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First published online May 31, 2007; 10.1105/tpc.107.050542

The Plant Cell 19:1590-1602 (2007)
© 2007 American Society of Plant Biologists

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The Arabidopsis ATNRT2.7 Nitrate Transporter Controls Nitrate Content in Seeds[W]

Franck Chopina, Mathilde Orselb, Marie-France Dorbea, Fabien Chardona, Hoai-Nam Truonga, Anthony J. Millerc, Anne Krappa and Françoise Daniel-Vedelea,1

a Institut National de la Recherche Agronomique, Institut Jean-Pierre Bourgin, Unité de la Nutrition Azotée des Plantes, F-78000 Versailles, France
b Amélioration des Plantes et Biotechnologies Végétales, Unité Mixte de Recherche 118, Institut National de la Recherche Agronomique–AgroCampus Rennes, 35653 Le Rheu Cedex, France
c Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom

1 To whom correspondence should be addressed. E-mail vedele{at}versailles.inra.fr; fax 33-3083-3096.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. ß-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Plant seeds are important for two reasons: one is to support human life as they are a vital component of man's diet, and the other is to ensure the successful propagation of the species. For the latter function, seeds must contain all the genetic material, macronutrients, and micronutrients needed to allow efficient germination and seedling establishment, even if the external environment is hostile. Seed composition and subsequent germination depend on the environmental conditions under which the seeds are produced and are controlled by developmental and hormonal signals. In addition, metabolic control can influence embryo development and seedling establishment. For example, disruption of the ACC1 gene, which codes for acetyl-CoA carboxylase, was lethal for the embryo (Baud et al., 2003Go), and mutants affected in key components of the glyoxylate cycle have demonstrated the importance of fatty acid metabolism for seedling establishment under nonfavorable conditions (Eastmond et al., 2000Go). Much research has been focused on the metabolism of major seed compounds, such as starch in cereals (James et al., 2003Go) and carbon and lipid in noncereals (Baud et al., 2002Go; Hills, 2004Go). Recently, a global metabolomic analysis was performed in the model species Arabidopsis thaliana (Fait et al., 2006Go), which, together with global transcriptomic analyses in the same species (Ruuska et al., 2002Go; Nakabayashi et al., 2005Go), gives an integrated view of seed development from early embryo morphogenesis through maturation to the early steps of germination. Surprisingly, there have been far fewer investigations of seed mineral accumulation. Seeds store minerals in the form of phytate and its cations, mostly Mg2+, K+, and Ca2+ (Lott et al., 1995Go), in two types of vacuoles and in the endoplasmic reticulum (Otegui et al., 2002Go). The seed iron is stored either as plastid ferritin in legume seeds (Lobreaux and Briat, 1991Go) or in globoids in the protein storage vacuoles of Arabidopsis (Lanquar et al., 2005Go). By contrast, nothing is known about the localization and the molecular basis of nitrate storage in seeds. N from nitrate represents less than one thousandth of seed nitrogen (our unpublished data), but endogenous nitrate levels can vary enormously even among seed batches from one species (Derckx and Karssen, 1993Go). In Sisymbrium officinale, seed nitrate was shown to be a necessary and limiting factor for light-induced germination in water (Hilhorst, 1990Go) and to be present both in seed coats (including the endosperm) and in the embryo, particularly in the axes and the radicle. The important role of nitrate alone or in combination with other factors, such as light, gibberellins, or ethylene, in breaking dormancy has also been shown in different species (Saini et al., 1985Go; Hilhorst and Karssen, 1988Go). It was recently demonstrated that the effect of nitrate in breaking dormancy in Arabidopsis is not nutritional but rather a signaling effect, whether it is provided exogenously during imbibition or accumulated during seed development (Alboresi et al., 2005Go). This effect may be of importance in agriculture because seed dormancy is generally an undesirable trait for the establishment of crops. However, a complete lack of dormancy may lead to germination of grains while still on the ear of the parent plant (preharvest sprouting), resulting in major losses to agriculture.

During plant vegetative growth, nitrate is taken up from soil solution by active transport across the plasma membrane of root cells. As a nutrient, nitrate can be reduced inside the cell by the nitrate reductase into nitrite, which is then further reduced into ammonium by nitrite reductase (Meyer and Stitt, 2001Go). To cope with low or high nitrate concentrations in soils, two uptake systems exist within plant roots: high-affinity nitrate transport systems (HATS) and low-affinity nitrate transport systems (LATS) (Glass and Siddiqi, 1995Go). During recent years, several genes involved in LATS and HATS have been isolated. On the basis of their deduced amino acid sequence, the corresponding proteins have been classified in two families: NITRATE TRANSPORTER1 (NRT1) and NRT2. The Arabidopsis genome contains 53 NRT1 and seven NRT2 family members (Arabidopsis Genome Initiative, 2000Go; Orsel et al., 2002aGo, 2002bGo).

