Plant Physiology Preview. Published on January 12, 2016, as DOI:10.1104/pp.15.01377 1 Running Title: Nitrate allocation and nitrogen use efficiency 2 3 Corresponding author: Zhen-hua Zhang 4 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, 5 National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources, 6 Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources 7 Use, Hunan Provincial Key Laboratory of Plant Nutrition in Common University, 8 College of Resources and Environmental Sciences, Hunan Agricultural University, PR. China, 9 [email protected]. 10 11 12 Title: Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots 13 of Brassica napus 14 15 Yong-Liang Han1*, Hai-Xing Song1*, Qiong Liao1*, Yin Yu1*, Shao-Fen Jian1*, Joe Eugene Lepo3, Qiang 16 Liu1, Xiang-Min Rong1, Chang Tian1, Jing Zeng1, Chun-Yun Guan1,2, Abdelbagi M. Ismail4, Zhen-Hua 17 Zhang1§ 18 19 1 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Hunan Agricultural 20 University, Changsha, China; 21 2 National Center of Oilseed Crops Improvement, Hunan Branch, Changsha, China; 22 3 Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, 23 Florida, 32514, United States of America; 24 4 Crop and Environment Sciences Division, International Rice Research Institute, DAPO 7777, Metro 25 Manila, Philippines 26 27 28 Decrease in VSC of NO3- in roots will enhance transport to shoot and essentially contribute to 29 higher NUE by promoting NO3- allocation to aerial parts 30 31 32 1 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Copyright 2016 by the American Society of Plant Biologists 33 Footnotes: We thank Dr. Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology, 34 35 Shanghai Institutes for Biological Sciences) for providing nrt1.5-3and nrt1.8-2 seeds. This 36 study was supported by the National Natural Science Foundation of China (Grant 37 No.31101596, 31372130); Hunan Provincial Recruitment Program of Foreign Experts; 38 National Oilseed Rape Production Technology System of China; “2011 Plan” supported by 39 The Ministry of Education in China; Open Novel Science Foundation of Hunan province 40 (13K062). National Key Laboratory of Plant Molecular Genetics and the "Twelfth Five-Year" 41 National Science and technology support program (2012BAD15BO4). 42 43 * These authors have contributed equally 44 § Corresponding author: e-mail: [email protected] 45 46 47 Author contributions: 48 ZHZ conceived the original screening and research plans; ZHZ supervised the experiments; 49 YLH, HXS, QL, YY, SFJ and ZHZ performed most of the experiments; CT and JZ provided 50 technical assistance to YLH, HXS, YY and ZHZ; YLH and ZHZ designed the experiments 51 and analyzed the data; QL, XMR, CYG and ZHZ interpreted the results, and JEL, AI and 52 ZHZ conceived the project and wrote the article with contributions of all the authors; JEL, AI 53 and ZHZ supervised and complemented the writing. All authors read and approved the final 54 manuscript. 55 56 57 58 59 60 61 62 63 64 65 66 2 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 67 68 Abstract Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target 69 70 to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the 71 environment. In Arabidopsis thaliana, allocation of more NO3- to shoots was associated with 72 higher NUE, however, the commonality of this process across plant species have not been 73 sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. 74 We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were 75 significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE 76 genotype (814); and consequently, less vacuolar NO3- was retained in roots of Xiangyou15. 77 Moreover, NO3- concentration in xylem sap, [15N] shoot:root (S:R) and [NO3-] S:R ratios 78 were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of 79 Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus 80 treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, 81 vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased. Expression of 82 NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3- 83 long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton 84 pumps was also significantly higher than in the wild type col-0. Taken together, these data 85 suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially 86 contribute to higher NUE by promoting NO3- allocation to aerial parts, likely through 87 coordinated regulation of NRT1.5 and NRT1.8. 88 89 Key words: Nitrogen use efficiency; Nitrate allocation; Proton-pumps; Vacuoles 90 91 92 93 94 95 96 97 98 3 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 99 Introduction 100 China is the largest consumer of nitrogen (N) fertilizer in the world, however, the 101 average N-fertilizer use efficiency (NUE) is only around 35%, suggesting considerable 102 potential for improvements (Shen et al., 2003; Wang et al., 2014). With the high amounts of 103 N-fertilizer being used, crop yields are declining in some areas, where application is 104 exceeding the optimum required for local field crops (Shen et al. 2003; Miller et al., 2008;Xu 105 et al., 2012). The extremely low NUE results in waste of resources and environmental 106 contamination, and also presents serious hazards for human health (Xu et al., 2012; Chen et 107 al., 2014). Consequently, exploiting the maximum potential for improving NUE in crop plants 108 will have practical significance for agriculture production and the environment (Zhang et al., 109 2010; Schroeder et al., 2013; Wang et al., 2014). Elucidating the genetic and physiological 110 regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with 111 higher NUE. 112 Ammonium (NH4+) and nitrate (NO3-) are the main N species absorbed and utilized by 113 crops, and NO3- accumulation and utilization are of major emphasis for N nutrient studies in 114 dry land crops, such as Brassica napus. Several studies revealed the close relationship 115 between NO3- content and NUE in plant tissues (Shen et al., 2003; Zhang et al., 2012; Tang et 116 al., 2013;Han et al., 2015a).When plants are sufficiently illuminated, NO3- assimilation 117 efficiency significantly increase in shoots compared with roots (Smirnoff et al., 1985; Tang et 118 al., 2013). Consequently, under daytime with optimal illumination, higher proportion of NO3- 119 in plant tissue is transported from root to shoot, as an advantageous physiological adaptation 120 that reduces the cost of energy for metabolism (Tang et al., 2013). NO3- assimilation in plant 121 shoots can therefore, take advantage of solar energy, while improving NUE (Smirnoff et al., 122 1985; Andrews et al., 1986; Tang et al., 2012; Tang et al., 2013). The NO3- long-distance transport and distribution between root and shoot is regulated by 123 124 two genes encoding long transport mechanisms. NRT1.5 is responsible for xylem NO3- 125 loading, while NRT1.8 is responsible for xylem NO3- unloading (Lin et al., 2008; Li et al., 126 2010). Expression of the two genes is influenced by NO3- concentration. NRT1.5 is strongly 127 induced by NO3- (Lin et al., 2008), while NRT1.8 expression is extremely up-regulated in 128 nrt1.5 mutants (Chen et al., 2012). A negative correlation between the extents of expression of 129 the two genes was observed when plants are subjected to abiotic stresses (Chen et al., 2012). 130 Moreover, expression of NRT1.5 is strongly inhibited by 1-aminocyclopropane-1-carboxylic 131 acid (ACC) and methyl jasmonate (MeJA), whereas the expression of NRT1.8 is significantly 4 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 132 up-regulated (Zhang et al., 2014). Based on these studies we argue that, the expression and 133 functioning of NO3- long-distance transport genes NRT1.5 and NRT1.8, are regulated by 134 cytosolic NO3- concentration. In addition, the vacuolar and cytosolic NO3- distribution is 135 likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase) 136 (Granstedt et al., 1982; Glass et al., 2002; Krebs et al., 2010). Therefore, NO3- use efficiency 137 must be affected by NO3- long-distant transport (between shoot and root), and short-distant 138 transport (between vacuole and cytosol). However the physiological mechanisms controlling 139 this regulation are still obscure. Previous studies showed that the chloride channel protein (CLCa) is mainly responsible 140 141 for vacuole NO3- short-distance transport, as it is the main channel for NO3-movement 142 between the vacuoles and cytosol (Angeli et al., 2006; Wege et al., 2014). The vacuole 143 proton-pumps (V-ATPase and V-PPase) located in the tonoplast, supply energy for active 144 transport of NO3- and accumulation within the vacuole (Gaxiola et al., 2001; Brux et al., 2008; 145 Krebs et al., 2010). Despite that about 90% of the volume of mature plant cells is occupied by 146 vacuoles, vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate 147 reductase (NR) is cytosolic (Shen et al., 2003; Han et al., 2015a). However, re-translocation of 148 NO3- from the vacuole to the cytosol will permit its immediate assimilation and utilization. Generally, NO3- concentrations in plant cell vacuoles and the cytoplasm are in the range 149 150 of 30-50 mol m-3 and 3-5 mol m-3; respectively (Martinoia et al., 1981; Martinoia et al., 2000). 151 Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant 152 tissues, their function in NO3- use efficiency cannot be ignored (Martinoia et al., 1981; Zhang 153 et al., 2012; Han et al., 2015b). NO3- assimilatory system in the cytoplasm is sufficient for its 154 assimilation when it is transported out of the vacuoles. Therefore, NO3- use efficiency could in 155 part, be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues 156 (Martinoia et al., 1981; Shen et al., 2003; Zhang et al., 2012; Han et al., 2015a). 157 Evidently, NO3- use efficiency is regulated by both NO3- long-distance transport from 158 root to shoot and short-distance transport and distribution between vacuoles and cytoplasm 159 within cells(Glass et al., 2002; Dechorgnat et al., 2011; Han et al., 2015a). Although vacuoles 160 compartment excess NO3- that accumulates in plant cells (Granstedt et al., 1982; Krebs et al., 161 2010), neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al., 2007; Han et al., 2015a) 162 nor the NO3- long-distance transport gene NRT1.5 (Lin et al., 2008), are regulated by vacuolar 163 NO3-, even though they are essential for NO3- assimilation. Only NO3- transported from the 164 vacuole to the cytosol can play a role in regulating NO3- inducible genes. Consequently we 165 argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are 5 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 166 regulated by cytosolic NO3- concentration. However, this hypothesis needs to be substantiated. 167 The mechanisms underlying both NO3- short-distance (Gaxiola et al., 2001; Angeli et al., 168 2006; Brux et al., 2008; Krebs et al., 2010), and long-distance transport (Lin et al., 2008; Li et 169 al., 2010) have been previously investigated, yet the underlying mechanisms regulating the 170 flux of NO3- and the obvious relationship between the two transport pathways, as well as their 171 relation to NUE are not well understood. The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles 172 173 (Chiu et al., 2004) and seed tissues (Chopin et al., 2007), whereas the proton pumps and 174 CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et 175 al., 2001; Angeli et al., 2006; Brux et al., 2008; Krebs et al., 2010). The vacuolar NO3- 176 short-distance transport system is spread throughout the plant tissues and is the principal 177 means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli 178 et al., 2006;Krebs et al., 2010). The NRT genes seem to work synergistically to control NO3- long-distance transport 179 180 between roots and shoots. NRT1.9 is responsible for NO3- loading into the phloem (Wang et 181 al., 2011), whereas NO3- loading and unloading into xylem are regulated by NRT1.5 and 182 NRT1.8, respectively (Lin et al., 2008; Li et al; 2010). Phloem transport mainly involves 183 organic N; the inorganic-N (NO3-) concentrations in the phloem sap are typically very low, 184 ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al., 185 2008;Fan et al., 2009). Therefore, this study focused on NO3- short-distance transport 186 mediated through the tonoplast proton-pumps and the CLCa system, and the long-distant 187 transport mechanisms responsible for xylem NO3- loading and unloading via NRT1.5 and 188 NRT1.8, respectively. Questions related to how long and short-distance transport of NO3- are coupled in plant 189 190 tissues and their role in determining NUE were addressed using a pair of high- and low-NUE 191 B. napus genotypes and Arabidopsis thaliana. Application of proton pump inhibitors and 192 ACC in the former, and use of mutants with defective proton pumps in the latter, allowed 193 experimental distinction of the physiological mechanisms regulating these processes. Data 194 presented here provide strong evidence from both model plants supporting this linkage and 195 strongly suggest that cytosolic NO3- concentration in roots regulates NO3- long-distance 196 transport from roots to shoots. We also investigated how NO3- concentration in plant tissues 197 would be affected by NO3- long-distance transport, vacuolar NO3- sequestration; and the 198 ensuing relationship with NO3- use efficiency. We also proposed the physiological 6 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 199 mechanisms likely to be important for enhancing NO3- use efficiency in plants. These findings 200 will provide scientific rationales for improving NUE in important industrial and food crops. 