1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate...

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Running Title Nitrate allocation and nitrogen use efficiency 1

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Corresponding author Zhen-hua Zhang 3

Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China 4

National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources 5

Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources 6

Use Hunan Provincial Key Laboratory of Plant Nutrition in Common University 7

College of Resources and Environmental Sciences Hunan Agricultural University PR China 8

zhzh1468163com 9

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Title Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots 12

of Brassica napus 13

14

Yong-Liang Han1 Hai-Xing Song1 Qiong Liao1 Yin Yu1 Shao-Fen Jian1 Joe Eugene Lepo3 Qiang 15

Liu1 Xiang-Min Rong1 Chang Tian1 Jing Zeng1 Chun-Yun Guan12 Abdelbagi M Ismail4 Zhen-Hua 16

Zhang1sect 17

18

1 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China Hunan Agricultural 19

University Changsha China 20

2 National Center of Oilseed Crops Improvement Hunan Branch Changsha China 21

3 Center for Environmental Diagnostics and Bioremediation University of West Florida Pensacola 22

Florida 32514 United States of America 23

4 Crop and Environment Sciences Division International Rice Research Institute DAPO 7777 Metro 24

Manila Philippines 25

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Decrease in VSC of NO3- in roots will enhance transport to shoot and essentially contribute to 28

higher NUE by promoting NO3- allocation to aerial parts 29

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Plant Physiology Preview Published on January 12 2016 as DOI101104pp1501377

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Footnotes 33

We thank Dr Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology 34

Shanghai Institutes for Biological Sciences) for providing nrt15-3and nrt18-2 seeds This 35

study was supported by the National Natural Science Foundation of China (Grant 36

No31101596 31372130) Hunan Provincial Recruitment Program of Foreign Experts 37

National Oilseed Rape Production Technology System of China ldquo2011 Planrdquo supported by 38

The Ministry of Education in China Open Novel Science Foundation of Hunan province 39

(13K062) National Key Laboratory of Plant Molecular Genetics and the Twelfth Five-Year 40

National Science and technology support program (2012BAD15BO4) 41

42

These authors have contributed equally 43

sect Corresponding author e-mail zhzh1468163com 44

45

46

Author contributions 47

ZHZ conceived the original screening and research plans ZHZ supervised the experiments 48

YLH HXS QL YY SFJ and ZHZ performed most of the experiments CT and JZ provided 49

technical assistance to YLH HXS YY and ZHZ YLH and ZHZ designed the experiments 50

and analyzed the data QL XMR CYG and ZHZ interpreted the results and JEL AI and 51

ZHZ conceived the project and wrote the article with contributions of all the authors JEL AI 52

and ZHZ supervised and complemented the writing All authors read and approved the final 53

manuscript 54

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Abstract 68

Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target 69

to reduce excessive use of chemical fertilizers with substantial benefits to farmers and the 70

environment In Arabidopsis thaliana allocation of more NO3- to shoots was associated with 71

higher NUE however the commonality of this process across plant species have not been 72

sufficiently studied Two Brassica napus genotypes were identified with high and low NUE 73

We found that activities of V-ATPase and V-PPase the two tonoplast proton-pumps were 74

significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE 75

genotype (814) and consequently less vacuolar NO3- was retained in roots of Xiangyou15 76

Moreover NO3- concentration in xylem sap [15N] shootroot (SR) and [NO3

-] SR ratios 77

were significantly higher in Xiangyou15 BnNRT15 expression was higher in roots of 78

Xiangyou15 compared with 814 while BnNRT18 expression was lower In both B napus 79

treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity 80

vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased Expression of 81

NRT15 was up-regulated but NRT18 was down-regulated driving greater NO3- 82

long-distance transport from roots to shoots NUE in Arabidopsis mutants impaired in proton 83

pumps was also significantly higher than in the wild type col-0 Taken together these data 84

suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially 85

contribute to higher NUE by promoting NO3- allocation to aerial parts likely through 86

coordinated regulation of NRT15 and NRT18 87

88

Key words Nitrogen use efficiency Nitrate allocation Proton-pumps Vacuoles 89

90

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92

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95

96

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98

4

Introduction 99

China is the largest consumer of nitrogen (N) fertilizer in the world however the 100

average N-fertilizer use efficiency (NUE) is only around 35 suggesting considerable 101

potential for improvements (Shen et al 2003 Wang et al 2014) With the high amounts of 102

N-fertilizer being used crop yields are declining in some areas where application is 103

exceeding the optimum required for local field crops (Shen et al 2003 Miller et al 2008Xu 104

et al 2012) The extremely low NUE results in waste of resources and environmental 105

contamination and also presents serious hazards for human health (Xu et al 2012 Chen et 106

al 2014) Consequently exploiting the maximum potential for improving NUE in crop plants 107

will have practical significance for agriculture production and the environment (Zhang et al 108

2010 Schroeder et al 2013 Wang et al 2014) Elucidating the genetic and physiological 109

regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 110

higher NUE 111

Ammonium (NH4+) and nitrate (NO3

-) are the main N species absorbed and utilized by 112

crops and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 113

dry land crops such as Brassica napus Several studies revealed the close relationship 114

between NO3- content and NUE in plant tissues (Shen et al 2003 Zhang et al 2012 Tang et 115

al 2013Han et al 2015a)When plants are sufficiently illuminated NO3- assimilation 116

efficiency significantly increase in shoots compared with roots (Smirnoff et al 1985 Tang et 117

al 2013) Consequently under daytime with optimal illumination higher proportion of NO3- 118

in plant tissue is transported from root to shoot as an advantageous physiological adaptation 119

that reduces the cost of energy for metabolism (Tang et al 2013) NO3- assimilation in plant 120

shoots can therefore take advantage of solar energy while improving NUE (Smirnoff et al 121

1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) 122

The NO3- long-distance transport and distribution between root and shoot is regulated by 123

two genes encoding long transport mechanisms NRT15 is responsible for xylem NO3- 124

loading while NRT18 is responsible for xylem NO3- unloading (Lin et al 2008 Li et al 125

2010) Expression of the two genes is influenced by NO3- concentration NRT15 is strongly 126

induced by NO3- (Lin et al 2008) while NRT18 expression is extremely up-regulated in 127

nrt15 mutants (Chen et al 2012) A negative correlation between the extents of expression of 128

the two genes was observed when plants are subjected to abiotic stresses (Chen et al 2012) 129

Moreover expression of NRT15 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 130

acid (ACC) and methyl jasmonate (MeJA) whereas the expression of NRT18 is significantly 131

5

up-regulated (Zhang et al 2014) Based on these studies we argue that the expression and 132

functioning of NO3- long-distance transport genes NRT15 and NRT18 are regulated by 133

cytosolic NO3- concentration In addition the vacuolar and cytosolic NO3

- distribution is 134

likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 135

(Granstedt et al 1982 Glass et al 2002 Krebs et al 2010) Therefore NO3- use efficiency 136

must be affected by NO3- long-distant transport (between shoot and root) and short-distant 137

transport (between vacuole and cytosol) However the physiological mechanisms controlling 138

this regulation are still obscure 139

Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140

for vacuole NO3- short-distance transport as it is the main channel for NO3

-movement 141

between the vacuoles and cytosol (Angeli et al 2006 Wege et al 2014) The vacuole 142

proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active 143

transport of NO3- and accumulation within the vacuole (Gaxiola et al 2001 Brux et al 2008 144

Krebs et al 2010) Despite that about 90 of the volume of mature plant cells is occupied by 145

vacuoles vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 146

reductase (NR) is cytosolic (Shen et al 2003 Han et al 2015a) However re-translocation of 147

NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization 148

Generally NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149

of 30-50 mol m-3 and 3-5 mol m-3 respectively (Martinoia et al 1981 Martinoia et al 2000) 150

Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 151

tissues their function in NO3- use efficiency cannot be ignored (Martinoia et al 1981 Zhang 152

et al 2012 Han et al 2015b) NO3- assimilatory system in the cytoplasm is sufficient for its 153

assimilation when it is transported out of the vacuoles Therefore NO3- use efficiency could in 154

part be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 155

(Martinoia et al 1981 Shen et al 2003 Zhang et al 2012 Han et al 2015a) 156

Evidently NO3- use efficiency is regulated by both NO3

- long-distance transport from 157

root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 158

within cells(Glass et al 2002 Dechorgnat et al 2011 Han et al 2015a) Although vacuoles 159

compartment excess NO3- that accumulates in plant cells (Granstedt et al 1982 Krebs et al 160

2010) neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al 2007 Han et al 2015a) 161

nor the NO3- long-distance transport gene NRT15 (Lin et al 2008) are regulated by vacuolar 162

NO3- even though they are essential for NO3

- assimilation Only NO3- transported from the 163

vacuole to the cytosol can play a role in regulating NO3- inducible genes Consequently we 164

argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 165

6

regulated by cytosolic NO3- concentration However this hypothesis needs to be substantiated 166

The mechanisms underlying both NO3- short-distance (Gaxiola et al 2001 Angeli et al 167

2006 Brux et al 2008 Krebs et al 2010) and long-distance transport (Lin et al 2008 Li et 168

al 2010) have been previously investigated yet the underlying mechanisms regulating the 169

flux of NO3- and the obvious relationship between the two transport pathways as well as their 170

relation to NUE are not well understood 171

The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172

(Chiu et al 2004) and seed tissues (Chopin et al 2007) whereas the proton pumps and 173

CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 174

al 2001 Angeli et al 2006 Brux et al 2008 Krebs et al 2010) The vacuolar NO3- 175

short-distance transport system is spread throughout the plant tissues and is the principal 176

means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 177

et al 2006Krebs et al 2010) 178

The NRT genes seem to work synergistically to control NO3- long-distance transport 179

between roots and shoots NRT19 is responsible for NO3- loading into the phloem (Wang et 180

al 2011) whereas NO3- loading and unloading into xylem are regulated by NRT15 and 181

NRT18 respectively (Lin et al 2008 Li et al 2010) Phloem transport mainly involves 182

organic N the inorganic-N (NO3-) concentrations in the phloem sap are typically very low 183

ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al 184

2008Fan et al 2009) Therefore this study focused on NO3- short-distance transport 185

mediated through the tonoplast proton-pumps and the CLCa system and the long-distant 186

transport mechanisms responsible for xylem NO3- loading and unloading via NRT15 and 187