The first gene isolated, ATNRT1.1, has been shown to be a dual-affinity transporter involved in both low- and high-affinity nitrate uptake (Liu et al., 1999Go), the molecular control of this switch being a protein phosphorylation/dephosphorylation mechanism (Liu and Tsay, 2003Go). Until now, the best characterized member of ATNRT2 family is the ATNRT2.1 gene, which was isolated either by a differential display approach (Filleur and Daniel-Vedele, 1999Go) or using degenerate primers (Zhuo et al., 1999Go). Analysis of the mutant atnrt2.1-1, disrupted in the ATNRT2.1 and ATNRT2.2 genes, showed that this plant is specifically deficient in the HATS but not the LATS (Cerezo et al., 2001Go; Filleur et al., 2001Go). Recent studies showed that the ATNRT2.1 protein requires a second gene, ATNAR2.1, for function (Okamoto et al., 2006Go) and possibly forms a complex with the NAR2 protein to transport nitrate efficiently (Orsel et al., 2006Go). Based on the sequence homology between to the ATNRT2.1 coding sequence, six other genes with various degrees of homology were identified (Orsel et al., 2002aGo). Except for the ATNRT2.2 gene (Li et al., 2007Go), the function of the other NRT2 genes has not been elucidated. Regarding the nitrate regulation of expression, some of the genes were found to be inducible (ATNRT2.1 and ATNRT2.2), while another was repressible (ATNRT2.5) (Okamoto et al., 2003Go). By contrast, the expression of the ATNRT2.3, ATNRT2.6, and ATNRT2.7 genes seemed to be insensitive to changes in nitrate supply. The ATNRT2 genes chiefly show expression in the roots, with the exception of ATNRT2.7, whose expression is higher in shoots than in roots (Orsel et al., 2002aGo; Okamoto et al., 2003Go). In this study, we focus on the ATNRT2.7 gene, and using several different approaches, we demonstrate its importance for the efficient storage of nitrate in seed vacuoles that can then influence the kinetics of seed germination.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
Expression of ATNRT2.7 in Different Organs
The AtGenExpress initiative, supported by different countries (Germany, Japan, the United States, and the UK), compiles data from >1300 microarrays and can be used as a tool to follow the expression of your favorite gene. When we analyzed these in silico data, ATNRT2.7 appeared to be highly expressed in seeds. The expression of ATNRT2.7 increases by a factor of 6.5 between the 7th and 8th stages and a factor of 15 between the 7th and 10th stages of seed development (https://genevestigator-1.ethz.ch/). We thus decided to validate these results by real-time PCR analysis alongside all the other members of the NRT2 family.

Two different stages of seed development were tested together with root and shoot extracts to compare with our previous results (Orsel et al., 2002aGo). Results of both experiments are summarized in Table 1 for the ATNRT2.1 and ATNRT2.7 genes. As found previously, the ATNRT2.1 gene is predominantly expressed in roots as compared with shoots, while the ATNRT2.7 gene is more expressed in shoots. However, the most striking point is the huge amount of ATNRT2.7 mRNA in seeds (nearly 200% of the EF1{alpha} control gene), especially at the end of seed maturation when the tissue is completely dry. Interestingly, the expression decreases after 6 h of imbibition. With the exception of ATNRT2.5, which reaches only 6% of the EF1{alpha} level in dry seeds, we could not detect significant expression of any other NRT2 family members in this organ in two independent experiments (data not shown).


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  		Table 1. Gene Expression Level in Different Organs

 
To further characterize the expression of ATNRT2.7 in seeds, we have used a fusion between a 2-kb fragment of the ATNRT2.7 promoter and either the ß-glucuronidase (GUS) or green fluorescent protein (GFP) reporter gene. These constructs were stably introduced into the Arabidopsis Wassilewskija (Ws) ecotype, and Figure 1 shows typical results from GUS staining of seeds harvested from one of these transformants. After 1 h of imbibition to enable better separation of embryos from seed coats, histochemical GUS staining was performed (Figures 1A and 1B). A uniform and strong GUS activity was detected in transgenic embryos, while wild-type embryos remained unstained (Figure 1A). The seed coat is characterized by a faint blue staining, which reveals a weak expression of ATNRT2.7 in the surrounding endosperm (Figure 1B). Homozygous lines were also obtained carrying the GFP reporter gene under the control of the same 2-kb fragment of the ATNRT2.7 promoter. In these lines, embryos were also dissected after 1 h of water imbibition and observed using confocal microscopy (Figure 1C). Membranes were stained with FM4-64 (red dye; Figure 1D), making the embryonic cells clearly visible. Strong green fluorescence was observed in all tissues, including the cotyledons and radicles (Figure 1E). Altogether, these data suggest strongly that the expression of ATNRT2.7 is developmentally regulated and that ATNRT2.7 transcripts accumulate in seeds undergoing dessication.


Figure 1
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  		Figure 1. Expression of ProATNRT2.7 in Mature Seed after 1 h of Imbibition.

(A) and (B) The ProATNRT2.7:GUS reporter gene.

(A) Embryo without seed coat.

(B) Seed coat of the embryo.

(C) to (E) The ProATNRT2.7:GFP reporter gene, with green indicating GFP fluorescence. Red indicates FM4-64 staining. Bars = 150 µm.

(C) GFP signal.

(D) FM4-64 dye where cytoplasmic membrane is stained in red.

(E) Merged image of GFP and FM4-64 signals.

 
ATNRT2.7 Nitrate Transport Activity
A large number of NRT2 gene sequences are now available, and homologous sequences are found in three clades: mosses, gymnosperms, and angiosperms (5, 5, and 26 genes, respectively; see Supplemental Table 1 online). When the evolutionary relationship between these sequences and those of two NRT2 genes from algae was investigated using a Bayesian method, the NRT2 genes appear to be grouped into well distinct classes for algae, mosses, and seed plants (Figure 2 ). In the seed plant clade, the NRT2 genes appeared to be clustered into two monophyletic groups, Group A (NRT2.1, NRT2.2, NRT2.3, NRT2.4, and NRT2.6) and Group B (NRT2.5 and NRT2.7). This phylogenetic analysis highlights the fact that the NRT2 genes that have been so far characterized as nitrate transporters in Arabidopsis (Filleur et al., 2001Go; Li et al., 2007Go), Nicotiana plumbaginifolia (Fraisier et al., 2000Go), or Hordeum vulgare (Tong et al., 2005Go) belong to Group A. The NRT2.7 gene is classified in a distinct monophyletic group within this best-characterized family of plant nitrate transporters.


Figure 2
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  		Figure 2. Rooted Phylogenetic Tree of the NRT2 Gene Family from Several Plant Species.

Support values for branches are shown and represent Bayesian posterior values. The NRT2 genes in seed plants were classed into monophyletic Groups A and B. Species abbreviations are as follows: At, Arabidopsis thaliana; Bn, Brassica napus; Cr, Chlamydomonas reinhardtii; Cs, Chlorella sorokiniana; Dc, Daucus carota; Fs, Fagus sylvatica; Gm, Glycine max; Hv, Hordeum vulgare; Sl, Solanum lycopersicum; Lj, Lotus japonicus; Np, Nicotiana plumbaginifolia; Nt, Nicotiana tabacum; Os, Oryza sativa; Pa, Phragmites australis; Pg, Picea glauca; Pp, Prunus persica; Ps, Physcomitrella patens; Pta, Pinus taeda; Ptr, Populus tremula; Ta, Triticum aestivum; Zm, Zea mays.