201 202 7 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 203 Results 204 B. napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- 205 in root tissues Our previous work identified high and low NUE B. napus genotypes (Han et al., 2015a; 206 207 Han et al., 2015b). NUE of B. napus, whether based on biomass or on grain yield (Table 1), 208 was significantly higher for the high-NUE genotype (H: Xiangyou15) than for the low-NUE 209 genotype (L: 814). That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase) 210 of root tissues in the H genotype were lower than the L genotype at both seedling (Fig. 1A) 211 and flowering stages (Fig. S1A). Given that proton pumps in the tonoplast supply energy for 212 vacuolar NO3- accumulation (Krebs et al., 2010), NO3- influx into the vacuole was 213 consequently much slower in the H genotype compared with the L genotype at both seedling 214 (Fig. 1B) and flowering stages (Fig. S1B). Moreover, the percentage of vacuolar NO3- relative 215 to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L 216 genotype at seedling (Fig. 1C) and flowering stages (Fig. S1C); and NO3- accumulation in the 217 cytosol increased significantly in the H genotype compared with the L genotype at seedling 218 (Fig. 1D) and flowering stages (Fig. S1D). 219 220 B. napus with higher NUE showed enhanced long-distance transport of NO3- from roots 221 to shoots The relative expression of BnNRT1.5 in roots of the H genotype was significantly higher 222 223 than that in the L genotype at both seedling and flowering stages, while the relative expression 224 of BnNRT1.8 in roots of the H genotype was lower than that in the L genotype at both 225 seedling (Fig. 2-A,B) and flowering stages (Fig. S2-A,B). As a consequence, total N 226 concentration (traced by 15N) in roots of the H genotype was significantly lower than that of 227 the L genotype at both seedling (Fig. 2C) and flowering stages (Fig. S2C), while shoot N 228 concentration in the H genotype was significantly higher than the L genotype at seedling stage 229 (Fig. 2C). This resulted in significantly higher [15N] S:R ratio in the H genotype compared 230 with the L genotype (Fig. 2D; Fig. S2D). No significant differences in total N per plant between the H and L genotypes were 231 232 observed at both seedling and flowering stages (Fig. S3). However, NO3- concentration in 233 roots of the H genotype was significantly lower than in the L genotype at both seedling and 234 flowering stages (Fig. 2E; Fig. S2E), while NO3- concentration in shoot tissues of this 8 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 235 genotype was significantly higher at seedling stage (Fig. 2E), resulting in higher [NO3-] S:R 236 ratios at both stages (Fig. 2F; Fig. S2F). 237 NO3- concentration in the xylem sap, xylem sap volume and total NO3- in xylem sap 238 were significantly higher in the H genotype than in the L genotype at seedling stage (Fig. 239 S4A,C,E); the amount of NO3- in xylem sap significantly increased in the H genotype relative 240 to the L genotype at flowering stage (Fig. S4F). Together, these data suggests greater 241 mobilization of NO3- from root to shoot in the H genotype. 242 243 NO3- up-regulates NRT1.5, but down-regulates NRT1.8 244 A previous study showed that increased NRT1.8 expression in nrt1.5 mutants (Chen et al., 245 246 2012) and the expression of NRT1.5 was induced by NO3- (Lin et al., 2008). We tested the 247 relationship between NRT1.5 and NRT1.8 expression in A. thaliana and B. napus. No 248 significant differences were observed in AtNRT1.5 expression in roots between wild type 249 (col-0) and mutant plants (nrt1.8-2) (Fig. 3A). However, AtNRT1.5 expression was 250 significantly up-regulated by NO3- in roots of nrt1.8-2 mutants (Fig. 3B). Expression of 251 AtNRT1.8 significantly increased in roots of nrt1.5-3 mutants, but not in col-0 plants, and 252 there were no significant differences in AtNRT1.8 expression with or without NO3- treatment 253 in roots of nrt1.5-3 mutants (Fig. 3C,D). In contrast, BnNRT1.5 expression in roots of the H genotype increased significantly with 254 255 NO3- treatment (Fig. 3E), while the reverse was observed in expression of BnNRT1.8, which 256 showed lower expression under NO3- treatment than control (Fig. 3F). This suggests that NO3- 257 induces the expression of NRT1.5, but down-regulates NRT1.8 in A. thaliana and B. napus. 258 Further studies are needed to elucidate the mechanisms regulating this reverse regulation. 259 260 Reduced VSC of NO3- in roots drives its long-distance transport from roots to shoots Our previous study showed that Bafi (Bafilomycin A1) inhibits V-ATPase; DCCD 261 262 (DCCD + Na2SO3) inhibits V-PPase; and a 1:1 combination of Bafi and DCCD (B+D) 263 inhibits both V-ATPase and V-PPase (Han et al., 2015a). These inhibitors were used to 264 control activities of the tonoplast proton pumps in the H B. napus plants. V-ATPase activity 265 significantly decreased under Bafil and B+D treatments relative to the control and DCCD 266 treatments, whereas V-PPase activity declined significantly under DCCD and B+D treatments 267 relative to that in the control and Bafil treatments (Fig. 4A). 9 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 268 The V-ATPase activity in roots of A. thaliana was significantly lower in the V-ATPase 269 mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig. 5A). 270 In contrast, the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower 271 than in the wild type (col-0) and V-ATPase mutants (vha-a2 and vha-a3) (Fig. 5A). NO3- influxes into vacuoles of the H B. napus plants significantly decreased when treated 272 273 with inhibitors of proton pumps (Bafil, DCCD, B+D) (Fig. 4B). Similarly, NO3- influxes into 274 the vacuole of the Arabidopsis mutants (vha-a2, vha-a3 and avp1) was significantly lower 275 than those found in the wild type (col-0) (Fig. 5B). Previous studies showed that VSC of NO3- 276 decreased when the activities of the proton pumps decline (Li et al., 2005; Krebs et al., 2010; 277 Han et al., 2015a). We further investigated NO3- distribution between the vacuole and cytosol as affected by 278 279 proton pump inhibition in the H genotype of B. napus and the mutants of A. thaliana 280 defective in vacuolar proton pumps. The percentage of vacuolar NO3- relative to total NO3- in 281 root protoplasts of the H genotype treated with inhibitors (Bafil, DCCD, B+D) was lower than 282 that observed in the control (Fig. 4C). Results were also similar when using A. thaliana 283 mutants, where the percentage of vacuolar NO3- relative to the total NO3- in protoplasts was 284 lower in the mutants (vha-a2, vha-a3 and avp1) than in the wild type (col-0) (Fig. 5C). 285 Consequently, NO3- accumulation in the cytosol showed a significant increase when energy 286 pumps were suppressed in roots of the H genotype (Fig. 4D); and NO3- accumulation in the 287 cytosol of root tissues of A. thaliana mutants (vha-a2, vha-a3 and avp1) similarly increased, 288 compared with col-0 (Fig. 5D). 289 Based on previous observations (Lin et al., 2008), expression of NRT1.5 is strongly 290 induced by NO3-, but expression of NRT1.8 is down-regulated (Fig.3; Chen et al., 2012). We 291 then hypothesized that expression of these two genes is contrastingly regulated by 292 concentration of NO3- in the cytosol. Our results were congruent with this hypothesis, the 293 expression of BnNRT1.5 in root tissues of the H genotype of B. napus was significantly higher 294 in plants treated with Bafil, DCCD or B+D, compared with the control (Fig. 4E), whereas the 295 expression of BnNRT1.8 decreased substantially (Fig. 4F). Similar results were also observed 296 in A. thaliana, where expression of AtNRT1.5 in roots of the mutants (vha-a2, vha-a3 and 297 avp1) were significantly higher than in the wild type (col-0), but expression of AtNRT1.8 in 298 the same mutants were considerably lower (Fig. 5E,F). NO3- concentrations in the xylem sap, the N-distribution between shoot and root (S:R 299 300 ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with 301 energy pumps’ inhibitors, were significantly higher than in the control (Fig. 4G,H; Fig. S5A). 10 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 302 Similar trends were also observed when using mutants of A. thaliana deficient in the energy 303 pumps’ activities. NO3- concentration in the xylem sap, the [15N] S:R ratios and [NO3-] S:R 304 ratios were all significantly higher in the mutants than in the wild type (Fig. 5G,H; Fig. S5B). 305 These data clearly showed that prevention of N sequestration in vacuoles would enhance its 306 translocation to shoot. Additional evidence linking NO3- long-distance transport and NO3- short-distance 307 308 distribution within cells were provided through experiments comparing A. thaliana wild type 309 (Ws) and mutant clca-2 (Fig. S6). The chloride channel (CLCa) is the main channel for 310 vacuolar anion accumulation. Vacuolar sequestration capacity of NO3-significantly declines in 311 clca mutants (Angeli et al., 2006). NO3- influx into the vacuolar space of Ws roots was 312 significantly higher than that observed in the clca-2 mutants (Fig. S6A). This resulted in a 313 smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3- in root tissue 314 protoplasts, as compared with Ws (Fig. S6B). Consequently, the accumulation of NO3- in the 315 cytosol was significantly higher in clca-2 than in Ws plants (Fig. S6C). The higher 316 NO3-concentration in the cytosol together with the higher expression of AtNRT1.5, coupled 317 with lower expression of AtNRT1.8 in clca-2 roots (Fig. S6D,E); resulted in significant 318 increase in xylem sap NO3- concentration, [15N] S:R ratios and [NO3-] S:R ratios compared 319 with Ws plants (Fig. S6F,G,H). 320 321 Increased NO3- translocation to shoots enhanced NUE NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al., 322 323 1985; Andrews et al., 1986; Tang et al., 2012; Tang et al., 2013). We therefore hypothesize 324 that, increased NO3- translocation to shoot and the consequent higher shoot:root ratio will 325 contribute to higher NUE. Our data support this hypothesis. The H B. napus genotype showed 326 enhanced long-distance transport of NO3- from roots to shoots (Fig. 2; Fig S2). This enhanced 327 NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate 328 (Tang et al., 2012; Tang et al., 2013). Our results showed that chlorophyll content, 329 intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than 330 the L genotype at both seedling and flowering stages (Table 2). Moreover, the NO3- 331 assimilating enzymes were strongly induced by NO3-, providing sufficient capacity for N 332 assimilation (Smirnoff et al., 1985; Andrews et al., 1986). Nitrate reductase (NR) and 333 glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than 334 in the L genotype both at seedling and flowering stages (Fig. S7). In contrast, NR activities in 11 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 335 shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig. 336 S7A,B); and GS activity in the H genotype was also higher than those of the L genotype at 337 seedling stage (Fig. S7C). 338 Experiments comparing A. thaliana wild type (col-0, Ws) with mutants defective in 339 vacuolar proton-pumps (vha-a2, vha-a3,avp1), and transport channel (clca-2) yielded similar 340 results, that is, increases in NO3- shoot:root ratio essentially contributed to enhanced NUE 341 (Fig. 6). The NO3- shoot:root ratios in A. thaliana mutants were higher than that in the wild 342 type (Fig. 5G,H). Generally, the efficiency of inorganic N (NO3-) assimilation into organic N 343 is higher in shoots than in roots (Smirnoff et al., 1985; Andrews et al., 1986), therefore the 344 NO3- concentration in A. thaliana mutants decreased (Fig. 6C,D), leading to higher NUE 345 compared with the wild type (Fig. 6A,B). 