Nitrate is an essential nutrient, and is involved in many adaptive responses of plants, such as localized proliferation of roots, flowering or stomatal movements. How such nitrate-specific mechanisms are regulated at the molecular level is poorly understood. Although the Arabidopsis ANR1 transcription factor appears to control stimulation of lateral root elongation in response to nitrate, no regulators of nitrate assimilation have so far been identified in higher plants. Legume-specific symbiotic nitrogen fixation is under the control of the putative transcription factor, NIN, in Lotus japonicus. Recently, the algal homologue NIT2 was found to regulate nitrate assimilation. Here we report that Arabidopsis thaliana NIN-like protein 7 (NLP7) knockout mutants constitutively show several features of nitrogen-starved plants, and that they are tolerant to drought stress. We show that nlp7 mutants are impaired in transduction of the nitrate signal, and that the NLP7 expression pattern is consistent with a function of NLP7 in the sensing of nitrogen. Translational fusions with GFP showed a nuclear localization for the NLP7 putative transcription factor. We propose NLP7 as an important element of the nitrate signal transduction pathway and as a new regulatory protein specific for nitrogen assimilation in non-nodulating plants.
Nitrogen (N) is an essential macronutrient for all living organisms (Lawler et al., 2001). Plants and fungi are the only multi-cellular organisms that are able to assimilate inorganic N, which, in higher plants, is mainly taken up by the roots as nitrate. Although the regulation of nitrate acquisition and utilization has been well described at the physiological level (Lawler et al., 2001), very little is known of the molecular mechanisms and molecular players that govern such responses in higher plants (Walch-Liu et al., 2005). In fungi and algae, however, several regulatory proteins of nitrate assimilation, such as NIT2 and NIT4 from Neurospora crassa, AREA and NIRA from Aspergillus nidulans and NIT2 from Chlamydomonas, have been identified using a simple selection system based on the resistance of mutants to chlorate, a toxic analogue of nitrate (Marzluf, 1997; Nichols and Syrett, 1978). Such screens have been performed on Arabidopsis mutants in order to identify regulators of nitrate assimilation in higher plants, but no transcription factors were isolated by this approach.
The molecular mechanisms controlling the adaptation of plants to N availability are also poorly understood (Walch-Liu et al., 2005). However, NLA (Nitrogen Limitation Adaptation), a RING-type ubiquitin ligase from Arabidopsis, was found to be a positive regulator of plant adaptation to N limitation (Peng et al., 2007). Furthermore, overexpression of a maize DOF transcription factor in Arabidopsis improved growth under low-nitrogen supply, accompanied by upregulation of multiple genes involved in carbon-skeleton production (Yanagisawa et al., 2004). The localized proliferation of lateral roots in nitrate-rich soil patches is under the control of the MADS box transcription factor ANR1 (Zhang and Forde, 1998). Moreover, the nitrate transporter NRT1.1 has been proposed to be a nitrate sensor that acts upstream of ANR1 in this signalling pathway (Remans et al., 2006).
Only legume species have evolved the capacity to deal with N limitation by developing a symbiotic interaction with the soil bacteria, rhizobiae. Under nitrogen-limiting conditions, rhizobiae colonize the roots, which then develop a new plant organ called a nodule. In this beneficial association, the bacteria fix and provide N to the plant, and the plant provides carbohydrate produced by photosynthesis to the bacteria. The initiation of nodule development has been shown to be dependent on nodule inception protein (NIN) (Borisov et al., 2003; Schauser et al., 1999). In addition to its positive role in bacterial entry and nodule organogenesis, NIN also represses spatial expression pattern of nodulation factors, which may control nodule number (Marsh et al., 2007). The most prominent feature of the NIN protein is a 60-amino-acid sequence (RWP-RK domain) that is similar to the DNA-binding and dimerization domains of the bZIP8 and bHLH/Z9 transcription factors (Schauser et al., 1999, 2005).
