OsNAR2.1 interaction with OsNIT1 and OsNIT2 functions in root … · 148 WT (cv. Nipponbare) using an OsNAR2.1-GST tag fusion protein (Supplemental 149 Figure S1A). The extracted

1

SHORT TITLE: Protein interaction in rice root nitrogen uptake 1

2

TITLE: 3

OsNAR2.1 interaction with OsNIT1 and OsNIT2 functions in 4

root-growth responses to nitrate and ammonium 5

6

AUTHORS: 7

Miaoquan Song1,2, Xiaorong Fan1,2, Jingguan Chen1,3, Hongye Qu1,2, Le Luo1,2 8

Guohua Xu1,2* 9 1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 10

Agricultural University, Nanjing 210095, China. 2MOA Key Laboratory of Plant 11

Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing 12

Agricultural University, Nanjing 210095, China. 3Agricultural Genomics Institute at 13

Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China. 14

* Corresponding author. Email: [email protected]; Tel: 0086-25-84396246 15

16

One-sentence summary: A nitrate transport accessory protein interacts with two 17

nitrilase proteins to regulate root-growth responses to nitrate and ammonium supply. 18

19

AUTHOR CONTRIBUTIONS 20

G.X. and X.F. conceived the research; M.S. performed the experiments; J.C., H.Q., 21

and L.L. provided technical assistance to the experiments; M.S and G.X. analyzed the 22

data and wrote the article. 23

24

# This study is financially supported by National Key Research and Development 25

Program of China (2016YFD0100700), National Natural Science Foundation of China 26

(31930101) and Fundamental Research Funds for the Central Universities 27

(KYZ201869). 28

Plant Physiology Preview. Published on February 18, 2020, as DOI:10.1104/pp.19.01364

Copyright 2020 by the American Society of Plant Biologists

www.plantphysiol.orgon June 19, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

2

29

ABSTRACT 30

The nitrate transport accessory protein OsNAR2 plays a critical role in root-growth 31

responses to nitrate and nitrate acquisition in rice (Oryza sativa). In this study, a 32

pull-down assay combined with yeast two-hybrid and co-immunoprecipitation 33

analyses revealed that OsNAR2.1 interacts with OsNIT1 and OsNIT2. Moreover, an 34

in vitro nitrilase activity assay indicated that indole-3-acetonitrile (IAN) is hydrolyzed 35

to indole-3-acetic acid (IAA) by OsNIT1, the activity of which was enhanced 3–36

4-fold by OsNIT2 and in excess of 5–8-fold by OsNAR2.1. Knockout (KO) of 37

OsNAR2.1 was accompanied by repressed expression of both OsNIT1 and OsNIT2, 38

whereas KO of OsNIT1 and OsNIT2 in the osnit1 and osnit2 mutant lines did not 39

affect expression of OsNAR2.1 or the root nitrate acquisition rate. osnit1 and osnit2 40

displayed decreased primary root length and lateral root density. Double KO of 41

OsNAR2.1 and OsNIT2 caused further decreases in lateral root density under nitrate 42

supply . Ammonium supply repressed OsNAR2.1 expression whereas it upregulated 43

OsNIT1 and OsNIT2 expression. Both osnit1 and osnit2 showed root growth 44

hypersensitivity to external ammonium; however, less root growth sensitivity to 45

external IAN, higher expression of three IAA-amido synthetase (GH3) genes, and a 46

lower rate of 3H-IAA movement towards the roots were observed. Taken together, we 47

conclude that the interaction of OsNIT1 and OsNIT2 activated by OsNAR2.1 and 48

nitrogen supply is essential for maintaining root growth possibly via altering the IAA 49

ratio of free to conjugate forms and facilitating its transportation. 50

51

Keywords: ammonium; auxin; indole-3-acetonitrile; nitrate; nitrilase; Oryza sativa; 52

roots. 53

54



3

55

INTRODUCTION 56

Plants display high root growth plasticity in primary root (PR) elongation, particularly 57

lateral root (LR) initiation and elongation, to adapt to variation in nutrient and water 58

supply (Drew et al., 1975; Gojon et al., 2011; Gruber et al., 2013). Two major forms 59

of nitrogen (N) available for plant root uptake are nitrate (NO3-) and ammonium 60

(NH4+). Nitrate is the major form of N available for dry land crop species and is also 61

an important source of N for paddy rice (Kirk and Kronzucker, 2005; Li et al., 2008; 62

Xu et al., 2012). Moreover, nitrate serves as a signaling molecule in various plant 63

developmental processes including the determination of root architecture (Perilli et al., 64

2012; Wierzba and Tax, 2013; Vidal et al., 2013; O’Brien et al., 2016). In Arabidopsis, 65

several key molecules have been identified that are involved in the regulation of LR 66

initiation and development by localized nitrate, such as the transcription factor ANR1 67

(Zhang et al., 1998), the nitrate transceptor NRT1.1 (Krouk et al., 2010), miR393, the 68

auxin receptor AFB3, and their downstream target NAC4 (Vidal et al., 2010; 2013). 69

The nac4 mutation results in altered LR growth but not PR growth in response to 70

nitrate (Vidal et al., 2010). Low N in roots represses the accumulation of auxin which 71

controls LR formation (Vidal et al., 2010; Wang et al., 2019). A gradual reduction in 72

auxin levels is closely related to enhanced differentiation of distal stem cells in root 73

tips in response to low nitrate (Wang et al., 2019). Moreover, NRT1.1 protein behaves 74

like a root auxin transporter under low nitrate supply (Krouk et al., 2010). 75

Unlike nitrate, ammonium commonly mediates inhibition of both PR and LR 76

elongation in most plant species (Li et al., 2010; Rogato et al., 2010; Li et al., 2014). 77

The presence of ammonium systematically alters root development, affecting 78

processes including elongation, gravitropism, and LR branching (Lima et al., 2010; Li 79

et al., 2014). The root lengths of the auxin-resistant mutants aux1, axr1, and axr2 are 80

less affected by high levels of ammonium compared with wild type (WT), indicating 81

that auxin may be involved in ammonium-induced root length reduction (Cao et al., 82

1993). However, several lines of evidence have suggested that the root growth 83

reduction induced by ammonium supply is associated with ammonium efflux in the 84



4

root elongation zone, independent of the auxin pathways (Li et al., 2010; Liu et al., 85

2013). The combinatorial effect of nitrate on ammonium-regulated root system 86

architecture suggests that ammonium regulates PR elongation and LR branching by 87

distinct pathways (Liu and von Wiren, 2017). 88

The main form of plant auxin is indole-3-acetic acid (IAA), for which several 89

pathways for tryptophan-dependent IAA biosynthesis have been proposed in plants 90

(Woodward et al., 2005; Sugawara et al., 2009; Korasick et al., 2013; Abu-Zaitoon, 91

2014). Indole-3-acetonitrile (IAN) has been identified as one of two key intermediates 92

in indole-3-acetaldoxime (IAOx)-mediated IAA biosynthesis (Sugawara et al., 2009). 93

IAN conversion during basal IAA production requires nitrilases for the hydrolysis of 94

nitriles (Müller et al., 1998; Park et al., 2003; Lehmann et al., 2017). Nitrilase 95

isogenes have been identified in almost all the major plant families (Piotrowski, 2008). 96

For example, the relatively small nitrilase family in Arabidopsis, comprising four 97

members (NIT1–4), has gained diverse biological functions in nitrile metabolism 98

(Piotrowski, 2008). Two groups of nitrilases that catalyze the conversion of IAN to 99

IAA, namely NIT1 in Arabidopsis and ZmNIT2 in maize (Zea mays) (NIT4 orthologs 100

of Arabidopsis), have been characterized thus far. Mutation of NIT1 was shown to 101

decrease both plant sensitivity to IAN treatment (Normanly et al., 1997) and total IAA 102

concentration without affecting the concentration of free IAA (Lehmann et al., 2017). 103

Over-expression of NIT1 resulted in drastic changes of both free IAA and IAN levels, 104

resulting in a phenotype characterized by shorter PR and increased LR number 105

(Lehmann et al., 2017). Maize kernels contain an endogenous nitrilase with activity 106

towards the substrate IAN (Park et al., 2003). ZmNIT2 can hydrolyze IAN to IAA 107

with 7–20-fold higher activity than AtNIT1/2/3 (Park et al., 2003). 108

As opposed to IAA production, the proposed major role of the nitrilase 4 (NIT4) 109

sub-family of angiosperms is catalyzing the conversion of β-cyanoalanine to aspartic 110

acid and asparagine for detoxification of hydrogen cyanide (Piotrowski et al., 2001; 111

Jenrich et al., 2007; Piotrowski, 2008). Poaceae possesses two different NIT4 112

orthologs (NIT4A and NIT4B). The rice (Oryza sativa) genome contains two NIT4 113

orthologs, namely OsNIT4A and OsNIT4B that were renamed as OsNIT1 and OsNIT2 114



5

by Ding et al (2008), which are investigated in this study. Changes in OsNIT1 and 115

OsNIT2 expression are similar to the changes in total IAA after bacterial infection 116

(Ding et al., 2008), indirectly suggesting that OsNIT1 and OsNIT2 affect IAA 117

accumulation. However, Sugawara et al. (2009) detected the presence of IAN in 118

Arabidopsis, but not in the seedlings of maize, rice, or tobacco (Nicotiana tabacum). 119

These data suggest a species- or organ-specific contribution of nitrilase to IAA 120

production in plants. 121

Nitrate Assimilation Related family (NAR2) proteins have been considered as 122

important components of nitrate transporters (NRT) in plants (Tong et al., 2005). In 123

Arabidopsis, six of the seven AtNRT2 members require AtNAR2.1 as a component of 124

high-affinity nitrate transporters (Kotur et al., 2012). Disruption of AtNAR2.1 causes 125

an almost complete loss of inducible high-affinity nitrate influx (Orsel et al., 2006). In 126

rice, OsNAR2.1 interacts with OsNRT2.1/2.2/2.3a and plays a broad role in root 127

acquisition of nitrate in response to both low- and high-nitrate supply (Feng et al., 128

2011; Yan et al., 2011; Liu et al., 2014). Knockout (KO) of OsNAR2.1 causes reduced 129

LR density and PR growth in nitrate medium (Huang et al., 2015). Notably, osnar2.1 130

mutants display decreased auxin distribution in the roots and the accompanying root 131

phenotype can be largely complemented by external NAA (Huang et al., 2015), 132

suggesting that OsNAR2.1 functions in both nitrate uptake and auxin-mediated nitrate 133

signaling. Therefore, the function of the OsNAR2.1 regulatory pathway in plant auxin 134

signaling is an intriguing question. 135

In this study, through a pull-down assay combined with yeast-two-hybrid and 136

co-immunoprecipitation analyses, we show that OsNAR2.1 directly interacts with the 137

two nitrilase proteins OsNIT1 and OsNIT2 in rice. In addition to demonstrating that 138

OsNIT1 and OsNIT2 potentially encode IAN hydrolysis enzymes, we provide a 139

number of physiological, biochemical, and genetic lines of evidence indicating that 140

OsNAR2.1 contributes to auxin-responsive, nitrate-regulated root growth via 141

activation of OsNIT1 and OsNIT2. 142

143

144



6

RESULTS 145

OsNAR2.1 interacts with OsNIT1 and OsNIT2 in roots under nitrate supply 146

We first conducted a preliminary pull-down assay of nitrate-treated seedlings of rice 147

WT (cv. Nipponbare) using an OsNAR2.1-GST tag fusion protein (Supplemental 148

Figure S1A). The extracted proteins revealed several known functional proteins that 149

potentially interact with OsNAR2.1 (Supplemental Table S1). Considering that 150

OsNAR2.1 is involved in root responses to nitrate and auxin (Huang et al., 2015), we 151

focused on two candidate nitrilases (EC 3.5.5.1), as this is the enzyme involved in 152

IAA synthesis and detoxification in Arabidopsis and maize (Piotrowski et al., 2008). 153

The predicted genes encoding nitrilase in the rice genome are OsNIT1 (OsNIT4A, 154

LOC_Os02g42350) and OsNIT2 (OsNIT4B, LOC_Os02g42330), which are orthologs 155

of Arabidopsis AtNITs and maize ZmNITs (Jenrich et al., 2007; Piotrowski, 2008). 156

We subsequently tested for interaction between OsNAR2.1 and OsNIT1/2 157

through yeast two-hybrid (Y2H) assays (Figure 1A) as well as 158

co-immunoprecipitation (Co-IP) assays (Figure 1B) and mass spectrometry analysis 159