346 347 BnNRT1.5/BnNRT1.8 in B. napus affects NO3- long-distance transport from roots to 348 shoots Both B. napus and A. thaliana are members of Cruciferae Family. The amino acid 349 350 sequence identity between BnNRT1.5 and AtNRT1.5 was 90% (Fig. S8A), the amino acid 351 sequence similarity between BnNRT1.8 and AtNRT1.8 was 90.8% (Fig. S8B). Comparisons of 352 nucleotide and amino acid sequences (Harper et al.,2012) of these genes 353 (http://brassica.nbi.ac.uk/cgi-bin/microarray_database.cgi) showed that BnNRT1.5 354 (EV220114) and BnNRT1.8 (EV116423) of B. napus are, respectively, highly homologous 355 with AtNRT1.5 and AtNRT1.8 and the two genes are mainly expressed in roots of both species, 356 showing similar organ-specificity (Lin et al., 2008; Li et al., 2010). 357 NO3- long-distance transport from roots to shoots is therefore, regulated by NRT1.5 and 358 NRT1.8, as reported before (Lin et al., 2008; Li et al., 2010). The [15N-traced] S:R ratios and 359 the [NO3-] S:R ratios were significantly lower in nrt1.5-3 mutants relative to the wild type 360 (col-0), while [15N] S:R and [NO3-] S:R ratios showed significant increase in nrt1.8-2mutant 361 relative to col-0 (Fig. S9C,D). A previous study showed that expression of NRT1.5 is 362 down-regulated by ACC and MeJA treatments, while the expression of NRT1.8 is strongly 363 up-regulated in A. thaliana (Zhang et al., 2014). Consequently, NO3- accumulated in plant 364 roots, probably as an adaptive measure for abiotic stresses (Chen et al., 2012). Both the H- 365 and L- B. napus genotypes showed significantly lower expression of BnNRT1.5 in roots when 366 treated with ACC (Fig. S10A), but the expression of BnNRT1.8 remained higher than in the 367 control plants (Fig. S10B). This resulted in significantly lower [15N] S:R ratios following 368 ACC treatment (Fig. S10C,D,E). These data indicate that BnNRT1.5 and BnNRT1.8 play 12 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 369 similar functions for NO3- long-distance transport between root and shoot in both B. napus 370 and A. thaliana (Lin et al., 2008; Li et al., 2010). 371 372 13 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 373 Discussion 374 Nitrate long-distance transport from root to shoot is regulated by cytosolic NO3- in roots 375 NO3- long-distance transport from root to shoot is controlled by NRT gene family. For 376 instance xylem NO3- loading and unloading are mainly controlled by NRT1.5 and NRT1.8, 377 respectively (Lin et al., 2008; Li et al., 2010). Whereas the NO3- short-distance transport 378 between the vacuole and cytosol is controlled by tonoplast proton-pumps (V-ATPase and 379 V-PPase) (Krebs et al., 2010) and by activity of the CLCa channel in the tonoplast membrane 380 (Angeli et al., 2006; Wege et al., 2014), tonoplast proton-pumps provide the energy required 381 for NO3- accumulation into the vacuole through CLCa (Gaxiola et al., 2001; Brux et al., 2008; 382 Krebs et al., 2010). The expression of NRT1.5 is up-regulated by NO3- in plant culture 383 solution (Lin et al., 2008), whereas regulation of NRT1.8 expression is contrary to that of 384 NRT1.5 under both control and abiotic stress conditions (Li et al., 2010; Chen et al., 2012; 385 Zhang et al., 2014). Despite the progress made in characterizing the genes involved in transport and 386 387 metabolism of NO3- in plants, the relationship between long- and short-distance transport and 388 how these two pathways are regulated have not been thoroughly investigated. We hypothesize 389 that, NRT1.5 expression and its role in long-distance transport is regulated by NO3- in plant 390 tissue as was observed in culture solution (Lin et al., 2008), and our data support this 391 hypothesis. Both BnNRT1.5 and AtNRT1.5 expression were up-regulated by increasing 392 cytosolic NO3- in roots (Fig. 4; Fig. 5). The increase in cytosolic NO3- was achieved through (i) 393 use of A. thaliana mutants defective in proton-pumps (V-ATPase and V-PPase) or CLCa 394 activities in the tonoplast, and (ii) inhibition of B. napus tonoplast proton-pumps. In both 395 model plants, the influx of NO3- into vacuoles was substantially reduced, resulting in higher 396 concentrations of NO3- in the cytosol (Fig. 4A-D; Fig. 5A-D; Fig. S6A-C). Under those 397 conditions, expression of BnNRT1.5 and AtNRT1.5 was up-regulated by the enhanced 398 cytosolic NO3-, while expression of BnNRT1.8 and AtNRT1.8 was down-regulated (Fig. 4E,F; 399 Fig. 5E,F; Fig.S6D,E). As a consequence, more NO3- was loaded into the xylem sap and 400 transported from root to shoot (Fig. S5A,B; Fig.S6F). This is also reflected as increased 401 shoot:root ratios of N, traced by [15N] and [NO3-] (Fig. 4G,H; Fig. 5G,H; Fig. S6G,H). 402 Interestingly, the expression of both BnNRT1.5 and AtNRT1.5 is up-regulated by 403 increased cytosolic NO3- concentration in roots, but were not affected by NO3- sequestered 404 into vacuoles (Fig. 4; Fig. 5; Fig. S6). This is because vacuolar NO3- is functionally separated 405 by the tonoplast and cannot be assimilated by enzymes that are localized in the cytosol. 14 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 406 Therefore, NO3--inducible genes, such as NR and NRT1.5, were not influenced by vacuolar 407 NO3- concentration (Martinoia et al., 1981; Martinoia et al., 2000; Zhang et al., 2012). In 408 addition, NO3- concentration in the cytosol is controlled by its short-distance transport through 409 the tonoplast, which is mediated through the tonoplast proton-pumps, providing the required 410 energy for its sequestration into vacuoles against concentration gradient (Gaxiola et al., 2001; 411 Brux et al., 2008; Krebs et al., 2010). The gradient of H+ between the inside of the vacuole 412 and the cytosol is maintained by an 2NO3-/1H+ antiporter mechanism that facilitates,NO3- 413 transport through CLCa (Angeli et al., 2006; Wege et al., 2014). Our data provide clear 414 evidence that NO3- long-distance transport from root to shoot is regulated by cytosolic NO3- 415 concentration in roots. Regulatory mechanisms that control the expression of NRT1.8 and NRT1.5 as mediated 416 417 by NO3- are still unknown, however, previous studies showed that their expression is also 418 affected by abiotic stresses, whereby NRT1.5 is down-regulated and NRT1.8 is up-regulated 419 (Li et al., 2010; Zhang et al., 2014). AtNRT1.8 expression in A. thaliana nrt1.5 mutants is 420 highly up-regulated (Chen et al., 2012), as was also observed here (Fig. 3C). Moreover, 421 AtNRT1.5 expression was enhanced by NO3- in culture solution and was not affected in 422 nrt1.8-2 mutant (Fig. 3A,B). On the other hand, AtNRT1.8 expression was not affected by 423 cytosolic NO3-, but down-regulated when AtNRT1.5 expression was up-regulated by NO3-(Fig. 424 S6D,E). Analogous results were also obtained with B. napus genotypes (Fig. 3E,F). Based on 425 these results we argue that NRT1.8 expression is not directly influenced by cytosolic NO3-, but 426 rather by NRT1.5 expression (Fig. 4, Fig. 5 and Fig. S6), suggesting NRT1.8 is probably 427 acting downstream of NRT1.5. Apparently the regulatory mechanisms of this NRT gene 428 family still awaits further studies to be elucidated. 429 430 Response of NUE to NO3- long-distance transport NO3- assimilation efficiency is higher in shoot than in root tissues, and can be further 431 432 enhanced by carbon assimilation (Smirnoff et al., 1985; Tang et al., 2013). Therefore, 433 translocation of higher proportion of NO3- from roots to shoots likely contributes to better 434 crop growth and higher NUE (Andrews et al., 1986; Tang et al., 2012). The present study 435 provides direct evidence for these results using contrasting B. napus genotype identified 436 before (Zhang et al., 2009; Han et al., 2015a). Our data confirmed the genetic variation in 437 NUE between these two genotypes (Table 1). We measured NO3- distribution in root and shoot of these genotypes to elucidate the 438 439 physiological mechanisms contributing to variation in NUE. The data were consistent with 15 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 440 our hypothesis that concentration of NO3- in the xylem sap and shoot would be significantly 441 higher in the H genotype than in the L genotype (Fig. 2E,F;Fig. S2E,F; Fig. S4). The NO3- 442 long-distance transport from root to shoot was affected by cytosolic NO3- in roots of both A. 443 thaliana and B. napus genotypes (Fig. 4-5; Fig. S6). The activities of the 444 proton-pumps in roots were significantly lower in the H genotype than in the L genotype (Fig. 445 1A,B). Consequently, less NO3- was sequestered into the vacuole and more NO3- was retained 446 in cytosol (Fig. 1C,D). BnNRT1.5 expression was then up-regulated, resulting in greater 447 loading into the xylem, while BnNRT1.8 was down-regulated (Fig. 2A,B), probably to 448 suppress NO3- unloading from xylem sap. Consequently, a higher proportion of NO3- was 449 transported from root to shoots in the H genotype(Fig. 2C-D; Fig. S4). tonoplast Moreover, photosynthetic carbon fixation (Table 2) and activities of both NR and GS 450 451 were higher in shoots of the H genotype (Fig. S7), both of which promote higher N 452 assimilation. These results agreed with previous reports (Smirnoff et al., 1985; Andrews et al., 453 1986; Tang et al., 2012; Tang et al., 2013), where higher NO3- transport from root to shoot, 454 higher photosynthetic rate and N assimilation efficiency in shoots were suggested as essential 455 mechanisms for high-NUE in crop plants. These qualities endowed the B. napus H genotype 456 Xiangyou15 with higher NUE as compared with the L genotype, 814. NO3- long-distance transport in B. napus is not only regulated by BnNRT1.5 and 457 458 BnNRT1.8 in root tissues (Fig. 4; Fig. 5; Fig. S6), but also by stomatal conductance and 459 transpiration, by indirectly controlling xylem sap flow and long-distance transport 460 (Dechorgnat et al., 2011; Krapp et al., 2014). Stomatal conductance and transpiration rates of 461 the H genotype were significantly higher than those of the L genotype (Table S1), a result 462 consistent with other studies describing characteristics of high-NUE plants (Daniel-Vedele et 463 al., 1998; Wilkinson et al., 2007; Dechorgnat et al., 2011; Krapp et al., 2014). 464 465 Response of NUE to NO3- short-distance transport NUE is also strongly controlled by NO3- short-distance distribution between the vacuole 466 467 and cytosol (Han et al., 2015a). A lower proportion of NO3- accumulating in vacuoles and a 468 higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et 469 al., 2015b). The energy required for vacuolar NO3- sequestration is provided by the tonoplast 470 proton-pumps (Gaxiola et al., 2001; Brux et al., 2008; Krebs et al., 2010), while the 471 distribution channel CLCa enables transport of NO3- across the vacuolar membrane (Angeli et 472 al., 2006; Wege et al., 2014). The activities of the tonoplast proton-pumps (V-ATPase and 473 V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at 16 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 474 flowering stages (Han et al., 2015a;Han et al., 2015b); thus we expected similar differences in 475 root tissues. Our results confirmed that the activities of tonoplast proton-pumps in root tissues 476 of B. napus H genotype and A. thaliana mutants were lower than those in the L genotype and 477 A. thaliana wild type, leading to less NO3- influx into vacuoles and more in the cytosol (Fig. 1; 478 Fig. 5A-D). Moreover, the enzymes catalyzing N assimilation are located in the cytoplasm 479 where NO3- could be assimilated (Martinoia et al., 1981; Han et al., 2015a), or immediately 480 loaded into xylem sap for long-distance transport from roots to shoots (Lin et al., 2008). 481 Consequently, the NO3- concentration in root tissues of the H genotype and A. thaliana 482 mutants defective in tonoplast proton-pumps was significantly lower than that in the L 483 genotype (Fig. 2E) and in A. thaliana wild type (Fig. 6C). Therefore, the higher proportion of 484 NO3- transported to shoot and the lower concentrations retained in root vacuoles contributed 485 to the higher NUE in Xiangyou15 and A. thaliana mutants (vha-a2, vha-a3 and avp1) (Table 486 1;Fig. 6A). Further studies are needed to assess the impacts of regulating vacuolar 487 sequestration of NO3- and rates of long-distance transport for enhancing NUE in other crop 488 species and genotypes. These data suggested that NO3- distribution between vacuoles and cytosol within cells 489 490 regulates NO3- long-distance transport from root to shoot, and ultimately affects NUE. NO3- 491 loading into the xylem sap is an active transport process (Lin et al., 2008), therefore, not 492 regulated by absolute concentration of NO3- in the cytosol but rather by genes involved in 493 NO3- long-distance transport (NRT1.5 and NRT1.8). Apparently, the expression of NRT1.5, the 494 gene responsible for xylem loading is regulated by NO3-concentration in the cytosol. 495 Therefore, both NO3- short-distance transport across the tonoplast and its long-distance 496 transport from root to shoot are important determinants of NUE in both B. napus and A. 497 thaliana (Table 1 and Fig. 6). 