Interestingly, the NIN gene family is found widely among higher plants and algae, including many species that do not fix gaseous nitrogen. The Chlamydomonas genome encodes two NIN-like proteins (NLPs). The first, MID, is necessary for the minus gamete development induced under limiting N growth conditions (Ferris and Goodenough, 1997); the second, NIT2, was identified as responsible for a chlorate resistance phenotype. Recently, NIT2 was shown to bind the nitrate reductase gene promoter in the presence of nitrate and to allow its transcription (Camargo et al., 2007).
The Arabidopsis genome encodes nine NLPs characterized by the highly homologous RWP-RK domain that is predicted to be a DNA-binding and dimerization domain. Another conserved domain shows strong homology to the PB1 domain, a protein–protein interaction domain enabling heterodimerization between PB1 domain-containing proteins (Schauser et al., 2005). Except for the invariant amino acids of these consensus sequences, homology within the Arabidopsis NLPs is rather low. However, the recent whole-genome duplication event in Arabidopsis created four AtNLP pairs of higher homology: NLP1 and 2, NLP4 and 5, NLP6 and 7, and NLP 8 and 9 (Schauser et al., 2005).
In the present work, we have developed a comparative approach, based on characterization of candidate genes, with the aim of identifying regulators of N metabolism in Arabidopsis. We provide evidence that the NIN-like protein 7 is a transcription factor that is involved in regulation of nitrate assimilation in higher plants.
nlp7 mutants show several features of a nitrogen-starved plant
To investigate the function of the AtNLP proteins, a systematic screen of T-DNA insertion mutants for most genes of the AtNLP family was undertaken. Preliminary studies revealed a visible phenotype for two homozygous allelic mutants of the NLP7 gene (At4g24020), nlp7-1 and nlp7-2 (Figure 1a). We therefore focused on the NLP7 gene, and further investigated the physiological impact of loss of function of NLP7. No NLP7 mRNA and only 6% of the wild-type NLP7 mRNA level were found in nlp7-1 and nlp7-2, respectively (Figure 1b). The nlp7 mutants developed a smaller rosette compared to the wild-type, especially when grown on full nutrient supply (Figure 1c), but the root fresh weight was not modified (data not shown). As a consequence, the shoot to root fresh weight ratio (S/R ratio), an important marker for nutrient starvation (Lawler et al., 2001), was lower for the nlp7 mutants grown hydroponically on 6 mm nitrate (Figure 1d). Moreover, mutants showed delayed growth and flowering when grown in the greenhouse (data not shown and Figure 1e). In addition, nlp7 mutants grown on nutrient-rich agar plates developed a longer primary root and had a higher lateral root density compared to wild-type (Figure 1f,g), whereas the root architecture was identical for wild-type and mutant plants grown on N-, P- or S-free agar plates (Figure 1f,g and Figure S1).
The allelic mutant nlp7-2 has a slightly less severe phenotype than nlp7-1. The T-DNA insertion in nlp7-2 is in the last exon of the NLP7 gene (Figure 1a), which may give rise to a small amount of protein, truncated only by the last few amino acids, including the PB1 protein-binding site. We have isolated a third knockout mutant, nlp7-3, which has a T-DNA insertion in the first intron and shows the same severe phenotype as nlp7-1 mutant (Figure S2).