NRT18 respectively 188

Questions related to how long and short-distance transport of NO3- are coupled in plant 189

tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 190

B napus genotypes and Arabidopsis thaliana Application of proton pump inhibitors and 191

ACC in the former and use of mutants with defective proton pumps in the latter allowed 192

experimental distinction of the physiological mechanisms regulating these processes Data 193

presented here provide strong evidence from both model plants supporting this linkage and 194

strongly suggest that cytosolic NO3- concentration in roots regulates NO3

- long-distance 195

transport from roots to shoots We also investigated how NO3- concentration in plant tissues 196

would be affected by NO3- long-distance transport vacuolar NO3

- sequestration and the 197

ensuing relationship with NO3- use efficiency We also proposed the physiological 198

7

mechanisms likely to be important for enhancing NO3- use efficiency in plants These findings 199

will provide scientific rationales for improving NUE in important industrial and food crops 200

201

202

8

Results 203

B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 204

in root tissues 205

Our previous work identified high and low NUE B napus genotypes (Han et al 2015a 206

Han et al 2015b) NUE of B napus whether based on biomass or on grain yield (Table 1) 207

was significantly higher for the high-NUE genotype (H Xiangyou15) than for the low-NUE 208

genotype (L 814) That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 209

of root tissues in the H genotype were lower than the L genotype at both seedling (Fig 1A) 210

and flowering stages (Fig S1A) Given that proton pumps in the tonoplast supply energy for 211

vacuolar NO3- accumulation (Krebs et al 2010) NO3

- influx into the vacuole was 212

consequently much slower in the H genotype compared with the L genotype at both seedling 213

(Fig 1B) and flowering stages (Fig S1B) Moreover the percentage of vacuolar NO3- relative 214

to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 215

genotype at seedling (Fig 1C) and flowering stages (Fig S1C) and NO3- accumulation in the 216

cytosol increased significantly in the H genotype compared with the L genotype at seedling 217

(Fig 1D) and flowering stages (Fig S1D) 218

219

B napus with higher NUE showed enhanced long-distance transport of NO3- from roots 220

to shoots 221

The relative expression of BnNRT15 in roots of the H genotype was significantly higher 222

than that in the L genotype at both seedling and flowering stages while the relative expression 223

of BnNRT18 in roots of the H genotype was lower than that in the L genotype at both 224

seedling (Fig 2-AB) and flowering stages (Fig S2-AB) As a consequence total N 225

concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 226

the L genotype at both seedling (Fig 2C) and flowering stages (Fig S2C) while shoot N 227

concentration in the H genotype was significantly higher than the L genotype at seedling stage 228

(Fig 2C) This resulted in significantly higher [15N] SR ratio in the H genotype compared 229

with the L genotype (Fig 2D Fig S2D) 230

No significant differences in total N per plant between the H and L genotypes were 231

observed at both seedling and flowering stages (Fig S3) However NO3- concentration in 232

roots of the H genotype was significantly lower than in the L genotype at both seedling and 233

flowering stages (Fig 2E Fig S2E) while NO3- concentration in shoot tissues of this 234

9

genotype was significantly higher at seedling stage (Fig 2E) resulting in higher [NO3-] SR 235

ratios at both stages (Fig 2F Fig S2F) 236

NO3- concentration in the xylem sap xylem sap volume and total NO3

- in xylem sap 237

were significantly higher in the H genotype than in the L genotype at seedling stage (Fig 238

S4ACE) the amount of NO3- in xylem sap significantly increased in the H genotype relative 239

to the L genotype at flowering stage (Fig S4F) Together these data suggests greater 240

mobilization of NO3- from root to shoot in the H genotype 241

242

NO3- up-regulates NRT15 but down-regulates NRT18 243

244

A previous study showed that increased NRT18 expression in nrt15 mutants (Chen et al 245

2012) and the expression of NRT15 was induced by NO3- (Lin et al 2008) We tested the 246

relationship between NRT15 and NRT18 expression in A thaliana and B napus No 247

significant differences were observed in AtNRT15 expression in roots between wild type 248

(col-0) and mutant plants (nrt18-2) (Fig 3A) However AtNRT15 expression was 249

significantly up-regulated by NO3- in roots of nrt18-2 mutants (Fig 3B) Expression of 250

AtNRT18 significantly increased in roots of nrt15-3 mutants but not in col-0 plants and 251

there were no significant differences in AtNRT18 expression with or without NO3- treatment 252

in roots of nrt15-3 mutants (Fig 3CD) 253

In contrast BnNRT15 expression in roots of the H genotype increased significantly with 254

NO3- treatment (Fig 3E) while the reverse was observed in expression of BnNRT18 which 255

showed lower expression under NO3- treatment than control (Fig 3F) This suggests that NO3

- 256

induces the expression of NRT15 but down-regulates NRT18 in A thaliana and B napus 257

Further studies are needed to elucidate the mechanisms regulating this reverse regulation 258

259

Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots 260

Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase DCCD 261

(DCCD + Na2SO3) inhibits V-PPase and a 11 combination of Bafi and DCCD (B+D) 262

inhibits both V-ATPase and V-PPase (Han et al 2015a) These inhibitors were used to 263

control activities of the tonoplast proton pumps in the H B napus plants V-ATPase activity 264

significantly decreased under Bafil and B+D treatments relative to the control and DCCD 265

treatments whereas V-PPase activity declined significantly under DCCD and B+D treatments 266

relative to that in the control and Bafil treatments (Fig 4A) 267

10

The V-ATPase activity in roots of A thaliana was significantly lower in the V-ATPase 268

mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig 5A) 269

In contrast the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 270

than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig 5A) 271

NO3- influxes into vacuoles of the H B napus plants significantly decreased when treated 272

with inhibitors of proton pumps (Bafil DCCD B+D) (Fig 4B) Similarly NO3- influxes into 273

the vacuole of the Arabidopsis mutants (vha-a2 vha-a3 and avp1) was significantly lower 274

than those found in the wild type (col-0) (Fig 5B) Previous studies showed that VSC of NO3- 275

decreased when the activities of the proton pumps decline (Li et al 2005 Krebs et al 2010 276

Han et al 2015a) 277

We further investigated NO3- distribution between the vacuole and cytosol as affected by 278

proton pump inhibition in the H genotype of B napus and the mutants of A thaliana 279

defective in vacuolar proton pumps The percentage of vacuolar NO3- relative to total NO3

- in 280

root protoplasts of the H genotype treated with inhibitors (Bafil DCCD B+D) was lower than 281

that observed in the control (Fig 4C) Results were also similar when using A thaliana 282

mutants where the percentage of vacuolar NO3- relative to the total NO3

- in protoplasts was 283

lower in the mutants (vha-a2 vha-a3 and avp1) than in the wild type (col-0) (Fig 5C) 284

Consequently NO3- accumulation in the cytosol showed a significant increase when energy 285

pumps were suppressed in roots of the H genotype (Fig 4D) and NO3- accumulation in the 286

cytosol of root tissues of A thaliana mutants (vha-a2 vha-a3 and avp1) similarly increased 287

compared with col-0 (Fig 5D) 288

Based on previous observations (Lin et al 2008) expression of NRT15 is strongly 289

induced by NO3- but expression of NRT18 is down-regulated (Fig3 Chen et al 2012) We 290

then hypothesized that expression of these two genes is contrastingly regulated by 291

concentration of NO3- in the cytosol Our results were congruent with this hypothesis the 292

expression of BnNRT15 in root tissues of the H genotype of B napus was significantly higher 293

in plants treated with Bafil DCCD or B+D compared with the control (Fig 4E) whereas the 294

expression of BnNRT18 decreased substantially (Fig 4F) Similar results were also observed 295

in A thaliana where expression of AtNRT15 in roots of the mutants (vha-a2 vha-a3 and 296

avp1) were significantly higher than in the wild type (col-0) but expression of AtNRT18 in 297

the same mutants were considerably lower (Fig 5EF) 298

NO3- concentrations in the xylem sap the N-distribution between shoot and root (SR 299

ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 300

energy pumpsrsquo inhibitors were significantly higher than in the control (Fig 4GH Fig S5A) 301

11

Similar trends were also observed when using mutants of A thaliana deficient in the energy 302

pumpsrsquo activities NO3- concentration in the xylem sap the [15N] SR ratios and [NO3

-] SR 303

ratios were all significantly higher in the mutants than in the wild type (Fig 5GH Fig S5B) 304

These data clearly showed that prevention of N sequestration in vacuoles would enhance its 305

translocation to shoot 306

Additional evidence linking NO3- long-distance transport and NO3

- short-distance 307

distribution within cells were provided through experiments comparing A thaliana wild type 308

(Ws) and mutant clca-2 (Fig S6) The chloride channel (CLCa) is the main channel for 309

vacuolar anion accumulation Vacuolar sequestration capacity of NO3-significantly declines in 310

clca mutants (Angeli et al 2006) NO3- influx into the vacuolar space of Ws roots was 311

significantly higher than that observed in the clca-2 mutants (Fig S6A) This resulted in a 312

smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3

- in root tissue 313

protoplasts as compared with Ws (Fig S6B) Consequently the accumulation of NO3- in the 314

cytosol was significantly higher in clca-2 than in Ws plants (Fig S6C) The higher 315

NO3-concentration in the cytosol together with the higher expression of AtNRT15 coupled 316

with lower expression of AtNRT18 in clca-2 roots (Fig S6DE) resulted in significant 317

increase in xylem sap NO3- concentration [15N] SR ratios and [NO3

-] SR ratios compared 318

with Ws plants (Fig S6FGH) 319

320

Increased NO3- translocation to shoots enhanced NUE 321

NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al 322

1985 Andrews et al 1986 Tang et al 2012 Tang et al 2013) We therefore hypothesize 323

that increased NO3- translocation to shoot and the consequent higher shootroot ratio will 324

contribute to higher NUE Our data support this hypothesis The H B napus genotype showed 325

enhanced long-distance transport of NO3- from roots to shoots (Fig 2 Fig S2) This enhanced 326

NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 327

(Tang et al 2012 Tang et al 2013) Our results showed that chlorophyll content 328

intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 329

the L genotype at both seedling and flowering stages (Table 2) Moreover the NO3- 330

assimilating enzymes were strongly induced by NO3- providing sufficient capacity for N 331

assimilation (Smirnoff et al 1985 Andrews et al 1986) Nitrate reductase (NR) and 332

glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 333

in the L genotype both at seedling and flowering stages (Fig S7) In contrast NR activities in 334