498 499 Roles of NRT1.5 and NRT1.8 in NO3- long-distance transport in B. napus and A. 500 thaliana. Our results (Figs. S9; S10) showed that the proportion of NO3- distributed from root to 501 502 shoot is strongly controlled by NRT1.5, in accord with previous studies (Lin et al. 2008; Li et 503 al. 2010). The data suggest that the high-NUE genotypes possess lower activities of tonoplast 504 proton-pumps (V-ATPase and V-PPase), resulting in less NO3- accumulation in vacuoles and 505 more NO3- retention in cytosol (Fig. 7). The higher NO3- in the cytosol then up-regulated 506 NRT1.5, which then down-regulates NRT1.8. As a result, more NO3- is loaded into the xylem 507 system by NRT1.5 mechanism, and less NO3- is unloaded via NRT1.8 (Fig. 7). Differential 17 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 508 regulation of these NRT genes in high-NUE plants increased transport of NO3- from root to 509 shoot through the xylem vascular tissues (Fig. 7). This coordinated NO3- long- and 510 short-distance transport likely fine-tunes NO3- allocation and modulates the balance of NO3- 511 distribution between roots and shoots (Fig. 7). Our research also revealed differences between A. thaliana and B. napus in the 512 513 expression of the wild-type NRT genes in response to NO3- in the protoplast. Moreover, using 514 A. thaliananrt1.5-3 mutants, we uncovered possible regulatory interactions between a 515 functioning (wild-type) NRT1.5 gene and NRT1.8 expression in response to NO3- 516 concentration. Because mutants defective in nrt1.5 are not available for B. napus, we could 517 not confirm that the respective NRT1.5 and NRT1.8 systems behave in an analogous way in 518 these plant species. Based on Harper et al. (2012) research database, our homology 519 comparisons and gene function annotations provided strong evidence for the roles of 520 BnNRT1.5 and BnNRT1.8 in B. napus. Finally, our studies were conducted using two model 521 dicotyledonous plant species, for which we had ample data on responses to specific inhibitors 522 (B. napus), or specific mutants were available, with results forming the bases for the model 523 presented in Figure 7. Further research using genetically diverse plant species (e.g., 524 monocotyledonous crops such as rice, wheat and corn), will help validate this model and its 525 broader application across species. The information generated in this study could have future 526 application for improving NUE in commercial crops, with consequent reduction in nitrogen 527 use and benefits to the environment. 528 529 Materials and Methods 530 Plant material 531 The two oilseed rape (B.napus) cultivars used in this study have been characterized 532 before as high- (Xiangyou15, referred to as H genotype hereafter) and low- (814, referred to 533 as L genotype) nitrogen use efficiency (NUE) genotypes (Zhang et al. 2009; Han et al. 2015b). 534 Here, we define NUE as the total biomass per unit of N uptake by the plant, with total 535 biomass includes roots, shoots and grains. The two genotypes were provided by the Hunan 536 Sub-Center of Improvement Center of National Oil Crops, Hunan, China. The Arabidopsis thaliana wild-type Columbia-0 (col-0) was used as control for 537 538 V-ATPase (vha-a2 and vha-a3), V-PPase (avp1), nrt1.5-3 and nrt1.8-2 mutants; whereas the A. 539 thaliana wild-type Wassilewskija (Ws) was used as control for clca-2 mutants. A. thaliana 18 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 540 mutants and transgenic lines (nrt1.5-3 and nrt1.8-2) have been described previously (Chen et 541 al., 2012; Zhang et al., 2014). The mutant lines vha-a2 (Salk_142642) and vha-a3 542 (Salk_122135) described by Krebs et al. (2010) were obtained from the Arabidopsis 543 Biological Resources Center (ABRC). The mutant line avp1 (GK-005004) described in Li et 544 al. (2005) was obtained from the European Arabidopsis Stock Centre, while the mutant line 545 clca-2 (FST 171A06) described in Angeli et al. (2006) was from the Institute National de la 546 Recherche Agronomique (INRA) collection in France, together with the T-DNA mutants used 547 for genotyping to select pure mutant lines. 548 549 Growth conditions B. napus plants were grown hydroponically in ceramic pots(20cm×15cm) filled with a 550 551 nutrient solution in the greenhouse under natural light as described in Han et al. (2015b). The 552 pots were arranged in a completely randomized design with six biological replications. The 553 nutrient solution was replaced every 3 days and its pH adjusted to 5.5 daily. Experiments were 554 conducted at the field station of Hunan Agricultural University, Southern China. 555 A. thaliana plants were grown in a nutrient solution in plastic pots as described in Arteca 556 et al. (2000) and Gong et al. (2003). The solution was changed every 3 days, with pH adjusted 557 daily to 5.8. Pots were arranged in a completely randomized design with six biological 558 replications. The nutrient solution used for both species consisted of 1.25mM KNO3, 559 0.625mM KH2PO4, 0.5mM MgSO4, 0.5mM Ca (NO3)2·4H2O, 0.025mM Fe-EDTA, 0.25 560 mlL-1 micronutrients (stock solution concentrations: 70mM B, 14mM Mn, 1mM Zn, 0.5mM 561 Cu and 0.2mM Mo). The experiments were conducted at Hunan Agricultural University in a 562 phytotron set at 70% relative humidity,16h-light/8h-dark cycle and constant temperature of 563 22°C. 564 565 Experimental treatments and phenotyping 566 All measurements made on B. napus plants were conducted either at the seedling stage 567 (2 months after transplanting) or at flowering (5 months after transplanting). The whole root 568 tissue was harvested and used for analyses at either stage. For shoot measurements, the fourth 569 leaf from the bottom was used for measurements made at seedling stage, whereas the 12th leaf 19 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 570 from the bottom was harvested for measurements made at flowering stage. For A. thaliana, 571 whole root and shoot of four-week-old plants were sampled and used for different assays. 572 573 Vacuolar proton-pumps inhibitor treatments 574 Inhibition of vacuolar proton pumps of B. napus (Xiangyou15) was conducted at the seedling 575 stage in hydroponic culture as described in Han et al. (2015a) with minor modifications. 576 Plants grown in hydroponic solution were used as control. Inhibitor treatment was conducted 577 using several chemicals: Bafi(25 nmol L-1 Bafilomycin A1), an inhibitor of V-ATPase; 578 DCCD(10 μmol L-1DCCD + 50 mmolL-1 Na2SO3), specifically inhibits V-PPase; and 579 Bafi+DCCD in a 1:1 ratio as inhibitor of both V-ATPase and V-PPase. The inhibitors were 580 applied in the hydroponic solutions for 24 h. Fresh plant tissue samples from all treatments 581 were collected for different assays. 582 583 NO3- induced gene expression 584 Four-week old A. thaliana mutants (nrt1.5-3,nrt1.8-2) were grown in hydroponics with 585 2.25mM (NH4)2succinate for 3 days and shifted to hydroponics with 4.5mM NO3- for 12 h; 586 then root tissues were collected to assess relative expression of AtNRT1.5 and AtNRT1.8 587 genes. Seedlings of B. napus (Xiangyou15) plants grown hydroponically were treated with 588 5mM (NH4)2succinate for 3 d and shifted to fresh hydroponic solution containing 15 mM 589 NO3- for 12 h. Root tissue was then collected to assess the relative expression of BnNRT1.5 590 and BnNRT1.8 genes using quantitative RT-PCR. 591 592 ACC treatment 593 Hydroponically grown seedlings of B. napus were subjected to ACC treatment as described in 594 Zhang et al. (2014) with minor modifications. Seedlings of the two genotypes, Xiangyou15 595 and 814 were transferred to a nutrient solution containing 0.02mM L-1 ACC for 6 h or grown 596 in normal hydroponic solution as control. Root tissues were then collected to assay relative 597 expression of BnNRT1.5 and BnNRT1.8 using quantitative RT-PCR. Another set of B. napus 598 plants were grown in ACC-containing hydroponic solution, but supplemented with 15NO3- (22% 599 15 N) replacing the unlabeled NO3-, for 1 h. Root and shoot tissues were collected separately 20 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 600 for 15N measurement using a continuous-flow isotope ratio mass spectrometer coupled with a 601 C-N elemental analyzer (ANCA-MS; PDZ Europa). 602 603 Biomass and N concentration 604 Grain yield, dry biomass and N concentration were determined in plant samples at 605 harvest. Plants were harvested (including senescing leaves) then dried in an oven for 30 min 606 at 105°C, then at 70°C to a constant weight and weighed. N concentration was determined 607 using the Kjeldahl method (Shi et al. 2010; Han et al. 2015b);0.2g of dried samples were 608 digested with H2SO4-H2O2 in a 100-ml Kjeldahl digestion flask, after which N concentration 609 was determined using a Foss Auto Analyser Unit (Kjeldahl 8400). 610 611 V-ATPase and V-PPase activities Roots (1.0 g) of B. napus were collected at seedling and flowering stages, and that of A. 612 613 thaliana (0.5 g) sampled at seedling stage; then used for determining the activities of 614 V-ATPase and V-PPase (Han et al., 2015b). Activities of these two energy pumps within 615 microsomal membranes were followed colorimetrically based on released Pi, as described by 616 Zhu et al. (2001) and Krebs et al. (2010). 617 618 Assay of NO3- flux in vacuoles Roots of both species were collected similarly as that used for V-ATPase and V-PPase 619 620 activities, then used for vacuole isolation and measurement of NO3- flux in vacuoles as 621 described by Robert et al. (2007), with minor modification (Han et al., 2015b). Net fluxes of 622 NO3- in vacuoles were measured non-invasively using SIET (scanning ion-selective electrode 623 technique, SIET system BIO-003A; Younger USA Science and Technology Corporation, 624 USA). An NO3--selective microelectrode used for the assay of NO3- flux was vibrated in the 625 measuring solution between two positions, 1 μm and 11 μm from the vacuole surface 626 (tonoplast), along an axis perpendicular to the tangent of the target vacuoles. Background 627 signal was recorded by vibrating the electrode in the measuring solution in the absence of 628 vacuoles. Ion flux was calculated by Fick’s law of diffusion: J = -D(dc/dx), where J represents 629 the ion flux (picomolescm-2s-1), dc/dx is the ion concentration gradient, and D is the ion 21 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 630 diffusion constant in a particular medium. The direction of the flux was derived from Fick’s 631 law of diffusion that relates the concentration gradient. 632 633 Isolation of intact protoplasts and vacuoles for determining NO3- concentration Similar to the above assays, root tissues (1.0 g) were collected from B. napus at seedling 634 635 and flowering stages and from A. thaliana (0.5 g) seedlings and used to isolate intact 636 protoplasts and vacuoles as described by Robert et al. (2007), with minor modifications as 637 outlined in Huang et al. (2012) and Han et al.(2015b). The purified protoplasts were divided 638 into two equal aliquots, with one of them used for isolation of vacuoles. The purified 639 protoplasts and vacuoles were then sub-sampled and used to determine NO3- concentrations 640 (Vogeli-Lange and Wagner, 1990) and for enzyme activity assays (Ma et al., 2005). 641 NO3-concentrations in protoplasts and vacuoles were measured by a continuous-flow 642 auto-analyzer (Auto Analyser 3, Bran and Luebbe, Germany) as described previously (Han et 643 al. 2015b).The activities of acid phosphatase (ACP) and cytochrome oxidase (COX) were 644 determined using plant ACP colorimetry- and COX-assay kits (GenMedSci Inc.) following 645 the manufacturer’s instructions. ACP activity specific to vacuoles was determined and used to 646 normalize NO3- accumulation. We measured NO3- in the protoplast outside the vacuole, which 647 includes the cytosol and organelles, e.g., mitochondria and Golgi Apparatus (Robert et 648 al.2007). Since most of the NO3- in the protoplast outside the vacuole is located in the cytosol 649 (Krebs et al., 2010), we refer to NO3- distribution between vacuoles and cytosol rather than 650 vacuole versus protoplast. 651 652 Quantitative RT-PCR Total RNA extracted from B. napus roots was prepared using Trizol reagent (Invitrogen). 653 654 cDNAs were synthesized using M-MLV reverse transcriptase (Promega) following 655 manufacturer’s protocol. The relative expression of BnNRT1.5 (EV220114), BnNRT1.8 656 (EV116423) and Bnactin (AF111812.1) genes in plant roots were determined by quantitative 657 RT-PCR, and run on a LightCycler instrument (Roche) with the SYBR Green Real-Time PCR 658 Master Mix Kit (TOYOBO, Japan) under the following conditions: 95°C for 2min, then 45 659 cycles of 95°C for 10s, 60°C for 10s and 72°C for 20s. The primer sequences of BnNRT1.5, 22 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 660 BnNRT1.8 and Bnactin genes were obtained from http://brassica.nbi.ac.uk as described in 661 Harper et al. (2012), and expression levels were normalized to Bnactin as control. Total RNA extracted from the 4-week-old A. thaliana roots under the treatments 662 663 conditions described above were prepared using Trizol reagent (Invitrogen). The cDNAs were 664 synthesized using M-MLV reverse transcriptase (Promega) based on the manufacturer’s 665 protocol and as described in Li et al. (2010).The primer sequences of quantitative RT-PCR 666 used in these assays are listed in Supplemental Table 2 and were described previously (Chen 667 et al. 2012; Zhang et al. 2014); and their expression was normalized to Atactin2 as control. 