(Supplemental Figure S1B, S1C). The results of these analyses confirmed that 160

OsNAR2.1 interacts with OsNIT1 and OsNIT2 at the protein level. 161

To further confirm the interaction of these proteins in rice, we detected their 162

tissue localization under the same experimental conditions. In-situ hybridization 163

analysis showed that OsNIT1, OsNIT2, and OsNAR2.1 were abundantly expressed in 164

root tips and LRs (Figure 1C). We have previously shown that OsNAR2.1 is expressed 165

mainly in the roots, especially in root tips and LRs under nitrate supply (Feng et al., 166

2011). In this study, we found that OsNIT1, OsNIT2, and OsNAR2.1 were each 167

transcriptionally upregulated in the roots by nitrate supply, and that gene expression 168

induction was further enhanced by increasing nitrate supply (Figure 1D). In addition, 169

sub-cellular localization analysis showed that OsNIT1 and OsNIT2 had a wide 170

cellular distribution (Supplemental Figure S2), which is similar to the localization of 171

OsNAR2.1 (Liu et al., 2014). 172

173

Knockout of OsNIT1 or OsNIT2 results in the same root phenotype as the 174



7

osnar2.1 mutant under nitrate supply 175

To examine the roles of OsNIT1 and OsNIT2 in root-growth responses to nitrate, we 176

examined the root phenotypes of osnit1 and osnit2 mutants generated by 177

CRISPR-Cas9 editing and T-DNA insertional mutagenesis. Three independent mutant 178

lines for each gene were selected for detailed analysis (Supplemental Figure S3). It 179

has been shown that osnar2.1 exhibits a phenotype of short PRs and low root density 180

under low nitrate supply (Huang et al., 2015). In this study, we observed that the root 181



8

phenotypes of osnit1, osnit2, and osnar2.1 mutants in the Nipponbare background 182

were similar under 0.25 mM nitrate supply. Under nitrate supply, inactivation of either 183

OsNAR2.1, OsNIT1, or OsNIT2 decreased PR growth by 15–25% and LR density by 184

16–26% (Figure 2A, 2C, 2D). OsNIT1 KO in the Hwayoung background (osnit1-1) 185

also resulted in significantly reduced PR length and LR density. Moreover, the 186



9

expression levels of OsNIT1 and OsNIT2 in the roots of the osnar2.1 mutant line were 187

largely repressed under nitrate supply (Figure 2B) relative to their expression levels in 188

WT, indicating that OsNAR2.1 is necessary for nitrate-regulated expression of OsNIT1 189

and OsNIT2 in rice roots. 190

To further confirm that OsNIT1 and OsNIT2 share the same function as 191

OsNAR2.1 in root responses to nitrate, we crossed osnit2-2 and osnar2.1-1 and 192

obtained a homozygous hybrid mutant line. Interestingly, under nitrate supply, double 193

KO of OsNIT2 and OsNAR2.1 resulted in similar PR length inhibition as seen in 194

osnar2.1, whereas considerably reduced LR density was observed compared to 195

osnar2.1 or osnit2 (Figure 2E and 2F). Due to unknown reasons, we could not isolate 196

a double mutant line of osnit1 and osnar2.1 in this study. The additive effect of 197

OsNIT2 on OsNAR2.1 function in nitrate regulation of root growth supports an 198

interaction between OsNAR2.1 and OsNIT2, and possibly OsNIT1 (Figure 1). 199

200

Knockout of OsNIT1 and OsNIT2 does not alter nitrate uptake rate per root unit 201

weight 202

We previously reported that inactivation of OsNAR2.1 limits root nitrate acquisition 203

under a broad range of nitrate supply, which is the result of OsNAR2.1 being an 204

essential partner protein for the function of several OsNRT2 nitrate transporters (Yan 205

et al., 2011). Therefore, we also investigated the roles of OsNIT1 and OsNIT2 in 206

OsNAR2.1 expression and root nitrate uptake. In contrast to the inactivation of 207

OsNAR2.1, which led to the repression of OsNIT1 and OsNIT2 expression (Figure 208

2B), no significant changes in expression levels of OsNAR2.1, OsNRT2.1, or 209

OsNRT2.3 were observed in either osnit1 or osnit2 mutants (Figure 2G). Moreover, 210

KO of OsNIT1 did not affect the expression of OsNIT2 and vice versa (Supplemental 211

Figure S4), indicating that both OsNIT1 and OsNIT2 are downstream components of 212

the OsNAR2.1 regulatory pathway. We further determined that the root unit weight 213

15NO3- influx rate of osnit1 and osnit2 lines were comparable with WT in a short-term 214

(5 min) assay (Figure 2H), even though the total amount of 15NO3- per plant in osnit1 215

and osnit2 was decreased by approximately 20% due to the smaller root size of the 216



10

mutants (Figure 2H). These results indicate that OsNAR2.1 is upstream of OsNIT1 and 217

OsNIT2 in a nitrate signaling pathway, and that OsNIT1 and OsNIT2 interaction with 218

OsNAR2.1 does not directly contribute to root nitrate uptake. 219

220

Knockout of OsNIT1 or OsNIT2 decreases root growth sensitivity to IAN, but not 221

to NAA 222

Since the presence and contribution of IAN to IAA synthesis in rice is questionable 223

(Sugawara et al., 2009), we analyzed the effect IAN and NAA (naphthylacetic acid, 224

IAA analogue) on rice root growth. In WT plants, PR length was slightly stimulated 225

by low (0.01 μM) IAN treatment but inhibited by high (10 μM) IAN and 0.1 μM NAA 226

treatment, whereas LR density and the expression levels of OsNIT1 and OsNIT2 did 227

not display distinct sensitivity to low external IAN and NAA treatment (Supplemental 228

Figures S5A, S5B, S5C). 229

The KO mutants of OsNIT1 showed less sensitivity to external IAN in both the 230

Nipponbare and Hwayoung backgrounds, and exhibited phenotypes of longer PRs and 231

reduced LR density (Figure 3A, 3B, 3C). The PR length and LR density in osnit1 was 232

approximately 140% longer and 40% less, respectively, than its Nipponbare WT 233

(Figure 3B, 3C). The osnit2 mutants showed a similar phenotype regarding changes in 234

PR length as osnit1, but the effect was less substantial. KO of OsNIT1 or OsNIT2 did 235

not affect root responses to external NAA (Figure 3B, 3C). These data indicate that 236

IAN, which can be hydrolyzed by NIT proteins, may play a regulatory role in rice 237

root growth. 238

239

Knockout of OsNIT1 or OsNIT2 decreases acropetal IAA transport, but does not 240

change root total IAA concentration 241

In view of the substantial responses of rice root growth to external IAN and that both 242

OsNIT1 and OsNIT2 were shown to be involved in these responses, we attempted to 243

analyze the effect of OsNIT1 or OsNIT2 KO on IAN and IAA concentration in rice. 244

We did not detect IAN in WT Nipponbare seedlings (Supplemental Figure S6A), 245

indicating that IAN was either not present, present at levels below our detection limits, 246



11

or that IAN is rapidly hydrolyzed in rice. In addition, the total concentration of IAA in 247

the roots of osnit1 and osnit2 mutants and WT was similar (Figure 4A), indicating that 248

OsNIT1 and OsNIT2 are not major influential factors for total IAA synthesis or 249

accumulation in rice seedlings. 250

We also compared [3H+]-IAA transport in the roots of WT and mutant plants 251

under nitrate supply. Notably, as shown by the amounts of [3H+]-IAA in 0–3 cm 252

root-tip sections, KO of OsNAR2.1 significantly restricted both root-ward and 253

shoot-ward movement of [3H+]-IAA (Figure 4B,C), whereas KO of OsNIT1 or 254

OsNIT2 caused inhibition of [3H+]-IAA root-ward movement alone (Figure 4B,C). 255

Since PR length in each of the three mutant lines was shorter than that in the WT 256

(Figure 2), the [3H+]-IAA transport distance from the site of application at the 257

root-shoot junction to the root tip was in effect shorter in the mutants, and therefore, 258



12

the effect of OsNIT1 or OsNIT2 disruption on acropetal transport of [3H+]-IAA in the 259

roots is theoretically larger than that implied by the data shown in Figure 4. 260

To confirm that the loss of OsNIT1 or OsNIT2 function can alter auxin 261

distribution or auxin forms, we analyzed the expression of five auxin efflux 262

transporter-encoding genes and three IAA-amido synthetase-encoding genes, which 263

may prevent free IAA accumulation in rice (Xu et al., 2005; Ding et al., 2008; Wang 264

et al., 2009; Zhang et al., 2009; Du et al., 2012; Wang et al., 2017). In comparison to 265

WT, both osnit1 and osnit2 lines exhibited repressed expression of OsPIN1c and 266



13

OsPIN1d an up-regulated expression of OsPIN2, OsGH3-2, OsGH3-8, and 267

OsGH3-13 in their roots (Figure 4D). The expression of OsPIN1a and OsPIN1b was 268

not significantly affected in the mutants (Supplemental Figure S6B). These data 269

support the hypothesis that OsNIT1 or OsNIT2 regulate root growth via alteration of 270

auxin root-ward transport and local auxin distribution. 271

272

OsNIT1 displays IAN hydrolysis activity, which is significantly improved by 273

co-expression of OsNIT2 and OsNAR2.1 274

It has been shown that some NIT enzymes like sorghum SbNIT4A and SbNIT4B2 275

may form heteromeric complexes to achieve high catalytic activity (Jenrich et al., 276

2007). Therefore, we tested whether the interaction between OsNIT1, OsNIT2, and 277

OsNAR2.1 caused enhanced IAN hydrolysis activity in vitro. 278

First, we observed that OsNIT1 and OsNIT2 interacted with each other in a Y2H 279

assay (Figure 5A), similar to the previously reported interaction between SbNIT4A 280

and SbNIT4B2 (Jenrich et al., 2007). A short-term in vitro assay of IAN conversion to 281

IAA quantified by liquid chromatography indicated that OsNIT1, but not OsNIT2 or 282

OsNAR2.1, performed IAN hydrolysis (Figure 5B). Remarkably, co-expression of 283

OsNIT1 and OsNIT2 enhanced OsNIT1 IAN hydrolysis activity by 3.2-fold, which 284

was further increased by 5.3-fold following the co-expression of OsNIT1 and OsNIT2 285

together with OsNAR2.1 (Figure 5B, Supplemental Figure S7). To confirm OsNIT1 286

function in hydrolyzing IAN to IAA and the activation of OsNIT1 enzymatic activity 287

by OsNIT2 and OsNAR2.1, we performed the conversion rate assay using four 288

different concentrations of IAN under the same experiment conditions except for an 289

extension of reaction time from 1 h to 2 h. The results showed that the 290

Michaelis-Menten equation could be used to predict the kinetics of OsNIT1 291

hydrolysis activity in the conversion of IAN to IAA (Supplemental Figure S8). The 292

additional presence of OsNIT2 or both OsNIT2 and OsNAR2.1 increased OsNIT1 293

enzyme affinity for IAN, resulting in a 3.8–4.7-fold or 5.6–8.2-fold increase in IAA 294

production rate, respectively (Supplemental Table S2). These results indicate the 295

biological significance of the interaction between OsNAR2.1 and the two NIT 296



14

proteins in rice nitrilase activity. 297

298

Unlike OsNAR2.1, OsNIT1 and OsNIT2 are upregulated by ammonium and 299

function in ammonium root-growth responses 300

We previously reported that expression of OsNAR2.1 is activated by nitrate whereas it 301

is repressed by ammonium (Feng et al., 2011). Moreover, there are no significant root 302

phenotypic differences between OsNAR2.1-mutant and WT plants under ammonium 303

supply (Yan et al., 2011; Liu et al., 2014; Huang et al., 2015). However, we noticed 304

that the expression levels of both OsNIT1 and OsNIT2 were upregulated in the roots 305

by ammonium (Figure 6A). To further confirm these expression dynamics, we 306

detected OsNIT1 and OsNIT2 tissue localization in rice under the same experimental 307

conditions. In-situ hybridization analyses showed that OsNIT1 and OsNIT2 were 308

abundantly expressed in root tips and LRs under ammonium supply, whereas 309

OsNAR2.1 expression was very faint in comparison (Figure 6B). 310

We subsequently supplied different genotypes with 0.25 mM NH4+ and observed 311

that the osnit1 and osnit2 mutants in the Nipponbare background consistently 312

displayed a phenotype of shorter PRs and lower LR root density, both of which were 313

approximately 35–45% less than in WT (Figure 6C). By contrast, KO of OsNAR2.1 in 314