12

shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig 335

S7AB) and GS activity in the H genotype was also higher than those of the L genotype at 336

seedling stage (Fig S7C) 337

Experiments comparing A thaliana wild type (col-0 Ws) with mutants defective in 338

vacuolar proton-pumps (vha-a2 vha-a3avp1) and transport channel (clca-2) yielded similar 339

results that is increases in NO3- shootroot ratio essentially contributed to enhanced NUE 340

(Fig 6) The NO3- shootroot ratios in A thaliana mutants were higher than that in the wild 341

type (Fig 5GH) Generally the efficiency of inorganic N (NO3-) assimilation into organic N 342

is higher in shoots than in roots (Smirnoff et al 1985 Andrews et al 1986) therefore the 343

NO3- concentration in A thaliana mutants decreased (Fig 6CD) leading to higher NUE 344

compared with the wild type (Fig 6AB) 345

346

BnNRT15BnNRT18 in B napus affects NO3- long-distance transport from roots to 347

shoots 348

Both B napus and A thaliana are members of Cruciferae Family The amino acid 349

sequence identity between BnNRT15 and AtNRT15 was 90 (Fig S8A) the amino acid 350

sequence similarity between BnNRT18 and AtNRT18 was 908 (Fig S8B) Comparisons of 351

nucleotide and amino acid sequences (Harper et al2012) of these genes 352

(httpbrassicanbiacukcgi-binmicroarray_databasecgi) showed that BnNRT15 353

(EV220114) and BnNRT18 (EV116423) of B napus are respectively highly homologous 354

with AtNRT15 and AtNRT18 and the two genes are mainly expressed in roots of both species 355

showing similar organ-specificity (Lin et al 2008 Li et al 2010) 356

NO3- long-distance transport from roots to shoots is therefore regulated by NRT15 and 357

NRT18 as reported before (Lin et al 2008 Li et al 2010) The [15N-traced] SR ratios and 358

the [NO3-] SR ratios were significantly lower in nrt15-3 mutants relative to the wild type 359

(col-0) while [15N] SR and [NO3-] SR ratios showed significant increase in nrt18-2mutant 360

relative to col-0 (Fig S9CD) A previous study showed that expression of NRT15 is 361

down-regulated by ACC and MeJA treatments while the expression of NRT18 is strongly 362

up-regulated in A thaliana (Zhang et al 2014) Consequently NO3- accumulated in plant 363

roots probably as an adaptive measure for abiotic stresses (Chen et al 2012) Both the H- 364

and L- B napus genotypes showed significantly lower expression of BnNRT15 in roots when 365

treated with ACC (Fig S10A) but the expression of BnNRT18 remained higher than in the 366

control plants (Fig S10B) This resulted in significantly lower [15N] SR ratios following 367

ACC treatment (Fig S10CDE) These data indicate that BnNRT15 and BnNRT18 play 368

13

similar functions for NO3- long-distance transport between root and shoot in both B napus 369

and A thaliana (Lin et al 2008 Li et al 2010) 370

371

372

14

Discussion 373

Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 374

NO3- long-distance transport from root to shoot is controlled by NRT gene family For 375

instance xylem NO3- loading and unloading are mainly controlled by NRT15 and NRT18 376

respectively (Lin et al 2008 Li et al 2010) Whereas the NO3- short-distance transport 377

between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 378

V-PPase) (Krebs et al 2010) and by activity of the CLCa channel in the tonoplast membrane 379

(Angeli et al 2006 Wege et al 2014) tonoplast proton-pumps provide the energy required 380

for NO3- accumulation into the vacuole through CLCa (Gaxiola et al 2001 Brux et al 2008 381

Krebs et al 2010) The expression of NRT15 is up-regulated by NO3- in plant culture 382

solution (Lin et al 2008) whereas regulation of NRT18 expression is contrary to that of 383

NRT15 under both control and abiotic stress conditions (Li et al 2010 Chen et al 2012 384

Zhang et al 2014) 385

Despite the progress made in characterizing the genes involved in transport and 386

metabolism of NO3- in plants the relationship between long- and short-distance transport and 387

how these two pathways are regulated have not been thoroughly investigated We hypothesize 388

that NRT15 expression and its role in long-distance transport is regulated by NO3- in plant 389

tissue as was observed in culture solution (Lin et al 2008) and our data support this 390

hypothesis Both BnNRT15 and AtNRT15 expression were up-regulated by increasing 391

cytosolic NO3- in roots (Fig 4 Fig 5) The increase in cytosolic NO3

- was achieved through (i) 392

use of A thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 393

activities in the tonoplast and (ii) inhibition of B napus tonoplast proton-pumps In both 394

model plants the influx of NO3- into vacuoles was substantially reduced resulting in higher 395

concentrations of NO3- in the cytosol (Fig 4A-D Fig 5A-D Fig S6A-C) Under those 396

conditions expression of BnNRT15 and AtNRT15 was up-regulated by the enhanced 397

cytosolic NO3- while expression of BnNRT18 and AtNRT18 was down-regulated (Fig 4EF 398

Fig 5EF FigS6DE) As a consequence more NO3- was loaded into the xylem sap and 399

transported from root to shoot (Fig S5AB FigS6F) This is also reflected as increased 400

shootroot ratios of N traced by [15N] and [NO3-] (Fig 4GH Fig 5GH Fig S6GH) 401

Interestingly the expression of both BnNRT15 and AtNRT15 is up-regulated by 402

increased cytosolic NO3- concentration in roots but were not affected by NO3

- sequestered 403

into vacuoles (Fig 4 Fig 5 Fig S6) This is because vacuolar NO3- is functionally separated 404

by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol 405

15

Therefore NO3--inducible genes such as NR and NRT15 were not influenced by vacuolar 406

NO3- concentration (Martinoia et al 1981 Martinoia et al 2000 Zhang et al 2012) In 407

addition NO3- concentration in the cytosol is controlled by its short-distance transport through 408

the tonoplast which is mediated through the tonoplast proton-pumps providing the required 409

energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al 2001 410

Brux et al 2008 Krebs et al 2010) The gradient of H+ between the inside of the vacuole 411

and the cytosol is maintained by an 2NO3-1H+ antiporter mechanism that facilitatesNO3

- 412

transport through CLCa (Angeli et al 2006 Wege et al 2014) Our data provide clear 413

evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3

- 414

concentration in roots 415

Regulatory mechanisms that control the expression of NRT18 and NRT15 as mediated 416

by NO3- are still unknown however previous studies showed that their expression is also 417

affected by abiotic stresses whereby NRT15 is down-regulated and NRT18 is up-regulated 418

(Li et al 2010 Zhang et al 2014) AtNRT18 expression in A thaliana nrt15 mutants is 419

highly up-regulated (Chen et al 2012) as was also observed here (Fig 3C) Moreover 420

AtNRT15 expression was enhanced by NO3- in culture solution and was not affected in 421

nrt18-2 mutant (Fig 3AB) On the other hand AtNRT18 expression was not affected by 422

cytosolic NO3- but down-regulated when AtNRT15 expression was up-regulated by NO3

-(Fig 423

S6DE) Analogous results were also obtained with B napus genotypes (Fig 3EF) Based on 424

these results we argue that NRT18 expression is not directly influenced by cytosolic NO3- but 425

rather by NRT15 expression (Fig 4 Fig 5 and Fig S6) suggesting NRT18 is probably 426

acting downstream of NRT15 Apparently the regulatory mechanisms of this NRT gene 427

family still awaits further studies to be elucidated 428

429

Response of NUE to NO3- long-distance transport 430

NO3- assimilation efficiency is higher in shoot than in root tissues and can be further 431

enhanced by carbon assimilation (Smirnoff et al 1985 Tang et al 2013) Therefore 432

translocation of higher proportion of NO3- from roots to shoots likely contributes to better 433

crop growth and higher NUE (Andrews et al 1986 Tang et al 2012) The present study 434

provides direct evidence for these results using contrasting B napus genotype identified 435

before (Zhang et al 2009 Han et al 2015a) Our data confirmed the genetic variation in 436

NUE between these two genotypes (Table 1) 437

We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438

physiological mechanisms contributing to variation in NUE The data were consistent with 439

16

our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 440

higher in the H genotype than in the L genotype (Fig 2EFFig S2EF Fig S4) The NO3- 441

long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A 442

thaliana and B napus genotypes (Fig 4-5 Fig S6) The activities of the tonoplast 443

proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig 444

1AB) Consequently less NO3- was sequestered into the vacuole and more NO3

- was retained 445

in cytosol (Fig 1CD) BnNRT15 expression was then up-regulated resulting in greater 446

loading into the xylem while BnNRT18 was down-regulated (Fig 2AB) probably to 447

suppress NO3- unloading from xylem sap Consequently a higher proportion of NO3

- was 448

transported from root to shoots in the H genotype(Fig 2C-D Fig S4) 449

Moreover photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450

were higher in shoots of the H genotype (Fig S7) both of which promote higher N 451

assimilation These results agreed with previous reports (Smirnoff et al 1985 Andrews et al 452

1986 Tang et al 2012 Tang et al 2013) where higher NO3- transport from root to shoot 453

higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 454

mechanisms for high-NUE in crop plants These qualities endowed the B napus H genotype 455

Xiangyou15 with higher NUE as compared with the L genotype 814 456

NO3- long-distance transport in B napus is not only regulated by BnNRT15 and 457

BnNRT18 in root tissues (Fig 4 Fig 5 Fig S6) but also by stomatal conductance and 458

transpiration by indirectly controlling xylem sap flow and long-distance transport 459

(Dechorgnat et al 2011 Krapp et al 2014) Stomatal conductance and transpiration rates of 460

the H genotype were significantly higher than those of the L genotype (Table S1) a result 461

consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 462

al 1998 Wilkinson et al 2007 Dechorgnat et al 2011 Krapp et al 2014) 463

464

Response of NUE to NO3- short-distance transport 465

NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466

and cytosol (Han et al 2015a) A lower proportion of NO3- accumulating in vacuoles and a 467

higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 468

al 2015b) The energy required for vacuolar NO3- sequestration is provided by the tonoplast 469

proton-pumps (Gaxiola et al 2001 Brux et al 2008 Krebs et al 2010) while the 470

distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 471

al 2006 Wege et al 2014) The activities of the tonoplast proton-pumps (V-ATPase and 472