 
Thus, to evaluate the capacity of the ATNRT2.7 protein to transport nitrate, we introduced the ATNRT2.7 cDNA driven by the 35S promoter into the atnrt2.1-1 mutant background (Filleur et al., 2001Go). As a positive control, we used transgenic plants expressing the Np NRT2.1 gene under the control of the RolD promoter that has been shown to restore nitrate influx in the Arabidopsis mutant back to 90% of that shown by the wild type (mR2 clone; Filleur et al., 2001Go). Homozygous transgenic plants were grown for 6 weeks in a hydroponic system on 0.2 mM NO3–, a N-limiting condition for the atnrt2.1-1 mutant (Orsel et al., 2004Go). Root nitrate influx was measured at an external concentration of 0.2 mM using 15NO3– (atom% 15N: 99%). At this concentration, the difference in the HATS activity between the wild type and the atnrt2.1-1 mutant was largest (Filleur et al., 2001Go). In two independent experiments, the overexpression of the two genes (ATNRT2.7 and Np NRT2.1) was correlated with an increase in the root 15NO3– influx (Figure 3A ). This influx in the transgenic plants represents 160% of that measured for the atnrt2.1-1 mutant for both genes, indicating a similar extent of phenotypic recovery as HATS activity was restored in both types of plants. Quantitative RT-PCR analyses confirmed that this restoration of nitrate influx in the transgenic plants was not due to altered expression or compensation by other members of the NRT2 family (Table 2 ).


Figure 3
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  		Figure 3. ATNRT2.7 Can Transport Nitrate.

(A) Root 15NO3– influx in atnrt2.1-1 and in Pro35S:ATNRT2.7 and ProRolD:Np NRT2.1 overexpressors. Plants were grown on 0.2 mM NO3– solution. Root 15NO3– influx was measured with complete nutrient solution containing 0.2 mM 15NO3–. The values are means ± SD of five replicates (pooling three to five plants). Asterisks indicate statistically significant differences between overexpressors and mutant (P < 0.0001).

(B) Uptake of 15N nitrate into oocytes injected with water or mRNA mixtures as indicated. Oocytes were incubated for 16 h in ND96 solution at pH 8.0 enriched with 5 mM 15NaNO3. The delta 15N values are means ± SD for five oocytes. The delta 15N was calculated as described previously (Tong et al., 2005Go). The asterisk indicates a statistically significant difference between RNA and water-injected oocytes (P < 0.05).

 

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  		Table 2. Relative Gene Expression Level of ATNRT2 and Np NRT2.1 Genes in Roots of the atnrt2.1-1 Mutant Complemented by Pro35S:ATNRT2.7 or ProRolD:Np NRT2.1 Constructs

 
In addition, to confirm the hypothesis that ATNRT2.7 is a nitrate transporter, we used a heterologous expression system. In this experiment, Xenopus laevis oocytes were injected with nuclease-free water or ATNRT2.7 mRNA. After 3 d, oocytes were incubated in a solution enriched with 5 mM Na15NO3 (atom% 15N: 98%), and the 15N enrichment of individual oocytes was measured after 16 h. These measurements showed that ATNRT2.7 mRNA–injected oocytes took up significantly more nitrate than water-injected controls (Figure 3B).

We next investigated the effect of a mutation in the nitrate transporter ATNRT2.7. T-DNA mutants were isolated from two independent T-DNA libraries, in the Columbia (Col-8) and Ws backgrounds, and homozygous lines were selected, called atnrt2.7-1 and atnrt2.7-2, respectively (see Methods). These mutants are knockouts for the NRT2.7 gene (data not shown), but no difference in root nitrate influx mediated by either the HATS or the LATS was found in both types of plant, whether grown with limiting (0.2 mM) or nonlimiting (6 mM) nitrate supply (Table 3 ). Nitrate and free amino acid contents in roots and shoots of both mutants were also measured and were found to be identical to the wild type under both levels of nitrate supply (Table 3). Taken together, these results suggest that the ATNRT2.7 protein is a functional nitrate transporter, but, possibly because of its expression site, it is not involved in the direct uptake of nitrate from soil by the root system or in nitrate distribution within the vegetative organs.


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  		Table 3. Physiological Characterization of the atnrt2.7 Mutants

 
Nitrate Contents in Seeds of Different ATNRT2.7 Genotypes
Due to its specific expression pattern, we asked if this gene could be involved in nitrate accumulation in seeds. In Arabidopsis, a recent study showed that N and C metabolites are accumulated in seeds in a specific way during seed formation and maturation (Baud et al., 2002Go), but to our knowledge, only a few studies have been devoted to nitrate storage in Arabidopsis seeds (Derckx and Karssen, 1993Go).

Six independently isolated plant lines per genotype were grown together in the greenhouse under long days, on the same soil type and under nonlimiting N conditions (watered with 10 mM nitrate nutrient solution). Seeds were harvested from individuals after complete maturation and senescence of the plants. The results of seed nitrate content determinations are shown in Figure 4A . The atnrt2.7-1 seeds had only 35% less nitrate than Ws wild-type controls. This decreased seed nitrate phenotype was even greater in the Col-8 ecotype, where a statistically significant 70% reduction of nitrate content in the mutant was observed. As the seed weights are very similar in both the genotypes, these differences are observed in nitrate content whether it is expressed on a per seed or on a milligram/dry weight (DW) basis (data not shown).


Figure 4
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  		Figure 4. Nitrate Content in Seeds.

(A) Seed nitrate content of atnrt2.7-1 and atnrt2.7-2 mutants and their wild-type controls. Mother plants were grown on 10 mM NO3– solution (values are means ± SD of four replicates). Letters indicate the statistically significant different classes (P < 0.05).

(B) Nitrate content of seeds of Ws, Pro35S:ATNRT2.7 overexpressor in Ws background, and atnrt2.7-2 mutants. Mother plants were grown on 10 and 50 mM NO3– solution 2 weeks after bolting (values are means ± SD of four replicates). Letters indicate the statistically significant different classes (P < 0.05).

