Nitrogen use efficiency is mediated by vacuolar nitrate sequestration

Plant Physiology Preview. Published on January 12, 2016, as DOI:10.1104/pp.15.01377
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Running Title: Nitrate allocation and nitrogen use efficiency
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Corresponding author: Zhen-hua Zhang
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Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China,
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National Engineering Laboratory for Efficient Utilization of Soil and Fertilizer Resources,
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Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources
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Use, Hunan Provincial Key Laboratory of Plant Nutrition in Common University,
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College of Resources and Environmental Sciences, Hunan Agricultural University, PR. China,
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[email protected]
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Title: Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots
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of Brassica napus
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Yong-Liang Han1*, Hai-Xing Song1*, Qiong Liao1*, Yin Yu1*, Shao-Fen Jian1*, Joe Eugene Lepo3, Qiang
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Liu1, Xiang-Min Rong1, Chang Tian1, Jing Zeng1, Chun-Yun Guan1,2, Abdelbagi M. Ismail4, Zhen-Hua
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Zhang1§
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1 Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Hunan Agricultural
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University, Changsha, China;
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2 National Center of Oilseed Crops Improvement, Hunan Branch, Changsha, China;
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3 Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola,
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Florida, 32514, United States of America;
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4 Crop and Environment Sciences Division, International Rice Research Institute, DAPO 7777, Metro
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Manila, Philippines
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Decrease in VSC of NO3- in roots will enhance transport to shoot and essentially contribute to
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higher NUE by promoting NO3- allocation to aerial parts
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Footnotes:
We thank Dr. Ji-Ming Gong (Shanghai Institute of Plant Physiology and Ecology,
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Shanghai Institutes for Biological Sciences) for providing nrt1.5-3and nrt1.8-2 seeds. This
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study was supported by the National Natural Science Foundation of China (Grant
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No.31101596, 31372130); Hunan Provincial Recruitment Program of Foreign Experts;
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National Oilseed Rape Production Technology System of China; “2011 Plan” supported by
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The Ministry of Education in China; Open Novel Science Foundation of Hunan province
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(13K062). National Key Laboratory of Plant Molecular Genetics and the "Twelfth Five-Year"
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National Science and technology support program (2012BAD15BO4).
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* These
authors have contributed equally
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§ Corresponding
author: e-mail: [email protected]
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Author contributions:
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ZHZ conceived the original screening and research plans; ZHZ supervised the experiments;
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YLH, HXS, QL, YY, SFJ and ZHZ performed most of the experiments; CT and JZ provided
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technical assistance to YLH, HXS, YY and ZHZ; YLH and ZHZ designed the experiments
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and analyzed the data; QL, XMR, CYG and ZHZ interpreted the results, and JEL, AI and
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ZHZ conceived the project and wrote the article with contributions of all the authors; JEL, AI
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and ZHZ supervised and complemented the writing. All authors read and approved the final
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manuscript.
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Abstract
Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target
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to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the
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environment. In Arabidopsis thaliana, allocation of more NO3- to shoots was associated with
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higher NUE, however, the commonality of this process across plant species have not been
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sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE.
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We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were
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significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE
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genotype (814); and consequently, less vacuolar NO3- was retained in roots of Xiangyou15.
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Moreover, NO3- concentration in xylem sap, [15N] shoot:root (S:R) and [NO3-] S:R ratios
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were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of
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Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus
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treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity,
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vacuolar sequestration capacity (VSC) of NO3- in roots substantially decreased. Expression of
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NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3-
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long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton
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pumps was also significantly higher than in the wild type col-0. Taken together, these data
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suggest that decrease in VSC of NO3- in roots will enhance transport to shoot and essentially
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contribute to higher NUE by promoting NO3- allocation to aerial parts, likely through
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coordinated regulation of NRT1.5 and NRT1.8.