V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 473

17

flowering stages (Han et al 2015aHan et al 2015b) thus we expected similar differences in 474

root tissues Our results confirmed that the activities of tonoplast proton-pumps in root tissues 475

of B napus H genotype and A thaliana mutants were lower than those in the L genotype and 476

A thaliana wild type leading to less NO3- influx into vacuoles and more in the cytosol (Fig 1 477

Fig 5A-D) Moreover the enzymes catalyzing N assimilation are located in the cytoplasm 478

where NO3- could be assimilated (Martinoia et al 1981 Han et al 2015a) or immediately 479

loaded into xylem sap for long-distance transport from roots to shoots (Lin et al 2008) 480

Consequently the NO3- concentration in root tissues of the H genotype and A thaliana 481

mutants defective in tonoplast proton-pumps was significantly lower than that in the L 482

genotype (Fig 2E) and in A thaliana wild type (Fig 6C) Therefore the higher proportion of 483

NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 484

to the higher NUE in Xiangyou15 and A thaliana mutants (vha-a2 vha-a3 and avp1) (Table 485

1Fig 6A) Further studies are needed to assess the impacts of regulating vacuolar 486

sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 487

species and genotypes 488

These data suggested that NO3- distribution between vacuoles and cytosol within cells 489

regulates NO3- long-distance transport from root to shoot and ultimately affects NUE NO3

- 490

loading into the xylem sap is an active transport process (Lin et al 2008) therefore not 491

regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 492

NO3- long-distance transport (NRT15 and NRT18) Apparently the expression of NRT15 the 493

gene responsible for xylem loading is regulated by NO3-concentration in the cytosol 494

Therefore both NO3- short-distance transport across the tonoplast and its long-distance 495

transport from root to shoot are important determinants of NUE in both B napus and A 496

thaliana (Table 1 and Fig 6) 497

498

Roles of NRT15 and NRT18 in NO3- long-distance transport in B napus and A 499

thaliana 500

Our results (Figs S9 S10) showed that the proportion of NO3- distributed from root to 501

shoot is strongly controlled by NRT15 in accord with previous studies (Lin et al 2008 Li et 502

al 2010) The data suggest that the high-NUE genotypes possess lower activities of tonoplast 503

proton-pumps (V-ATPase and V-PPase) resulting in less NO3- accumulation in vacuoles and 504

more NO3- retention in cytosol (Fig 7) The higher NO3

- in the cytosol then up-regulated 505

NRT15 which then down-regulates NRT18 As a result more NO3- is loaded into the xylem 506

system by NRT15 mechanism and less NO3- is unloaded via NRT18 (Fig 7) Differential 507

18

regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 508

shoot through the xylem vascular tissues (Fig 7) This coordinated NO3- long- and 509

short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3

- 510

distribution between roots and shoots (Fig 7) 511

Our research also revealed differences between A thaliana and B napus in the 512

expression of the wild-type NRT genes in response to NO3- in the protoplast Moreover using 513

A thaliananrt15-3 mutants we uncovered possible regulatory interactions between a 514

functioning (wild-type) NRT15 gene and NRT18 expression in response to NO3- 515

concentration Because mutants defective in nrt15 are not available for B napus we could 516

not confirm that the respective NRT15 and NRT18 systems behave in an analogous way in 517

these plant species Based on Harper et al (2012) research database our homology 518

comparisons and gene function annotations provided strong evidence for the roles of 519

BnNRT15 and BnNRT18 in B napus Finally our studies were conducted using two model 520

dicotyledonous plant species for which we had ample data on responses to specific inhibitors 521

(B napus) or specific mutants were available with results forming the bases for the model 522

presented in Figure 7 Further research using genetically diverse plant species (eg 523

monocotyledonous crops such as rice wheat and corn) will help validate this model and its 524

broader application across species The information generated in this study could have future 525

application for improving NUE in commercial crops with consequent reduction in nitrogen 526

use and benefits to the environment 527

528

Materials and Methods 529

Plant material 530

The two oilseed rape (Bnapus) cultivars used in this study have been characterized 531

before as high- (Xiangyou15 referred to as H genotype hereafter) and low- (814 referred to 532

as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al 2009 Han et al 2015b) 533

Here we define NUE as the total biomass per unit of N uptake by the plant with total 534

biomass includes roots shoots and grains The two genotypes were provided by the Hunan 535

Sub-Center of Improvement Center of National Oil Crops Hunan China 536

The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537

V-ATPase (vha-a2 and vha-a3) V-PPase (avp1) nrt15-3 and nrt18-2 mutants whereas the A 538

thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants A thaliana 539

19

mutants and transgenic lines (nrt15-3 and nrt18-2) have been described previously (Chen et 540

al 2012 Zhang et al 2014) The mutant lines vha-a2 (Salk_142642) and vha-a3 541

(Salk_122135) described by Krebs et al (2010) were obtained from the Arabidopsis 542

Biological Resources Center (ABRC) The mutant line avp1 (GK-005004) described in Li et 543

al (2005) was obtained from the European Arabidopsis Stock Centre while the mutant line 544

clca-2 (FST 171A06) described in Angeli et al (2006) was from the Institute National de la 545

Recherche Agronomique (INRA) collection in France together with the T-DNA mutants used 546

for genotyping to select pure mutant lines 547

548

Growth conditions 549

B napus plants were grown hydroponically in ceramic pots(20cmtimes15cm) filled with a 550

nutrient solution in the greenhouse under natural light as described in Han et al (2015b) The 551

pots were arranged in a completely randomized design with six biological replications The 552

nutrient solution was replaced every 3 days and its pH adjusted to 55 daily Experiments were 553

conducted at the field station of Hunan Agricultural University Southern China 554

A thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 555

et al (2000) and Gong et al (2003) The solution was changed every 3 days with pH adjusted 556

daily to 58 Pots were arranged in a completely randomized design with six biological 557

replications The nutrient solution used for both species consisted of 125mM KNO3 558

0625mM KH2PO4 05mM MgSO4 05mM Ca (NO3)2middot4H2O 0025mM Fe-EDTA 025 559

mlL-1 micronutrients (stock solution concentrations 70mM B 14mM Mn 1mM Zn 05mM 560

Cu and 02mM Mo) The experiments were conducted at Hunan Agricultural University in a 561

phytotron set at 70 relative humidity16h-light8h-dark cycle and constant temperature of 562

22degC 563

564

Experimental treatments and phenotyping 565

All measurements made on B napus plants were conducted either at the seedling stage 566

(2 months after transplanting) or at flowering (5 months after transplanting) The whole root 567

tissue was harvested and used for analyses at either stage For shoot measurements the fourth 568

leaf from the bottom was used for measurements made at seedling stage whereas the 12th leaf 569

20

from the bottom was harvested for measurements made at flowering stage For A thaliana 570

whole root and shoot of four-week-old plants were sampled and used for different assays 571

572

Vacuolar proton-pumps inhibitor treatments 573

Inhibition of vacuolar proton pumps of B napus (Xiangyou15) was conducted at the seedling 574

stage in hydroponic culture as described in Han et al (2015a) with minor modifications 575

Plants grown in hydroponic solution were used as control Inhibitor treatment was conducted 576

using several chemicals Bafi(25 nmol L-1 Bafilomycin A1) an inhibitor of V-ATPase 577

DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3) specifically inhibits V-PPase and 578

Bafi+DCCD in a 11 ratio as inhibitor of both V-ATPase and V-PPase The inhibitors were 579

applied in the hydroponic solutions for 24 h Fresh plant tissue samples from all treatments 580

were collected for different assays 581

582

NO3- induced gene expression 583

Four-week old A thaliana mutants (nrt15-3nrt18-2) were grown in hydroponics with 584

225mM (NH4)2succinate for 3 days and shifted to hydroponics with 45mM NO3- for 12 h 585

then root tissues were collected to assess relative expression of AtNRT15 and AtNRT18 586

genes Seedlings of B napus (Xiangyou15) plants grown hydroponically were treated with 587

5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 588

NO3- for 12 h Root tissue was then collected to assess the relative expression of BnNRT15 589

and BnNRT18 genes using quantitative RT-PCR 590

591

ACC treatment 592

Hydroponically grown seedlings of B napus were subjected to ACC treatment as described in 593

Zhang et al (2014) with minor modifications Seedlings of the two genotypes Xiangyou15 594

and 814 were transferred to a nutrient solution containing 002mM L-1 ACC for 6 h or grown 595

in normal hydroponic solution as control Root tissues were then collected to assay relative 596

expression of BnNRT15 and BnNRT18 using quantitative RT-PCR Another set of B napus 597

plants were grown in ACC-containing hydroponic solution but supplemented with 15NO3- (22 598

15N) replacing the unlabeled NO3- for 1 h Root and shoot tissues were collected separately 599

21

for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 600

C-N elemental analyzer (ANCA-MS PDZ Europa) 601

602

Biomass and N concentration 603

Grain yield dry biomass and N concentration were determined in plant samples at 604

harvest Plants were harvested (including senescing leaves) then dried in an oven for 30 min 605

at 105degC then at 70degC to a constant weight and weighed N concentration was determined 606

using the Kjeldahl method (Shi et al 2010 Han et al 2015b)02g of dried samples were 607

digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask after which N concentration 608

was determined using a Foss Auto Analyser Unit (Kjeldahl 8400) 609

610

V-ATPase and V-PPase activities 611

Roots (10 g) of B napus were collected at seedling and flowering stages and that of A 612

thaliana (05 g) sampled at seedling stage then used for determining the activities of 613

V-ATPase and V-PPase (Han et al 2015b) Activities of these two energy pumps within 614

microsomal membranes were followed colorimetrically based on released Pi as described by 615

Zhu et al (2001) and Krebs et al (2010) 616

617

Assay of NO3- flux in vacuoles 618

Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619

activities then used for vacuole isolation and measurement of NO3- flux in vacuoles as 620

described by Robert et al (2007) with minor modification (Han et al 2015b) Net fluxes of 621

NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 622

technique SIET system BIO-003A Younger USA Science and Technology Corporation 623

