout lines grown in soil. 610

(A, B) Metal concentrations in the straw (A) and brown rice (B). Both the wild-type 611

rice and two independent OsZIP9 knockout lines were grown in soil under flooded 612

conditions until maturity. The concentration of different metals was determined by 613

ICP-MS. Data are means ±SD of three biological replicates. Statistical comparison was 614


significant difference (p<0.01). All data for each element were compared with the 616



23

wild-type rice. 617

618

Figure 7. Short-term labeling experiment with 67Zn. 619

(A) Concentration of Δ67Zn in the roots and shoots. (B) Uptake of Δ67Zn. (C) Root to 620

shoot translocation of Δ67Zn. The wild-type rice and two independent OsZIP9 621

knockout lines grown in 0.02 μM Zn conditions for 17 days were exposed to a solution 622

containing 0.4 µM 67Zn for 24 h. (D-E) Kinetic study of 67Zn uptake. Seedlings grown 623

in Zn-deficient solution for 7 days were exposed to a solution containing different 624

concentrations of 67Zn for 30 min at 25°C or 4°C. Net uptake (E) was calculated by 625

subtracting the apparent uptake at 4°C from that at 25°C. Data are means ±SD of three 626

biological replicates. Different letters and asterisks indicate significant difference 627

(p<0.01). Statistical comparison was performed by ANOVA followed by 628

Tukey-Kramer’s test. 629

630



0

2

4

6

8

10

0 20 40 60 80 100 120

ΔZn c

onc. in

yeast (μg/g

DW

)

VC

OsZIP9

Time (min)

(A) (B)

(C)

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120

ΔZn c

onc. in

yeast (μg/g

DW

) VC

OsZIP9

Time (min)

**

**

** **

0

20

40

60

80

100

120

140

160

67Zn 57Fe 63Cu

VC (Gal)

OsZIP9 (Gal)

67Zn 57Fe 65Cu

ΔMetal conc. in

yeast (µ

g/g

DW

)

**

** *

Figure 1. Transport activity of OsZIP9 for metals in yeast cells.

(A-B) Time-dependent uptake of OsZIP9 for 67Zn in the presence of glucose (A) and galactose (B). Zn uptake defective yeast cells (ZHY3)

expressing OsZIP9 or empty vector (VC) were exposed to a solution containing 5 µM 67Zn for different time periods. (C) Transport activity

for different metals. Wild-type yeast cells (BY4741) expressing OsZIP9 or empty vector (VC) were exposed to a solution containing 5 µM of 67Zn, 57Fe, or 65Cu for two hours in the presence of galactose. The concentration of stable metal isotopes was determined by isotope mode of

ICP-MS. ΔMetal was calculated by subtracting the natural abundance of each metal isotope. Data are means ±SD of three biological

replicates. The asterisks indicate significant differences (*p<0.05 or**p<0.01 by T-test). All data were compared with VC in each part.



0.0

0.2

0.4

0.6

0.8

1.0

1.2

OsZ

IP9

expre

ssio

n le

vel

Distance from root tip (cm)

(B) (C)

0

1

2

3

4

5

6

7

6 week 9 week

(Tilling)

12 week

(Booting)

14 week

(Flowering)

16 week

(Grain filling) O

sZ

IP9

expre

ssio

n level

(A)

15

25

35

45

55

OsZ

IP9

expre

ssio

n

0

2

4

6

Control -Zn -Fe -Mn -Cu

Treatment

C C

C

a

b

Figure 2. Expression pattern of OsZIP9.