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Key words: Nitrogen use efficiency; Nitrate allocation; Proton-pumps; Vacuoles
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Introduction
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China is the largest consumer of nitrogen (N) fertilizer in the world, however, the
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average N-fertilizer use efficiency (NUE) is only around 35%, suggesting considerable
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potential for improvements (Shen et al., 2003; Wang et al., 2014). With the high amounts of
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N-fertilizer being used, crop yields are declining in some areas, where application is
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exceeding the optimum required for local field crops (Shen et al. 2003; Miller et al., 2008;Xu
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et al., 2012). The extremely low NUE results in waste of resources and environmental
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contamination, and also presents serious hazards for human health (Xu et al., 2012; Chen et
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al., 2014). Consequently, exploiting the maximum potential for improving NUE in crop plants
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will have practical significance for agriculture production and the environment (Zhang et al.,
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2010; Schroeder et al., 2013; Wang et al., 2014). Elucidating the genetic and physiological
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regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with
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higher NUE.
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Ammonium (NH4+) and nitrate (NO3-) are the main N species absorbed and utilized by
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crops, and NO3- accumulation and utilization are of major emphasis for N nutrient studies in
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dry land crops, such as Brassica napus. Several studies revealed the close relationship
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between NO3- content and NUE in plant tissues (Shen et al., 2003; Zhang et al., 2012; Tang et
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al., 2013;Han et al., 2015a).When plants are sufficiently illuminated, NO3- assimilation
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efficiency significantly increase in shoots compared with roots (Smirnoff et al., 1985; Tang et
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al., 2013). Consequently, under daytime with optimal illumination, higher proportion of NO3-
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in plant tissue is transported from root to shoot, as an advantageous physiological adaptation
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that reduces the cost of energy for metabolism (Tang et al., 2013). NO3- assimilation in plant
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shoots can therefore, take advantage of solar energy, while improving NUE (Smirnoff et al.,
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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
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two genes encoding long transport mechanisms. NRT1.5 is responsible for xylem NO3-
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loading, while NRT1.8 is responsible for xylem NO3- unloading (Lin et al., 2008; Li et al.,
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2010). Expression of the two genes is influenced by NO3- concentration. NRT1.5 is strongly
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induced by NO3- (Lin et al., 2008), while NRT1.8 expression is extremely up-regulated in
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nrt1.5 mutants (Chen et al., 2012). A negative correlation between the extents of expression of
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the two genes was observed when plants are subjected to abiotic stresses (Chen et al., 2012).
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Moreover, expression of NRT1.5 is strongly inhibited by 1-aminocyclopropane-1-carboxylic
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acid (ACC) and methyl jasmonate (MeJA), whereas the expression of NRT1.8 is significantly
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up-regulated (Zhang et al., 2014). Based on these studies we argue that, the expression and
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functioning of NO3- long-distance transport genes NRT1.5 and NRT1.8, are regulated by
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cytosolic NO3- concentration. In addition, the vacuolar and cytosolic NO3- distribution is
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likely regulated by proton-pumps located within the tonoplast (V-ATPase and V-PPase)
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(Granstedt et al., 1982; Glass et al., 2002; Krebs et al., 2010). Therefore, NO3- use efficiency
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must be affected by NO3- long-distant transport (between shoot and root), and short-distant
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transport (between vacuole and cytosol). However the physiological mechanisms controlling
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this regulation are still obscure.
Previous studies showed that the chloride channel protein (CLCa) is mainly responsible
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for vacuole NO3- short-distance transport, as it is the main channel for NO3-movement
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between the vacuoles and cytosol (Angeli et al., 2006; Wege et al., 2014). The vacuole
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proton-pumps (V-ATPase and V-PPase) located in the tonoplast, supply energy for active
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transport of NO3- and accumulation within the vacuole (Gaxiola et al., 2001; Brux et al., 2008;
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Krebs et al., 2010). Despite that about 90% of the volume of mature plant cells is occupied by
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vacuoles, vacuolar NO3- cannot be efficiently assimilated because the enzyme nitrate
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reductase (NR) is cytosolic (Shen et al., 2003; Han et al., 2015a). However, re-translocation of
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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
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of 30-50 mol m-3 and 3-5 mol m-3; respectively (Martinoia et al., 1981; Martinoia et al., 2000).