USA) An NO3--selective microelectrode used for the assay of NO3

- flux was vibrated in the 624

measuring solution between two positions 1 μm and 11 μm from the vacuole surface 625

(tonoplast) along an axis perpendicular to the tangent of the target vacuoles Background 626

signal was recorded by vibrating the electrode in the measuring solution in the absence of 627

vacuoles Ion flux was calculated by Fickrsquos law of diffusion J = -D(dcdx) where J represents 628

the ion flux (picomolescm-2s-1) dcdx is the ion concentration gradient and D is the ion 629

22

diffusion constant in a particular medium The direction of the flux was derived from Fickrsquos 630

law of diffusion that relates the concentration gradient 631

632

Isolation of intact protoplasts and vacuoles for determining NO3- concentration 633

Similar to the above assays root tissues (10 g) were collected from B napus at seedling 634

and flowering stages and from A thaliana (05 g) seedlings and used to isolate intact 635

protoplasts and vacuoles as described by Robert et al (2007) with minor modifications as 636

outlined in Huang et al (2012) and Han et al(2015b) The purified protoplasts were divided 637

into two equal aliquots with one of them used for isolation of vacuoles The purified 638

protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 639

(Vogeli-Lange and Wagner 1990) and for enzyme activity assays (Ma et al 2005) 640

NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 641

auto-analyzer (Auto Analyser 3 Bran and Luebbe Germany) as described previously (Han et 642

al 2015b)The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 643

determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc) following 644

the manufacturerrsquos instructions ACP activity specific to vacuoles was determined and used to 645

normalize NO3- accumulation We measured NO3

- in the protoplast outside the vacuole which 646

includes the cytosol and organelles eg mitochondria and Golgi Apparatus (Robert et 647

al2007) Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 648

(Krebs et al 2010) we refer to NO3- distribution between vacuoles and cytosol rather than 649

vacuole versus protoplast 650

651

Quantitative RT-PCR 652

Total RNA extracted from B napus roots was prepared using Trizol reagent (Invitrogen) 653

cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 654

manufacturerrsquos protocol The relative expression of BnNRT15 (EV220114) BnNRT18 655

(EV116423) and Bnactin (AF1118121) genes in plant roots were determined by quantitative 656

RT-PCR and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 657

Master Mix Kit (TOYOBO Japan) under the following conditions 95degC for 2min then 45 658

cycles of 95degC for 10s 60degC for 10s and 72degC for 20s The primer sequences of BnNRT15 659

23

BnNRT18 and Bnactin genes were obtained from httpbrassicanbiacuk as described in 660

Harper et al (2012) and expression levels were normalized to Bnactin as control 661

Total RNA extracted from the 4-week-old A thaliana roots under the treatments 662

conditions described above were prepared using Trizol reagent (Invitrogen) The cDNAs were 663

synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturerrsquos 664

protocol and as described in Li et al (2010)The primer sequences of quantitative RT-PCR 665

used in these assays are listed in Supplemental Table 2 and were described previously (Chen 666

et al 2012 Zhang et al 2014) and their expression was normalized to Atactin2 as control 667

668

Determination of nitrate distribution using 15NO3- 669

Hydroponically grown B napus (seedling and flowering stages) and A thaliana 670

(seedling) were transferred to 01mM CaSO4 solution for 1min then to their respective 671

hydroponic nutrient solutions with15NO3- (22 excess) replacing unlabeled NO3

- for 1h 672

Plants were then transferred to 01mM CaSO4 solution for 1min after which roots were 673

washed with deionized water Root and shoot samples were separated and dried at 105degC for 674

30min followed by 70degC for 3 d then grounded and used for assaying 15N content using a 675

continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 676

analyzer (ANCA-MS PDZ Europa) 677

678

Xylem sap collection and assay of nitrate concentration 679

We used the method of Tang et al (2012) for collecting xylem sap Plants were cut 680

leaving1cm segments with intact roots at seedling and flowering stages for B napus and at 681

seedling stage for A thaliana Roots were immediately immersed in their respective nutrition 682

solutions Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h 683

The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 684

the weight gain of the cotton Xylem sap was then squeezed from the cotton using a syringe 685

and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 686

(Auto Analyser 3 Bran and Luebbe Germany) 687

To determine nitrate concentration samples (10 g fresh root and shoot at seedling and 688

flowering of B napus and 05 g fresh root and shoot of 4-week-old A thaliana) were frozen 689

in liquid N2 and ground with a mortar and pestle the powder was then transferred to a beaker 690

24

containing 70 ml deionized water and boiled for 30 min to extract nitrate then cooled and 691

made to 100 ml volume and 02 g activated carbon added to eliminate the effect of 692

chlorophyll The mixture was filtered and nitrate in the filtrate was determined using a 693

continuous-flow auto-analyser (Atuo-Analyser 3 Bran and Luebbe Germany) 694

695

Measurements of photosynthesis 696

All measurements of photosynthesis and related parameters were conducted at 1000 h 697

using intact leaves The 4th leaf from the bottom in B napus at seedling stage and the 12th leaf 698

from the bottom at flowering stage were used for determining chlorophyll concentration 699

photosynthetic rate intercellular CO2 concentration transpiration rate and stomatal 700

conductance Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co 701

Ltd Japan) (Luo et al 2006 Yandeau-Nelson et al 2011) Photosynthetic rate intercellular 702

CO2 concentration transpiration rate and stomatal conductance were measured using LI-6400 703

Portable Photosynthesis System (Li-Cor Biosciences Co Ltd USA) set at flow rate of 500 704

μmol s-1 photosynthetic photon flux density of 1000 μmol m-2 s-1 relative humidity of 65 705

CO2 concentration of 400ppm and ambient temperatures 706

707

NR and GS activities 708

Leaves with similar age as those used for photosynthesis in B napus were used for the 709

assay of nitrate reductase (NR) and glutamine synthetase (GS) activities NR activity was 710

measured by the modified method of Fan et al (2007) Fresh samples (05g) were frozen in 711

liquid N2 ground into powder in the presence of acid-washed sand and homogenized with 4 712

ml of extraction buffer (0025mol L-1phosphate buffer pH87 1211g L-1 cysteine 0372g L-1 713

EDTA) Homogenates were centrifuged at 30000timesg for 15 min at 4degC and the supernatants 714

were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 715

determination of nitrite as described by Fan et al (2007) 716

GS activity was determined using a modified reverseγ-glutamyltransferase method 717

(Wang et al2014) which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 718

from glutamine and hydroxylamine Fresh samples were frozen for 30 min at -20degC then 719

ground in 10ml Tris-HCl buffer and acid-washed sand The homogenates were filtered 720

through two layers of gauze and centrifuged at 8000timesg for 15 min at 4degC About 12ml of the 721

25

supernatant was added in a reaction mixture and treated as described (Wang et al2014) After 722

the GS-catalyzed reaction was stopped and the solution centrifuged glutamyl-γ723

-hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 724

concentration was determined using a standard curve 725

726

Statistical analyses 727

We used the SPSS software (Statistical Product and Service Solutions V130 USA) for 728

ANOVA and mean separation of main-effects and interactions using Duncanrsquos multiple range 729

test at Plt005Values are presented as means and SD of three or six replicates from three 730

independent experiments Different letters or an asterisk () associated with specific data (eg 731

at the top of histogram bars in figures or within tables) denote significant differences at 732

Plt005 733

734

Supplemental Data 735

Supplemental Figure 1 B napus with higher NUE showed lower vacuolar sequestration 736

capacity (VSC) for NO3-in roots at flowering stage 737

Supplemental Figure 2 B napus with higher NUE showed enhanced long-distance transport 738

of NO3- from roots to shoots at flowering stage 739

Supplemental Figure 3 The two B napus (H and L genotypes) showed the same total N per 740

plant at seedling stage (A) and flowering stage (B) 741

Supplemental Figure 4 B napus with higher NUE showed increased NO3- concentration in 742

the xylem sap at seedling and flowering stages 743

Supplemental Figure 5 NO3- concentration in the xylem sap as affected by inhibitor 744

treatments in B napus and in the energy pumpsrsquo mutants of A thaliana (col-0 vha-a2 vha-a3 745

avp1) 746

Supplemental Figure 6 Reduced VSC for NO3- in roots drives long-distance transport of 747

NO3- from roots to shoots in the A thaliana wild type (Ws) and mutant (clca-2) 748

Supplemental Figure 7 Differences of NR and GS activities between the two B napus (H 749

and L genotypes) at seedling and flowering stages 750

Supplemental Figure 8 Amino acid sequences of BnNRT15 and BnNRT18 751

Supplemental Figure 9 Functions of AtNRT15 and AtNRT18 genes in root tissues of A 752

thaliana in controlling NO3- long-distance transport from root to shoots 753

26

Supplemental Figure 10 B napus BnNRT15 and BnNRT18 genes in roots were 754

coordinately modulated to facilitate NO3- long distance transport from roots to shoots 755

Supplemental Table 1 Differences in stomatal conductance and transpiration rate between 756

the two B napus genotypes 757

Supplemental Table 2 Sequences of primera used for qRT-PCR 758

759

Acknowledgements 760

Shanghai Institutes for Biological Sciences) for providing nrt15-3 and nrt18-2 seeds 762

This study was supported by the National Natural Science Foundation of China (Grant 763

(13K062) the National Key Laboratory of Plant Molecular Genetics and the Twelfth 767

Five-Year National Science and technology support program (2012BAD15BO4) 768

769

Figure 1 B napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 770

NO3- in roots at the seedling stage 771

H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 772

genotype 814 Specific activities of the tonoplast proton pumps are expressed as μmol Pi 773

released mg-1 protein h-1 NO3- fluxes are expressed as pmol NO3

- cm-2 S-1 Mature vacuoles 774

were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 775

measuring solution between the two positions 1 μm and 11 μm from the vacuole surface 776

(tonoplast) along an axis perpendicular to the tangent of the target vacuoles recording the 777

stable reading data The background was recorded by vibrating the electrode in measuring 778

solution without vacuoles 779

Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 780

for NO3- content and NO3

- accumulation normalized against the specific activity of the 781

vacuole acid phosphatase (ACP) as described in Materials and Methods and plotted as μmol 782

NO3- per μmol p-nitrophenol the end product of ACP assay 783

Proton pump activities in root tissues between H and L genotypes are shown at the seedling 784

stage (A) Different letters at the top of the histogram bars denote significant differences of 785