15

both the Hwayoung and Nipponbare backgrounds did not alter the expression of 315

OsNIT1 and OsNIT2 (Figure 6D) or root growth under ammonium supply (Figure 6E, 316

6F). Moreover, double mutation of OsNAR2.1 and OsNIT2 resulted in a similar root 317

phenotype as osnit2 (Figure 6G, 6H), confirming that OsNAR2.1 is not involved in 318

the role that NIT proteins play in root responses to ammonium supply. 319



16

To further verify that OsNIT1 and OsNIT2 function in regulating root responses 320

to ammonium, we investigated acropetal transport of [3H+]-IAA in the corresponding 321

mutant lines. In agreement with the repression of OsNAR2.1 expression by 322

ammonium supply, there was no significant difference in either acropetal or basipetal 323

transport of IAA in osnar2.1 compared to WT (Figure 6I, 6J). Moreover, inactivation 324

of OsNIT1 or OsNIT2 did not significantly affect IAA basipetal (shoot-ward) transport 325

in roots under ammonium supply (Figure 6J), comparable to that seen in roots under 326

nitrate supply (Figure 4C). Remarkably, in osnit1 and osnit2, the amount of [3H+]-IAA 327

transported from its site of application at the root-shoot junction to the 0–3mm root tip 328

section was less than that in WT (Figure 6I). Since the ammonium-supplied mutant 329

lines showed much shorter PR length than WT (Figure 6C, 6E), these data confirm 330

that OsNIT1 and OsNIT2 play regulatory roles in root responses to both ammonium 331

and nitrate. 332

333

334



17

DISCUSSION 335

For efficient acquisition of N with varied forms and concentrations, plants develop 336

sophisticated regulatory pathways in altering root morphology, architecture, N 337

transport and assimilation. We have previously reported that OsNAR2.1 in rice is 338

activated by nitrate and inhibited by ammonium (Feng et al., 2011). As a 339

component of nitrate transporters, OsNAR2.1 contributes to both root nitrate 340

acquisition and nitrate regulated root growth (Yan et al., 2011; Liu et al., 2014; 341

Huang et al., 2015). In this study, we identified that OsNIT1 and OsNIT2 are two 342

new interacting proteins of OsNAR2.1 and found that they are the down-stream 343

components of OsNAR2.1 regulation pathway in the root response to nitrate 344

supply. Moreover, ammonium supply could activate the interaction of OsNIT1 and 345

OsNIT2 for maintaining root growth in rice. 346

347

The role of OsNIT1 and OsNIT2 as the components of nitrilase enzyme in 348

catalyzing IAN conversion and IAA distribution 349

Previously, two groups of nitrilases that can catalyze the conversion of IAN to IAA, 350

NIT1 orthologs in Brassicaceae and NIT4 orthologs in maize (Zea mays) have been 351

characterized (Osswald et al., 2002; Park et al., 2003; Ishikawa et al., 2007; 352

Kriechbaumer et al., 2007). In this study, we detected that OsNIT1 alone belonging to 353

NIT4 sub-family could also have the activity to hydrolyze IAN (Figure 5B; 354

Supplemental Figure S8; Supplemental Table S2), whereas the velocity was slow and 355

similar to that catalyzed previously by NIT1 and NIT4 in Arabidopsis, maize, 356

Brassica rapa, and Sorghum (Osswald et al., 2002; Park et al., 2003; Ishikawa et al., 357

2007; Jenrich et al., 2007; Kriechbaumer et al., 2007). In addition, the Km of 358

SbNIT4A/B2 with IAN was 0.16 mM (Jenrich et al., 2007), which is comparable to 359

Km (0.65 mM) of OsNIT1/2/OsNAR2.1 (Supplemental Figure S8), indicating the 360

similar NIT enzyme activity in rice and Sorghum bicolor. Moreover, as showed in 361

Arabidopsis, nit1 mutants were resistant to external IAN, indicating that the slow IAN 362

hydrolysis activity of NIT1 was sufficient to produce an auxin-overproduction 363

phenotype (Normanly et al., 1997). Similarly, we observed that osnit2, in particular 364



18

osnit1, displayed a stimulated PR growth and a reduced LR growth as compared to 365

WT in the presence of exogenous IAN (Figure 3A, 3B), indicating that inactivation of 366

OsNIT1 or OsNIT2 could prevent auxin-overproduction in rice (Supplemental Figure 367

S5B). Notably, IAN was not detectable in the seedlings of rice (cv. Nipponbare) in 368

this study (Supplemental Figure S6A), the same as reported by Sugawara et al (2009). 369

In addition, KO of OsNIT1 or OsNIT2 did not significantly affect total IAA content in 370

the roots of seedlings (Figure 4A). These results indicated that nitrilases may not be 371

the key enzymes in IAA biosynthesis as proposed by Piotrowski et al (2008) but 372

affect auxin-related regulatory pathways in plants. 373

Sorghum bicolor contains three NIT4 isoforms SbNIT4A, SbNIT4B1, and 374

SbNIT4B2 (Jenrich et al., 2007). Interestingly, each isoform of SbNIT4 did not 375

possess the enzymatic activity in hydrolyzing β-cyanoalanine whereas the heteromeric 376

complexes of SbNIT4A/B1 and SbNIT4A/B2 showed high activity in catalyzing the 377

hydrolysis of β-cyanoalanine (Jenrich et al., 2007). The SbNIT4A/B2 complex could 378

also catalyze the conversion of IAN to IAA (Jenrich et al., 2007). The assay of 379

site-specific mutagenesis of the active cysteine residue demonstrates that hydrolysis 380

of β-cyanoalanine is catalyzed by the SbNIT4A isoform in both complexes whereas 381

hydrolysis of IAN occurs at the SbNIT4B2 isoform (Jenrich et al., 2007). In maize, 382

ZmNIT2 is expressed in auxin synthesizing tissues and shows efficient activity in 383

hydrolyzing IAN to IAA (Kriechbaumer et al., 2007). Notable, ZmNIT2 could have 384

an additional enzymatic function in turnover of β-cyanoalanine when it forms 385

heteromers with the orthologs ZmNIT1 (Kriechbaumer et al., 2007). Interestingly, the 386

IAN hydrolysis activity of OsNIT1 was also strongly enhanced by presence of 387

OsNIT2 (Figure 5A, 5B). Taken together, we predict that the basic function of 388

Poaceae nitrilase in catalyzing either IAN to IAA or hydrolyzing β-cyanoalanine is 389

determined by one NIT component and enhanced by the other. 390

It has been shown that inactivation of NIT1 resulted in decrease of total IAA, but 391

not free IAA in the seedlings of Arabidopsis (Lehmann et al., 2017). In this study, we 392

found that KO of OsNIT1 or OsNIT2 did not change the concentration of total IAA 393

(Figure 4A), but confined the acropetal transport of 3H-IAA in the roots (Figure 4B). 394



19

In comparison to WT, osnit1 and osnit2 mutants showed upregulated expression of 395

three IAA-amido synthetase (GH3) genes (Figure 4D) and down-regulation of two 396

putative IAA efflux transporter genes OsPIN1c and OsPIN1d expression in the nitrate 397

supplied roots (Figure 4D). Notably, OsPIN1c and 1d are orthologs genes of AtPIN1 398

expressed in Arabidopsis vascular tissue (Gälweiler et al., 1998). Double mutation of 399

OsPIN1c and OsPIN1d resulted in a short PR phenotype (Li et al., 2019). Therefore, 400

we hypothesize that OsPIN1c and OsPIN1d are also involved in rice root IAA 401

distribution. It can be deduced that the activation of OsNIT1 and OsNIT2 mediates 402

root growth partially via altering the IAA ratio of free to conjugate forms and 403

transportation. 404

405

Interaction between OsNIT1 and OsNIT2 and activation by OsNAR2.1 406

contribute to nitrate regulated root growth in rice 407

Root architecture is shaped through N interactions with PR and LRs. It is known that 408

interactions with auxin signaling are important to N regulation of root branching 409

(Lavenus et al., 2013; Forde, 2014). In this study, we found that KO of either OsNIT1 410

or OsNIT2 resulted in shorter length of PR and lower LR density at nitrate supply 411

condition (Figure 2). In comparison to their WT, the mutants showed less sensitivity 412

of the root growth to external IAN (Figure 3A, 3B) and lower rate of root-ward 413

movement of 3H-IAA (Figure 4B). However, the contribution of OsNIT1 and OsNIT2 414

to maintain root growth seems different to their putative orthologs in Arabidopsis. KO 415

of AtNIT1 and AtNIT2 did not affect the length of PR whereas over-expression of 416

AtNIT1 resulted in shorter PR and higher LR density (Lehmann et al., 2017). 417

Notably, the impaired root growth of both osnit1 and osnit2 mutants was similar 418

to that of osnar2.1 mutant (Figure 2A, 2C and 2D). Previously, we have shown that 419

knockdown of OsNAR2.1 inhibited LR formation under nitrate supply (Huang et al., 420

2015). The osnar2.1 mutant showed significant less LRs than WT co-occurring with 421

decreased auxin transport from shoots to roots even at similar N concentrations in 422

their roots (Huang et al., 2015), suggesting that OsNAR2.1 probably functions in 423

nitrate-signaling in addition to nitrate uptake (Yan et al., 2011; Liu et al., 2014). In 424



20

this study, we found that OsNAR2.1, OsNIT1 and OsNIT2 are co-localized in the root 425

tissues (Figure 1C) and all of them showed nitrate-enhanced expression in the roots 426

(Figure 1D). We provide robust evidence that the three proteins can interact with each 427

other. The presence of OsNAR2.1 and OsNIT2 enhanced the enzyme activity of 428

OsNIT1 (Figure 5B). The preliminary pull-down assay (Supplemental Figure S1) 429

together with yeast-two-hybrid assay (Figure 1A), co-immunoprecipitation assay 430

(Figure 1B) and its Mass Spectrometry analysis (Supplemental Figure S1B, S1C) all 431

support this notion. 432

We also found that OsNAR2.1 is at nitrate regulatory upstream of OsNIT1 and 433

OsNIT2. KO of OsNAR2.1 repressed the expression of OsNIT1 and OsNIT2 (Figure 434

2B), whereas inactivation of OsNIT1 or OsNIT2 did not affect the expression of 435

OsNAR2.1 (Figure 2G). OsNAR2.1 enhanced the enzyme activity of OsNIT1 and 436

OsNIT2 (Figure 5B) and functions in root nitrate acquisition at wide range (Yan et al., 437

2011; Liu et al., 2014), whereas OsNIT1 and OsNIT2 mutations did not affect root 438

nitrate uptake rate (Figure 2H). Nevertheless, the double mutation of OsNAR2.1 and 439

OsNIT2 (osnit2-2×osnar2.1-1) showed a smaller LR density than the two parental 440

mutants under nitrate conditions (Figure 2F). Since OsNAR2.1 is essential to activate 441

the nitrate uptake functions of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in rice (Yan et 442

al., 2011; Liu et al., 2014), it is not so surprised that the regulatory role of OsNAR2.1 443

in occurrence of LRs is broader than that of OsNIT2, and probably OsNIT1 as well. 444

Therefore, our results demonstrated that OsNAR2.1 could regulate root formation not 445

only by interacting with OsNIT1 and OsNIT2 but also through the nitrate uptake 446

(Figure 7). 447

OsNIT1 and OsNIT2 also play a regulatory role on root growth that is partially 448

independent from OsNAR2.1. In osnar2.1 mutant, both the activities of NIT1 and the 449

NIT1+NIT2 complex might be impaired but remain significant (Figure 2B). Since 450

OsNIT1 alone showed an IAN to IAA conversion activity and OsNIT2 could enhance 451

the OsNIT1 activity in hydrolyzing IAN (Figure 5A), it is not surprising that osnar2.1 452

or osnit2 single mutant has a weaker phenotype than their double osnit2×osnar2.1 453



21

mutant (Figure 2F). 454

Remarkably, the role of OsNAR2.1 in nitrate uptake was independent from 455

OsNIT1 and OsNIT2 (Figure 2G and 2H), whereas the role of OsNIT1 and OsNIT2 in 456

the regulation of root growth can be enhanced by OsNAR2.1 (Figure 5B, 457

Supplemental Figure S7 and Supplemental Table S2). We found that OsNAR2.1 may 458

have other interacting proteins besides nitrilase and nitrate transporters as shown in 459

pull down and Co-IP assay (Supplemental Figure S1 and Supplemental Table S1). 460