 
We next compared seed nitrate content in over- and underexpressing ATNRT2.7 plants. As the Pro35S:ATNRT2.7 plants were obtained in the Ws genotype, we increased the amount of nitrate in the nutrient solution up to 50 mM after flowering to enhance the small difference already observed on 10 mM between the mutant and Ws wild type (Figure 4A). Results of a typical experiment are shown in Figure 4B. As expected, the amount of nitrate in seeds of wild-type plants supplied with 50 mM is higher than with 10 mM nitrate (82 and 58 nmol/mg DW, respectively). When comparing seed nitrate content after either 10 to 50 mM nitrate supply, the amount in the atnrt2.7-2 mutant had increased only slightly, leading to twofold less nitrate compared with the wild type (Figure 4B), and this difference was statistically significant. These data support the idea that ATNRT2.7 plays a major role in seed nitrate accumulation and agrees with the result obtained when seed nitrate contents were compared between overexpressor and wild-type plants. Nitrate contents in Pro35S:ATNRT2.7-overexpressing plants reached 160 and 310% of the nitrate in Ws and atnrt2.7-2 seeds, respectively.

Germination Kinetics of the Different Genotypes
Nitrate was demonstrated to be a signal relieving seed dormancy in Arabidopsis (Alboresi et al., 2005Go). In this study, we used the same batch of seeds as those used for nitrate determination to test the impact of endogenous nitrate content on the seed germination of our different genotypes.

Freshly harvested seeds were sown on agar medium containing only distilled water, and the percentage of germination was scored at different times after sowing. In the same experiment, the viability of seeds was compared after stratification on media containing 1 mM nitrate, and in this case, the percentage of germination of all genotypes had increased to 100%. However, just 2 d after sowing on water, both mutants showed a delay in germination compared with their respective wild-type controls (Figure 5A ). The strongest phenotype was observed for the atnrt2.7-2 mutant, which displayed significantly fewer germinated seeds throughout the 7 d of measurements compared with its Ws control. By contrast, the Col-8 wild-type background showed less dormancy, and the germination difference with the corresponding atnrt2.7-1 mutant was only significantly different 2 d after sowing.


Figure 5
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  		Figure 5. Kinetics of Seed Germination during 7 d.

(A) Kinetics of germination of atnrt2.7 mutants during 7 d. Mother plants were grown on 10 mM NO3– solution. Values are means ± SD of four replicates, and letters indicate the statistically significant different classes (P < 0.05).

(B) Kinetics of germination of Ws, Pro35S:ATNRT2.7 overexpressor in Ws background, and atnrt2.7-2 during 7 d. Mother plants were grown on 50 mM NO3– solution (values are means ± SD of four replicates). Letters indicate the statistically significant different classes (P < 0.05).

 
This correlation between nitrate content and seed dormancy was further highlighted by measuring the kinetics of germination of freshly harvested seeds from over- and underexpressing plants. The same batches of seeds that had already been tested for their nitrate content (Figure 4B) were sown on water-containing medium, and germination was scored as described above (Figure 5B). As found when plants are grown on 10 mM nitrate, the percentage of germination of seeds of the atnrt2.7-2 mutant grown on 50 mM nitrate was always lower when compared with wild-type seeds. By contrast, seeds from the Pro35S:ATNRT2.7 overexpressors showed a significantly higher percentage of germinated seeds only at 2 d after sowing.

Subcellular Localization of the ATNRT2.7 Protein
To localize ATNRT2.7 at the subcellular level, we used a chimeric protein where GFP6 was fused to the N-terminal part of ATNRT2.7. The capacity of the GFP-tagged protein to transport nitrate was first tested. We compared the Pro35S:ATNRT2.7- with the Pro35S:GFP-ATNRT2.7-overexpressing lines in the atnrt21.1-1 mutant background and found similar effects on the root nitrate influx of 15NO3– and on the seed nitrate contents (Table 4 ). In addition, the dormancy of the overexpressors was tested, and GFP-tagged plants were less dormant than the wild type, like the Pro35S:ATNRT2.7 lines (data not shown). Altogether, these results demonstrate that the ATNRT2.7 protein was still active when it was fused to GFP.


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  		Table 4. Nitrate Transport Activity of the Pro35S:GFP-ATNRT2.7 Fusion

 
Confocal microscopy revealed an ATNRT2.7-dependent green fluorescent labeling that coincided with the tonoplast in the mature part of the root (Figure 6A ). This tonoplast localization was even more evident in the root tip (Figure 6D), where this subcellular compartment was present as little vacuoles that fused to give the large central vacuole. This localization was further confirmed by staining lipid membranes using the red fluorescent probe, FM4-64 (Figures 6B and 6E). By merging the two pictures, GFP and FM4-64, the tonoplast was clearly distinguishable in the root meristem (Figure 6F) and in the mature part of the root where formation of the main central vacuole occurs (Figure 6C). In embryos (Figure 6G), although GFP imaging in dry seeds led to a diffuse labeling (data not shown), 12 h of water imbibition allowed us to clearly distinguish two different intracellular patterns: a tonoplast localization surrounding the vacuoles that corresponded to seed protein bodies and another more punctuated localization (Figure 6H).


Figure 6
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  		Figure 6. Expression of Translational Fusion between ATNRT2.7 and the GFP Reporter Gene.

Green indicates GFP fluorescence, and red indicates FM4-64 staining.

(A) to (C) Epidermal cells in the mature root of 4-d-old Pro35S:GFP-ATNRT2.7 plantlets. The plasma membrane is stained red. Bars = 11.33 µm.

(D) to (F) Epidermal cells near the root tip of young plantlets. Bars = 7.18 µm.

(G) Overview of mature seeds after 12 h of imbibition. Bar = 142.82 µm.