V-ATPase in root tissues between H and L genotypes (Plt005) an asterisk () at the top of the 786

27

histogram bars indicates significant difference in V-PPase activity in root tissues between H 787

and L genotypes (Plt005) Vertical bars on the figures indicate SD (n=6) 788

Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 789

genotypes are shown at the seedling stage (B) Different letters at the top of the histogram 790

bars denote significant differences of NO3- flux between H and L genotypes (Plt005) Vertical 791

bars on the figures indicate SD (n=6) 792

Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 793

seedling stage (C) Values above the bars represent the percentage of vacuolar NO3- relative 794

to the total NO3- in protoplasts 795

NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D) P-V is the 796

total NO3-in the cytosol and was calculated as total NO3

- in protoplasts ndash total NO3- in 797

vacuole Different letters at the top of histogram bars denote significant differences of total 798

NO3- in cytosol between H and L genotypes (Plt005) Vertical bars on the figures indicate SD 799

(n=6) 800

801

Figure 2 B napus with higher NUE showed enhanced long-distance transport of NO3- from 802

roots to shoots at the seedling stage 803

H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814 804

Expression of the BnNRT15 (A) and BnNRT18 (B) genes relative to that of the actin gene in 805

the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 806

as described in Materials and Methods a value of 10 is equivalent to levels of expression of 807

the Bnactin gene Vertical bars on the figures indicate SD (n=3) different letters at the top of 808

the histogram bars denote significant differences at Plt005 809

Hydroponically grown B napus plants were subjected to 15N-labeling treatment as described 810

in Materials and Methods The 15N concentration in the root and shoot tissues of the two 811

genotypes is shown at the seedling stage (C) The [15N] SR ratios in the root and shoot tissues 812

of the two genotypes are shown at the seedling stage (D)Vertical bars on the figures indicate 813

SD (n=3) different letters at the top of the histogram bars denote significant differences at 814

Plt005 level 815

The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 816

at the seedling stage (E) The [NO3-] SR ratios in the root and shoot tissues of the two 817

genotypes are shown at the seedling stage (F) Vertical bars on the figures indicate SD (n=3) 818

different letters at the top of the histogram bars denote significant differences at Plt005 819

820

28

Figure 3 NRT15 gene expression is up-regulated by NO3- and might affect downstream 821

regulation of the NRT18 gene 822

Culture conditions and plant materials are defined in the Materials and Methods Seedling 823

stage A thaliana mutant plants (nrt18-2 and nrt15-3) were cultured hydroponically with 824

225 mM (NH4)2succinate for 3 d and shifted to hydroponics with 45 mM NO3- for 12 h after 825

which root tissues were collected for analysis Expression of AtNRT15 or AtNRT18 genes 826

were assessed by quantitative RT-PCR as described in Materials and Methods and presented 827

relative to that of the Atactin2 gene 828

B napus (Xiangyou15) was cultured in hydroponics with 75 mM (NH4)2succinate for 3 d and 829

shifted to hydroponics with 15 mM NO3- for 12 h after which root tissues were collected for 830

analysis as described in the Materials and Methods 831

Relative expressions of the AtNRT15 genes are shown at the seedling stage in root tissues of 832

col-0 and nrt18-2 (A) Relative expression of the AtNRT15 gene is shown in root tissues of 833

nrt18-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 834

for 12 h at the seedling stage (B) Relative expressions of the AtNRT18 genes are shown at 835

the seedling stage in root tissues of col-0 and nrt15-3 (C) Panel (D) show results for the 836

relative expression of the AtNRT18 genes under the same conditions as in panel (B) 837

Relative expression of BnNRT15 (E) and BnNRT18 (F) genes in root tissues of B napus 838

(Xiangyou15) are shown at the seedling stage Different letters at the top of the histogram 839

bars denote significant differences (Plt005) Vertical bars on the figures indicate SD (n=6) 840

841

Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3

- from roots to 842

shoots in the B napus (Xiangyou15) 843

Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were conducted at 844

the seedling stage Control normal hydroponics solution Bafi(25 nmolmiddotL-1Bafilomycin A1) 845

an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD + 50 mmolmiddotL-1Na2SO3) which together 846

specifically inhibits V-PPase and B+D a 11 combination of Bafi and DCCD which inhibits 847

both V-ATPase and V-PPase The inhibitors were applied at the seedling stage in hydroponic 848

solutions for 24 h Mature vacuoles were collected from the root tissues of the plant materials 849

at the seedling stage The NO3- flux measurement is described in the legend to Figure 1 850

Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 851

NO3- accumulation plotted as μmol NO3

- per μmol p-nitrophenol as described in the legend to 852

Figure 1 and in Materials and Methods 853

29

Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B 854

napus as affected by inhibitor treatments (A) Different letters at the top of histogram bars 855

denote significant differences in V-ATPase activities in root tissues between inhibitor 856

treatments (Plt005) asterisk () at the top of the histogram bars denote significant differences 857

in V-PPase activities in root tissues between inhibitor treatments (Plt005) Vertical bars on 858

the figures indicate SD (n=6) 859

NO3- fluxes within the vacuoles of root tissues are shown for B napus between inhibitor 860

treatments (B) Different letters at the top of the histogram bars denote significant differences 861

of NO3- flux between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD 862

(n=6) 863

Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B 864

napus (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 865

total NO3- in protoplasts Vertical bars indicate SD (n=6) 866

Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is the total 867

NO3- in the cytosol calculated as total NO3

- in protoplasts ndash total NO3- in vacuoles Different 868

letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 869

outside of the protoplast (Plt005) Vertical bars on the figures indicate SD (n=6) 870

Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown relative to that 871

of the actin gene assessed by quantitative RT-PCR as described in Materials and Methods a 872

value of 10 is equivalent to levels of expression of the Bnactin gene Different letters at the 873

top of the histogram bars denote significant differences in gene expression (Plt005) Vertical 874

bars indicate SD (n=3) 875

Growth conditions for hydroponically grown plants with 15N treatmentare described in 876

Materials and Methods The [15N] SR ratios as affected by inhibitor treatments are depicted 877

for B napus (G)The [NO3-] SRratioas affected by inhibitor treatments are shown for B 878

napus (H) Different letters at the top of the histogram bars denote significant differences 879

(Plt005) Vertical bars on the figures indicate SD (n=3) 880

881

- from roots to 882

shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1) 883

Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-a2 vha-a3) and 884

V-PPase gene mutant plant (avp1) were used as the model plant materials Mature vacuoles 885

were collected from the root tissues of the plant materials at the seedling stage The NO3- flux 886

measurement is described in the legend to Figure 1 Protoplasts and vacuoles isolated from 887

30

roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 888

NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 889

Methods 890

Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 891

different A thaliana plant materials (A) Different letters at the top of the histogram bars 892

denote significant differences in V-ATPase activities in root tissues between A thaliana plant 893

materials (Plt005) asterisk () at the top of the histogram bars denote significant differences 894

of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD (n=6) 895

NO3- fluxes within the vacuoles of root tissues are shown for A thaliana between different 896

mutants (B) Different letters at the top of the histogram bars denote significant differences in 897

NO3- flux between Arabidopsis plant materials (Plt005) Vertical bars indicate SD (n=6) 898

Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A 899

thaliana (C) Values above the bars represent the percentage of vacuolar NO3- relative to the 900

Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D) P-V is the total 902

letters at the top of the histogram bars denote significant differences in total NO3-in the 904

cytosol (Plt005) Vertical bars indicate SD (n=6) 905

Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown relative to that of the 906

actin gene assessed by quantitative RT-PCR a value of 10 is equivalent to levels of 907

expression of the Atactin2 gene Different letters at the top of the histogram bars denote 908

significant differences in gene expression (Plt005) Vertical bars on the figures indicate SD 909

(n=3) 910

for various A thaliana mutants (G) The [NO3-] SR ratio as affected by inhibitor treatments 913

are depicted for various A thaliana mutants (H) Different letters at the top of the histogram 914

916

Figure 6 Increased NO3- shootsroots ratio essentially contributed to enhancing NUE in A 917

thaliana 918

Plants grown hydroponically were sampled for further analysis at seedling stage and at 919

harvest Conditions for hydroponics culture and characteristics of the various A thaliana 920

genotypes are defined in Materials and Methods The wild-type Columbia-0 (col-0) plants 921

31

were used as control for V-ATPase mutants (vha-a2 and vha-a3) V-PPase mutants (avp1) 922

nrt15-3 mutants and nrt18-2 mutants A thaliana wild-type Wassilewskija (Ws) plants were 923

used as control for clca-2 mutants 924

Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 925

wild-type (col-0) and mutants nrt15-3 nrt18-2 vha-a2 vha-a3 and avp1 926

Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A 927

thaliana wild-type plants Ws and clca-2 Different letters at the top of the histogram bars 928

denote significant differences between the Arabidopsis genotypes (Plt005) Vertical bars on 929

931

Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues 932

Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 933

in root tissues Red lines display the route for regulation pathway 934

935

936

Figure 1 B napus with higher NUE showed lower vacuolar

sequestration capacity (VSC) for NO3- in roots at the seedling stage

H refers to the high-NUE oilseed rape genotype Xiangyou15 and L

refers to the low-NUE genotype 814 Specific activities of the tonoplast

proton pumps are expressed as μmol Pi released mg-1 protein h-1 NO3-

fluxes are expressed as pmol NO3- cm-2 S-1 Mature vacuoles were

collected from the root tissues at seedling stage and a microelectrode

was vibrated in the measuring solution between the two positions 1

μm and 11 μm from the vacuole surface (tonoplast) along an axis

perpendicular to the tangent of the target vacuoles recording the stable

reading data The background was recorded by vibrating the electrode

in measuring solution without vacuoles

Protoplasts and vacuoles isolated from roots of hydroponically grown

plants were measured for NO3- content and NO3

- accumulation

normalized against the specific activity of the vacuole acid phosphatase

(ACP) as described in Materials and Methods and plotted as μmol

NO3- per μmol p-nitrophenol the end product of ACP assay

Proton pump activities in root tissues between H and L genotypes are

shown at the seedling stage (A) Different letters at the top of the

histogram bars denote significant differences of V-ATPase in root

tissues between H and L genotypes (Plt005) an asterisk () at the top

of the histogram bars indicates significant difference in V-PPase

activity in root tissues between H and L genotypes (Plt005) Vertical

bars on the figures indicate SD (n=6)