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Because vacuoles are obviously the organelle for high NO3- accumulation and storage in plant
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tissues, their function in NO3- use efficiency cannot be ignored (Martinoia et al., 1981; Zhang
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et al., 2012; Han et al., 2015b). NO3- assimilatory system in the cytoplasm is sufficient for its
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assimilation when it is transported out of the vacuoles. Therefore, NO3- use efficiency could in
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part, be dependent on vacuolar-cytosolic NO3- short-distance transport in plant tissues
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(Martinoia et al., 1981; Shen et al., 2003; Zhang et al., 2012; Han et al., 2015a).
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Evidently, NO3- use efficiency is regulated by both NO3- long-distance transport from
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root to shoot and short-distance transport and distribution between vacuoles and cytoplasm
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within cells(Glass et al., 2002; Dechorgnat et al., 2011; Han et al., 2015a). Although vacuoles
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compartment excess NO3- that accumulates in plant cells (Granstedt et al., 1982; Krebs et al.,
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2010), neither NO3- inducible NR genes (NIA1and NIA2) (Fan et al., 2007; Han et al., 2015a)
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nor the NO3- long-distance transport gene NRT1.5 (Lin et al., 2008), are regulated by vacuolar
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NO3-, even though they are essential for NO3- assimilation. Only NO3- transported from the
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vacuole to the cytosol can play a role in regulating NO3- inducible genes. Consequently we
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argue that both NO3- assimilation in cells and its long-distance transport from root to shoot are
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regulated by cytosolic NO3- concentration. However, this hypothesis needs to be substantiated.
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The mechanisms underlying both NO3- short-distance (Gaxiola et al., 2001; Angeli et al.,
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2006; Brux et al., 2008; Krebs et al., 2010), and long-distance transport (Lin et al., 2008; Li et
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al., 2010) have been previously investigated, yet the underlying mechanisms regulating the
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flux of NO3- and the obvious relationship between the two transport pathways, as well as their
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relation to NUE are not well understood.
The NRT family of genes play a partial role in vacuolar NO3- accumulation in petioles
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(Chiu et al., 2004) and seed tissues (Chopin et al., 2007), whereas the proton pumps and
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CLCa system in the tonoplast play a major role in accumulating NO3- in vacuoles (Gaxiola et
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al., 2001; Angeli et al., 2006; Brux et al., 2008; Krebs et al., 2010). The vacuolar NO3-
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short-distance transport system is spread throughout the plant tissues and is the principal
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means by which vacuolar NO3- short-distance transport and distribution is controlled (Angeli
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et al., 2006;Krebs et al., 2010).
The NRT genes seem to work synergistically to control NO3- long-distance transport
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between roots and shoots. NRT1.9 is responsible for NO3- loading into the phloem (Wang et
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al., 2011), whereas NO3- loading and unloading into xylem are regulated by NRT1.5 and
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NRT1.8, respectively (Lin et al., 2008; Li et al; 2010). Phloem transport mainly involves
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organic N; the inorganic-N (NO3-) concentrations in the phloem sap are typically very low,
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ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al.,
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2008;Fan et al., 2009). Therefore, this study focused on NO3- short-distance transport
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mediated through the tonoplast proton-pumps and the CLCa system, and the long-distant
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transport mechanisms responsible for xylem NO3- loading and unloading via NRT1.5 and
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NRT1.8, respectively.