Therefore, the nitrate signaling role of OsNAR2.1 not only relies on its interaction 461

with the OsNITs and nitrate transporters, but also other unknown regulators. 462

463

Independent role of OsNIT1 and OsNIT2 in maintaining ammonium-supplied 464

root growth 465

It has been shown that ammonium trigged LR branching and inhibition of PR and LR 466

elongation (Liu et al., 2013; Zou et al., 2013), which was the same as that we 467

observed in this study (Figure 6). The repression of PIN2 altered auxin distribution in 468

the root apices exposed to ammonium suggested the auxin involvement in the 469

ammonium repression of root growth (Liu et al., 2013; Zou et al., 2013). Since 470

OsNAR2.1 was repressed when ammonium was provided as only N source (Feng et 471



22

al., 2011; Figure 6A), it was not surprising that KO of OsNAR2.1 did not significantly 472

affect the root phenotypes at ammonium supply condition (Figure 6C-H). However, 473

ammonium supply enhanced expression of both OsNIT1 and OsNIT2, and their 474

inactivation repressed acropetal transport of 3H-IAA and limited both PR length and 475

LR densities (Figure 6). The data clearly demonstrated that OsNIT1 and OsNIT2 have 476

independent roles via activation of nitrilase in maintaining root growth which is 477

basically not regulated by OsNAR2.1. There might be other ammonium-induced 478

proteins which activate the enzyme activity of OsNIT1 and/or OsNIT2 for altering the 479

form and transportation of IAA. This speculation is worth to be further characterized 480

in future. 481

482

MATERIALS AND METHODS 483

Plant Materials and Growth Conditions. Rice (Oryza sativa L. ssp. japonica) of the 484

Nipponbare background was used for physiological experiments and rice 485

transformation (mutant constructed by Crispr Cas9 system). The osnit1-1 T-DNA 486

insertion mutant (Line ID PFG_1C-01739.R) with a genetic background of japonica 487

cv. Hwayoung was obtained from PFG. For hydroponic experiments of nearly 488

1-month duration, which formed the basis of most of the experiments in this paper 489

(except for the 10-day hydroponic experiment described below), rice seeds were 490

surface sterilized in a 30% (v/v) NaClO solution for 30 min, washed, and germinated 491

on 1/2 MS medium for 3 days at 25°C in darkness and for another 5 days in 492

growth-chamber conditions as follows: 14-h light/10-h dark photo cycle, day/night 493

temperatures of 30°C/24°C, and relative humidity of approximately 60%. The air in 494

the growth room was refreshed every 6 h. Hydroponic experiments were performed 495

using the rice normal nutrient solution from IRRI. Nitrate treatment used Ca(NO3)2 496

instead of NH4NO3, whereas ammonium treatment used (NH4)2SO4 instead of 497

NH4NO3. Twenty seedlings were grown in each culture vessel containing 7.5 L 498

nutrient solution, and the solution was changed every two days. Following 499

germination for 8 days, rice seedlings were initially treated with 1/2 strength nutrient 500

solution for 6 days, then transferred to full-strength culture solution containing either 501



23

1.25 mM NH4NO3, 1.25 mM Ca(NO3)2, or 1.25 mM (NH4)2SO4 for another 7 days 502

before treatments or sampling. For 10-day hydroponic experiments, rice seeds were 503

surface sterilized in a 30% (v/v) NaClO solution for 30 min, washed, and germinated 504

in deionized water for 3 days at 25°C in darkness. Seeds displaying a comparable 505

extent of germination (just had a white tip) were transferred to hydroponic media 506

containing either 0.125 mM Ca(NO3)2, 0.125 mM (NH4)2SO4, or other experimental 507

treatments as described in the figure legends for another 7 days, and maintained in the 508

growth-chamber conditions described above. Thirty seedlings were grown in each 509

culture vessel containing nearly 3 L solution, and the solution was changed every two 510

days. 511

512

Pull Down assay. Details of OsNAR2.1-GST tag fusion protein generation are 513

provided below in the ‘Preparation of recombinant proteins’ section. Target proteins 514

were purified using ProteinIsoTM GST Resin (TRANSGEN BIOTECH, DP201, 515

Beijing). Total protein of rice (cv. Nipponbare) roots which were treated with nitrate 516

was extracted with Plant Protein Extraction Kit (CWBIO, CW0885S, Beijing). Total 517

protein was incubated with the OsNAR2.1-GST fusion protein for 4 h at 4°C. Bound 518

proteins were collected and subjected to two-dimensional electrophoresis (2-DE). The 519

gel was recovered and analyzed for each gel point protein. 520

521

Yeast two-hybrid assay. The interactions between OsNAR2.1 and either OsNIT1 or 522

OsNIT2 were tested using the DUAL membrane pairwise interaction kit (Yan et al., 523

2011). HIS3 and ADE2 were used as reporter genes in the yeast strain NMY51, with 524

each strain carrying a pair of bait and prey plasmids (pBT3-C and pPR3-N are the 525

control vectors with no cloned cDNA). 526

Full-length cDNA of OsNAR2.1 was cloned into pBT3-C (LEU2, KanR) (Liu et al., 527

2014) and OsNIT1 and OsNIT2 cDNAs were cloned into pPR3-N (TRP1, AmpR) 528

(primers detailed in Supplemental Table S3), and expression vectors were 529

co-transformed into yeast strain NMY51 (MATa his3 trp1 leu2 ade2 LYS2::HIS3 530

ura3::lacZ ade2::ADE2 GAL4) using the DS Yeast transformation kit (Dualsystems 531



24

Biotech, Shanghai). Transformed colonies were selected in SD-LW medium and 532

incubated for growth of positive transformants. For growth assays, several 533

independent positive transformants were selected and grown in SD-LW liquid 534

medium at 30°C overnight. Culture concentrations were adjusted to OD546 = 0.8 and 535

diluted 10, 100, and 1000 times. Five microliters of each dilution was spotted on to 536

SD-LW and SD-AHLW solid media, respectively, and incubated at 30 °C for 2.5 days. 537

538

Co-IP assay. Root protein was extracted from rice (cv. Nipponbare) OsNAR2.1+His 539

and His over-expression lines using a Plant Protein Extraction Kit (CWBIO, 540

CW0885S, Beijing). Total root protein was passed through ProteinIso Ni-NTA Resin 541

(TRANSGEN, DP101, Beijing) to obtain binding protein (Elution Buffer: 300 mM 542

NaCl, 50 mM NaH2PO4, 400 mM imidazole, 10 mM Tris base, pH 8.0). Anti-His 543

analysis for testing OsNAR2.1 interactiion with other proteins after IP) was 544

performed by Native Page and all other analyses were SDS-PAGE. anti-NIT2 was 545

purified from a rabbit injected with a specific peptide chain of OsNIT2 (amino acid 546

sequence EKNSAAKSDGISRT). A portion of the sample was subjected to 547

immunoblot analysis using indicated antibodies, and the remaining sample was 548

incubated with 10 mM DTT, 55 mM ammonium iodoacetate, and 1 µg trypsin for 549

enzymatic hydrolysis overnight. Afterwards, the polypeptide was desalted by a C18 550

column, then drained and dissolved with 15 ul of Loading Buffer (0.1%(v/v) formic 551

acid, 3%(v/v) acetonitrile). The peptide was analyzed by LC-MS/MS (ekspertTM 552

nanoLC; AB Sciex Triple TOF 5600-plus). 553

554

RNA In Situ Hybridization. Longitudinal sections of root tips and mature roots of 555

WT (cv. Nipponbare) seedlings with a length of about 10 mm were fixed in FAA 556

solution (1.85% (v/v) formaldehyde, 5% (v/v) acetic acid, and 63% (v/v) ethanol), 557

dehydrated with a mixture of ethanol and 1-butanol, and then embedded in paraffin. 558

The embedded sections were sliced (10-µm thickness) using a microtome (LEICA 559

RM2235). The full-length cDNA sequences of OsNAR2.1, OsNIT1, and OsNIT2 were 560

cloned into pENTR-D-TOPO (primers detailed in Supplemental Table S3). Digoxin 561



25

(DIG)-labeled RNA probes in antisense orientation were synthesized using T7 RNA 562

polymerase, with each linearized plasmid DNA used as a template, using the DIG 563

RNA labeling kit as described previously (Ishiyama et al., 2004). RNA in situ 564

hybridization with DIG-labeled RNA probes was performed as previously described 565

(Ishiyama et al., 2004). 566

567

RNA extraction, cDNA synthesis, and RT-qPCR. Total RNA was extracted from 568

plant samples using Trizol reagent (Invitrogen, Shanghai) according to the 569

manufacturer’s instructions. First-strand cDNA was synthesized from total RNA using 570

the HIScript II Reverse Transcriptase with gDNA wiper (Vazyme, R223-01, Nanjing). 571

Reverse transcription quantitative PCR (RT-qPCR) was performed using the AceQ 572

qPCR SYBR Green Master Mix (Vazyme, Q111-01, Nanjing) on the QuantStudio 6 573

Flex Real-Time PCR System (Applied Biosystems, Shanghai) according to the 574

manufacturer’s instructions. Relative expression level of each sample was determined 575

by normalizing it to the amount of OsActin1 (LOC_Os03g50885) detected in the same 576

sample and presented as 2-△CT. All primers used for RT-qPCR are detailed in 577

Supplemental Table S3. 578

579

Determination of 15N-NO3- uptake rate. Rice seedlings were first planted in IRRI 580

solution containing 1.25 mM NH4NO3, then they were deprived of N for 3 days. Next, 581

plants were first transferred into 0.1 mM CaSO4 for 1 min, then to a complete nutrient 582

solution containing 0.25 mM 15NO3- (Ca(15NO3

-)2) for 5 min, and finally to 0.1 mM 583

CaSO4 for 1 min. Then, we used paper to blot excess water from the plants. The 584

shoots and roots were separated. Root samples were placed in an oven at 105°C for 30 585

min to inactivate the enzymes, and further dried to a constant weight at 70°C. After 586

recording their dry weights, the samples were ground into powder using a ball mill. 587

The Isotope Ratio Mass Spectrometer system (Flash 2000 HT, Thermo Fisher 588

Scientific, Germany) was used to determine the 15N content of the samples. 589

590

Determination of total IAA and IAN. Samples were ground to a power with liquid 591



26

nitrogen and freeze-dried. IAA measurement was performed as described below. The 592

above dry powder was added with 1 ng of [13C]6-IAA and extracted three times with 593

80% (v/v) methanol. After the extraction was concentrated, ethyl acetate containing 5% 594

(v/v) acetic acid was added, and the extraction was collected again. Extractions were 595

freeze-dried once more, following which 10 μl of pyridine and 40 μl of BSTFA were 596

added and reacted at 80°C for 30 min. Finally, 50 μl of n-hexane was added for mass 597

spectrometry using the method of Hor-A-MRM.M. The instrument used was GC-QqQ 598

MS (7890a-5975b, Agilent) and the column was DB-5ms (30 m × 0.25 mm × 0.10 μm, 599

Agilent) (Novák et al., 2012). IAN measurement: The above dry powder was 600

extracted three times with methanol, and the extraction was concentrated, and then 601

subjected to liquid phase analysis by the method of GB/T 16631-2008 high 602

performance liquid chromatography. The instrument used was the Agilent 1200 high 603

performance liquid chromatograph (Sugawara et al., 2009). 604

605

3H-IAA transport assay. Acropetal and basipetal auxin transport was assayed in 606

excised seminal roots as described by Lewis and Munday (2009) with minor 607

modifications. For acropetal auxin transport, agar mixtures containing 0.7% (w/v) 608

agar, 0.04% (v/v) [3H]-IAA (26.0 Ci/mmol), 10 μM cold IAA, 2% DMSO, and 25 609

mM MES (pH 5.5) were prepared in a scintillation vial. Following shoot excision 1 610

cm above the root-shoot junction, 20-μl agar droplets of the [3H]-IAA solution was 611

applied to the cut surface. After a 6-h incubation in 60–70% humidity at 25°C in 612

darkness, root segments were excised at distances of 0–1, 1–2, and 2–3 cm from the 613

root apex and weighed. Then the root segments were immediately placed in 614

scintillation solution (3 ml) for 12 h. For basipetal auxin transport, 20-μl agar droplets 615