Mean rates of NO3- flux during 160 s within vacuoles of root tissues

between H and L genotypes are shown at the seedling stage (B)

Different letters at the top of the histogram bars denote significant

differences of NO3- flux between H and L genotypes (Plt005) Vertical

Accumulation of NO3- inside the vacuole and in the protoplasts of root

tissues is shown at the seedling stage (C) Values above the bars

represent the percentage of vacuolar NO3- relative to the total NO3

- in

protoplasts

NO3- accumulation in the cytosol of root tissues is shown at seedling

stage (D) P-V is the total NO3-in the cytosol and was calculated as

total NO3- in protoplasts ndash total NO3

- in vacuole Different letters at the

top of histogram bars denote significant differences of total NO3- in

cytosol between H and L genotypes (Plt005) Vertical bars on the

figures indicate SD (n=6)

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

Figure 2 B napus with higher NUE showed enhanced

long-distance transport of NO3- from roots to shoots at

the seedling stage

H refers to the high-NUE genotype Xiangyou15 and L

refers to the low-NUE genotype 814 Expression of the

BnNRT15 (A) and BnNRT18 (B) genes relative to that

of the actin gene in the root tissues of the two

genotypes at seedling stage was assessed by

quantitative RT-PCR as described in Materials and

Methods a value of 10 is equivalent to levels of

expression of the Bnactin gene Vertical bars on the

figures indicate SD (n=3) different letters at the top of

the histogram bars denote significant differences at

Plt005

Hydroponically grown B napus plants were subjected

to 15N-labeling treatment as described in Materials and

Methods The 15N concentration in the root and shoot

tissues of the two genotypes is shown at the seedling

stage (C) The [15N] SR ratios in the root and shoot

tissues of the two genotypes are shown at the seedling

stage (D)Vertical bars on the figures indicate SD

(n=3) different letters at the top of the histogram bars

denote significant differences at Plt005 level

The NO3- concentration (μg g-1 FW) in root and shoot

stage (E) The [NO3-] SR ratios in the root and shoot

stage (F) Vertical bars on the figures indicate SD

denote significant differences at Plt005 wwwplantphysiolorgon May 17 2018 - Published by Downloaded from

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

(NH4)2succinate KNO3

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

col-0 nrt15-3 (NH4)2succinate KNO3

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

Figure 3 NRT15 gene expression is up-regulated by NO3-

and might affect downstream regulation of the NRT18

gene

Culture conditions and plant materials are defined in the

Materials and Methods Seedling stage A thaliana mutant

plants (nrt18-2 and nrt15-3) were cultured hydroponically

with 225 mM (NH4)2succinate for 3 d and shifted to

hydroponics with 45 mM NO3- for 12 h after which root

tissues were collected for analysis Expression of AtNRT15

or AtNRT18 genes were assessed by quantitative RT-PCR

as described in Materials and Methods and presented

relative to that of the Atactin2 gene

B napus (Xiangyou15) was cultured in hydroponics with

75 mM (NH4)2succinate for 3 d and shifted to hydroponics

with 15 mM NO3- for 12 h after which root tissues were

collected for analysis as described in the Materials and

Methods

Relative expressions of the AtNRT15 genes are shown at

the seedling stage in root tissues of col-0 and nrt18-2 (A)

Relative expression of the AtNRT15 gene is shown in root

tissues of nrt18-2 plants treated with either

(NH4)2succinate for 3 d or shifted to hydroponics with NO3-

for 12 h at the seedling stage (B) Relative expressions of

the AtNRT18 genes are shown at the seedling stage in root

tissues of col-0 and nrt15-3 (C) Panel (D) show results for

the relative expression of the AtNRT18 genes under the

same conditions as in panel (B)

Relative expression of BnNRT15 (E) and BnNRT18 (F)

genes in root tissues of B napus (Xiangyou15) are shown

at the seedling stage Different letters at the top of the

histogram bars denote significant differences (Plt005)

a

bb

c

0

001

002

003

004

Control Bafil DCCD B+D

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

control Bafi DCCD B+D

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

from roots to shoots in the B napus (Xiangyou15)

Inhibitor treatments of B napus (Xiangyou15) hydroponics-grown plants were

conducted at the seedling stage Control normal hydroponics solution Bafi(25

nmolmiddotL-1Bafilomycin A1) an inhibitor of V-ATPase DCCD(10 μmolmiddotL-1DCCD

+ 50 mmolmiddotL-1Na2SO3) which together specifically inhibits V-PPase and B+D a

11 combination of Bafi and DCCD which inhibits both V-ATPase and V-PPase

The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h

Mature vacuoles were collected from the root tissues of the plant materials at the

seedling stage The NO3- flux measurement is described in the legend to Figure 1

Protoplasts and vacuoles isolated from roots of hydroponically grown plants were

assessed for NO3- accumulation plotted as μmol NO3

- per μmol p-nitrophenol as

described in the legend to Figure 1 and in Materials and Methods

Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root

tissues of B napus as affected by inhibitor treatments (A) Different letters at the

top of histogram bars denote significant differences in V-ATPase activities in

root tissues between inhibitor treatments (Plt005) asterisk () at the top of the

histogram bars denote significant differences in V-PPase activities in root tissues

between inhibitor treatments (Plt005) Vertical bars on the figures indicate SD

(n=6)

NO3- fluxes within the vacuoles of root tissues are shown for B napus between

inhibitor treatments (B) Different letters at the top of the histogram bars denote

significant differences of NO3- flux between inhibitor treatments (Plt005)

Vertical bars on the figures indicate SD (n=6)

Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is

shown for B napus (C) Values above the bars represent the percentage of

vacuolar NO3- relative to the total NO3

- in protoplasts Vertical bars indicate SD

(n=6)

Accumulation of cytosolic NO3 -in root tissues is shown for B napus (D) P-V is

the total NO3- in the cytosol calculated as total NO3

- in protoplasts ndash total NO3-

in vacuoles Different letters at the top of the histogram bars denote significant

differences in total NO3- in vacuoles outside of the protoplast (Plt005) Vertical

Expression levels of the BnNRT15 gene (E) and BnNRT18gene (F) are shown

relative to that of the actin gene assessed by quantitative RT-PCR as described in

Materials and Methods a value of 10 is equivalent to levels of expression of the

Bnactin gene Different letters at the top of the histogram bars denote significant

differences in gene expression (Plt005) Vertical bars indicate SD (n=3)

Growth conditions for hydroponically grown plants with 15N treatmentare

described in Materials and Methods The [15N] SR ratios as affected by inhibitor

treatments are depicted for B napus (G)The [NO3-] SRratioas affected by

inhibitor treatments are shown for B napus (H) Different letters at the top of the

histogram bars denote significant differences (Plt005) Vertical bars on the

D

b

a

0

2

4

6

8

col-0 vha-a2 vha-a3 avp1

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

avp1 vha-a3 vha-a2 col-0

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

45Vacuole Protoplast

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of

NO3- from roots to shoots in the A thaliana (col-0 vha-a2 vha-a3 avp1)

Wild type A thaliana plants (col-0) V-ATPase gene mutant plants (vha-

a2 vha-a3) and V-PPase gene mutant plant (avp1) were used as the model

plant materials Mature vacuoles were collected from the root tissues of the

plant materials at the seedling stage The NO3- flux measurement is

described in the legend to Figure 1 Protoplasts and vacuoles isolated from

roots of hydroponically grown plants were assessed for NO3- accumulation

plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to

Figure 1 and in Materials and Methods

Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in

root tissues from different A thaliana plant materials (A) Different letters

at the top of the histogram bars denote significant differences in V-ATPase

activities in root tissues between A thaliana plant materials (Plt005)

asterisk () at the top of the histogram bars denote significant differences

of V-PPase activities in root tissues (Plt005) Vertical bars indicate SD

(n=6)

NO3- fluxes within the vacuoles of root tissues are shown for A thaliana

between different mutants (B) Different letters at the top of the histogram

bars denote significant differences in NO3- flux between Arabidopsis plant

materials (Plt005) Vertical bars indicate SD (n=6)

tissues is shown for A thaliana (C) Values above the bars represent the

percentage of vacuolar NO3- relative to the total NO3

- in protoplasts

Vertical bars indicate SD (n=6)

Accumulation of cytosol NO3- in root tissues is shown for A thaliana (D)

P-V is the total NO3- in the cytosol calculated as total NO3

- in protoplasts

ndash total NO3- in vacuoles Different letters at the top of the histogram bars

denote significant differences in total NO3-in the cytosol (Plt005) Vertical

bars indicate SD (n=6)

Expression of the AtNRT15 gene (E) and AtNRT18 gene (F) are shown

relative to that of the actin gene assessed by quantitative RT-PCR a value

of 10 is equivalent to levels of expression of the Atactin2 gene Different

letters at the top of the histogram bars denote significant differences in

gene expression (Plt005) Vertical bars on the figures indicate SD (n=3)

described in Materials and Methods The [15N] SR ratios as affected by

inhibitor treatments are depicted for various A thaliana mutants (G) The

[NO3-] SR ratio as affected by inhibitor treatments are depicted for various

A thaliana mutants (H) Different letters at the top of the histogram bars

denote significant differences (Plt005) Vertical bars on the figures

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

Figure 6 Increased NO3- shootsroots

ratio essentially contributed to enhancing

NUE in A thaliana

Plants grown hydroponically were

sampled for further analysis at seedling

stage and at harvest Conditions for

hydroponics culture and characteristics

of the various A thaliana genotypes are

defined in Materials and Methods The

wild-type Columbia-0 (col-0) plants

were used as control for V-ATPase

mutants (vha-a2 and vha-a3) V-PPase

mutants (avp1) nrt15-3 mutants and

nrt18-2 mutants A thaliana wild-type

Wassilewskija (Ws) plants were used as

control for clca-2 mutants

Differences in NUE based on biomass

(A) and NO3- concentration (C) are

shown for the wild-type (col-0) and

mutants nrt15-3 nrt18-2 vha-a2 vha-

a3 and avp1

Differences of NUE based on biomass

(B) and NO3-concentration (D) are

shown between A thaliana wild-type

plants Ws and clca-2 Different letters at

the top of the histogram bars denote

significant differences between the

Arabidopsis genotypes (Plt005) Vertical

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

Figure 7 Simplified model for NO3- long-distance transport in xylem of

vascular tissues

Long-distance transport is regulated by NO3- distribution between the vacuole

and the cytosol in root tissues Red lines display the route for regulation

pathway wwwplantphysiolorgon May 17 2018 - Published by Downloaded from

Table 1 Comparison of NUE between the two B napus genotype

aNUE values were calculated based on either total biomass (shoot root and grains) per total N

uptake or as grain yield per total N uptake bH represents the high NUE genotype (Xiangyou15) and L represents the low NUE genotype