668 669 Determination of nitrate distribution using 15NO3Hydroponically grown B. napus (seedling and flowering stages) and A. thaliana 670 671 (seedling) were transferred to 0.1mM CaSO4 solution for 1min, then to their respective 672 hydroponic nutrient solutions with15NO3- (22% excess) replacing unlabeled NO3-, for 1h. 673 Plants were then transferred to 0.1mM CaSO4 solution for 1min, after which roots were 674 washed with deionized water. Root and shoot samples were separated and dried at 105°C for 675 30min, followed by 70°C for 3 d, then grounded and used for assaying 15N content using a 676 continuous-flow isotope ratio mass spectrometer coupled with a carbon-nitrogen elemental 677 analyzer (ANCA-MS; PDZ Europa). 678 679 Xylem sap collection and assay of nitrate concentration We used the method of Tang et al. (2012) for collecting xylem sap. Plants were cut 680 681 leaving1cm segments with intact roots at seedling and flowering stages for B. napus and at 682 seedling stage for A. thaliana. Roots were immediately immersed in their respective nutrition 683 solutions. Weighed cotton was put on the cut surface to absorb extruding xylem sap for 1 h. 684 The cotton was then wrapped in plastic film and the volume of xylem sap was calculated as 685 the weight gain of the cotton. Xylem sap was then squeezed from the cotton using a syringe, 686 and used for subsequent assay of nitrate concentration using a continuous-flow auto-analyzer 687 (Auto Analyser 3, Bran and Luebbe, Germany). 688 To determine nitrate concentration, samples (1.0 g fresh root and shoot at seedling and 689 flowering of B. napus and 0.5 g fresh root and shoot of 4-week-old A. thaliana) were frozen 690 in liquid N2 and ground with a mortar and pestle; the powder was then transferred to a beaker 23 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 691 containing 70 ml deionized water and boiled for 30 min to extract nitrate, then cooled and 692 made to 100 ml volume; and 0.2 g activated carbon added to eliminate the effect of 693 chlorophyll. The mixture was filtered and nitrate in the filtrate was determined using a 694 continuous-flow auto-analyser (Atuo-Analyser 3, Bran and Luebbe, Germany). 695 696 Measurements of photosynthesis All measurements of photosynthesis and related parameters were conducted at 1000 h 697 698 using intact leaves. The 4th leaf from the bottom in B. napus at seedling stage, and the 12th leaf 699 from the bottom at flowering stage were used for determining chlorophyll concentration, 700 photosynthetic rate, intercellular CO2 concentration, transpiration rate and stomatal 701 conductance. Chlorophyll concentration was determined by SPAD-502 (Minolta Camera Co., 702 Ltd., Japan) (Luo et al. 2006; Yandeau-Nelson et al. 2011). Photosynthetic rate, intercellular 703 CO2 concentration, transpiration rate and stomatal conductance were measured using LI-6400 704 Portable Photosynthesis System (Li-Cor Biosciences Co., Ltd., USA), set at flow rate of 500 705 μmol s-1, photosynthetic photon flux density of 1000 μmol m-2 s-1, relative humidity of 65%, 706 CO2 concentration of 400ppm, and ambient temperatures. 707 708 NR and GS activities Leaves with similar age as those used for photosynthesis in B. napus were used for the 709 710 assay of nitrate reductase (NR) and glutamine synthetase (GS) activities. NR activity was 711 measured by the modified method of Fan et al. (2007). Fresh samples (0.5g) were frozen in 712 liquid N2, ground into powder in the presence of acid-washed sand and homogenized with 4 713 ml of extraction buffer (0.025mol L-1phosphate buffer, pH8.7, 1.211g L-1 cysteine, 0.372g L-1 714 EDTA). Homogenates were centrifuged at 30,000×g for 15 min at 4°C, and the supernatants 715 were treated with sulfanilamide and α-naphthylamine reagents for colorimetric (540nm) 716 determination of nitrite as described by Fan et al. (2007). 717 GS activity was determined using a modified reverseγ-glutamyltransferase method 718 (Wang et al.2014), which measures the GS-catalyzed formation of glutamyl-γ-hydroxamate 719 from glutamine and hydroxylamine. Fresh samples were frozen for 30 min at -20°C, then 720 ground in 10ml Tris-HCl buffer and acid-washed sand. The homogenates were filtered 721 through two layers of gauze, and centrifuged at 8000×g for 15 min at 4°C. About 1.2ml of the 24 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 722 supernatant was added in a reaction mixture and treated as described (Wang et al.2014). After 723 the GS-catalyzed reaction was stopped and the solution centrifuged, glutamyl-γ 724 -hydroxamate was quantified colorimetrically in the supernatant (at 485 nm) and the 725 concentration was determined using a standard curve. 726 727 Statistical analyses We used the SPSS software (Statistical Product and Service Solutions V13.0, USA) for 728 729 ANOVA and mean separation of main-effects and interactions using Duncan’s multiple range 730 test at P<0.05.Values are presented as means and SD of three or six replicates from three 731 independent experiments. Different letters or an asterisk (*) associated with specific data (e.g., 732 at the top of histogram bars in figures or within tables) denote significant differences at 733 P<0.05. 734 735 Supplemental Data 736 Supplemental Figure 1. B. napus with higher NUE showed lower vacuolar sequestration 737 capacity (VSC) for NO3-in roots at flowering stage. 738 Supplemental Figure 2. B. napus with higher NUE showed enhanced long-distance transport 739 of NO3- from roots to shoots at flowering stage. 740 Supplemental Figure 3. The two B. napus (H and L genotypes) showed the same total N per 741 plant at seedling stage (A) and flowering stage (B). 742 Supplemental Figure 4. B. napus with higher NUE showed increased NO3- concentration in 743 the xylem sap at seedling and flowering stages. 744 Supplemental Figure 5. NO3- concentration in the xylem sap as affected by inhibitor 745 treatments in B. napus and in the energy pumps’ mutants of A. thaliana (col-0, vha-a2, vha-a3, 746 avp1). 747 Supplemental Figure 6. Reduced VSC for NO3- in roots drives long-distance transport of 748 NO3- from roots to shoots in the A. thaliana wild type (Ws) and mutant (clca-2). 749 Supplemental Figure 7. Differences of NR and GS activities between the two B. napus (H 750 and L genotypes) at seedling and flowering stages. 751 Supplemental Figure 8. Amino acid sequences of BnNRT1.5 and BnNRT1.8. 752 Supplemental Figure 9. Functions of AtNRT1.5 and AtNRT1.8 genes in root tissues of A. 753 thaliana in controlling NO3- long-distance transport from root to shoots. 25 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 754 Supplemental Figure 10. B. napus BnNRT1.5 and BnNRT1.8 genes in roots were 755 coordinately modulated to facilitate NO3- long distance transport from roots to shoots. 756 Supplemental Table 1. Differences in stomatal conductance and transpiration rate between 757 the two B. napus genotypes. 758 Supplemental Table 2. Sequences of primera used for qRT-PCR. 759 760 Acknowledgements We thank Dr. Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology, 761 762 Shanghai Institutes for Biological Sciences) for providing nrt1.5-3 and nrt1.8-2 seeds. This study was supported by the National Natural Science Foundation of China (Grant 763 764 No.31101596, 31372130); Hunan Provincial Recruitment Program of Foreign Experts; 765 National Oilseed Rape Production Technology System of China; “2011 Plan” supported by 766 The Ministry of Education in China; Open Novel Science Foundation of Hunan province 767 (13K062), the National Key Laboratory of Plant Molecular Genetics and the "Twelfth 768 Five-Year" National Science and technology support program (2012BAD15BO4). 769 770 Figure 1 B. napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for 771 NO3- in roots at the seedling stage. 772 H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE 773 genotype 814. Specific activities of the tonoplast proton pumps are expressed as μmol Pi 774 released mg-1 protein h-1. NO3- fluxes are expressed as pmol NO3- cm-2 S-1. Mature vacuoles 775 were collected from the root tissues at seedling stage and a microelectrode was vibrated in the 776 measuring solution between the two positions, 1 μm and 11 μm from the vacuole surface 777 (tonoplast), along an axis perpendicular to the tangent of the target vacuoles recording the 778 stable reading data. The background was recorded by vibrating the electrode in measuring 779 solution without vacuoles. 780 Protoplasts and vacuoles isolated from roots of hydroponically grown plants were measured 781 for NO3- content and NO3- accumulation normalized against the specific activity of the 782 vacuole acid phosphatase (ACP) as described in Materials and Methods; and plotted as μmol 783 NO3- per μmol p-nitrophenol, the end product of ACP assay. 784 Proton pump activities in root tissues between H and L genotypes are shown at the seedling 785 stage (A). Different letters at the top of the histogram bars denote significant differences of 786 V-ATPase in root tissues between H and L genotypes (P<0.05); an asterisk (*) at the top of the 26 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 787 histogram bars indicates significant difference in V-PPase activity in root tissues between H 788 and L genotypes (P<0.05). Vertical bars on the figures indicate SD (n=6). 789 Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L 790 genotypes are shown at the seedling stage (B). Different letters at the top of the histogram 791 bars denote significant differences of NO3- flux between H and L genotypes (P<0.05). Vertical 792 bars on the figures indicate SD (n=6). 793 Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the 794 seedling stage (C). Values above the bars represent the percentage of vacuolar NO3- relative 795 to the total NO3- in protoplasts. 796 NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D). P-V is the 797 total NO3-in the cytosol and was calculated as: total NO3- in protoplasts – total NO3- in 798 vacuole. Different letters at the top of histogram bars denote significant differences of total 799 NO3- in cytosol between H and L genotypes (P<0.05). Vertical bars on the figures indicate SD 800 (n=6). 801 802 Figure 2 B. napus with higher NUE showed enhanced long-distance transport of NO3- from 803 roots to shoots at the seedling stage. 804 H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814. 805 Expression of the BnNRT1.5 (A) and BnNRT1.8 (B) genes relative to that of the actin gene in 806 the root tissues of the two genotypes at seedling stage was assessed by quantitative RT-PCR 807 as described in Materials and Methods; a value of 1.0 is equivalent to levels of expression of 808 the Bnactin gene. Vertical bars on the figures indicate SD (n=3); different letters at the top of 809 the histogram bars denote significant differences at P<0.05. 810 Hydroponically grown B. napus plants were subjected to 15N-labeling treatment as described 811 in Materials and Methods. The 15N concentration in the root and shoot tissues of the two 812 genotypes is shown at the seedling stage (C). The [15N] S:R ratios in the root and shoot tissues 813 of the two genotypes are shown at the seedling stage (D).Vertical bars on the figures indicate 814 SD (n=3), different letters at the top of the histogram bars denote significant differences at 815 P<0.05 level. 816 The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown 817 at the seedling stage (E). The [NO3-] S:R ratios in the root and shoot tissues of the two 818 genotypes are shown at the seedling stage (F). Vertical bars on the figures indicate SD (n=3), 819 different letters at the top of the histogram bars denote significant differences at P<0.05. 820 27 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 821 Figure 3 NRT1.5 gene expression is up-regulated by NO3- and might affect downstream 822 regulation of the NRT1.8 gene. 823 Culture conditions and plant materials are defined in the Materials and Methods. Seedling 824 stage A. thaliana mutant plants (nrt1.8-2 and nrt1.5-3) were cultured hydroponically with 825 2.25 mM (NH4)2succinate for 3 d and shifted to hydroponics with 4.5 mM NO3- for 12 h, after 826 which root tissues were collected for analysis. Expression of AtNRT1.5 or AtNRT1.8 genes 827 were assessed by quantitative RT-PCR as described in Materials and Methods and presented 828 relative to that of the Atactin2 gene. 829 B. napus (Xiangyou15) was cultured in hydroponics with 7.5 mM (NH4)2succinate for 3 d and 830 shifted to hydroponics with 15 mM NO3- for 12 h, after which root tissues were collected for 831 analysis as described in the Materials and Methods. 