(agar mixtures as described above) of the [3H]-IAA solution was applied to root 616

segments excised at distances of 0–3, 3–6, 6–9, 9–12, and 12–15 mm distance from 617

the root apex. The root segments were digested with perchloric acid and then 618

immediately placed in scintillation solution (3 ml) for 12 h. The amount of 619

radioactivity of [3H] in each sample was determined using a Beckman LS6500. 620

621



27

Preparation of recombinant proteins. To obtain recombinant proteins, the plasmids 622

MBP-OsNIT1, OsNIT2-6×His, and OsNAR2.1-6×His (primers detailed in 623

Supplemental Table S3) were transformed into E. coli strain Transetta (DE3) 624

(TRANSGEN BIOTECH, CD801-01, Beijing). The bacterial cells were cultivated at 625

37°C shaking at 150–200 rpm. At an A600 of 0.6–0.8, the bacterial cells were induced 626

by 0.5 mM IPTG at 16°C for 16 h. Cells were collected and disrupted using ultrasonic 627

cell breakers (Fisher Scientific, Model 120, Shanghai) on ice. The recombinant 628

proteins were affinity-purified with amylose resin high flow (NEB, E8022, Beijing) or 629

ProteinIsoTM Ni-NTA Resin (TRANSGEN, DP101, Beijing) according to 630

manufacturer’s instructions. 631

632

Nitrilase activity assays. Assays were carried out in a volume of 50 μl solution 633

containing 50 mM KPi (pH 8.0), 1 mM DTT, 3 mM ATP (important), 3 mM substrate 634

IAN, and 2 μg for each purified enzyme except for OsNIT1, for which 6 μg was used 635

since the western blot result showed many bands. IAN conversion rate (consumed 636

IAN content / total IAN content × 100%) was used as a measure of enzymatic activity 637

as analyzed by HPLC. Reaction time was 1–2 h at 28°C, with 200 μl methanol added 638

to stop reactions. An aliquot (15 μl) of the diluted sample was injected into the HPLC 639

system (Agilent 1200LC) equipped with a ZORBAX C18 SB-Aq column. The flow 640

rate was 0.8 ml/min, and the sample was eluted with 0.1% (v/v) H3PO4 (5 min), 641

followed by a linear methanol gradient to 40% (v/v) methanol in 7 min and holding at 642

this composition for an additional 18 min. The column effluent was monitored at 280 643

nm. Under these conditions, the retention times of IAA and IAN were 23.161 and 644

28.684 min, respectively. 645

646

Subcellular Localization. For subcellular localization constructs, the full-length open 647

reading frames (ORFs) of OsNIT1 and OsNIT2 were amplified and subcloned into the 648

intermediate vectors pSAT6A-EGFP-N1 and pSAT6-EGFP-C1 to generate 649

OsNIT1-GFP, GFP-OsNIT1, OsNIT2-GFP, and GFP-OsNIT2 vectors. All vectors 650

were introduced into the final expression vector pRCS2-ocs-nptII with the rare cutter 651



28

PI-PspI. The constructs were transformed into rice protoplasts by the polyethylene 652

glycol-mediated method. The isolation and transformation of rice protoplast was 653

performed as described previously (Jia et al., 2011). In brief, 10 μg plasmid DNA of 654

each construct was transformed into 0.2-ml protoplast suspension. HDEL fusion 655

mCherry served as an endoplasmic reticulum marker. After incubation at 28°C in 656

darkness for 12–15 h, fluorescence signals in rice protoplasts were detected. Confocal 657

microscopy images were taken using a TCS SP8 X confocal laser scanning 658

microscope (Leica). Excitation/emission wavelengths were 488 nm/495–556 nm for 659

GFP and 587 nm/600–650 nm for mCherry. 660

661

Accession Numbers 662

Sequence data from this article can be found in the GenBank/EMBL data libraries 663

under accession numbers: LOC_Os02g42350 (OsNIT1), LOC_Os02g42330 (OsNIT2), 664

LOC_Os02g38230 (OsNAR2.1), LOC_Os02g02170 (OsNRT2.1), LOC_Os01g50820 665

(OsNRT2.3), LOC_Os06g12610 (OsPIN1a), LOC_Os02g50960 (OsPIN1b), 666

LOC_Os11g04190 (OsPIN1c), LOC_Os12g04000 (OsPIN1d), LOC_Os06g44970 667

(OsPIN2), LOC_Os01g55940 (OsGH3-2), LOC_Os07g40290 (OsGH3-8), 668

LOC_Os11g32520 (OsGH3-13). 669

670

SUPPLEMENTAL DATA 671

Supplemental Figure S1. OsNAR2.1 interacts with OsNIT1 and OsNIT2. 672

Supplemental Figure S2. Subcellular Localization Analysis of OsNIT1 and OsNIT2. 673

Supplemental Figure S3. Characterization of different genotypes used in this paper. 674

675

Supplemental Figure S4. Gene expression pattern of OsNIT1 in osnit2 and OsNIT2 676

in osnit1. 677

Supplemental Figure S5. Selection of appropriate IAN treatment concentration. 678

Supplemental Figure S6. Extracted IAN was not detectable in the seedlings of 679

Nipponbare wild-type and expression levels of OsPIN1a and OsPIN1b were 680

unchanged in osnit1 or osnit2. 681



29

Supplemental Figure S7. The enzyme activity of OsNIT1 and OsNIT2 for IAN 682

hydrolysis. 683

Supplemental Figure S8. The kinetics of IAN conversion rate to IAA by NIT 684

enzyme. 685

Supplemental Table S1. List of proteins identified by pull-down assay. 686

Supplemental Table S2. The enzyme activity for conversion of IAN to IAA at four 687

substrate concentration ranges after a 2-h reaction time. 688

Supplemental Table S3. The primers used in this paper. 689

690

ACKNOWLEDGMENTS 691

This work was supported by the National Key Research and Development Program of 692

China (2016YFD0100700), National Natural Science Foundation of China 693

(31930101), Jiangsu Collaborative Innovation Center for Solid Organic Waste 694

Resource Utilization, the Innovative Research Team Development Plan of the 695

Ministry of Education of China (Grant No. IRT_17R56, KYT201802). 696

697

FIGURE LEGENDS 698

Figure 1. OsNAR2.1 interacts with OsNIT1 and OsNIT2 following nitrate supply 699

to roots. 700

A, Interaction test of OsNAR2.1 with OsNIT1 and OsNIT2 using the DUAL 701

membrane pairwise interaction kit. B, Interaction test of OsNAR2.1 with OsNIT1 and 702

OsNIT2 using Co-IP. OE11 and OE16, two independent lines over-expressing 703

OsNAR2.1 with 6×His tag; Negative, control line segregated from OsNAR2.1 with 704

6×His tag over-expression lines; pUbi::His, control line over-expressing 6×His tag 705

only. Immunoblots were developed with anti-His antibody to detect OsNAR2.1 706

expression and with anti-NIT2 to detect OsNIT2 expression. Anti-HSP used as 707

positive control. C and D, Analysis of OsNAR2.1, OsNIT1, and OsNIT2 expression 708

patterns in the roots of rice seedings (cv. Nipponbare) supplied with different 709

concentration of Ca(NO3)2. C, Expression patterns of OsNAR2.1, OsNIT1, and 710



30

OsNIT2 revealed by RNA in situ hybridization in root tip and mature root area 711

supplied with 2.5 mM nitrate. All detection probes were based on the whole anti-sense 712

strand. Negative control materials were incubated with water. Scale bar in mature is 713

500 μm; scale bar in root tip is 200 μm. D, Relative expression levels of OsNAR2.1, 714

OsNIT1, and OsNIT2 in the roots treated with different nitrate concentrations as 715

quantified by RT-qPCR. OsActin (LOC_Os03g50885) was used as an internal control. 716

The values in D represent means ± SE of three biological replicates. 717

718

Figure 2. Knockout of OsNIT1 or OsNIT2 results in the same root phenotype as 719

the osnar2.1 mutant following nitrate supply but does not alter root nitrate 720

uptake rate. 721

A, B, C, and D, Seeds were first germinated in deionized water for 3 days, then were 722

transferred to hydroponic media containing 0.125 mM Ca(NO3)2 for a further 7 days. 723

HY, Hwayoung; Nip, Nipponbare; WT, wild type; nit1-1, the knockout (KO) mutant 724

of OsNIT1 in the Hwayoung genetic background; nit1-2, nit1-3, nit2-1, nit2-2, nit2-3, 725

nar2.1-1, nar2.1-2, and nar2.1-3, individual KO mutant lines of OsNIT1, OsNIT2, 726

and OsNAR2.1 genes in the Nipponbare genetic background. A, Root phenotype of 727

different lines. Scale bar, 1 cm. B, Effect of OsNAR2.1 KO on expression levels of 728

OsNIT1 and OsNIT2 in roots analyzed by RT-qPCR. OsActin was used as an internal 729

control. C and D, PR, primary root; LR, lateral root. Others are same to A. The values 730

in B, C, and D represent means ± SE of three (B) and six (C and D) biological 731

replicates, respectively (**P≤0.01). E and F, Seeds were germinated and grown under 732

the same conditions as in A. nit2×nar2.1, homozygous hybrid of nit2-2 (abbreviated 733

as nit2 in E and F) and nar2.1-1 (abbreviated as nar2.1 in E and F). Others are the 734

same to C and D. Scale bar in E is 1 cm. The values in F represent means ± SE of 735

seven biological replicates (*P≤0.05, ** P≤0.01). G, Relative expression levels of 736

OsNAR2.1, OsNRT2.1, and OsNRT2.3 in the roots of Nipponbare wild-type (WT) and 737

the OsNIT1 and OsNIT2 KO mutants supplied with 1.25 mM Ca(NO3)2 for one week. 738

H, The 15N absorption rate per root unit weight and total 15N content of WT, osnit1, 739

and osnit2 mutants supplied with 0.125 mM Ca(15NO3)2 for 5 minutes. The values 740



31

represent means ± SE of three biological replicates (*P≤0.05); ns: not significant. 741

742

Figure 3. Knockout of OsNIT1 or OsNIT2 decreases root growth sensitivity to 743

indole-3-acetonitrile, but not to naphthylacetic acid. 744

Seeds were first germinated in deionized water for three days, then were transferred to 745

the hydroponic media containing 0.125 mM Ca(NO3)2 with 10 μM IAN or 0.1 μM 746

NAA for another 7 days before being photographed and sampled for the measurement 747

of root growth and LR density. The genotypes are the same as those used and 748

described in Figure 2A. IAN: indole-3-acetonitrile; NAA: naphthylacetic acid. Scale 749

bar, 1 cm. Others are same to Figure 2. The data in B and C represent means ± SE of 750

six biological replicates (*P≤0.05, ** P≤0.01); ns: not significant. 751

752

Figure 4. Knockout of OsNIT1 or OsNIT2 alters expression of auxin efflux 753

transporter OsPIN genes and IAA amido synthetase OsGH3 genes, and decreases 754

acropetal IAA transport, but does not affect root total IAA concentration. 755

A and D, Wild type (WT; cv. Nipponbare), osnit1, and osnit2 plants were grown in 756

IRRI solution containing 1.25 mM Ca(NO3)2 for one week before being sampled for 757

gene expression and IAA concentration analyses. A, Total IAA concentration in the 758

roots. The roots of five seedlings were taken and mixed as one sample. B and C, WT, 759

osnit1, osnit2, and osnar2.1 plants were grown in IRRI solution containing 1.25 mM 760

Ca(NO3)2 for 10 days before being sampled for [3H]IAA transport analysis. B, 761

Acropetal transport; C, Basipetal transport. D, Relative expression levels of auxin 762

efflux transporter OsPIN genes and IAA amido synthetase OsGH3 genes quantified 763

by RT-qPCR. OsActin was used as an internal control. Data represent means ± SE of 764

three biological replicates (* and lowercase letters represent statistically significant 765

difference at P≤0.05 level); ns, not significant. 766

767

Figure 5. Co-presence of OsNAR2.1, OsNIT1m, and OsNIT2 improves 768

OsNIT1-mediated IAN hydrolysis. 769

A, Interaction test between OsNIT1 and OsNIT2 using the DUAL membrane pairwise 770



32

interaction kit. B, OsNIT1 and OsNIT2 enzyme activity. IAN conversion rate 771

(consumed IAN content / total IAN content × 100%) was used as a measure of 772

enzymatic activity by HPLC. Reaction time was 1 h at 28°C. Both the 6×His and 773