(H) Cotyledon cells of an embryo after 30 min of FM4-64 staining. Bar = 6.9 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
The sequence of the Arabidopsis genome identified >600 predicted open reading frames that encode potential membrane-bound transporters (Arabidopsis Genome Initiative, 2000Go). These proteins were classified into different families of transporters each playing specific roles in uptake or the control of nutrient and metabolite distribution. The ATNRT2.7 protein is the NRT2 family member that shows the lowest degree of homology with the other NRT2 proteins (55% similarity with ATNRT2.1) (Orsel et al., 2002aGo; this work). The gene organ expression pattern and its absence of regulation by nitrate suggest that ATNRT2.7 is a specialized member of this family and implies that it has a very precise role in nitrate distribution within the plant.

We first demonstrated that ATNRT2.7 exhibits a strong seed-specific pattern of expression. In dry seeds, only two members of the ATNRT2 family are significantly expressed, ATNRT2.5 and ATNRT2.7, the latter gene being expressed 20 times more strongly than the former. During seed imbibition, the expression of both of these NRT2 genes decreases and becomes hardly detectable for ATNRT2.5. Dry mature seeds contain a large number of mRNA species. Stored RNAs were first described in cotton (Gossypium hirsutum) but are universal in plant species. Recently, a genome-wide profiling of stored RNAs in dry seeds and imbibed germinating seeds was performed in Arabidopsis (Nakabayashi et al., 2005Go). Expression analyses during late embryogenesis and subsequent germination have shown that these developmental phases occur gradually (Nambara et al., 2000Go); therefore, stored RNAs contain transcripts necessary for both phases (Hughes and Galau, 1991Go). Data from the AtGenExpress initiative (https://genevestigator-1.ethz.ch/) show that ATNRT2.7 expression is dramatically enhanced between stages 7 and 8, and this increase is maintained until seed maturation, leading to a huge accumulation of transcripts for the gene in dry seeds (Table 1). The ATNRT2.7 transcript abundance was found to decline during seed imbibition (Table 1), which may suggest a potential role of the protein during late embryogenesis rather than during subsequent germination. Genome-wide profiling has identified a G-box–like CACGTG sequence as the most prominent abscisic acid–responsive element in the dry seed transcriptome (Nakabayashi et al., 2005Go). However, no abscisic acid–responsive element element was found in the ATNRT2.7 promoter.

If ATNRT2.7 is important during late embryogenesis, is its function still related to nitrate transport? To address this question, we used a heterologous system and we took advantage of the atnrt2.1-1 mutant phenotype. Xenopus oocytes have already been used to measure the nitrate uptake capacity of several NRT1 and some NRT2 genes. In the latter case, the coexpression of two components, one NRT2 gene and one NAR2 gene was found to be necessary to obtain an efficient nitrate uptake for Chamydomonas reinhardtii (Zhou et al., 2000Go), barley (Tong et al., 2005Go), and Arabidopsis (Orsel et al., 2006Go). Our experiments with ATNRT2.7 mRNA-injected oocytes show that this protein alone is able to take up nitrate from the external solution. This result is in agreement with the expression pattern of the two NAR2 genes in Arabidopsis. In complete contrast with ATNAR2.1 and ATNRT2.1, which show identical tissue expression patterns (Okamoto et al., 2006Go), ATNRT2.7 has a shoot- and seed-specific pattern that is very different from that shown by ATNAR2.1. We also confirmed the capacity of the ATNRT2.7 protein to transport nitrate in planta by atnrt2.1-1 mutant complementation. This mutant is affected in the ATNRT2.1 and ATNRT2.2 genes and, as a consequence, exhibits only 20% of the wild-type nitrate uptake capacity (Cerezo et al., 2001Go; Filleur et al., 2001Go). We showed that the ATNRT2.7 protein, when expressed ectopically under the control of a strong promoter, is able to increase by a factor of 2 the nitrate uptake mediated by the HATS system. This increase is not due to compensation by enhanced expression of other NRT2 genes still present in the mutant, as was found to be the case for ammonium transporters (Kaiser et al., 2002Go). However, this result is quite surprising because of the tonoplastic localization of the ATNRT2.7 protein. The overexpression of the GFP-ATNRT2.7 fusion protein may lead to some targeting of the protein to the plasma membrane. Similarly, strong expression in Xenopus oocytes may result in default targeting to the plasma membrane. On the other hand, we cannot rule out the possibility that an enhanced flux of nitrate through the tonoplast of overexpressors could direct an increase in root influx mediated by other unidentified channels or transporters. These coordinated fluxes would participate in the homeostasis of cytosolic nitrate (der Leij et al., 1998Go). Although the ATNRT2.7 may participate in high-affinity nitrate transport (Figure 3A), the concentration of nitrate in xylem (Andrews, 1986Go) and cytoplasm (Cookson et al., 2006Go) is likely to be in the low mM range, and the seed dessication process will further increase the local concentration. The oocyte experiments used a 5 mM concentration to demonstrate nitrate transport (Figure 3B), but the affinity range of ATNRT2.7 has yet to be determined.

If the ATNRT2.7 protein is able to transport nitrate and this activity mainly takes place during late embryogenesis, then the impact of a modification of the expression of ATNRT2.7 should lead to a variation in seed nitrate content. To assess this possible role, we studied two allelic atnrt2.7-1 and atnrt2.7-2 mutants in Col-8 and Ws backgrounds, respectively. Both mutants shared the same phenotype, namely, normal growth under greenhouse conditions and no difference from wild-type nitrogen metabolism during the vegetative phase, but both had a lower nitrate content in their mature seeds. Moreover, an increase in seed nitrate content was observed in ATNRT2.7-overexpressing plants. As we demonstrated that the ATNRT2.7 protein was able to take up external nitrate when it was expressed ectopically in roots, we cannot rule out that this could simply explain the increase in seed nitrate content. We observed a strong interaction between the genetic background and the effect of a modification of ATNRT2.7 expression and the seed nitrate content. The Col-8 genotype was more sensitive than Ws, and it was easier to show more significant results in the former than the latter ecotype (Figures 4A and 4B). Whether or not these results reflect the presence of unknown transporters, more active in Ws than Col-8 in seed nitrate accumulation, remains to be demonstrated. Alternatively, this may reflect natural variability between these ecotype backgrounds, the ATNRT2.7 protein itself could be less efficient in Ws than in Col-8, leading to a less dramatic phenotype in a null mutation. In Arabidopsis, seed development can be divided in three stages. After an early period of morphogenesis, the maturation phase includes both the synthesis of carbon (lipids) and nitrogen storage compounds as seed storage proteins (Heath et al., 1986Go). Recently, an integrated overview of seed development revealed two peaks of amino acid accumulation during the maturation and late maturation phases, explained by enrichment in Ser and Gly (Baud et al., 2002Go). Nothing is known, however, about the time course of nitrate storage in seeds. Nitrate content in leaves is higher during the vegetative rather than the reproductive stages, but in Arabidopsis, contrary to cereals, this storage pool does not significantly contribute to the bulk flow of resources to the seeds (Schulze et al., 1994Go). Nitrate in seeds seems to come directly from that taken up by roots, and this result is in agreement with the observed net increase in seed nitrate content when wild-type plants are transferred from 10 to 50 mM nitrate after bolting (Figure 4B).