To identify which processes are deregulated in nlp7 mutants, we performed transcriptome analyses on nlp7-1 and wild-type plants grown hydroponically under low-N conditions (0.2 mm nitrate). Growth of wild-type plants is slightly limited under these conditions, but no N starvation arises as the nutrient solution is replaced regularly (Orsel et al., 2004). Developmental defects of the nlp7-1 mutant were less pronounced under these conditions (Figure 1c,d), which allowed us to study changes in gene expression directly related to the mutation. Around 500 genes were differentially expressed in the mutant compared to the wild-type under these conditions, and most of them were downregulated in the mutant (data not shown). However, only 145 genes had more than a twofold modified expression, and 121 of them were specifically deregulated in the roots of the mutant (Figure S3). Comparing these data to the transcriptome data for wild-type plants starved for nitrogen (CATdb, http://urgv.evry.inra.fr/CATdb/; project GNP3-B4), it was obvious that many marker genes for N starvation had modified expression in nlp7-1 roots (Figure 2). The left panel of Figure 2 shows the expression level of 12 selected genes that are downregulated by N starvation in wild-type plants. These genes also show lower expression in roots of the mutant, compared to the wild-type, even in the presence of N. Similarly, eight genes that are upregulated by N starvation in the wild-type are upregulated even in the presence of N in the nlp7-1 mutant. Marker genes for sulphate and phosphate starvation that are either up- or downregulated in wild-type plants (data from Nikiforova et al. (2003) and Müller et al. (2007), respectively) (Figure 2, right panel) showed no obvious expression changes relative to wild-type plants in the nlp7-1 mutant. The fact that only expression of marker genes for N starvation was modified in the nlp7-1 mutant reinforced our hypothesis that the mutant phenotype is a constitutive response to N starvation. However, the total gene expression pattern of the mutant did not match perfectly the pattern of a wild-type N-starved plant. Although 11 of the 16 genes that are induced in nlp7-1 roots are also induced by N starvation in the wild-type, only 16% of the genes repressed in the roots of the mutants are also repressed by N starvation. This indicates that only specific parts of the response to N starvation are modified in the nlp7 mutants.
Both nitrate assimilation and nitrate sensing are impaired in nlp7 mutants
We analyzed nitrate assimilation and the nitrate responsiveness of nlp7 mutants in order to determine the origin of the mutant’s N-starved-like phenotype. Nitrate content was increased in the mutants, but high-affinity nitrate uptake and total amino acid content were slightly lower (Table 1 and Figure S4a). However, no changes occurred in the pattern of individual amino acids (Table S1). These results led us to investigate the enzymatic activity of key enzymes of the nitrate assimilation pathway. The nitrate reductase and glutamine synthetase enzyme activities were two to three times lower in the mutants compared to the wild-type (Table 1), which could explain nitrate accumulation and lower levels of amino acids. However, the percentage of total N remained unchanged in the nlp7 mutants (Figure S4c), indicating that their physiological status was not reminiscent of N starvation per se. The mutants were therefore not nitrogen-starved, but seem to respond to N starvation signals.
Table 1. Nitrate and amino acid contents, nitrate reductase and glutamine synthetase activities in the leaves of nlp7 mutants and wild-type plants grown on 6 mm nitrate solution in hydroponic culture
Data are means ± standard deviation of four individual plants.
Nitrate content (μmol g−1 FW)
113.3 ± 4.9
146.5 ± 2.8
137 ± 13.6
Amino acid content (μmol g−1 FW)
10.3 ± 0.7
8.1 ± 0.2
7.6 ± 1.3
Nitrate reductase activity (μmol g−1 FW min−1)
0.187 ± 0.020
0.062 ± 0.006
0.054 ± 0.006
Glutamine synthetase activity (μmol g−1 FW min−1)
1.14 ± 0.09
0.59 ± 0.10
0.43 ± 0.12
The responses of the nlp7 mutants to nitrate were studied by following steady-state amounts of root transcripts for four known nitrate-responsive genes (high-affinity nitrate transporters NRT2.1 and NRT2.2, and the NIA1 and NIA2 genes encoding nitrate reductase) after re-supply of nitrate to N-starved plants in hydroponic culture. The wild-type and the mutants had a similar expression level for these genes after a short period of N starvation (time point zero in Figure 3). However, the expression pattern after re-supply of nitrate was different in the nlp7 mutants when compared to the wild-type (Figure 3a–d). In the wild-type, all four genes were highly induced by nitrate supply, but the induction was much lower in the nlp7 mutants, especially after 2 h of induction. At this time point, NIA1 and NIA2 expression levels in nlp7-1 were 89 and 64% lower than in the wild-type, respectively. A similar reduction was observed for the nlp7-2 mutant (84% and 62%, respectively). The induction of the nitrate transporter genes NRT2.1 and NRT2.2 was also impaired in the nlp7 mutants. Compared to the wild-type, NRT2.1 expression was two times lower in the nlp7 mutants, and NRT2.2 expression was reduced by 85 and 82% in the nlp7-1 and nlp7-2 mutants, respectively.