832 Relative expressions of the AtNRT1.5 genes are shown at the seedling stage in root tissues of 833 col-0 and nrt1.8-2 (A). Relative expression of the AtNRT1.5 gene is shown in root tissues of 834 nrt1.8-2 plants treated with either (NH4)2succinate for 3 d or shifted to hydroponics with NO3- 835 for 12 h at the seedling stage (B). Relative expressions of the AtNRT1.8 genes are shown at 836 the seedling stage in root tissues of col-0 and nrt1.5-3 (C). Panel (D) show results for the 837 relative expression of the AtNRT1.8 genes under the same conditions as in panel (B). 838 Relative expression of BnNRT1.5 (E) and BnNRT1.8 (F) genes in root tissues of B. napus 839 (Xiangyou15) are shown at the seedling stage. Different letters at the top of the histogram 840 bars denote significant differences (P<0.05). Vertical bars on the figures indicate SD (n=6). 841 842 Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3- from roots to 843 shoots in the B. napus (Xiangyou15). 844 Inhibitor treatments of B. napus (Xiangyou15) hydroponics-grown plants were conducted at 845 the seedling stage. Control: normal hydroponics solution; Bafi:(25 nmol·L-1Bafilomycin A1), 846 an inhibitor of V-ATPase; DCCD:(10 μmol·L-1DCCD + 50 mmol·L-1Na2SO3), which together 847 specifically inhibits V-PPase; and B+D: a 1:1 combination of Bafi and DCCD, which inhibits 848 both V-ATPase and V-PPase. The inhibitors were applied at the seedling stage in hydroponic 849 solutions for 24 h. Mature vacuoles were collected from the root tissues of the plant materials 850 at the seedling stage. The NO3- flux measurement is described in the legend to Figure 1. 851 Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for 852 NO3- accumulation plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to 853 Figure 1 and in Materials and Methods. 28 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 854 Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues of B. 855 napus as affected by inhibitor treatments (A). Different letters at the top of histogram bars 856 denote significant differences in V-ATPase activities in root tissues between inhibitor 857 treatments (P<0.05); asterisk (*) at the top of the histogram bars denote significant differences 858 in V-PPase activities in root tissues between inhibitor treatments (P<0.05). Vertical bars on 859 the figures indicate SD (n=6). 860 NO3- fluxes within the vacuoles of root tissues are shown for B. napus between inhibitor 861 treatments (B). Different letters at the top of the histogram bars denote significant differences 862 of NO3- flux between inhibitor treatments (P<0.05). Vertical bars on the figures indicate SD 863 (n=6). 864 Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for B. 865 napus (C). Values above the bars represent the percentage of vacuolar NO3- relative to the 866 total NO3- in protoplasts. Vertical bars indicate SD (n=6). 867 Accumulation of cytosolic NO3 -in root tissues is shown for B. napus (D). P-V is the total 868 NO3- in the cytosol; calculated as total NO3- in protoplasts – total NO3- in vacuoles. Different 869 letters at the top of the histogram bars denote significant differences in total NO3- in vacuoles 870 outside of the protoplast (P<0.05). Vertical bars on the figures indicate SD (n=6). 871 Expression levels of the BnNRT1.5 gene (E) and BnNRT1.8gene (F) are shown relative to that 872 of the actin gene, assessed by quantitative RT-PCR as described in Materials and Methods; a 873 value of 1.0 is equivalent to levels of expression of the Bnactin gene. Different letters at the 874 top of the histogram bars denote significant differences in gene expression (P<0.05). Vertical 875 bars indicate SD (n=3). 876 Growth conditions for hydroponically grown plants with 15N treatmentare described in 877 Materials and Methods. The [15N] S:R ratios as affected by inhibitor treatments are depicted 878 for B. napus (G).The [NO3-] S:Rratioas affected by inhibitor treatments are shown for B. 879 napus (H). Different letters at the top of the histogram bars denote significant differences 880 (P<0.05). Vertical bars on the figures indicate SD (n=3). 881 882 Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3- from roots to 883 shoots in the A. thaliana (col-0, vha-a2, vha-a3, avp1). 884 Wild type A. thaliana plants (col-0), V-ATPase gene mutant plants (vha-a2, vha-a3) and 885 V-PPase gene mutant plant (avp1) were used as the model plant materials. Mature vacuoles 886 were collected from the root tissues of the plant materials at the seedling stage. The NO3- flux 887 measurement is described in the legend to Figure 1. Protoplasts and vacuoles isolated from 29 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 888 roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol 889 NO3- per μmol p-nitrophenol as described in the legend to Figure 1 and in Materials and 890 Methods. 891 Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root tissues from 892 different A. thaliana plant materials (A). Different letters at the top of the histogram bars 893 denote significant differences in V-ATPase activities in root tissues between A. thaliana plant 894 materials (P<0.05); asterisk (*) at the top of the histogram bars denote significant differences 895 of V-PPase activities in root tissues (P<0.05). Vertical bars indicate SD (n=6). 896 NO3- fluxes within the vacuoles of root tissues are shown for A. thaliana between different 897 mutants (B). Different letters at the top of the histogram bars denote significant differences in 898 NO3- flux between Arabidopsis plant materials (P<0.05). Vertical bars indicate SD (n=6). 899 Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown for A. 900 thaliana (C). Values above the bars represent the percentage of vacuolar NO3- relative to the 901 total NO3- in protoplasts. Vertical bars indicate SD (n=6). 902 Accumulation of cytosol NO3- in root tissues is shown for A. thaliana (D). P-V is the total 903 NO3- in the cytosol; calculated as total NO3- in protoplasts – total NO3- in vacuoles. Different 904 letters at the top of the histogram bars denote significant differences in total NO3-in the 905 cytosol (P<0.05). Vertical bars indicate SD (n=6). 906 Expression of the AtNRT1.5 gene (E) and AtNRT1.8 gene (F) are shown relative to that of the 907 actin gene, assessed by quantitative RT-PCR; a value of 1.0 is equivalent to levels of 908 expression of the Atactin2 gene. Different letters at the top of the histogram bars denote 909 significant differences in gene expression (P<0.05). Vertical bars on the figures indicate SD 910 (n=3). 911 Growth conditions for hydroponically grown plants with 15N treatmentare described in 912 Materials and Methods. The [15N] S:R ratios as affected by inhibitor treatments are depicted 913 for various A. thaliana mutants (G). The [NO3-] S:R ratio as affected by inhibitor treatments 914 are depicted for various A. thaliana mutants (H). Different letters at the top of the histogram 915 bars denote significant differences (P<0.05). Vertical bars on the figures indicate SD (n=3). 916 917 Figure 6 Increased NO3- shoots:roots ratio essentially contributed to enhancing NUE in A. 918 thaliana. 919 Plants grown hydroponically were sampled for further analysis at seedling stage and at 920 harvest. Conditions for hydroponics culture and characteristics of the various A. thaliana 921 genotypes are defined in Materials and Methods. The wild-type Columbia-0 (col-0) plants 30 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 922 were used as control for V-ATPase mutants (vha-a2 and vha-a3), V-PPase mutants (avp1), 923 nrt1.5-3 mutants and nrt1.8-2 mutants; A. thaliana wild-type Wassilewskija (Ws) plants were 924 used as control for clca-2 mutants. 925 Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the 926 wild-type (col-0) and mutants nrt1.5-3, nrt1.8-2, vha-a2, vha-a3 and avp1. 927 Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A. 928 thaliana wild-type plants Ws and clca-2. Different letters at the top of the histogram bars 929 denote significant differences between the Arabidopsis genotypes (P<0.05). Vertical bars on 930 the figures indicate SD (n=6). 931 932 Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues. 933 Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol 934 in root tissues. Red lines display the route for regulation pathway. 935 936 31 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. μmol NO3- /μmol p-nitrophenol C μmol NO3- /μmol p-nitrophenol μmol Pi mg-1 protein h-1 NO3- flux (pmol cm-2 s-1) A Figure 1 B. napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3- in roots at the seedling stage. H refers to the high-NUE oilseed rape genotype Xiangyou15 and L refers to the low-NUE genotype 814. Specific activities of the tonoplast proton pumps are expressed as μmol Pi released mg-1 protein h-1. NO3fluxes are expressed as pmol NO3- cm-2 S-1. Mature vacuoles were B collected from the root tissues at seedling stage and a microelectrode was vibrated in the measuring solution between the two positions, 1 μm and 11 μm from the vacuole surface (tonoplast), along an axis perpendicular to the tangent of the target vacuoles recording the stable reading data. The background was recorded by vibrating the electrode in measuring solution without vacuoles. Protoplasts and vacuoles isolated from roots of hydroponically grown Influx plants were measured for NO3- content and NO3- accumulation normalized against the specific activity of the vacuole acid phosphatase H L (ACP) as described in Materials and Methods; and plotted as μmol Root NO3- per μmol p-nitrophenol, the end product of ACP assay. Proton pump activities in root tissues between H and L genotypes are Seedling stage Seedling stage shown at the seedling stage (A). Different letters at the top of the histogram bars denote significant differences of V-ATPase in root D tissues between H and L genotypes (P<0.05); an asterisk (*) at the top of the histogram bars indicates significant difference in V-PPase activity in root tissues between H and L genotypes (P<0.05). Vertical bars on the figures indicate SD (n=6). Mean rates of NO3- flux during 160 s within vacuoles of root tissues between H and L genotypes are shown at the seedling stage (B). Different letters at the top of the histogram bars denote significant differences of NO3- flux between H and L genotypes (P<0.05). Vertical bars on the figures indicate SD (n=6). Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is shown at the seedling stage (C). Values above the bars represent the percentage of vacuolar NO3- relative to the total NO3- in protoplasts. Seedling stage Seedling stage NO3- accumulation in the cytosol of root tissues is shown at seedling stage (D). P-V is the total NO3-in the cytosol and was calculated as: total NO3- in protoplasts – total NO3- in vacuole. Different letters at the top of histogram bars denote significant differences of total NO3- in cytosol between H and L genotypes (P<0.05). Vertical bars on the Downloaded from on July 31, 2017 - Published by www.plantphysiol.org figures indicate SD (n=6). Copyright © 2016 American Society of Plant Biologists. All rights reserved. A B BnNRT1.8 relative expression BnNRT1.5 relative expression Seedling stage 15 15 -1 [ N] S:R ratios mmol N g DW C Seedling stage - -1 (μg g FW) - [NO3 ] S:R ratios E NO3 concentration Figure 2 B. napus with higher NUE showed enhanced long-distance transport of NO3- from roots to shoots at the seedling stage. H refers to the high-NUE genotype Xiangyou15 and L refers to the low-NUE genotype 814. Expression of the BnNRT1.5 (A) and BnNRT1.8 (B) genes relative to that of the actin gene in the root tissues of the two genotypes at seedling stage was assessed by Seedling stage quantitative RT-PCR as described in Materials and D Methods; a value of 1.0 is equivalent to levels of expression of the Bnactin gene. Vertical bars on the figures indicate SD (n=3); different letters at the top of the histogram bars denote significant differences at P<0.05. Hydroponically grown B. napus plants were subjected to 15N-labeling treatment as described in Materials and Methods. The 15N concentration in the root and shoot tissues of the two genotypes is shown at the seedling stage (C). The [15N] S:R ratios in the root and shoot Seedling stage tissues of the two genotypes are shown at the seedling stage (D).Vertical bars on the figures indicate SD F (n=3), different letters at the top of the histogram bars denote significant differences at P<0.05 level. The NO3- concentration (μg g-1 FW) in root and shoot tissues of the two genotypes are shown at the seedling stage (E). The [NO3-] S:R ratios in the root and shoot tissues of the two genotypes are shown at the seedling stage (F). Vertical bars on the figures indicate SD (n=3), different letters at the top of the histogram bars Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rightsdenote reserved. significant differences at P<0.05. Seedling stage Seedling stage 1.0 0.5 0.0 AtNRT1.8 relative expression C 12 8 4 E 2.0 BnNRT1.5 relative expression 0 1.5 1.0 0.5 0.0 B 1.6 AtNRT1.5 AtNRT1.5 relative expression 1.5 Figure 3 NRT1.5 gene expression is up-regulated by NO3a and might affect downstream regulation of the NRT1.8 a 1.2 gene. a Culture conditions and plant materials are defined in the 0.8 Materials and Methods. Seedling stage A. thaliana mutant plants (nrt1.8-2 and nrt1.5-3) were cultured hydroponically b 0.4 with 2.25 mM (NH4)2succinate for 3 d and shifted to hydroponics with 4.5 mM NO3- for 12 h, after which root 0 tissues were collected for analysis. Expression of AtNRT1.5 (NH4)2succinate KNO3 nrt1.8-2 col-0 or AtNRT1.8 genes were assessed by quantitative RT-PCR nrt1.8-2 Root Root as described in Materials and Methods and presented relative to that of the Atactin2 gene. D 12 AtNRT1.8 AtNRT1.8 a B. napus (Xiangyou15) was cultured in hydroponics with a a 7.5 mM (NH4)2succinate for 3 d and shifted to hydroponics with 15 mM NO3- for 12 h, after which root tissues were 8 collected for analysis as described in the Materials and Methods. 