Questions related to how long and short-distance transport of NO3- are coupled in plant
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tissues and their role in determining NUE were addressed using a pair of high- and low-NUE
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B. napus genotypes and Arabidopsis thaliana. Application of proton pump inhibitors and
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ACC in the former, and use of mutants with defective proton pumps in the latter, allowed
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experimental distinction of the physiological mechanisms regulating these processes. Data
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presented here provide strong evidence from both model plants supporting this linkage and
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strongly suggest that cytosolic NO3- concentration in roots regulates NO3- long-distance
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transport from roots to shoots. We also investigated how NO3- concentration in plant tissues
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would be affected by NO3- long-distance transport, vacuolar NO3- sequestration; and the
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ensuing relationship with NO3- use efficiency. We also proposed the physiological
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mechanisms likely to be important for enhancing NO3- use efficiency in plants. These findings
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will provide scientific rationales for improving NUE in important industrial and food crops.
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Results
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B. napus with higher NUE showed lower vacuolar sequestration capacity (VSC) for NO3-
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in root tissues
Our previous work identified high and low NUE B. napus genotypes (Han et al., 2015a;
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Han et al., 2015b). NUE of B. napus, whether based on biomass or on grain yield (Table 1),
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was significantly higher for the high-NUE genotype (H: Xiangyou15) than for the low-NUE
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genotype (L: 814). That the activities of the tonoplast proton-pumps (V-ATPase and V-PPase)
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of root tissues in the H genotype were lower than the L genotype at both seedling (Fig. 1A)
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and flowering stages (Fig. S1A). Given that proton pumps in the tonoplast supply energy for
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vacuolar NO3- accumulation (Krebs et al., 2010), NO3- influx into the vacuole was
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consequently much slower in the H genotype compared with the L genotype at both seedling
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(Fig. 1B) and flowering stages (Fig. S1B). Moreover, the percentage of vacuolar NO3- relative
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to the total NO3- in protoplasts of root tissues in the H genotype was lower than in the L
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genotype at seedling (Fig. 1C) and flowering stages (Fig. S1C); and NO3- accumulation in the
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cytosol increased significantly in the H genotype compared with the L genotype at seedling
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(Fig. 1D) and flowering stages (Fig. S1D).
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B. napus with higher NUE showed enhanced long-distance transport of NO3- from roots
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to shoots
The relative expression of BnNRT1.5 in roots of the H genotype was significantly higher
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than that in the L genotype at both seedling and flowering stages, while the relative expression
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of BnNRT1.8 in roots of the H genotype was lower than that in the L genotype at both
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seedling (Fig. 2-A,B) and flowering stages (Fig. S2-A,B). As a consequence, total N
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concentration (traced by 15N) in roots of the H genotype was significantly lower than that of
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the L genotype at both seedling (Fig. 2C) and flowering stages (Fig. S2C), while shoot N
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concentration in the H genotype was significantly higher than the L genotype at seedling stage
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(Fig. 2C). This resulted in significantly higher [15N] S:R ratio in the H genotype compared
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with the L genotype (Fig. 2D; Fig. S2D).
No significant differences in total N per plant between the H and L genotypes were
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observed at both seedling and flowering stages (Fig. S3). However, NO3- concentration in
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roots of the H genotype was significantly lower than in the L genotype at both seedling and
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flowering stages (Fig. 2E; Fig. S2E), while NO3- concentration in shoot tissues of this
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genotype was significantly higher at seedling stage (Fig. 2E), resulting in higher [NO3-] S:R
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ratios at both stages (Fig. 2F; Fig. S2F).
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NO3- concentration in the xylem sap, xylem sap volume and total NO3- in xylem sap
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were significantly higher in the H genotype than in the L genotype at seedling stage (Fig.
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S4A,C,E); the amount of NO3- in xylem sap significantly increased in the H genotype relative
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to the L genotype at flowering stage (Fig. S4F). Together, these data suggests greater
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mobilization of NO3- from root to shoot in the H genotype.