(814)

Different letters associated with data represent significant differences at P＜005 n=3

bGenotypes

Physiological parameters aNUE

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Based on grain yield

(gg-1)

H 13944plusmn677a 3126plusmn050a 261plusmn008a 5354plusmn240a 1201plusmn044a

L 10873plusmn305b 2084plusmn144b 245plusmn015a 4440plusmn264b 853plusmn108b

Table 2 Variation inchlorophyll content intercellular CO2 concentration and photosynthetic rate

between high (H) and low (L) nitrogen use efficiency genotypes of B napus

aChlorophyll content intercellular CO2 concentration and photosynthetic rate were measured at

1000 h using a LI-6400 Portable Photosynthesis System Measurements were conducted using the

4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering

Different letters between the H and L genotypes denote a significant difference at Plt005 level

Data are means plusmnSD (n=6)

Genotypes

Seedling stage Flowering stage

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

H 4865plusmn129a 27184plusmn764a 2331plusmn096a 6311plusmn208a 28399plusmn2084a 1753plusmn058a

L 4586plusmn208b 24636plusmn878b 2102plusmn076b 5740plusmn300b 23368plusmn2750b 1622plusmn067b

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Parsed Citations
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Page 2: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

2

Footnotes 33

Shanghai Institutes for Biological Sciences) for providing nrt15-3and nrt18-2 seeds This 35

study was supported by the National Natural Science Foundation of China (Grant 36

(13K062) National Key Laboratory of Plant Molecular Genetics and the Twelfth Five-Year 40

National Science and technology support program (2012BAD15BO4) 41

42

These authors have contributed equally 43

sect Corresponding author e-mail zhzh1468163com 44

45

46

Author contributions 47

ZHZ conceived the original screening and research plans ZHZ supervised the experiments 48

YLH HXS QL YY SFJ and ZHZ performed most of the experiments CT and JZ provided 49

technical assistance to YLH HXS YY and ZHZ YLH and ZHZ designed the experiments 50

and analyzed the data QL XMR CYG and ZHZ interpreted the results and JEL AI and 51

ZHZ conceived the project and wrote the article with contributions of all the authors JEL AI 52

and ZHZ supervised and complemented the writing All authors read and approved the final 53

manuscript 54

55

56

57

58

59

60

61

62

63

64

65

66

3

67

Abstract 68

-] SR ratios 77

88

90

91

92

93

94

95

96

97

98

4

Introduction 99

higher NUE 111

5

-movement 141

6

- long-distance 195

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
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TWO
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FOUR
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Page 3: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

3

67

Abstract 68

-] SR ratios 77

88

90

91

92

93

94

95

96

97

98

4

Introduction 99

higher NUE 111

5

-movement 141

6

- long-distance 195

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 4: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

4

Introduction 99

higher NUE 111

5

-movement 141

6

- long-distance 195

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 5: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

5

-movement 141

6

- long-distance 195

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 6: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

6

- long-distance 195

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 7: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

7

201

202

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 8: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

8

Results 203

in root tissues 205

219

to shoots 221

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 9: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

9

- in xylem sap 237

242

244

- 256

259

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 10: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

10

- in 280

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 11: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

11

-] SR 303

320

12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
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SEVEN
T-ONE
T-TWO
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12

346

shoots 348

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 13: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

13

371

372

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 14: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

14

Discussion 373

- sequestered 403

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
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SEVEN
T-ONE
T-TWO
Parsed Citations

Page 15: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

15

- 412

- 414

-(Fig 423

429

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 16: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

16

- was retained 445

- was 448

464

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 17: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

17

- 490

498

thaliana 500

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 18: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

18

- 510

528

Plant material 530

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 19: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

19

548

22degC 563

564

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 20: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

20

572

582

591

ACC treatment 592

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 21: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

21

602

610

617

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 22: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

22

632

651

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 23: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

23

668

- for 1h 672

678

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 24: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

24

695

707

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 25: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

25

726

Plt005 733

734

avp1) 746

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 26: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

26

759

769

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
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SEVEN
T-ONE
T-TWO
Parsed Citations

Page 27: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

27

(n=6) 800

801

Plt005 level 815

820

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 28: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

28

841

- from roots to 842

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 29: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

29

(n=6) 863

881

- from roots to 882

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 30: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

30

Methods 890

(n=3) 910

916

thaliana 918

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 31: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

31

931

935

936

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
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ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 32: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

- accumulation

- in

protoplasts

A B

C D

μm

ol

Pi

mg

-1

pro

tein

h-1

Seedling stage

Influx

H L

Root

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

μm

ol

NO

3- μ

mol

p-n

itro

phen

ol

Seedling stage

NO

3- f

lux

(pm

ol

cm-2

s-1

)

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
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ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

Page 33: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

NO

3

- concentr

ati

on

(μg g

-1

FW

)

Seedling stage

[NO

3

- ] S

R r

atio

s

Seedling stage

C

mm

ol1

5

N g

-1

DW

Seedling stage

E

[15

N]

SR

rati

os

Seedling stage

BnN

RT

15

rela

tiv

e

expre

ssio

n

Seedling stage

BnN

RT

18

rela

tiv

e

expre

ssio

n

Seedling stage

A B

D

F

the seedling stage

Plt005

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
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Page 34: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

aa

00

05

10

15

20 A

tNR

T1

5 r

elat

ive

exp

ress

ion

col-0 nrt18-2

Root

b

a

0

04

08

12

16

AtN

RT

15

rel

ativ

e ex

pre

ssio

n

nrt18-2 Root

AtNRT15 AtNRT15 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

0

4

8

12

a a

0

4

8

12AtNRT18 AtNRT18 A

tNR

T1

8 r

elat

ive

exp

ress

ion

b

a

00

05

10

15

20

Bn

NR

T1

5 r

elat

ive

exp

ress

ion

Root nrt15-3 Root

a

b

0

002

004

006

008

Root Root

A B

C D

E F

Bn

NR

T1

8 r

elat

ive

exp

ress

ion

BnNRT15 BnNRT18

gene

Methods

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
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Page 35: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

a

bb

c

0

001

002

003

004

Root

BnNRT18

c

bb

a

0

2

4

6

Root

BnNRT15

a

b

a b

0

5

10

15

20

25

Root

V-ATPase V-PPaseμ

mol

Pi

mg

-1 p

rote

in h

-1

A B

a

b

c

-350

-280

-210

-140

-70

0

C D

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

932

852 821 745

0

2

4

6

8

Root

Vacuole Protoplast

c

b

a

0

04

08

12

Root

P-V

μm

ol

NO

3-

μm

ol

p-n

itro

phen

ol

BnN

RT

15

rel

ativ

e ex

pre

ssio

n

BnN

RT

18

rel

ativ

e ex

pre

ssio

n

Influx

[1

5N

] S

R r

atio

c

b b

a

000

025

050

075

100

G

[NO

3- ]

SR

rat

io

c

b b

a

0

2

4

6

8

10

H

Root

E F

NO

3- f

lux

(pm

ol

cm-2

s-1

)

-

(n=6)

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
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FOUR
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Page 36: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

D

b

a

0

2

4

6

8

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

P-V

AtN

RT

15

rela

tive

ex

pres

sion

b

aa a

00

05

10

15

20

25

Root

AtNRT15 E

Root

A

a

bb

a

0

01

02

03

04V-ATPase V-PPase

μm

ol P

i m

g-1 p

rote

in h

-1

Root

B

NO

3- flu

x

(pm

ol c

m-2

s-1

)

a

b

c

-2000

-1500

-1000

-500

0

C

940

826780

791

0

15

30

μm

ol N

O3- μ

mol

p-

nitr

ophe

nol

Root

H

[NO

3- ] S

R r

atio

cb

b

a

00

25

50

G

c

bb

a

0

02

04

06

08

[15

N]

SR

rat

io

a

b b

c

0000

0005

0010

0015

0020

0025

AtN

RT

18

rela

tive

ex

pres

sion

AtNRT18 F

Root

Influx

(n=6)

- in protoplasts

indicate SD (n=3)

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
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SEVEN
T-ONE
T-TWO
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Page 37: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

b

c

aa a a

0

10

20

30

a

b bb

0

1000

2000

3000

4000

b

a

0

10

20

30

NU

E b

ased

on

b

iom

ass

(gg

)

col-

0

nrt

15

-3

nrt

18

-2

Ws

vha

-a2

vha

-a3

avp

1

clca-2 N

UE

bas

ed o

n

bio

mas

s (g

g)

a

b

0

1000

2000

3000

4000

Ws clca-2

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

NO

3

- co

nce

ntr

atio

n

(μg

g-1

FW

)

A B

C D

NUE in A thaliana

a3 and avp1

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
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SEVEN
T-ONE
T-TWO
Parsed Citations

Page 38: 1 Running Title: Nitrate allocation and nitrogen use ... · PDF file1 Running Title: Nitrate allocation and nitrogen use efficiency ... (Smirnoff et al., 1985; Tang et al., ... take

Vascular tissues

Vacuole

Cytoplasm

V-PPase

H+

V-ATPase

1 H+

CLCa

2 NO3- 2 NO3

-

Xy

lem

NO

3-

Ph

loem

NRT18

Unloading

NO3-

NRT15

Loading

NO3-

Shoot

Root

En

erg

y

vascular tissues

(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
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ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
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(814)

bGenotypes

Biomass

(g plant-1)

Grain yield

(g plant-1)

Total N in plant

(g plant-1)

Based on biomass

(gg-1)

Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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Genotypes

Chlorophyll

contenta(SP

AD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Chlorophyll

content (SPAD

readings)

Intercellular

CO2

concentration

(micromol

CO2mol-1)

Photosynthetic

rate (micromol

CO2 m-2 s-1)

Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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Parsed Citations
Reviewer PDF
ONE
TWO
THREE
FOUR
FIVE
SIX
SEVEN
T-ONE
T-TWO
Parsed Citations

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