(A) Growth stage- and organ-dependent expression

of OsZIP9. Samples of various organs were taken

from rice grown in the field at different growth

stages. (B) Response of OsZIP9 expression to metal

deficiency. Rice seedlings were grown in the 1/2

Kimura B solution with or without Cu, Zn, Fe, or

Mn for three days. (C) Spatial expression pattern of

OsZIP9 in roots. Different root segments (0–0.5,

0.5–1.0, 1.0–1.5, 1.5–2.0, 2.0–2.5, and 2.5–3.0 cm

from the root tip) were collected from roots of 5-d-

old seedlings. The expression level of OsZIP9 was

determined by RT-qPCR. Histone H3 (A, B) and

Actin (C) were used as internal controls. The

expression relative to root at 6 weeks (A), control

condition (B), and the root segment of 2.5–3.0 cm

(C) are shown. Data are means ±SD of three

biological replicates. Statistical comparison was

performed by ANOVA followed by Tukey-

Kramer’s test. Different letters indicate significant

difference (p<0.01). www.plantphysiol.orgon June 24, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.


-Zn +Zn

en

ex

ex

en

(A) (C)

(D)

(B)

(E) (F)

(G) (H)

Figure 3. Tissue specificity of OsZIP9 expression.

Two-week-old plants of transgenic lines carrying the OsZIP9 promoter fused with GFP were exposed to a solution containing Zn (A, D)

or not (B, E) for 5 days. The root cross sections from the root tip (0.2 cm from the tip) (A-C) and mature region (1.5 cm from the tip) (D-F)

were prepared and used for immunostaining with an anti-GFP antibody. (G, H) Magnified image of orange box area in (E). (C, F) Wild-

type rice roots as a negative control. Red color shows signal from the anti-GFP antibody and blue color from auto fluorescence of cell wall.

ex, exodermis; en, endodermis. Scale bar, 25 µm.



0.02 µM Zn

WT oszip9-1 oszip9-2

0.2 µM Zn

WT oszip9-1 oszip9-2

2.0 µM Zn

WT oszip9-1 oszip9-2 WT oszip9-1 oszip9-2

0.02 µM Zn

a

a

a

b

a

a

b

a a

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.02 0.2 2

Shoot

fresh w

eig

ht

(g)

Zn concentration (µM)

WT

oszip9-1

oszip9-2

(E)

0.02 0.2 2.0

(A) (B) (C) (D)

a

a

a

a

a

a

a

a a

0.0

0.2

0.4

0.6

0.8

1.0

0.02 0.2 2

Root

fresh w

eig

ht

(g)


WT

oszip9-1

oszip9-2

(F)

0.02 0.2 2.0

Figure 4. Phenotypic analysis of OsZIP9

knockout lines in hydroponic solution.

(A-C) Phenotype of the wild-type rice

and two OsZIP9 knockout lines (oszip9-1

and oszip9-2). Scale bar, 10 cm. (D) Zn-

deficiency symptom of new leaf. Scale

bar, 2.5 cm. (E-F) Fresh weight of shoots

(E) and roots (F). The plants were grown

in a nutrient solution containing 0.02 (A,

D), 0.2 µM (B) and 2 µM (C) Zn for 17

days. Data in E and F are means ±SD of

three biological replicates. Statistical

comparison was performed by ANOVA

followed by Tukey-Kramer’s test.

Different letters indicate significant

difference (p<0.01). www.plantphysiol.orgon June 24, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.


a

a

a

b

b

a

b

b

a

0

20

40

60

80

100

120

0.02 0.2 2.0

Shoot

Zn c

onc. (m

g/k

g D

W)


WT

oszip9-1

oszip9-2a

a

a

0

40

80

120

160

200

0.02 0.2 2.0

Root

Zn c

onc. (m

g/k

g D

W)


WT

oszip9-1

oszip9-2

a a

b

a

b

a

0

5

10

15

20

25

0.02 0.2

(B) (A)

0

10

20

30

40

50

60

0.02 0.2 2.0

Shoot

Zn c

ont. (

µg)

Zn pretreatment concentation (µM)

WToszip9-1oszip9-2

a b b

a

b b

a a

a

0

2

4

6

8

10

12

14

0.02 0.2 2.0

Root

Zn c

ont. (

µg)

Zn pretreatment concentation (µM)

WToszip9-1oszip9-2

a b b

a a a

a a

a

0.0

0.5

1.0

1.5

0.02 0.2

a b b

a a a

(D) (C)

Figure 5. Zn concentrations and contents in wild-type rice and OsZIP9 knockout lines.