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NO3- up-regulates NRT1.5, but down-regulates NRT1.8
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A previous study showed that increased NRT1.8 expression in nrt1.5 mutants (Chen et al.,
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2012) and the expression of NRT1.5 was induced by NO3- (Lin et al., 2008). We tested the
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relationship between NRT1.5 and NRT1.8 expression in A. thaliana and B. napus. No
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significant differences were observed in AtNRT1.5 expression in roots between wild type
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(col-0) and mutant plants (nrt1.8-2) (Fig. 3A). However, AtNRT1.5 expression was
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significantly up-regulated by NO3- in roots of nrt1.8-2 mutants (Fig. 3B). Expression of
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AtNRT1.8 significantly increased in roots of nrt1.5-3 mutants, but not in col-0 plants, and
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there were no significant differences in AtNRT1.8 expression with or without NO3- treatment
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in roots of nrt1.5-3 mutants (Fig. 3C,D).
In contrast, BnNRT1.5 expression in roots of the H genotype increased significantly with
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NO3- treatment (Fig. 3E), while the reverse was observed in expression of BnNRT1.8, which
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showed lower expression under NO3- treatment than control (Fig. 3F). This suggests that NO3-
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induces the expression of NRT1.5, but down-regulates NRT1.8 in A. thaliana and B. napus.
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Further studies are needed to elucidate the mechanisms regulating this reverse regulation.
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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
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(DCCD + Na2SO3) inhibits V-PPase; and a 1:1 combination of Bafi and DCCD (B+D)
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inhibits both V-ATPase and V-PPase (Han et al., 2015a). These inhibitors were used to
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control activities of the tonoplast proton pumps in the H B. napus plants. V-ATPase activity
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significantly decreased under Bafil and B+D treatments relative to the control and DCCD
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treatments, whereas V-PPase activity declined significantly under DCCD and B+D treatments
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relative to that in the control and Bafil treatments (Fig. 4A).
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The V-ATPase activity in roots of A. thaliana was significantly lower in the V-ATPase
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mutants (vha-a2 and vha-a3) than the wild type (col-0) and V-PPase mutants (avp1) (Fig. 5A).
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In contrast, the V-PPase activity in roots of V-PPase mutant (avp1) were significantly lower
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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
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with inhibitors of proton pumps (Bafil, DCCD, B+D) (Fig. 4B). Similarly, NO3- influxes into
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the vacuole of the Arabidopsis mutants (vha-a2, vha-a3 and avp1) was significantly lower
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than those found in the wild type (col-0) (Fig. 5B). Previous studies showed that VSC of NO3-
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decreased when the activities of the proton pumps decline (Li et al., 2005; Krebs et al., 2010;
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Han et al., 2015a).
We further investigated NO3- distribution between the vacuole and cytosol as affected by
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proton pump inhibition in the H genotype of B. napus and the mutants of A. thaliana
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defective in vacuolar proton pumps. The percentage of vacuolar NO3- relative to total NO3- in
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root protoplasts of the H genotype treated with inhibitors (Bafil, DCCD, B+D) was lower than
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that observed in the control (Fig. 4C). Results were also similar when using A. thaliana
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mutants, where the percentage of vacuolar NO3- relative to the total NO3- in protoplasts was
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lower in the mutants (vha-a2, vha-a3 and avp1) than in the wild type (col-0) (Fig. 5C).
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Consequently, NO3- accumulation in the cytosol showed a significant increase when energy
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pumps were suppressed in roots of the H genotype (Fig. 4D); and NO3- accumulation in the
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cytosol of root tissues of A. thaliana mutants (vha-a2, vha-a3 and avp1) similarly increased,
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compared with col-0 (Fig. 5D).