MBP tag proteins were used as negative controls. The data represent means ± SE of 774

four biological replicates (different lowercase letters represents statistically significant 775

difference at P≤0.05 level). 776

777

Figure 6. Knockout of OsNIT1 and OsNIT2 decreases both primary root and 778

lateral root growth following supply of ammonium. 779

A and B, Expression levels and patterns of OsNAR2.1, OsNIT1, and OsNIT2 in the 780

roots of rice seedings (cv. Nipponbare) in response to growth in IRRI solution 781

containing 1.25 mM NH4NO3 (AN) or 1.25 mM (NH4)2SO4 (A). A, Relative gene 782

expression levels under the above experimental conditions quantified by RT-qPCR. 783

OsActin was used as an internal control. The values in A represent means ± SE of 784

three biological replicates. B, Expression patterns of OsNAR2.1, OsNIT1, and OsNIT2 785

determined by RNA in situ hybridization in root tips and mature root area supplied 786

with 2.5 mM ammonium. All detection probes were based on the whole anti-sense 787

strand. Scale bar in mature is 500 μm; scale bar in root tip is 200 μm. C, D, E, and F, 788

Seeds were first germinated in deionized water for three days, then transferred to 789

hydroponic media containing 0.125 mM (NH4)2SO4 for another 7 days. Genotypes are 790

the same as those used and described in Figure 2A. C, Scale bar, 1 cm. D, Effect of 791

OsNAR2.1 knockout (KO) on expression levels of OsNIT1 and OsNIT2 in roots 792

determined by RT-qPCR. OsActin was used as an internal control. C and D, PR, 793

primary root; LR, lateral root. Others are the same to Figure 2A. The values in D, E, 794

and F represent means ± SE of three (D) and six (E and F) biological replicates 795

(**P≤0.01). G and H, Seeds were germinated and grown under the same conditions as 796

described for C. Genotypes are the same as those used and described in Figure 2E. 797

Others are same to Figure 2C and 2D. Scale bar in G is 1 cm. The values in H 798

represent means ± SE of seven biological replicates (**P≤0.01); ns: not significant. I 799

and J, Wild type (WT; cv. Nipponbare) and KO mutant plants were grown in IRRI 800



33

solution containing 1.25 mM (NH4)2SO4 for 10 days before being sampled for 801

[3H]IAA transport analysis. I, Acropetal transport; J, Basipetal transport. Data 802

represent means ± SE of three biological replicates (P≤0.05); ns: not significant. 803

804

Figure 7. Model for the role of OsNAR2.1, OsNIT1, and OsNIT2 in mediating 805

root growth in rice. 806

Interaction of OsNAR2.1 with OsNRT2.1, OsNRT2.2, and OsNRT2.3a and their 807

effect on root nitrate uptake were previously reported (Feng et al., 2011; Yan et al., 808

2011; Liu et al., 2014). All other noted functions are demonstrated in this study. 809

810

811



Parsed CitationsAbu-Zaitoon, Y. M. (2014). Phylogenetic analysis of putative genes involved in the tryptophan-dependent pathway of auxin biosynthesisin rice. Applied Biochemistry and Biotechnology, 172(5): 2480-2495.

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

Cao, Y., Glass, A. D., & Crawford, N. M. (1993). Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and byauxin resistance mutations aux1, axr1, and axr2. Plant Physiology, 102(3): 983-989.


Ding, X., Cao, Y., Huang, L., Zhao, J., Xu, C., Li, X., & Wang, S. (2008). Activation of the indole-3-acetic acid–amido synthetase GH3-8suppresses expansin expression and promotes salicylate-and jasmonate-independent basal immunity in rice. The Plant Cell, 20(1): 228-240.


Drew, M. C., & Saker, L. R. (1975). Nutrient supply and the growth of the seminal root system in barley: II. Localized, compensatoryincreases in lateral root growth and rates op nitrate uptake when nitrate supply is restricted to only part of the root system. Journal ofExperimental Botany, 26(1): 79-90.


Du, H., Wu, N., Fu, J., Wang, S., Li, X., Xiao, J., & Xiong, L. (2012). A GH3 family member, OsGH3-2, modulates auxin and abscisic acidlevels and differentially affects drought and cold tolerance in rice. Journal of Experimental Botany, 63(18): 6467-6480.


Feng, H., Yan, M., Fan, X., Li, B., Shen, Q., Miller, A. J., & Xu, G. (2011). Spatial expression and regulation of rice high-affinity nitratetransporters by nitrogen and carbon status. Journal of Experimental Botany, 62(7): 2319-2332.


Forde, B. G. (2014). Nitrogen signalling pathways shaping root system architecture: an update. Current Opinion in Plant Biology, 21: 30-36.


Gälweiler, L., Guan, C., Müller, A., Wisman, E., Mendgen, K., Yephremov, A., & Palme, K. (1998). Regulation of polar auxin transport byAtPIN1 in Arabidopsis vascular tissue. Science, 282(5397): 2226-2230.


Gojon, A., Krouk, G., Perrine-Walker, F., & Laugier, E. (2011). Nitrate transceptor (s) in plants. Journal of Experimental Botany, 62(7):2299-2308.


Gruber, B. D., Giehl, R. F., Friedel, S., & von Wirén, N. (2013). Plasticity of the Arabidopsis root system under nutrient deficiencies.Plant physiology, 163(1): 161-179.


Huang, S., Chen, S., Liang, Z., Zhang, C., Yan, M., Chen, J., Xu, G., Fan, X., & Zhang, Y. (2015). Knockdown of the partner proteinOsNAR2. 1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner. Scientific Reports, 5:18192.


Ishikawa, T., Okazaki, K., Kuroda, H., Itoh, K., Mitsui, T., & Hori, H. (2007). Molecular cloning of Brassica rapa nitrilases and theirexpression during clubroot development. Molecular Plant Pathology, 8(5): 623-637.


Ishiyama, K., Inoue, E., Watanabe-Takahashi, A., Obara, M., Yamaya, T., & Takahashi, H. (2004). Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis. Journal of Biological Chemistry, 279(16): 16598-16605.


Jenrich, R., Trompetter, I., Bak, S., Olsen, C. E., Møller, B. L., & Piotrowski, M. (2007). Evolution of heteromeric nitrilase complexes inPoaceae with new functions in nitrile metabolism. Proceedings of the National Academy of Sciences, 104(47): 18848-18853.


https://www.ncbi.nlm.nih.gov/pubmed?term=Abu%2DZaitoon%2C Y%2E M%2E %282014%29%2E Phylogenetic analysis of putative genes involved in the tryptophan%2Ddependent pathway of auxin biosynthesis in rice%2E Applied Biochemistry and Biotechnology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Abu-Zaitoon,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Phylogenetic analysis of putative genes involved in the tryptophan-dependent pathway of auxin biosynthesis in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Phylogenetic analysis of putative genes involved in the tryptophan-dependent pathway of auxin biosynthesis in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Abu-Zaitoon,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Cao%2C Y%2E%2C Glass%2C A%2E D%2E%2C %26 Crawford%2C N%2E M%2E %281993%29%2E Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1%2C axr1%2C and axr2%2E Plant Physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cao,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cao,&as_ylo=1993&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ding%2C X%2E%2C Cao%2C Y%2E%2C Huang%2C L%2E%2C Zhao%2C J%2E%2C Xu%2C C%2E%2C Li%2C X%2E%2C %26 Wang%2C S%2E %282008%29%2E Activation of the indole%2D3%2Dacetic acid%26%23x2013%3Bamido synthetase GH3%2D8 suppresses expansin expression and promotes salicylate%2Dand jasmonate%2Dindependent basal immunity in rice%2E The Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ding,&hl=en&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Drew%2C M%2E C%2E%2C %26 Saker%2C L%2E R%2E %281975%29%2E Nutrient supply and the growth of the seminal root system in barley%3A II%2E Localized%2C compensatory increases in lateral root growth and rates op nitrate uptake when nitrate supply is restricted to only part of the root system%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Drew,&hl=en&c2coff=1

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https://scholar.google.com/scholar?as_q=Nutrient supply and the growth of the seminal root system in barley: II&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Drew,&as_ylo=1975&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Du%2C H%2E%2C Wu%2C N%2E%2C Fu%2C J%2E%2C Wang%2C S%2E%2C Li%2C X%2E%2C Xiao%2C J%2E%2C %26 Xiong%2C L%2E %282012%29%2E A GH3 family member%2C OsGH3%2D2%2C modulates auxin and abscisic acid levels and differentially affects drought and cold tolerance in rice%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Du,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Du,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Feng%2C H%2E%2C Yan%2C M%2E%2C Fan%2C X%2E%2C Li%2C B%2E%2C Shen%2C Q%2E%2C Miller%2C A%2E J%2E%2C %26 Xu%2C G%2E %282011%29%2E Spatial expression and regulation of rice high%2Daffinity nitrate transporters by nitrogen and carbon status%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Feng,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Feng,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Forde%2C B%2E G%2E %282014%29%2E Nitrogen signalling pathways shaping root system architecture%3A an update%2E Current Opinion in Plant Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Forde,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrogen signalling pathways shaping root system architecture: an update&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=G�lweiler%2C L%2E%2C Guan%2C C%2E%2C M�ller%2C A%2E%2C Wisman%2C E%2E%2C Mendgen%2C K%2E%2C Yephremov%2C A%2E%2C %26 Palme%2C K%2E %281998%29%2E Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue%2E Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gälweiler,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Gojon%2C A%2E%2C Krouk%2C G%2E%2C Perrine%2DWalker%2C F%2E%2C %26 Laugier%2C E%2E %282011%29%2E Nitrate transceptor %28s%29 in plants%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gojon,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate transceptor (s) in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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https://www.ncbi.nlm.nih.gov/pubmed?term=Gruber%2C B%2E D%2E%2C Giehl%2C R%2E F%2E%2C Friedel%2C S%2E%2C %26 von Wir�n%2C N%2E %282013%29%2E Plasticity of the Arabidopsis root system under nutrient deficiencies%2E Plant physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Gruber,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Plasticity of the Arabidopsis root system under nutrient deficiencies&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Plasticity of the Arabidopsis root system under nutrient deficiencies&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Gruber,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Huang%2C S%2E%2C Chen%2C S%2E%2C Liang%2C Z%2E%2C Zhang%2C C%2E%2C Yan%2C M%2E%2C Chen%2C J%2E%2C Xu%2C G%2E%2C Fan%2C X%2E%2C %26 Zhang%2C Y%2E %282015%29%2E Knockdown of the partner protein OsNAR2%2E 1 for high%2Daffinity nitrate transport represses lateral root formation in a nitrate%2Ddependent manner%2E Scientific Reports&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Huang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Knockdown of the partner protein OsNAR2&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Knockdown of the partner protein OsNAR2&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huang,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ishikawa%2C T%2E%2C Okazaki%2C K%2E%2C Kuroda%2C H%2E%2C Itoh%2C K%2E%2C Mitsui%2C T%2E%2C %26 Hori%2C H%2E %282007%29%2E Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development%2E Molecular Plant Pathology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ishikawa,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ishikawa,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ishiyama%2C K%2E%2C Inoue%2C E%2E%2C Watanabe%2DTakahashi%2C A%2E%2C Obara%2C M%2E%2C Yamaya%2C T%2E%2C %26 Takahashi%2C H%2E %282004%29%2E Kinetic properties and ammonium%2Ddependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis%2E Journal of Biological Chemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ishiyama,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ishiyama,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1



Jia HF, Ren HY, Gu M, Zhao JN, Sun SB, Zhang X, Chen JY, Wu P, Xu GH (2011) The phosphate transporter gene OsPht1;8 is involvedin phosphate homeostasis in rice. Plant Physiol. 156: 1164-1175


Kirk GJD, Kronzucker HJ. (2005). The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modellingstudy. Annals of Botany. 96: 639-646.


Korasick, D. A., Enders, T. A., & Strader, L. C. (2013). Auxin biosynthesis and storage forms. Journal of Experimental Botany, 64(9):2541-2555.


Kotur Z, Mackenzie N, Ramesh S, Tyerman SD, Kaiser BN, Glass ADM. (2012). Nitrate transport capacity of the Arabidopsis thalianaNRT2 family members and their interactions with AtNAR2.1. New Phytologist, 194: 724–731.


Kriechbaumer, V., Park, W. J., Piotrowski, M., Meeley, R. B., Gierl, A., & Glawischnig, E. (2007). Maize nitrilases have a dual role in auxinhomeostasis and β-cyanoalanine hydrolysis. Journal of Experimental Botany, 58(15-16): 4225-4233.