Pattern of NLP7 expression
Using quantitative RT-PCR, we have shown that the NLP7 gene is expressed in all plant organs, but chiefly in roots (Figure S5). We studied tissue-specific expression of NLP7 using in situ mRNA and whole-mount hybridization (Figure 4a–c). NLP7 mRNA was detected in root hairs and emerging secondary roots (Figure 4a). NLP7 RNA was also detected in vascular tissue of stems (Figure 4b,c), close to the xylem. These results were confirmed by expression of the GUS reporter gene under the control of the NLP7 promoter, which was detected in the root tip and vascular tissue (Figure 4d,e, 1 h of staining) and to a lesser extent in whole roots and root hairs (Figure 4f, 16 h of staining), leaf parenchyma cells and stomata (Figure 4g, 16 h of staining). Taken together, the NLP7 gene is highly expressed in tissues or cells that are involved in transport and sensing of nutrients and assimilates.
The putative transcription factor NLP7 is targeted to the nucleus
NLPs have been described as putative transcription factors, with bipartite SV40-like nuclear localization signals (Schauser et al., 1999). Up to now, however, no proof of nuclear localization has been obtained for proteins of this gene family. To investigate the subcellular localization of the NLP7 protein, we cloned the NLP7 cDNA in-frame with the GFP reporter gene at either the C- or N-terminal position (35S::NLP7-GFP and 35S::GFP-NLP7). These constructs permitted the localization of NLP7 by transient expression and generation of stable lines expressing the corresponding fusion proteins. Both constructs restored the wild-type phenotype when expressed in the nlp7-1 mutant genetic background, which indicates that the fusion proteins are correctly processed and are functional in planta (Figure S6a,b). Using both transient and stable expression, we showed that NLP7 is indeed located in the nucleus (Figure 4h–j and Figure S7a–d), which is in agreement with the function of a transcriptional regulator. In addition to the clear nuclear staining, slight GFP fluorescence also occurred in the cytosol. These results were confirmed using the 1.6 kb NLP7 promoter region in front of the fusion proteins instead of the 35S promoter (Figures S6c and S7e,f).
The nlp7-1 mutant has reduced water loss and is drought-tolerant
The GUS staining observed in the guard cells of the pNLP7::GUS lines led us to further investigate the involvement of NLP7 in stomatal function. We therefore performed water-loss experiments on both detached leaves and whole nlp7-1 and wild-type plants. Interestingly, water loss form excised leaves was slower for the nlp7-1 mutant compared to the wild-type (Figure 5a). Moreover, nlp7-1 mutant plants subjected to drought stress for 14 days showed less damage (leaf senescence, loss of turgor) than the wild-type, and were able to recover after 5 days of water re-supply, whereas the wild-type could not (Figure 5b). These results suggest that NLP7 may play a role in stomatal movements and drought resistance.