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Based on previous observations (Lin et al., 2008), expression of NRT1.5 is strongly
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induced by NO3-, but expression of NRT1.8 is down-regulated (Fig.3; Chen et al., 2012). We
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then hypothesized that expression of these two genes is contrastingly regulated by
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concentration of NO3- in the cytosol. Our results were congruent with this hypothesis, the
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expression of BnNRT1.5 in root tissues of the H genotype of B. napus was significantly higher
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in plants treated with Bafil, DCCD or B+D, compared with the control (Fig. 4E), whereas the
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expression of BnNRT1.8 decreased substantially (Fig. 4F). Similar results were also observed
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in A. thaliana, where expression of AtNRT1.5 in roots of the mutants (vha-a2, vha-a3 and
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avp1) were significantly higher than in the wild type (col-0), but expression of AtNRT1.8 in
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the same mutants were considerably lower (Fig. 5E,F).
NO3- concentrations in the xylem sap, the N-distribution between shoot and root (S:R
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ratios based on [15N]) and the [NO3-] in shoots relative to roots of the H genotype treated with
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energy pumps’ inhibitors, were significantly higher than in the control (Fig. 4G,H; Fig. S5A).
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Similar trends were also observed when using mutants of A. thaliana deficient in the energy
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pumps’ activities. NO3- concentration in the xylem sap, the [15N] S:R ratios and [NO3-] S:R
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ratios were all significantly higher in the mutants than in the wild type (Fig. 5G,H; Fig. S5B).
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These data clearly showed that prevention of N sequestration in vacuoles would enhance its
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translocation to shoot.
Additional evidence linking NO3- long-distance transport and NO3- short-distance
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distribution within cells were provided through experiments comparing A. thaliana wild type
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(Ws) and mutant clca-2 (Fig. S6). The chloride channel (CLCa) is the main channel for
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vacuolar anion accumulation. Vacuolar sequestration capacity of NO3-significantly declines in
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clca mutants (Angeli et al., 2006). NO3- influx into the vacuolar space of Ws roots was
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significantly higher than that observed in the clca-2 mutants (Fig. S6A). This resulted in a
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smaller proportion of vacuolar NO3- in clca-2 plants relative to the total NO3- in root tissue
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protoplasts, as compared with Ws (Fig. S6B). Consequently, the accumulation of NO3- in the
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cytosol was significantly higher in clca-2 than in Ws plants (Fig. S6C). The higher
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NO3-concentration in the cytosol together with the higher expression of AtNRT1.5, coupled
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with lower expression of AtNRT1.8 in clca-2 roots (Fig. S6D,E); resulted in significant
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increase in xylem sap NO3- concentration, [15N] S:R ratios and [NO3-] S:R ratios compared
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with Ws plants (Fig. S6F,G,H).
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Increased NO3- translocation to shoots enhanced NUE
NO3- assimilation efficiency is known to be higher in shoots than in roots (Smirnoff et al.,
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1985; Andrews et al., 1986; Tang et al., 2012; Tang et al., 2013). We therefore hypothesize
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that, increased NO3- translocation to shoot and the consequent higher shoot:root ratio will
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contribute to higher NUE. Our data support this hypothesis. The H B. napus genotype showed
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enhanced long-distance transport of NO3- from roots to shoots (Fig. 2; Fig S2). This enhanced
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NO3- transport requires higher carbon skeleton provided through higher photosynthetic rate
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(Tang et al., 2012; Tang et al., 2013). Our results showed that chlorophyll content,
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intercellular CO2 concentration and photosynthetic rate were significantly higher in the H than
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the L genotype at both seedling and flowering stages (Table 2). Moreover, the NO3-
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assimilating enzymes were strongly induced by NO3-, providing sufficient capacity for N
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assimilation (Smirnoff et al., 1985; Andrews et al., 1986). Nitrate reductase (NR) and
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glutamine synthetase (GS) activities in roots of the H genotype were significantly lower than
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in the L genotype both at seedling and flowering stages (Fig. S7). In contrast, NR activities in
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shoots of the H genotype were significantly higher than in the L genotype at both stages (Fig.
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S7A,B); and GS activity in the H genotype was also higher than those of the L genotype at
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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).
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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