The physiological role of nitrate in seeds could be as a nutrient, either to participate to reserve synthesis during the maturation phase or to sustain growth of young seedlings after germination. The high levels of mRNAs coding for genes involved in amino acid biosynthesis during seed maturation and during the dessication period suggest that amino acid biosynthesis takes place during these developmental stages (Fait et al., 2006Go). However, even if photosynthesis efficiency is maintained until the dessication period (Fait et al., 2006Go), providing the reducing power necessary for nitrate reduction, the mRNAs of the two key enzymes of the pathway, nitrate and nitrite reductase, are hardly detectable during these two phases (https://genevestigator-1.ethz.ch). On the other hand, an increase in the levels of many metabolites is observed very early in postimbibition germination. This reactivation of metabolic pathways must require the availability of key internal precursors. Among the genes involved in nitrogen assimilation and metabolism, the mRNA level of the nitrite reductase is multiplied by a factor of 4 after 3 h of imbibition (Fait et al., 2006Go). It is thus possible that the nitrate storage pool is used during the very early hours after imbibition to sustain the metabolic boost that precedes the mobilization of storage compounds. The role of ATNRT2.7 may be to secure a supply of nitrate in the seed, ensuring cytosolic homeostasis during very early growth, until the young root can access external sources.

However, the total amount of nitrate in the seeds is negligible compared with organic N (<1 in 1000). This prompts us to propose that, rather than a nutritional function in seed maturation, this nitrate plays either a signaling or osmotic role during the first steps of imbibition. The role of nitrate in the osmotic control of plant development has already been suggested (McIntyre, 1997Go). In barley seeds, exogenous nitrate increased water uptake by the seedling (Lieffering et al., 1996Go). The seed nitrate storage pool, mediated by ATNRT2.7 during seed development, may act as an osmoticum to drive water uptake during imbibition and germination and so may have an osmotic, rather than a nutritional, function. Nitrate could also act as a signal to turn on or off the expression of the numerous nitrate-regulated genes (Wang et al., 2003Go, 2004Go) or, as it has been demonstrated recently, to release seed dormancy (Alboresi et al., 2005Go). Bethke et al. (2006)Go proposed a simple four-component model that explains how nitric oxide (NO) derived from nitrogen-containing compounds can reduce seed dormancy. Nitrate reductase was shown to catalyze the reduction of nitrite to NO under anaerobic conditions or darkness (Meyer et al., 2005Go; Crawford, 2006Go). Possibly, during the very early hours of imbibition, the level of NO could increase due to both the presence of both nitrate in the seed vacuole and increasing expression of the nia1 gene coding for one of the two nitrate reductase isoforms (https://genevestigator-1.ethz.ch). Here, 2 d after sowing the genotypes lacking ATNRT2.7 expression were more dormant than their wild-type controls and the reverse was found for ATNRT2.7 overexpressors (Figure 5). We can thus probably link the relationship between ATNRT2.7 expression and seed dormancy through seed nitrate content.

GFP-tagged ATNRT2.7 proteins seem to be located in the vacuolar membrane (Figure 6). In 5-d-old plantlets, ATNRT2.7 was localized in the tonoplast of the main central vacuole, while when dry seeds are imbibed during 12 h, it is present on small vacuole bodies or as punctuate structures (Figures 6C and 6H). Both lytic vacuoles and protein storage vacuoles have been postulated to coexist in some plant embryos (Jiang et al., 2001Go), but the situation is less clear in Arabidopsis. Results of Otegui et al. (2006)Go using different markers suggest that only protein storage vacuoles occur at the late bent-cotyledon and mature embryo stages. However, the localization analysis of the iron transporters At NRAMP3 and At NRAMP4 has led to the hypothesis that seed storage vacuoles have within them a membrane-bound compartment that surrounds globoids and is a marker for the lytic vacuole (Lanquar et al., 2005Go). The authors proposed that during germination, the membrane surrounding storage vacuoles, where the ATNRT2.7 protein could mediate the accumulation of nitrate, rapidly acquires the properties of a lytic vacuole.

Many transporters have been found to be targeted to the tonoplast, such as ammonium transporters (Loque et al., 2005Go), sulfate transporters (Kataoka et al., 2004Go), and sucrose transporter (Endler et al., 2006Go). Depending on their substrate and mechanism, these transport proteins can be involved in influx or efflux. The SULTR4-type transporters actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm (Kataoka et al., 2004Go), and At NRAMP3 and At NRAMP4 are essential for mobilization of vacuolar iron during seed germination on low iron (Lanquar et al., 2005Go). By contrast, a nitrate/proton antiporter, At CLCa, was recently found to mediate nitrate accumulation in Arabidopsis vacuoles, showing for the first time how these anions channels acted as ion exchangers (De Angeli et al., 2006Go). However, this gene is not expressed in seeds (https://genevestigator-1.ethz.ch). We demonstrated in this work that ATNRT2.7 expression increases during seed maturation and subsequently decreases during germination and atnrt2.7 mutants show lower seed nitrate content; therefore, we can postulate that the ATNRT2.7 transporter acts in a similar manner, mediating nitrate loading in the seed vacuole.