Krouk, G., Lacombe, B., Bielach, A., Perrine-Walker, F., Malinska, K., et al. (2010). Nitrate-regulated auxin transport by NRT1.1 defines amechanism for nutrient sensing in plants. Developmental Cell, 18(6): 927-937.


Lavenus, J., Goh, T., Roberts, I., Guyomarc'h, S., Lucas, M., et al. (2013). Lateral root development in Arabidopsis: fifty shades of auxin.Trends in Plant Science, 18(8): 450-458.


Lehmann, T., Janowitz, T., Sánchez-Parra, B., Alonso, M. M. P., Trompetter, I., Piotrowski, M., & Pollmann, S. (2017). ArabidopsisNITRILASE 1 contributes to the regulation of root growth and development through modulation of auxin biosynthesis in seedlings.Frontiers in Plant Science, 8: 36.


Lewis, D. R., & Muday, G. K. (2009). Measurement of auxin transport in Arabidopsis thaliana. Nature Protocols, 4(4): 437.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li, B., Li, G., Kronzucker, H. J., Baluška, F., & Shi, W. (2014). Ammonium stress in Arabidopsis: signaling, genetic loci, and physiologicaltargets. Trends in Plant Science, 19(2): 107-114.


Li, Q., LI, B. H., Kronzucker, H. J., & SHI, W. M. (2010). Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and islinked to NH4+ efflux and GMPase activity. Plant, Cell & Environment, 33(9): 1529-1542.


Li, Y. L., Fan, X. R., & Shen, Q. R. (2008). The relationship between rhizosphere nitrification and nitrogen‐use efficiency in rice plants.Plant, Cell & Environment, 31(1): 73-85.


Li, Y., Zhu, J., Wu, L., Shao, Y., Wu, Y., & Mao, C. (2019). Functional divergence of PIN1 paralogous genes in rice. Plant and CellPhysiology, 60(12): 2720-2732.


Lima, J. E., Kojima, S., Takahashi, H., & von Wirén, N. (2010). Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUMTRANSPORTER1; 3-dependent manner. The Plant Cell, 22(11): 3621-3633.


Liu, X., Huang, D., Tao, J., Miller, A. J., Fan, X., & Xu, G. (2014). Identification and functional assay of the interaction motifs in the partnerprotein OsNAR2.1 of the two‐component system for high‐affinity nitrate transport. New Phytologist, 204(1): 74-80. www.plantphysiol.orgon June 19, 2020 - Published by Downloaded from

Copyright © 2020 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Jenrich%2C R%2E%2C Trompetter%2C I%2E%2C Bak%2C S%2E%2C Olsen%2C C%2E E%2E%2C M�ller%2C B%2E L%2E%2C %26 Piotrowski%2C M%2E %282007%29%2E Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism%2E Proceedings of the National Academy of Sciences&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jenrich,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jenrich,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Jia HF%2C Ren HY%2C Gu M%2C Zhao JN%2C Sun SB%2C Zhang X%2C Chen JY%2C Wu P%2C Xu GH %282011%29 The phosphate transporter gene OsPht1%3B8 is involved in phosphate homeostasis in rice%2E Plant Physiol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jia&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jia&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kirk GJD%2C Kronzucker HJ%2E %282005%29%2E The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants%3A a modelling study%2E Annals of Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kirk&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modelling study&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modelling study&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kirk&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Korasick%2C D%2E A%2E%2C Enders%2C T%2E A%2E%2C %26 Strader%2C L%2E C%2E %282013%29%2E Auxin biosynthesis and storage forms%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Korasick,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin biosynthesis and storage forms&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin biosynthesis and storage forms&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Korasick,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kotur Z%2C Mackenzie N%2C Ramesh S%2C Tyerman SD%2C Kaiser BN%2C Glass ADM%2E %282012%29%2E Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2%2E1%2E New Phytologist%2C 194%3A 724%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kotur&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kotur&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kriechbaumer%2C V%2E%2C Park%2C W%2E J%2E%2C Piotrowski%2C M%2E%2C Meeley%2C R%2E B%2E%2C Gierl%2C A%2E%2C %26 Glawischnig%2C E%2E %282007%29%2E Maize nitrilases have a dual role in auxin homeostasis and β%2Dcyanoalanine hydrolysis%2E Journal of Experimental Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kriechbaumer,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kriechbaumer,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Krouk%2C G%2E%2C Lacombe%2C B%2E%2C Bielach%2C A%2E%2C Perrine%2DWalker%2C F%2E%2C Malinska%2C K%2E%2C et al%2E %282010%29%2E Nitrate%2Dregulated auxin transport by NRT1%2E1 defines a mechanism for nutrient sensing in plants%2E Developmental Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Krouk,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate-regulated auxin transport by NRT1&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate-regulated auxin transport by NRT1&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Krouk,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lavenus%2C J%2E%2C Goh%2C T%2E%2C Roberts%2C I%2E%2C Guyomarc%27h%2C S%2E%2C Lucas%2C M%2E%2C et al%2E %282013%29%2E Lateral root development in Arabidopsis%3A fifty shades of auxin%2E Trends in Plant Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lavenus,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Lateral root development in Arabidopsis: fifty shades of auxin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Lateral root development in Arabidopsis: fifty shades of auxin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lavenus,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lehmann%2C T%2E%2C Janowitz%2C T%2E%2C S�nchez%2DParra%2C B%2E%2C Alonso%2C M%2E M%2E P%2E%2C Trompetter%2C I%2E%2C Piotrowski%2C M%2E%2C %26 Pollmann%2C S%2E %282017%29%2E Arabidopsis NITRILASE 1 contributes to the regulation of root growth and development through modulation of auxin biosynthesis in seedlings%2E Frontiers in Plant Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lehmann,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis NITRILASE 1 contributes to the regulation of root growth and development through modulation of auxin biosynthesis in seedlings&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis NITRILASE 1 contributes to the regulation of root growth and development through modulation of auxin biosynthesis in seedlings&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lehmann,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lewis%2C D%2E R%2E%2C %26 Muday%2C G%2E K%2E %282009%29%2E Measurement of auxin transport in Arabidopsis thaliana%2E Nature Protocols&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lewis,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Measurement of auxin transport in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Measurement of auxin transport in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lewis,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C B%2E%2C Li%2C G%2E%2C Kronzucker%2C H%2E J%2E%2C Baluška%2C F%2E%2C %26 Shi%2C W%2E %282014%29%2E Ammonium stress in Arabidopsis%3A signaling%2C genetic loci%2C and physiological targets%2E Trends in Plant Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium stress in Arabidopsis: signaling, genetic loci, and physiological targets&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium stress in Arabidopsis: signaling, genetic loci, and physiological targets&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C Q%2E%2C LI%2C B%2E H%2E%2C Kronzucker%2C H%2E J%2E%2C %26 SHI%2C W%2E M%2E %282010%29%2E Root growth inhibition by NH4%2B in Arabidopsis is mediated by the root tip and is linked to NH4%2B efflux and GMPase activity%2E Plant%2C Cell %26 Environment&dopt=abstract


https://scholar.google.com/scholar?as_q=Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C Y%2E L%2E%2C Fan%2C X%2E R%2E%2C %26 Shen%2C Q%2E R%2E %282008%29%2E The relationship between rhizosphere nitrification and nitrogen�?�use efficiency in rice plants%2E Plant%2C Cell %26 Environment&dopt=abstract


https://scholar.google.com/scholar?as_q=The relationship between rhizosphere nitrification and nitrogenâ�?�use efficiency in rice plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The relationship between rhizosphere nitrification and nitrogenâ�?�use efficiency in rice plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C Y%2E%2C Zhu%2C J%2E%2C Wu%2C L%2E%2C Shao%2C Y%2E%2C Wu%2C Y%2E%2C %26 Mao%2C C%2E %282019%29%2E Functional divergence of PIN1 paralogous genes in rice%2E Plant and Cell Physiology&dopt=abstract


https://scholar.google.com/scholar?as_q=Functional divergence of PIN1 paralogous genes in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Functional divergence of PIN1 paralogous genes in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lima%2C J%2E E%2E%2C Kojima%2C S%2E%2C Takahashi%2C H%2E%2C %26 von Wir�n%2C N%2E %282010%29%2E Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1%3B 3%2Ddependent manner%2E The Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lima,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1; 3-dependent manner&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1; 3-dependent manner&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lima,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1



Liu, Y., Lai, N., Gao, K., Chen, F., Yuan, L., & Mi, G. (2013). Ammonium inhibits primary root growth by reducing the length of meristemand elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana. PLoS One, 8(4): e61031.


Liu, Y., & von Wirén, N. (2017). Ammonium as a signal for physiological and morphological responses in plants. Journal of ExperimentalBotany, 68(10): 2581-2592.


Müller, A., Hillebrand, H., & Weiler, E. W. (1998). Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana.Planta, 206(3): 362-369.


Normanly, J., Grisafi, P., Fink, G. R., & Bartel, B. (1997). Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile aredefective in the nitrilase encoded by the NIT1 gene. The Plant Cell, 9(10): 1781-1790.


Novák, O., Hényková, E., Sairanen, I., Kowalczyk, M., Pospíšil, T., & Ljung, K. (2012). Tissue‐specific profiling of the Arabidopsisthaliana auxin metabolome. The Plant Journal, 72(3): 523-536.


O'Brien, J. A., Vega, A., Bouguyon, E., Krouk, G., Gojon, A., Coruzzi, G., & Gutiérrez, R. A. (2016). Nitrate transport, sensing, andresponses in plants. Molecular Plant, 9(6): 837-856.


Orsel M, Chopin F, Leleu O, Smith SJ, Krapp A, Daniel-Vedele F, Miller AJ. 2006. Characterization of a two-component high-affinitynitrate uptake system in Arabidopsis physiology and protein–protein interaction. Plant Physiology. 142: 1304–1317.


Osswald, S., Wajant, H., & Effenberger, F. (2002). Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsisthaliana. European Journal of Biochemistry, 269(2): 680-687.


Park, W. J., Kriechbaumer, V., Müller, A., Piotrowski, M., Meeley, R. B., Gierl, A., & Glawischnig, E. (2003). The nitrilase ZmNIT2 convertsindole-3-acetonitrile to indole-3-acetic acid. Plant Physiology, 133(2): 794-802.


Perilli, S., Di Mambro, R., & Sabatini, S. (2012). Growth and development of the root apical meristem. Current Opinion in Plant Biology,15(1): 17-23.


Piotrowski, M., Schönfelder, S., & Weiler, E. W. (2001). The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β-cyano-L-alanine hydratase/nitrilase. Journal of Biological Chemistry, 276(4): 2616-2621.


Piotrowski, M. (2008). Primary or secondary? Versatile nitrilases in plant metabolism. Phytochemistry, 69(15): 2655-2667.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rogato, A., D'Apuzzo, E., Barbulova, A., Omrane, S., Parlati, A., et al. (2010). Characterization of a developmental root response causedby external ammonium supply in Lotus japonicus. Plant Physiology, 154(2): 784-795.


Sugawara, S., Hishiyama, S., Jikumaru, Y., Hanada, A., Nishimura, T., Koshiba, T., Zhao, Y., Kamiya, Y., & Kasahara, H. (2009).Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis. Proceedings of the National Academy ofSciences, 106(13): 5430-5435.


Tong Y P, Zhou JJ，，Li Z S, and Miller A J. (2005). A two-component high-affinity nitrate uptake system in barley. The Plant Journal.10:13651374. www.plantphysiol.orgon June 19, 2020 - Published by Downloaded from

Copyright © 2020 American Society of Plant Biologists. All rights reserved.