The NLP proteins have been proposed to be involved in N-regulated processes (Schauser et al., 2005). The Arabidopsis NLP family contains nine members that show rather low homology outside the characteristic DNA-binding domain (Figure S8 and Schauser et al., 2005). A survey of mutants in most Arabidopsis NLP genes and an in-depth study of nlp7 mutants allowed us to identify NLP7 as an important factor in N-regulated processes. nlp7 mutant lines (nlp7-1nlp7-2) and nlp7-3 showed typical phenotypes of nitrogen-starved plants, irrespective of N supply, and transcriptome analysis confirmed the hypothesis of a N-starvation phenotype. Knowledge of the molecular players governing the adaptation of plants to N starvation is still rather scarce. For example, the nla mutant, which is deficient in a RING-type ubiquitin ligase, is hypersensitive to N starvation, but develops normally on full nitrate supply (Peng et al., 2007). We show that nlp7 mutants are impaired in transduction of the nitrate signal, as they fail to completely induce nitrate-responsive genes after a short nitrogen starvation followed by nitrate re-supply. We therefore propose that NLP7 belongs to the nitrate signalling pathway. Interestingly, the growth defects of nlp7 mutants are less pronounced when plants are supplied with limiting N, which is the opposite phenotype to that of nla mutants. NLA has also been proposed to be involved in the ubiquitination-mediated degradation of negative regulators in the N-limitation signalling pathway. The degradation of negative regulators results in a positive effect with respect to adaptation of Arabidopsis to N limitation (Peng et al., 2007). In the absence of NLP7, plants always appear to respond to N starvation, whether it actually exists or not. The presence of NLP7 therefore seems to inhibit adaptive responses to N starvation. It is tempting to formulate a hypothesis that NLP7 may be one of the NLA targets. However, an additional feature of the nlp7 mutants is that induction of gene expression by nitrate is disturbed. This suggests that NLP7 also functions as a positive regulator of N metabolism. A dual positive and negative role for LjNIN of Lotus japonicus has already been proposed by Schauser et al. (1999). Interestingly, a dual function in the regulation of N metabolism has also been suggested for the Chlamydomonas NIT2 protein, an algal homologue of the AtNLPs (Camargo et al., 2007).
The assimilation of nitrate is also impaired in nlp7 mutants. They show lower activities of key enzymes of the nitrate reduction pathway, which leads to accumulation of nitrate and lower amount of amino acids. Arabidopsis mutants with reduced nitrate reductase activity have been described previously (Wilkinson and Crawford, 1993). These lines carry mutations in the NIA genes, which encode the nitrate reductase enzyme. However, even though nitrate assimilation is dramatically impaired in the double mutant nia1 nia2, the response to the nitrate signal is still present (Wang et al., 2004), in contrast to the nlp7 mutants. Again, these data suggest that nlp7 mutants are not able to activate nitrate assimilation genes, due to a defect in transmission of the signal arising from perception of nitrate.
NLP7 mRNA was detected in roots and roots hairs and in the perivascular tissues of the stem. The expression pattern of the GUS reporter gene driven by the NLP7 promoter is consistent with the mRNA localization in the root endodermis, root hairs and in leaves and stomata after extended staining. Interestingly, the first events in the legume nodulation process induced by N starvation also take place in root hairs (Oldroyd and Downie, 2008). Moreover, roots and root hairs are the organs that first sense N avaiability and are important for the morphological adaptation to N deficiency (Walch-Liu et al., 2005). It is known that communication between the shoot and root takes place for N-regulated processes (Forde, 2002). N metabolites are transported in the vascular system, and therefore this tissue would be a strategic point to sense N availability and to initiate regulatory mechanisms in which NLP7 may be involved. In addition, the xylem-adjacent pericycle cells form lateral roots, and it is tempting to postulate a signalling mechanism between the xylem and pericycle that is necessary to induce lateral root formation in response to N availability. Interestingly, nlp7 mutants have an increased lateral root density, suggesting that NLP7 might play a role in lateral root development in response to nitrate availability.