With these findings we provide additional information on the complementary roles of two members of the NRT2 family for the distribution of nitrate within the plant. One gene, ATNRT2.1, is involved in root uptake and entry of nitrate into the plant at the beginning, while the other gene, ATNRT2.7, is important at the final destination for nitrate loading into the vacuole during seed maturation.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Plant Material and Growth Conditions
The SALK_073582 line (atnrt2.7-1) was obtained from the ABRC and was derived from a T-DNA–mutagenized population of the Col-8 ecotype (Alonso et al., 2003Go), while the atnrt2.7-2 line was derived from a T-DNA–mutagenized population of the Ws ecotype (Bechtold et al., 1993Go; Bouchez et al., 1993Go). Homozygous mutant plants were isolated by PCR with the primers 2.7A (5'-CGGTATCTCTCAGCTCCTTATGCCTCTC-3') and 2.7B (5'-GATGACGAAGGCCCACAACACATTCCAC-3'). Both of the T-DNA insertions were located in the first exon of ATNRT2.7. The absence of transcript downstream from the insertion sites was checked by quantitative RT-PCR with the primers 7E (5'-CCTTCATCCTCGTCCGTTTC-3') and 7F (5'-AATTCGGCTATGGTGGAGTA-3'). Wild-type Arabidopsis thaliana ecotypes Col-8 and Ws as well as mutants or transgenic plants were grown either in the greenhouse or under hydroponic conditions in a Sanyo growth chamber with an 8-h-light/16-h-dark cycle at 21°C/17°C, respectively, 80% relative humidity, and 150 µmol m–2 s–1 irradiation. In greenhouse conditions, seeds were stratified for 48 h at 4°C in the dark in a 0.1% agar solution and then sown and cultivated as already described (Loudet et al., 2003Go). The nutrient solutions contained 10 mM nitrate, and in some cases, 2 weeks after bolting (flower initiation), the nitrate regime of plants was changed to 50 mM nitrate.

Phylogenetic Analysis of the NRT2 Gene Family
Nucleotide sequences were manually aligned by first translating into proteins and then using the sequence alignment editor software BioEdit. Only regions where the assessment of primary homology appeared reasonable were kept, generating a 1517-nucleotide position matrix. A phylogenetic tree was reconstructed using MrBayes version 3.1 (Ronquist and Huelsenbeck, 2003Go) after back-translating to nucleotide sequences. The model of evolution used is the general time reversible model estimating separate rates of change for each type of nucleotide substitution with gamma distribution shape parameter estimated to account for rate heterogeneity. Analysis was run with the following parameters: number of generations = 500,000; sampling frequency = 10 generations; number of chains = 4. Posterior probabilities were computed after discarding 200,000 generations (burn-in period) and computing a consensus of trees saved in the remaining generations. The tree was visualized using MEGA3.1 (Kumar et al., 2004Go).

Quantitative RT-PCR
RNA was extracted with the Gen Elute Mammalian Total RNA kit from Sigma-Aldrich, modified by adding a DNase step, which was performed with the Qiagen RNase-free DNase kit. First strands were synthesized according to Daniel-Vedele and Caboche (1993)Go using M-MLV reverse transcriptase and oligo(dT) 15 primers (Promega). The PCR was performed on a LightCycler instrument (Roche) with the LightCycler-FastStart DNA Master SYBR Green I kit for PCR (Roche) according to the manufacturer's protocol. Each reaction was performed on a 1:20 dilution of the first cDNA strands, synthesized as described above, in a total reaction of 20 µL. With this dilution, the SYBR green signal was linear. Specific primer sets were used for the seven ATNRT2 genes, as previously described (Orsel et al., 2002aGo). The experiment was performed twice on independent biological samples.

Complementation of the atnrt2.1-1 Mutant
In this experiment, the full-length ATNRT2.7 cDNA was amplified by RT-PCR from total RNAs extracted from Ws leaves using the start primer (5'-CGAGAGTAATGGAGCCATCTCAACG-3'), the stop primer (5'-CGCACATCAACAAACGGGACGTAGA-3'), and the high-fidelity Taq enzyme (Roche) and cloned into pGEM-T Easy vector (Promega). The nucleotide sequence of the insert was checked before transferring the cDNA first in the pRT103 vector (Töpfer et al., 1987Go) downstream from the 35S promoter, and this whole chimeric gene was then transferred into a pGREEN vector (Hellens et al., 2000Go) already containing the 35S terminator. Binary vectors were introduced into Agrobacterium tumefaciens strain C58C1 (pMP90). The atnrt2.1-1 mutant was transformed by the in planta method using the surfactant Silwet L-77, and transformants were selected on 20 µg/mL of hygromycin B.

Root 15N Influx
Influx of 15NO3– was assayed as previously described (Orsel et al., 2004Go). The plants were transferred first to 0.1 mM CaSO4 for 1 min and then to complete nutrient solution containing 0.2 mM 15NO3– (atom% 15N: 99%) for 5 min and finally to 0.1 mM CaSO4 for 1 min (300 mL for plants grown in hydroponics and 20 mL for plants grown in vitro). After homogenization, an aliquot of the frozen powder was dried overnight at 80°C and analyzed using the ANCA-MS system (PDZ Europa). Influx of 15NO3– was calculated from the total N and 15N content of the roots (1 mg DW).

Xenopus laevis Oocyte Expression System
The pGEM-T Easy vector containing the full-length AtNRT2.7 cDNA was fully digested with NotI. The cDNA fragment was blunted using the klenow enzyme and subcloned into the EcoRV site of the pT7TS expression vector containing the 5'- and 3'-untranslated regions of the Xenopus ß-globin gene (Cleaver et al., 1996Go). For in vitro synthesis of mRNA, pT7TS clones were linearized by digestion with BamHI. Synthesis of capped full-length mRNAs and Xenopus oocyte preparation were performed as previously described (Orsel et al., 2006Go). Healthy oocytes at stage V or VI were injected with 50 nL of water (nuclease free) or ATNRT2.7 mRNAs at 1 µg·µL–1. After 3 d incubation at 18°C, 5 to 10 oocytes were incubated in 3 mL of ND96 solution enriched with 5 mM Na15NO3 (atom% 15N: 98%) during 16 h at 18°C. The oocytes were then thoroughly washed four times with ice-cooled 5 mM NaNO3 ND96 solution and dried at 60°C. The 15N/14N ratio of the single dried oocyte was measured as previously described (Orsel et al., 2006Go). The values are means ± SD of five replicates; results from a representative experiment are shown.