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C X%2E%2C Huang%2C D%2E%2C Tao%2C J%2E%2C Miller%2C A%2E J%2E%2C Fan%2C X%2E%2C %26 Xu%2C G%2E %282014%29%2E Identification and functional assay of the interaction motifs in the partner protein OsNAR2%2E1 of the two�?�component system for high�?�affinity nitrate transport%2E New Phytologist&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liu,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Identification and functional assay of the interaction motifs in the partner protein OsNAR2&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Identification and functional assay of the interaction motifs in the partner protein OsNAR2&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C Y%2E%2C Lai%2C N%2E%2C Gao%2C K%2E%2C Chen%2C F%2E%2C Yuan%2C L%2E%2C %26 Mi%2C G%2E %282013%29%2E Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana%2E PLoS One%2C 8%284%29%3A e&dopt=abstract


https://scholar.google.com/scholar?as_q=Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C Y%2E%2C %26 von Wir�n%2C N%2E %282017%29%2E Ammonium as a signal for physiological and morphological responses in plants%2E Journal of Experimental Botany&dopt=abstract


https://scholar.google.com/scholar?as_q=Ammonium as a signal for physiological and morphological responses in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ammonium as a signal for physiological and morphological responses in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=M�ller%2C A%2E%2C Hillebrand%2C H%2E%2C %26 Weiler%2C E%2E W%2E %281998%29%2E Indole%2D3%2Dacetic acid is synthesized from L%2Dtryptophan in roots of Arabidopsis thaliana%2E Planta&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Müller,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Müller,&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Normanly%2C J%2E%2C Grisafi%2C P%2E%2C Fink%2C G%2E R%2E%2C %26 Bartel%2C B%2E %281997%29%2E Arabidopsis mutants resistant to the auxin effects of indole%2D3%2Dacetonitrile are defective in the nitrilase encoded by the NIT1 gene%2E The Plant Cell&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Normanly,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Normanly,&as_ylo=1997&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Novák%2C O%2E%2C Hényková%2C E%2E%2C Sairanen%2C I%2E%2C Kowalczyk%2C M%2E%2C Pospíšil%2C T%2E%2C %26 Ljung%2C K%2E %282012%29%2E Tissue�?�specific profiling of the Arabidopsis thaliana auxin metabolome%2E The Plant Journal&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nov�?¡k,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Tissueâ�?�specific profiling of the Arabidopsis thaliana auxin metabolome&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Tissueâ�?�specific profiling of the Arabidopsis thaliana auxin metabolome&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nov�?¡k,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=O%27Brien%2C J%2E A%2E%2C Vega%2C A%2E%2C Bouguyon%2C E%2E%2C Krouk%2C G%2E%2C Gojon%2C A%2E%2C Coruzzi%2C G%2E%2C %26 Guti�rrez%2C R%2E A%2E %282016%29%2E Nitrate transport%2C sensing%2C and responses in plants%2E Molecular Plant&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=O'Brien,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate transport, sensing, and responses in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate transport, sensing, and responses in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=O'Brien,&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Orsel M%2C Chopin F%2C Leleu O%2C Smith SJ%2C Krapp A%2C Daniel%2DVedele F%2C Miller AJ%2E 2006%2E Characterization of a two%2Dcomponent high%2Daffinity nitrate uptake system in Arabidopsis physiology and protein%26%23x2013%3Bprotein interaction%2E Plant Physiology%2E 142%3A 1304%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Orsel&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis physiology and protein?protein interaction&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis physiology and protein?protein interaction&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Orsel&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Osswald%2C S%2E%2C Wajant%2C H%2E%2C %26 Effenberger%2C F%2E %282002%29%2E Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana%2E European Journal of Biochemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Osswald,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Osswald,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Park%2C W%2E J%2E%2C Kriechbaumer%2C V%2E%2C M�ller%2C A%2E%2C Piotrowski%2C M%2E%2C Meeley%2C R%2E B%2E%2C Gierl%2C A%2E%2C %26 Glawischnig%2C E%2E %282003%29%2E The nitrilase ZmNIT2 converts indole%2D3%2Dacetonitrile to indole%2D3%2Dacetic acid%2E Plant Physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Park,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Park,&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Perilli%2C S%2E%2C Di Mambro%2C R%2E%2C %26 Sabatini%2C S%2E %282012%29%2E Growth and development of the root apical meristem%2E Current Opinion in Plant Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Perilli,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Growth and development of the root apical meristem&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Growth and development of the root apical meristem&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Perilli,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Piotrowski%2C M%2E%2C Schönfelder%2C S%2E%2C %26 Weiler%2C E%2E W%2E %282001%29%2E The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β%2Dcyano%2DL%2Dalanine hydratase%2Fnitrilase%2E Journal of Biological Chemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Piotrowski,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode �?²-cyano-L-alanine hydratase/nitrilase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode �?²-cyano-L-alanine hydratase/nitrilase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Piotrowski,&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Piotrowski%2C M%2E %282008%29%2E Primary or secondary%3F Versatile nitrilases in plant metabolism%2E Phytochemistry&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Piotrowski,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Primary or secondary&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Primary or secondary&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Piotrowski,&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Rogato%2C A%2E%2C D%27Apuzzo%2C E%2E%2C Barbulova%2C A%2E%2C Omrane%2C S%2E%2C Parlati%2C A%2E%2C et al%2E %282010%29%2E Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus%2E Plant Physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rogato,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rogato,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Sugawara%2C S%2E%2C Hishiyama%2C S%2E%2C Jikumaru%2C Y%2E%2C Hanada%2C A%2E%2C Nishimura%2C T%2E%2C Koshiba%2C T%2E%2C Zhao%2C Y%2E%2C Kamiya%2C Y%2E%2C %26 Kasahara%2C H%2E %282009%29%2E Biochemical analyses of indole%2D3%2Dacetaldoxime%2Ddependent auxin biosynthesis in Arabidopsis%2E Proceedings of the National Academy of Sciences&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sugawara,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Biochemical analyses of indole-3-acetaldoxime-dependent auxin biosynthesis in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sugawara,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1



Vidal, E. A., Araus, V., Lu, C., Parry, G., Green, P. J., Coruzzi, G. M., & Gutiérrez, R. A. (2010). Nitrate-responsive miR393/AFB3regulatory module controls root system architecture in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 107(9):4477-4482.


Vidal, E. A., Moyano, T. C., Riveras, E., Contreras-López, O., & Gutiérrez, R. A. (2013). Systems approaches map regulatory networksdownstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots. Proceedings of the National Academy ofSciences, 110(31): 12840-12845.


Wang, J. R., Hu, H., Wang, G. H., Li, J., Chen, J. Y., & Wu, P. (2009). Expression of PIN genes in rice (Oryza sativa L.): tissue specificityand regulation by hormones. Molecular Plant, 2(4): 823-831.


Wang, Y., Gong, Z., Friml, J., & Zhang, J. (2019). Nitrate modulates the differentiation of root distal stem cells. Plant Physiology, 180(1):22.


Wang, L., Guo, M., Li, Y., Ruan, W., Mo, X., et al. (2017). LARGE ROOT ANGLE1, encoding OsPIN2, is involved in root systemarchitecture in rice. Journal of Experimental Botany, 69(3): 385-397.


Wierzba, M. P., & Tax, F. E. (2013). Notes from the underground: Receptor‐like kinases in Arabidopsis root development. Journal ofIntegrative Plant Biology, 55(12): 1224-1237.


Woodward, A. W., & Bartel, B. (2005). Auxin: regulation, action, and interaction. Annals of Botany, 95(5): 707-735.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu G, Fan X, Miller AJ. (2012). Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology. 63: 153-182.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, M., Zhu, L., Shou, H., & Wu, P. (2005). A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence andtillering in rice. Plant and Cell Physiology, 46(10): 1674-1681.


Yan, M., Fan, X., Feng, H., Miller, A. J., Shen, Q., & Xu, G. (2011). Rice OsNAR2. 1 interacts with OsNRT2. 1, OsNRT2. 2 and OsNRT2. 3anitrate transporters to provide uptake over high and low concentration ranges. Plant, Cell & Environment, 34(8): 1360-1372.


Zhang, H., & Forde, B. G. (1998). An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science,279(5349): 407-409.


Zhang, S. W., Li, C. H., Cao, J., Zhang, Y. C., Zhang, S. Q., Xia, Y. F., Sun, D. Y., Sun Y. (2009). Altered architecture and enhanceddrought tolerance in rice via the down-regulation of indole-3-acetic acid by TLD1/OsGH3. 13 activation. Plant Physiology, 151(4): 1889-1901.


Zou, N., Li, B., Chen, H., Su, Y., Kronzucker, H. J., Xiong, L., Baluška, F. & Shi, W. (2013). GSA‐1/ARG 1 protects root gravitropism inArabidopsis under ammonium stress. New Phytologist, 200(1): 97-111.



https://www.ncbi.nlm.nih.gov/pubmed?term=Tong Y P%2C Zhou JJ�?Li Z S%2C and Miller A J%2E %282005%29%2E A two%2Dcomponent high%2Daffinity nitrate uptake system in barley%2E The Plant Journal&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tong&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A two-component high-affinity nitrate uptake system in barley&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A two-component high-affinity nitrate uptake system in barley&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tong&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vidal%2C E%2E A%2E%2C Araus%2C V%2E%2C Lu%2C C%2E%2C Parry%2C G%2E%2C Green%2C P%2E J%2E%2C Coruzzi%2C G%2E M%2E%2C %26 Guti�rrez%2C R%2E A%2E %282010%29%2E Nitrate%2Dresponsive miR393%2FAFB3 regulatory module controls root system architecture in Arabidopsis thaliana%2E Proceedings of the National Academy of Sciences&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vidal,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidal,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vidal%2C E%2E A%2E%2C Moyano%2C T%2E C%2E%2C Riveras%2C E%2E%2C Contreras%2DL�pez%2C O%2E%2C %26 Guti�rrez%2C R%2E A%2E %282013%29%2E Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots%2E Proceedings of the National Academy of Sciences&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vidal,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidal,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang%2C J%2E R%2E%2C Hu%2C H%2E%2C Wang%2C G%2E H%2E%2C Li%2C J%2E%2C Chen%2C J%2E Y%2E%2C %26 Wu%2C P%2E %282009%29%2E Expression of PIN genes in rice %28Oryza sativa L%2E%29%3A tissue specificity and regulation by hormones%2E Molecular Plant&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Expression of PIN genes in rice (Oryza sativa L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Expression of PIN genes in rice (Oryza sativa L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang%2C Y%2E%2C Gong%2C Z%2E%2C Friml%2C J%2E%2C %26 Zhang%2C J%2E %282019%29%2E Nitrate modulates the differentiation of root distal stem cells%2E Plant Physiology&dopt=abstract


https://scholar.google.com/scholar?as_q=Nitrate modulates the differentiation of root distal stem cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Nitrate modulates the differentiation of root distal stem cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang,&as_ylo=2019&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wang%2C L%2E%2C Guo%2C M%2E%2C Li%2C Y%2E%2C Ruan%2C W%2E%2C Mo%2C X%2E%2C et al%2E %282017%29%2E LARGE ROOT ANGLE1%2C encoding OsPIN2%2C is involved in root system architecture in rice%2E Journal of Experimental Botany&dopt=abstract



https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wang,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wierzba%2C M%2E P%2E%2C %26 Tax%2C F%2E E%2E %282013%29%2E Notes from the underground%3A Receptor�?�like kinases in Arabidopsis root development%2E Journal of Integrative Plant Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wierzba,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Notes from the underground: Receptorâ�?�like kinases in Arabidopsis root development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Notes from the underground: Receptorâ�?�like kinases in Arabidopsis root development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wierzba,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Woodward%2C A%2E W%2E%2C %26 Bartel%2C B%2E %282005%29%2E Auxin%3A regulation%2C action%2C and interaction%2E Annals of Botany&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Woodward,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin: regulation, action, and interaction&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Auxin: regulation, action, and interaction&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Woodward,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xu G%2C Fan X%2C Miller AJ%2E %282012%29%2E Plant nitrogen assimilation and use efficiency%2E Annual Review of Plant Biology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xu&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Plant nitrogen assimilation and use efficiency&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Plant nitrogen assimilation and use efficiency&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xu%2C M%2E%2C Zhu%2C L%2E%2C Shou%2C H%2E%2C %26 Wu%2C P%2E %282005%29%2E A PIN1 family gene%2C OsPIN1%2C involved in auxin%2Ddependent adventitious root emergence and tillering in rice%2E Plant and Cell Physiology&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xu,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yan%2C M%2E%2C Fan%2C X%2E%2C Feng%2C H%2E%2C Miller%2C A%2E J%2E%2C Shen%2C Q%2E%2C %26 Xu%2C G%2E %282011%29%2E Rice OsNAR2%2E 1 interacts with OsNRT2%2E 1%2C OsNRT2%2E 2 and OsNRT2%2E 3a nitrate transporters to provide uptake over high and low concentration ranges%2E Plant%2C Cell %26 Environment&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yan,&hl=en&c2coff=1

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OsNAR2.1 interaction with OsNIT1 and OsNIT2 functions in root … · 148 WT (cv. Nipponbare) using an OsNAR2.1-GST tag fusion protein (Supplemental 149 Figure S1A). The extracted

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