Expression in root endodermis and stomata has also been found for the nitrate transporter NRT1.1, which has also been proposed to be a nitrate sensor (Guo et al., 2003; Remans et al., 2006). Both NRT1.1 and NLP7 appear to belong to nitrate signalling pathways, and nrt1.1 mutants and nlp7 mutants show common features, such as a delayed flowering and a reduced rosette size (Guo et al., 2001). The NRT1.1 protein regulates stomatal opening by controlling the entry of nitrate in guard cells. Indeed, nrt1.1 mutants show nitrate-dependent drought resistance caused by a reduced stomatal aperture (Guo et al., 2003). At the cellular level, nrt1.1 mutants show reduced nitrate accumulation in guard cells during stomatal opening and fail to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induces depolarization, and nitrate concentrations increase threefold during stomatal opening (Guo et al., 2003). However, the direct relationship between nitrate content and stomatal opening remains unclear. The nlp7-1 mutant also shows reduced leaf water loss and higher resistance to drought stress. We therefore postulate that NLP7 is also involved in stomatal movement. The fact that the nitrate signal cannot be transmitted in the nlp7 mutant and that nitrate content is lower in the nrt1.1 mutant might explain the drought-resistant phenotype.
NLPs have been described as putative transcription factors, because they carry a conserved RWP-RK putative DNA-binding domain (Schauser et al., 2005 and Figure S8). In this paper, we describe localization in the nucleus for a protein of this family. Moreover, Camargo et al. (2007) recently reported a role as a transcriptional activator for the Chlamydomonas NIT2 protein, which is a homologue of the AtNLPs. These data strongly support the hypothesis that NLP7 is a transcription factor. Moreover, the NIT2 protein binds directly to the NIA gene promoter and promotes NIA gene expression in the presence of nitrate (Camargo et al., 2007). Given these results and the low nitrate reductase activity observed in nlp7 mutants, it is possible that the Arabidopsis NIA genes may also be targets of NLP7. The expression of NIA1 (http://www.genevestigator.ethz.ch/) is very similar to that of NLP7, making NIA1 a good candidate target gene. ChIP studies are planned to identify the target genes of the NLP7 protein. No consensus sequence has been identified in the promoter sequences of the ten genes that show the most modified expression in the nlp7 mutants. However, up to now, no consensus sequences for nitrate-inducible promoters have been identified either (Das et al., 2007). Transcriptome analysis after short-term nitrate induction should provide a more exhaustive view of the modified gene induction by nitrate in nlp7 mutants.
Interestingly, NLP7 expression is not regulated by the N source or by the presence of nitrate (Figure S9). In Lotus japonicus, NIN expression in response to Nod factors is regulated by the N source (Barbulova et al., 2007). Furthermore, in Chlamydomonas, the NIT2 gene is highly regulated by ammonium, and the presence of nitrate stabilizes the transcript (Camargo et al., 2007). It is possible that other members of the AtNLP family are functionally more closely related to the NIT2 gene. It has already been found that AtNLP3 is highly induced by nitrate (Scheible et al., 2004). Indeed, the functions of the other eight NLP proteins, especially NLP6 (At1g64530), the closest homologue to NLP7, may also be important to N nutrition. However, we isolated a NLP6 knockout mutant (nlp6-1) that shows no obvious phenotype under our experimental conditions (Figure S10). These data indicate no functional redundancy between NLP6 and NLP7 for the N-related features that have been investigated.
Taken together, we have identified a higher-plant transcription factor that regulates the nitrate assimilation pathway in response to nitrate signals. NLP7 could be an important regulator involved in several nitrate-dependent processes, and, given the important role of nitrate as a signal for plants, could therefore have an impact on many metabolic and developmental processes (Figure 6). The functions of the NLPs NIT2 and MID from Chlamydomonas suggest that the increase in complexity of the N-starvation response during the evolution of multi-cellular vascular plants was achieved, at least in part, via development of functions such as root growth and tissue communication under the control of a regulatory system inherited from unicellular photosynthetic ancestors, as in the phosphate-starvation response (Rubio et al., 2001). We report here a new example of a higher-plant gene controlling N metabolism, which was then recruited as an essential factor controlling nodulation.