4 Relative expressions of the AtNRT1.5 genes are shown at the seedling stage in root tissues of col-0 and nrt1.8-2 (A). b Relative expression of the AtNRT1.5 gene is shown in root 0 col-0 nrt1.5-3 (NH4)2succinate KNO3 tissues of nrt1.8-2 plants treated with either Root nrt1.5-3 Root (NH4)2succinate for 3 d or shifted to hydroponics with NO3for 12 h at the seedling stage (B). Relative expressions of F 0.08 BnNRT1.5 BnNRT1.8 the AtNRT1.8 genes are shown at the seedling stage in root a a tissues of col-0 and nrt1.5-3 (C). Panel (D) show results for 0.06 the relative expression of the AtNRT1.8 genes under the b same conditions as in panel (B). 0.04 b Relative expression of BnNRT1.5 (E) and BnNRT1.8 (F) genes in root tissues of B. napus (Xiangyou15) are shown 0.02 at the seedling stage. Different letters at the top of the 0 histogram bars denote significant differences (P<0.05). (NH succinate 31, - Published by www.plantphysiol.org (NH4)2succinate KNO3 Downloaded from on July KNO 4)22017 3 Copyright © 2016 American Society of Plant Biologists. All rightsVertical reserved. bars on the figures indicate SD (n=6). AtNRT1.5 AtNRT1.8 relative expression 2.0 BnNRT1.8 relative expression AtNRT1.5 relative expression A Root Root A B 5 0 C μmol NO3- /μmol p-nitrophenol 8 6 4 2 0 E BnNRT1.5 relative expression 6 4 2 0 G 0.75 0.50 15 [ N] S:R ratio 1.00 0.25 0.00 [NO3-] S:R ratio 10 NO3- flux (pmol cm-2 s-1) 15 0 V-PPa se μmol NO3- /μmol p-nitrophenol 20 Figure 4 Reduced VSC of NO3- in roots drives long-distance transport of NO3from roots to shoots in the B. napus (Xiangyou15). -70 * Inhibitor treatments of B. napus (Xiangyou15) hydroponics-grown plants were * conducted at the seedling stage. Control: normal hydroponics solution; Bafi:(25 -140 c nmol·L-1Bafilomycin A1), an inhibitor of V-ATPase; DCCD:(10 μmol·L-1DCCD a a b -210 b + 50 mmol·L-1Na2SO3), which together specifically inhibits V-PPase; and B+D: a b b 1:1 combination of Bafi and DCCD, which inhibits both V-ATPase and V-PPase. -280 a The inhibitors were applied at the seedling stage in hydroponic solutions for 24 h. Influx -350 Mature vacuoles were collected from the root tissues of the plant materials at the Control Ba fil DCCD B+D control Bafi DCCD B+D seedling stage. The NO3- flux measurement is described in the legend to Figure 1. Root Root Protoplasts and vacuoles isolated from roots of hydroponically grown plants were assessed for NO3- accumulation plotted as μmol NO3- per μmol p-nitrophenol as D 1.2 93.2 described in the legend to Figure 1 and in Materials and Methods. Vacuole Protoplast a P-V Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in root b 85.2 82.1 tissues of B. napus as affected by inhibitor treatments (A). Different letters at the 74.5 0.8 b top of histogram bars denote significant differences in V-ATPase activities in c root tissues between inhibitor treatments (P<0.05); asterisk (*) at the top of the 0.4 histogram bars denote significant differences in V-PPase activities in root tissues between inhibitor treatments (P<0.05). Vertical bars on the figures indicate SD 0 (n=6). Control Bafil DCCD B+D Control Bafil DCCD B+D NO3- fluxes within the vacuoles of root tissues are shown for B. napus between Root Root inhibitor treatments (B). Different letters at the top of the histogram bars denote significant differences of NO3- flux between inhibitor treatments (P<0.05). F 0.04 Vertical bars on the figures indicate SD (n=6). a a BnNRT1.5 BnNRT1.8 Accumulation of NO3- inside the vacuole and in the protoplasts of root tissues is 0.03 b shown for B. napus (C). Values above the bars represent the percentage of b b vacuolar NO3- relative to the total NO3- in protoplasts. Vertical bars indicate SD 0.02 b c (n=6). c 0.01 Accumulation of cytosolic NO3 -in root tissues is shown for B. napus (D). P-V is the total NO3- in the cytosol; calculated as total NO3- in protoplasts – total NO30 in vacuoles. Different letters at the top of the histogram bars denote significant Control Ba fil DCCD B+D Control Bafil DCCD B+D differences in total NO3- in vacuoles outside of the protoplast (P<0.05). Vertical Root Root bars on the figures indicate SD (n=6). Expression levels of the BnNRT1.5 gene (E) and BnNRT1.8gene (F) are shown H relative to that of the actin gene, assessed by quantitative RT-PCR as described in 10 a Materials and Methods; a value of 1.0 is equivalent to levels of expression of the a b b 8 Bnactin gene. Different letters at the top of the histogram bars denote significant b b c differences in gene expression (P<0.05). Vertical bars indicate SD (n=3). 6 c Growth conditions for hydroponically grown plants with 15N treatmentare 4 described in Materials and Methods. The [15N] S:R ratios as affected by inhibitor 2 treatments are depicted for B. napus (G).The [NO3-] S:Rratioas affected by inhibitor treatments are shown for B. napus (H). Different letters at the top of the Downloaded from0 on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society ofDCCD Plant Biologists. All rights reserved. Control Bafil B+D histogram bars denote significant differences (P<0.05). Vertical bars on the Control Bafil DCCD B+D figures indicate SD (n=3). V-ATPa se BnNRT1.8 relative expression μmol Pi mg-1 protein h-1 25 V-ATPase V-PPase Figure 5 Reduced VSC of NO3- in roots drives long-distance transport of NO3- from roots to shoots in the A. thaliana (col-0, vha-a2, vha-a3, avp1). Wild type A. thaliana plants (col-0), V-ATPase gene mutant plants (vha0.3 -500 a2, vha-a3) and V-PPase gene mutant plant (avp1) were used as the model plant materials. Mature vacuoles were collected from the root tissues of the a c 0.2 -1000 a plant materials at the seedling stage. The NO3- flux measurement is b b b described in the legend to Figure 1. Protoplasts and vacuoles isolated from b 0.1 -1500 roots of hydroponically grown plants were assessed for NO3- accumulation Influx a plotted as μmol NO3- per μmol p-nitrophenol as described in the legend to -2000 0 Figure 1 and in Materials and Methods. avp1 col-0 vha-a2 vha-a3 col-0 vha-a2 vha-a3 avp1 Tonoplast proton-pump (V-ATPase and V-PPase) activities are shown in Root Root root tissues from different A. thaliana plant materials (A). Different letters C D Vacuole Protoplast at the top of the histogram bars denote significant differences in V-ATPase 45 a 8 94.0 P-V 82.6 activities in root tissues between A. thaliana plant materials (P<0.05); a a 78.0 79.1 6 asterisk (*) at the top of the histogram bars denote significant differences 30 of V-PPase activities in root tissues (P<0.05). Vertical bars indicate SD 4 (n=6). b 15 NO3- fluxes within the vacuoles of root tissues are shown for A. thaliana 2 between different mutants (B). Different letters at the top of the histogram bars denote significant differences in NO3- flux between Arabidopsis plant 0 0 vha-a3 avp1 avp1 col-0 vha-a2 materials (P<0.05). Vertical bars indicate SD (n=6). col-0 vha-a2 vha-a3 Root Root Accumulation of NO3- inside the vacuole and in the protoplasts of root F 0.025 tissues is shown for A. thaliana (C). Values above the bars represent the E a AtNRT1.5 AtNRT1.8 2.5 a a percentage of vacuolar NO3- relative to the total NO3- in protoplasts. a 0.020 2.0 Vertical bars indicate SD (n=6). Accumulation of cytosol NO3- in root tissues is shown for A. thaliana (D). 0.015 1.5 P-V is the total NO3- in the cytosol; calculated as total NO3- in protoplasts b 0.010 1.0 – total NO3- in vacuoles. Different letters at the top of the histogram bars b b denote significant differences in total NO3-in the cytosol (P<0.05). Vertical c 0.005 0.5 bars indicate SD (n=6). 0.000 0.0 Expression of the AtNRT1.5 gene (E) and AtNRT1.8 gene (F) are shown vha-a2 vha-a3 avp1 col-0 vha-a2 vha-a3 avp1 col-0 relative to that of the actin gene, assessed by quantitative RT-PCR; a value Root Root of 1.0 is equivalent to levels of expression of the Atactin2 gene. Different H G 5.0 0.8 letters at the top of the histogram bars denote significant differences in a a gene expression (P<0.05). Vertical bars on the figures indicate SD (n=3). b 0.6 b b Growth conditions for hydroponically grown plants with 15N treatmentare c b described in Materials and Methods. The [15N] S:R ratios as affected by 2.5 0.4 c inhibitor treatments are depicted for various A. thaliana mutants (G). The [NO3-] S:R ratio as affected by inhibitor treatments are depicted for various 0.2 A. thaliana mutants (H). Different letters at the top of the histogram bars Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. 0.0 denote significant differences (P<0.05). Vertical bars on the figures 0 col-0 vha-a2 vha-a3 avp1 col-0 vha-a2 vha-a3 avp1 indicate SD (n=3). A * * * B μmol NO3 /μmol p-nitrophenol - AtNRT1.8 relative expression [NO3-] S:R ratio 15 [ N] S:R ratio AtNRT1.5 relative expression μmol NO3- /μmol p-nitrophenol NO3- flux (pmol cm-2 s-1) μmol Pi mg-1 protein h-1 0.4 0 30 a NUE based on biomass (g/g) b a a B a 30 a NUE based on biomass (g/g) A c 20 10 avp1 vha-a3 vha-a2 nrt1.8-2 nrt1.5-3 col-0 clca-2 D a b - 2000 1000 (μg g FW) b -1 b a -1 3000 4000 NO3 concentration 4000 (μg g FW) 10 Ws C - b 0 0 NO3 concentration 20 3000 b 2000 1000 0 0 col-0 vha-a2 vha-a3 avp1 Ws clca-2 Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Figure 6 Increased NO3- shoots:roots ratio essentially contributed to enhancing NUE in A. thaliana. Plants grown hydroponically were sampled for further analysis at seedling stage and at harvest. Conditions for hydroponics culture and characteristics of the various A. thaliana genotypes are defined in Materials and Methods. The wild-type Columbia-0 (col-0) plants were used as control for V-ATPase mutants (vha-a2 and vha-a3), V-PPase mutants (avp1), nrt1.5-3 mutants and nrt1.8-2 mutants; A. thaliana wild-type Wassilewskija (Ws) plants were used as control for clca-2 mutants. Differences in NUE based on biomass (A) and NO3- concentration (C) are shown for the wild-type (col-0) and mutants nrt1.5-3, nrt1.8-2, vha-a2, vhaa3 and avp1. Differences of NUE based on biomass (B) and NO3-concentration (D) are shown between A. thaliana wild-type plants Ws and clca-2. Different letters at the top of the histogram bars denote significant differences between the Arabidopsis genotypes (P<0.05). Vertical bars on the figures indicate SD (n=6). Cytoplasm Energy NO3Unloading NRT1.8 2 NO3- 2 NO3- CLCa 1 H+ Phloem V-ATPase H+ V-PPase Xylem NO3- Shoot NRT1.5 Loading NO3- Vacuole Root Vascular tissues Figure 7 Simplified model for NO3- long-distance transport in xylem of vascular tissues. Long-distance transport is regulated by NO3- distribution between the vacuole and the cytosol inDownloaded root tissues. Red lines display the route for regulation from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. pathway. Table 1. Comparison of NUE between the two B. napus genotype. a Physiological parameters NUE b Genotypes Biomass Grain yield Total N in plant Based on biomass Based on grain yield (g plant-1) (g plant-1) (g plant-1) (gg-1) (gg-1) H 139.44±6.77a 31.26±0.50a 2.61±0.08a 53.54±2.40a 12.01±0.44a L 108.73±3.05b 20.84±1.44b 2.45±0.15a 44.40±2.64b 8.53±1.08b aNUE values were calculated based on either total biomass (shoot, root and grains) per total N uptake or as grain yield per total N uptake. bH represents the high NUE genotype (Xiangyou15), and L represents the low NUE genotype (814). Different letters associated with data represent significant differences at P<0.05, n=3. Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Table 2. Variation inchlorophyll content, intercellular CO2 concentration and photosynthetic rate between high (H) and low (L) nitrogen use efficiency genotypes of B. napus. Seedling stage Flowering stage Chlorophyll contenta(SP AD readings) Intercellular CO2 concentration (µmol CO2mol-1) Photosynthetic rate (µmol CO2 m-2 s-1) H 48.65±1.29a 271.84±7.64a 23.31±0.96a 63.11±2.08a 283.99±20.84a 17.53±0.58a L 45.86±2.08b 246.36±8.78b 21.02±0.76b 57.40±3.00b 233.68±27.50b 16.22±0.67b Genotypes Chlorophyll content (SPAD readings) aChlorophyll Intercellular CO2 concentration (µmol CO2mol-1) Photosynthetic rate (µmol CO2 m-2 s-1) content, intercellular CO2 concentration and photosynthetic rate were measured at 1000 h using a LI-6400 Portable Photosynthesis System. Measurements were conducted using the 4th leaf from the bottom at seedling stage and the 12thleaf from the bottom up at flowering. Different letters between the H and L genotypes denote a significant difference at P<0.05 level. Data are means ±SD (n=6). Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Parsed Citations Andrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environment 9:511519 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Angeli AD, Monachello D, Ephritikhine G, Frachisse JM, Thomine S, Gambale F, Barbier-Brygoo H (2006) The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442: 939-942 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Arteca RN, Arteca JM (2000) A novel method for growing Arabidopsis thaliana plants hydroponically. Physiol Plantarum 108: 188-193 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Brux A, Liu TY, Krebs M, Stierhof YD, Lohmann JU, Miersch O, Wasternack C, Sschumacher K (2008) Reduced V-ATPPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in arabidopsis. The Plant Cell 20: 1088-1100 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Chen CZ, Lv XF, Li JY, Yi HY, Gong JM (2012) Arabidopsis NRT1.5 is another essential component in the regulation of nitrate reallocation and stress tolerance. Plant Physiology 159: 1582-1590 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Chen XP, Cui ZL, Fan MS, Vitousek P, Zhao M, Ma WQ, Wang ZL, Zhang WJ, Yan XY, Yang JC, Deng XP, Gao Q, Zhang Q, Guo SW, Ren J, Li SQ, Ye YL, Wang ZH, Huang JL, Tang QY, Sun YX, Peng XL, Zhang JW, He MR, Zhu YJ, Xue JQ, Wang GL, Wu L, An N, Wu LQ, Ma L, Zhang WF, Zhang FS (2014) Producing more grain with lower environmental costs. Nature 514: 486-489 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Chiu CC, Lin CS, Hsia AP, Su RC, Lin HL, Tsay YF (2004) Mutation of a nitrate transporter, AtNRT1.4, results in a reduced petiole nitrate content and altered leaf development. Plant Cell Physiology 45: 1139-1148 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Chopin F, Orsel M, Dorbe MF, Chardon F, Truong HN, Miller AJ, Krapp A, Daniel-VedeleF (2007) The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds. Plant Cell 19: 1590-1602 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Daniel-Vedele F, Filleur S, CabocheM (1998) Nitrate transport: a key step in nitrate assimilation. Current Opinion in Plant Biology 1: 235-239 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds:the long journey of nitrate in plants. Journal of Experimental Botany 62: 1349-1359 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fan SC, Lin CS, Hsu PK, Lin SH, TsayYF (2009) The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell 21:2750-2761 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Fan XR, Jia L, Li Y, Smith SJ, Miller AJ, Shen QR (2007) Comparing nitrate storage and remobilization in two rice cultivars that differ in their nitrogen use efficiency. Journal of Experimental Botany 58(7):1729-1740 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Gaxiola RA, Li JS, Undurraga S, Dang LM, Allen GJ, Alper SL, Flink GR (2001) Drought and salt tolerant plants result from over expression of the AVP1 H+-pump. PNAS 98(20): 11444-11449 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Glass ADM, Britto DT, Kaiser BN, Kinghorn JR, Kronzucker HJ, Kumar A, Okamoto M, Rawat S, Siddiqi MY, Unkles SE, Vidmar JJ (2002) The regulation of nitrate and ammonium transport systems in plants. Journal of Experimental Botany 53:855-864 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Gong JM, Lee DA, Schroeder JI (2003) Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis. PNAS 100: 10118-10123 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Granstedt RC, Huffaker RC (1982) Identification of the leaf vacuole as a major nitrate storage pool. Plant Physiology70:410-413 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Han YL, Liao Q, Yu Y, Song HX, Liu Q, Rong XM, Gu JD, Lepo JE, Guan CY, Zhang ZH (2015a) Nitrate reutilization mechanisms in the tonoplast of two Brassica napus genotypes with different nitrogen use efficiency. Acta Physiologia Planturum 37:42 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Han YL, Liu Q, Gu JD, Gong JM, Guan CY, Lepo JE, Rong XR, Song HX, Zhang ZH (2015b) V-ATPase and V-PPase at the Tonoplast affect NO3- Content in Brassica napus by Controlling Distribution of NO3- between the Cytoplasm and Vacuole. Journal of Plant Growth Regulation 2015, 34: 22-34 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Harper AL, Trick M, Higgins J, Fraser F, Clissold L, Wells R, Hattori C, Werner P, Bancroft I (2012) Associative transcriptomics of traits in the polyploidy crop species Brassica napus. Nature Biotechnology 30: 798-802 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Huang J, Zhang Y, Peng JS, Zhong C, Yi HY, Ow DW, Gong JM (2012) Fission yeast HMT1 lowers seed Cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis. Plant Physiology 158(4): 1779-1788 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Krapp A, David LC, Chardin C, Cirin T, Marmagne A, Leprince AC, Chaillou S, Ferrario-Mery S, Meyer C, Daniel-VedeleF (2014) Nitrate transport and signaling in Arabidopsis. Journal of Experiment Botany 65:789-798 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Krebs M, Beyhl D, Gorlich E, Al-Rasheid KA, Marten I, Stierhof YD, Hedrich R, Schumacher K (2010) Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. PNAS107:3251-3256 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Leran S, Varala K, Boyer JC, Chiurazzi M, Crawford N, Daniel-Vedele J, David L, Rebecca D (2014) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends in Plant Science 19(1): 5-9 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Lin SH, Kou HF, Canivenc G, Lin CS, Lepetit M, Hsu PK, Tillard P, Lin HL, Wang YY, Tsai CB, Gojon A, Tsay YF (2008) Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. The Plant Cell 20:2514-2528 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Li JY, Fu YL, Pike SM, Bao J, Tian W, Zhang Y, Chen CZ, Zhang Y, Li HM, Huang J, Li LG, Schroeder J, Gassmann W, Gong JM (2010) The arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. The Plant Cell 22: 1633-1646 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Li JS, Yang HB, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards E, Krizek B, Murphy AS, Gilroy S, Gaxiola R (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310 (121): 121-125 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Luo JK, Sun SB, Jia LJ, Chen W, Shen QR (2006) The mechanism of nitrate accumulation in Pakchoi [Brassica campestrisL.ssp.Chinensis (L.)]. Plant and Soil 282:291-300 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Ma JF, Ueno D, Zhao FJ, McGrath SP (2005) Subcellular localization of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspicaerulescens.Planta 220: 731-736 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Martinoia E, Massonneau A, Frangne N (2000) Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiology 41(11):1175-1186 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Martinoia E, Heck U, Wiemken A (1981) Vacuoles as storage compartments for nitrate in barley leaves. Nature 289: 292-294 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Miller AJ, Smith SJ (2008) Cytosolic nitrate ion homeostasis, could it have a role in sensing nitrogen status. Annals of Botany,101:485-489. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Robert S, Zouhar J, Carter C, Raikhel N (2007) Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts. Nature Protocols 2(2):259-262 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Schroeder JL, Delhaize E, Frommer WB, Guerinot ML, Harrison MJ, Herrera-Estrella L, Horie T, Kochian LV, Munns R, Nishizawa NK, Tsay YF, Sanders D (2013) Using membrane transporters to improve crops for sustainable food production. Nature 497: 60-66 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Shi WM, ,Xu WF, Li SM, Zhao XQ, Dong GQ (2010) Responses of two rice cultivars differing in seedling-stage nitrogen use efficiency to growth under low-nitrogen conditions. Plant and Soil 326:291-302 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Shen QR, Tang L, Xu YC (2003) A review on the behavior of nitrate in vacuoles of plants. ActaPedologicaSinica40(3):465-470. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Smirnoff N, Stewart G (1985) Nitrate assimilation and translocation by higher plants: Comparative physiology and ecological consequences. Physiologia Plantarum 64: 133-140 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Tang TF, Sun XC, Hu CX, Tan QL, Zhao XH (2013) Genotypic differences in nitrate uptake, translocation and assimilation of two Chinese cabbage cultivars [Brassica campestris L. ssp. Chinesnsis (L.)]. Plant Physiology and Biochemistry70:14-20 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Tang Z, Fan XR, Li Qing, Feng HM, Miller AJ, Shen QR, Xu GH (2012) Knockdown of a rice stellar nitrate transporter alters longdistance translocation but not root influx. Plant Physiology 160:2052-2063 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Vogeli-Lange R, Wagner GJ (1990) Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves: implication of a transport function for cadmium-binding peptides. Plant Physiology 92:1086-1093 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Wang GL, Ding GD, Li L, Cai HM, Ye XS, Zou J, Xu FS (2014) Identification and characterization of improved nitrogen efficiency in interspecific hybridized new-type Brassica napus. Annals of Botany 114(3): 549-559 Pubmed: Author and Title CrossRef: Author and Title Downloaded Google Scholar: Author Only Title Only Author and Title from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved. Wang YY, Tsay YF (2011) Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport. The Plant Cell 23:19451957 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Wege S, Angeli AD, Droillard MJ, Kroniewicz L, Merlot S, Cornu D, Gambale F, Martinoia E, Barbier-Brygoo H, Thomine S, Leonhardt N, Filleur S (2014) Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid. Science Signalling 7(333), ra65 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Wilkinson S, Bacon MA, Davies WJ (2007) Nitrate signaling to stomatal and growing leaves: interactions with soil drying, ABA, and xylem sap pH in maize. Journal of Experimental Botany 58(7): 1705-1716 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Xu GH, Fan XR, Miller AJ (2012) Plant nitrogen assimilation and use efficiency. The Annual Review of Plant Biology 63:153-182 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Yandeau-Nelson MD, Laurens L, Shi Z, Xia H, Smith AM, Guiltinan MJ (2011) Starch-Branching Enzyme IIa Is Required for Proper Diurnal Cycling of Starch in Leaves of Maize [OA]. Plant Physiology 156:479-490 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Zhang GB, Yi HY, Gong JM (2014) The Arabidopsis Ethylene/Jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaption. The Plant Cell 26(10):3984-3998 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Zhang ZH, Song HX, Liu Qing, Rong XM, Xie GX, Peng JW, Zhang YP (2009) Study on differences of nitrogen efficiency and nitrogen response in different oilseed rape (Brassica napus L.) varieties. Asian Journal of Crop Science 1(2):105-112 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Zhang ZH, Song HX, Liu Q, Rong XM, Peng JW, Xie GX, Zhang YP, Guan CY (2010) Nitrogen redistribution characteristics of oilseed rape varieties with different nitrogen use efficiencies during later growth period. Communications in Soil Science and Plant Analysis 41(14): 1693-1706 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Zhang ZH, Huang HT, Song HX, Liu Q, Rong XM, Peng JW, Xie GX, Zhang YP, Guan CY (2012) Research advances on nitrate nitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency. Australian Journal of Crop Science 6(9): 1377-1382 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Zhu ZJ, Qian YR, Pfeiffer W (2001) Effect of nitrogen form on the activity of tonoplast pyrophosphatase in tomato roots. Acta Botanica Sinica 43(11): 1146-1149 Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title Downloaded from on July 31, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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