(A-B) Zn concentrations in root (A) and shoot (B) of wild-type rice and knockout lines. (C-D) Zn contents in the roots and shoots. The

plants were grown in a nutrient solution containing 0.02, 0.2, or 2 μM Zn for 17 days. Data are means ±SD of three biological replicates.

Statistical comparison was performed by ANOVA followed by Tukey-Kramer’s test. Different letters indicate significant difference

(p<0.01). All data were compared with the wild-type rice in each treatment.



0

20

40

60

80

100

Zn Cu Fe Mn

Ele

men

t con

c. in

bro

wn r

ice

(mg

/kg D

W)

WT

oszip9-1

oszip9-2

b b

a

a ab b

a

a a

a a

a

0

200

400

600

800

1,000

1,200

1,400

Zn Cu Fe Mn

Ele

men

t con

c. in

str

aw

(m

g/k

g D

W)

WT

oszip9-1

oszip9-2

0

10

20

30

40

50

Zn Cu

b b a a a

b a

ab

b

a a

a a a

a

b b

a

(A) (B)

Figure 6. Comparison of metal accumulation between wild-type rice and two independent OsZIP9 knockout lines grown in soil.

(A, B) Metal concentrations in the straw (A) and brown rice (B). Both the wild-type rice and two independent OsZIP9 knockout lines were

grown in soil under flooded conditions until maturity. The concentration of different metals was determined by ICP-MS. Data are means

±SD of three biological replicates. Statistical comparison was performed by ANOVA followed by Tukey-Kramer’s test. Different letters

indicate significant difference (p<0.01). All data for each element were compared with the wild-type rice.



0

5

10

15

20

0.0 0.5 1.0 1.5 2.0

Net ∆

67Z

n c

onc. (m

g/k

g r

oot D

W)

67Zn concentration (µM)

WT

oszip9-1

oszip9-2

** **

**

**

**

**

**

**

a

b b

0

10

20

30

40

50

60

∆6

7Z

n u

pta

ke (

mg/k

g r

oot D

W)

a

a

b

b

b

b

0

5

10

15

20

25

30

Root Shoot

∆6

7Z

n c

onc. (m

g/k

g D

W)

WT

oszip9-1

oszip9-2

(A) (B) (C)

0

5

10

15

20

25

0.0 0.5 1.0 1.5 2.0

∆6

7Z

n c

onc. (m

g/k

g r

oot D

W)

67Zn concentration (µM)

WT （25℃）

oszip9-1（25℃）

oszip9-2（25℃）

WT（4℃）

oszip9-1（4℃）

oszip9-2（4℃）

**

**

**

**

**

**

(4℃)

(4℃)

(25℃)

(25℃)

** **

(D) (E)

a a a

0

10

20

30

40

50

60

70

∆6

7Z

n r

oot to

shoot tr

anslo

cation (

%)

Figure 7. Short-term labeling experiment with 67Zn.

(A) Concentration of Δ67Zn in the roots and shoots. (B) Uptake of Δ67Zn. (C) Root to shoot translocation of Δ67Zn. The wild-type rice and two independent

OsZIP9 knockout lines grown in 0.02 μM Zn conditions for 17 days were exposed to a solution containing 0.4 µM 67Zn for 24 h. (D-E) Kinetic study of 67Zn

uptake. Seedlings grown in Zn-deficient solution for 7 days were exposed to a solution containing different concentrations of 67Zn for 30 min at 25°C or

4°C. Net uptake (E) was calculated by subtracting the apparent uptake at 4°C from that at 25°C. Data are means ±SD of three biological replicates.

Different letters and asterisks indicate significant difference (p<0.01). Statistical comparison was performed by ANOVA followed by Tukey-Kramer’s test.



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1 Short title: Zn uptake transporter in rice2 31 KAKENHI Grant Number 16H06296 to J.F.M.). 32 33...

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Transcript of 1 Short title: Zn uptake transporter in rice2 31 KAKENHI Grant Number 16H06296 to J.F.M.). 32 33...