Nitrate Content Measurements
Nitrate contents of seeds were determined as described previously (Alboresi et al., 2005Go). For the extraction, 50 seeds were homogenized in 500 µL 80% (v/v) ethanol at 4°C and extracted for 120 min. Nitrate content of seeds was analyzed by high-performance liquid chromatography on a DX-120 analyzer (Dionex). Nitrate and free amino acid contents in leaves or roots were measured as previously described (Orsel et al., 2004Go).

Germination Test
For each experiment, all genotypes were harvested on the same day. Four independent batches of 50 to 80 mature seeds were sown on 0.5% agarose plates (Litex agarose; FCM A/C) sometimes containing an additional 1 mM potassium nitrate. The plates were incubated in a growth chamber (Percival; Cu-36L6) at 25°C, with 16 h of light (100 µmol m–2 s–1), 8 h of dark, and 60% relative humidity. Germination was scored as positive when the radicle protruded from the seed. A cold treatment at 4°C for 3 d was also performed to test the viability of mature seeds.

GUS Staining
Histochemical GUS staining was performed according to the method described by Jefferson (1987)Go, with some modifications. Dry seeds were first sown on imbibed filter paper during 1 h, and the seed coat was removed before staining embryos. Embryos were vacuum-infiltrated for 1 h in a 50 mM potassium phosphate buffer, pH 7.2 (1% Triton, 0.5 to 5 mM ferro/ferricyanide, and 2 mM X-glucuronide). Subsequently, samples were incubated overnight in the dark at 37°C. Stained embryos was cleared by incubation in a chloralhydrate solution (8:2:1 [w/v/v] of chloralhydrate, water, and glycerol, respectively) and then observed under a light microscope (Axioplan 2; Zeiss).

Construction of GUS and GFP Fusions
Binary vectors containing GUS or GFP fusions with the ATNRT2.7 promoter or coding sequences were obtained using Gateway technology (Curtis and Grossniklaus, 2003Go). A genomic Arabidopsis ATNRT2.7 region, starting from position –2,000 bp upstream of the translation initiation site and terminating before the ATG codon, was amplified from ecotype Ws by PCR with primers NRT2.7 PGW5' (5'-AAAAAAGCAGGCCTCAGGTTGACTTCATCATTGG-3') and NRT2.7PGW3' (5'-AAGAAAGCTGGGTACGACTCTTACTTACACGAC-3'). Amplification was performed using the Expand high-fidelity PCR system (Roche), and the amplified fragment was cloned in front of the GFP and GUS coding sequence in the pBI101 derived gateway vector (Divol et al., 2006Go). The binary plasmids were transferred to A. tumefaciens strain C58C1 (pMP90) by triparental mating.

First primers AttB1-ATNRT2.7 start (AttB1, 5'-GAGCCATCTCAACGCAAC-3') and AttB2-ATNRT2.7 End-Stop (AttB2, 5'-AACAAACGGGACGTAGACTACC-3') were used to amplify a complete ATNRT2.7 cDNA from our previously isolated clone (see above). PCR products were obtained with the Expand high-fidelity PCR system and amplified with the universal U3-endstop (5'-AGATTGGGGACCACTTTGTACAAGAAAGCTGGGTCTCCACCTCCGGATC-3') and U5 primers (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATG-3') to create the recombinant site AttB. The product of recombination reactions (BP reactions) was used to transform competent Escherichia coli, strain TOP10 (Invitrogen), by heat shock. LR clonase reactions to T-DNA fragments from the entry clone to the destination binary vector pMDC 43 (Curtis and Grossniklaus, 2003Go) were performed. The vectors pMDC43/ATNRT2.7 were generated with GFP6 in N-terminal fusion, and the binary vectors, containing the Pro35S:GFP-ATNRT2.7 construct, were sequenced before transformation of A. tumefaciens.

Arabidopsis plants, the wild type or the atnrt2.1-1 mutant, were transformed according to the in planta method using the surfactant Silwet L-77 (Clough and Bent, 1998Go). Transgenic plants were selected on Estelle and Sommerville media (Estelle and Sommerville, 1987Go) containing 50 µg L–1 of kanamycin.

Confocal Microscopy Analyses
Seven-day-old transformed plantlets grown on Estelle and Sommerville media were observed with the TCSNT confocal microscope (Leica) equipped with an argon/krypton laser (Omnichrome). Cells in living roots and embryo were stained with 0.1 µg/mL of FM4-64 (Molecular Probes). The different fluorochromes were detected using laser lines 488 nm (Alexa 488, GFP, and FM4-64) and 543 nm (Alexa 568). The images were coded green (fluorescent isothiocyannate and GFP) and red (Alexa 568 and FM4-64), giving yellow colocalization in merged images. The samples were washed twice after staining before observation with the confocal microscope. Each image shown represents a single focal plane.

Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession numbers listed in Supplemental Table 1 online.

Supplemental Data
The following material is available in the online version of this article.

Supplemental Table 1. The NRT2 Gene Family in Plants and Algae.


   Acknowledgments
 
We thank our colleagues Olivier Grandjean and Samantha Vernetthes for their useful advice on confocal analyses, Pascal Tillard for 15N analyses, and Christian Meyer and Eugene Diatloff for critical reading of the manuscript. Rothamsted Research is grant-aided by the Biotechnology and Biological Sciences Research Council of the UK.


   Footnotes
 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Françoise Daniel-Vedele (vedele{at}versailles.inra.fr).

[W] Online version contains Web-only data. Back

www.plantcell.org/cgi/doi/10.1105/tpc.107.050542

Received January 18, 2007; Revision received April 30, 2007. accepted May 14, 2007.


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