The nlp7-1 (SALK_26134) and nlp7-2 (SALK_114886) lines were obtained from a Nottingham Arabidopsis Stock Centre T-DNA-mutagenized population of the Col-8 Arabidopsis ecotype. Homozygous mutant plants were identified by PCR using the primers listed in Table S2. Transgenic plants carrying 35S::NLP7cDNA-GFP or 35S::GFP-NLP7cDNA constructs or a 1.6 kb NLP7 promoter fused to the GUS reporter gene (pNLP7::GUS) were obtained by floral dipping (Clough and Bent, 1998) of Col-8 Arabidopsis plants after cloning into Gateway technology-compatible (Invitrogen, http://www.invitrogen.com/) pMDC83, pMDC43 and pGWB3 plasmids, respectively.
Plant growth conditions
Plants were grown for 42 days in hydroponic culture (Orsel et al., 2004) (8 h light at 20°C, 150 μmol m−2 sec−1/16 h dark at 18°C) on limiting (0.2 mm NO3−) or non-limiting (6 mm NO3−) nitrate supply. The solutions were changed three times per week during the first 5 weeks of the culture and then changed daily. For nitrate induction experiments, mutant and wild-type plants were grown under the same conditions for 21 days on 6 mm NO3− supply, starved for 5 days (0 mm NO3−), and re-supplied with 6 mm NO3− for 0, 30, 60 or 120 min. Plants were harvested 2 h after the start of the light period, and immediately frozen in liquid N2.
In vitro root architecture analysis
Plants were grown in vitro for 12 days on vertical agar plates containing either 9 mm nitrate (Estelle and Somerville, 1987) or no nitrogen. For the medium without nitrogen, the ion balance was adjusted by adding KCl (5 mm) and CaCl2 (4 mm). The culture was performed at 18°C under long-day conditions (16 h light/8 h dark), with a light intensity of 70 μmol m−2 sec−1. The plates were photographed, and the primary root length and lateral root number were determined using Photolite software (Photonic Science, http://www.photonic-science.co.uk).
Water loss and drought resistance experiments
nlp7-1 and wild-type plants were grown in the greenhouse for 49 days. Water loss from excised leaves was measured by weighing leaves placed under a laminar air flow. For drought resistance experiments, plants were grown for 23 days on soil in the greenhouse (watered twice a week), drought-stressed (no irrigation) for 14 days and, then re-supplied with nutrient solution for 5 days (watered every second day).
N metabolite contents and enzyme activity measurements
Plants were grown in hydroponic culture as described above. After ethanolic extraction, nitrate and amino acid contents were determined in leaf and root samples (Orsel et al., 2004). A further aliquot of the same frozen plant material was used for determination of the maximum in vitro activities of nitrate reductase (Ferrario-Mery et al., 1998) and glutamine synthetase (O’Neal and Joy, 1973). Means and standard errors were calculated from three biological repetitions of three plants each.
Total RNAs were isolated from shoots and roots using Trizol reagent (Invitrogen), according to the manufacturer’s protocols.
Microarray analysis was performed using the complete Arabidopsis transcriptome micro array (CATMA) array, containing 24 576 gene-specific tags from Arabidopsis thaliana (Crowe et al., 2003; Hilson et al., 2004). RNA samples from two independent biological replicates were used for each comparison, with dye-swap technical replicates (i.e. four hybridizations per comparison). The labelling, hybridizations and scanning were performed as described previously (Lurin et al., 2004). The statistical analysis was based on two dye swaps (Lurin et al., 2004). To determine differentially expressed genes, we performed a paired t test on the log ratios, assuming that the variance of the log ratios was the same for all genes. The raw P values were adjusted by the Bonferroni method, which controls the family-wise error rate. Genes with a family-wise error rate of 5% were considered as differentially expressed. Using the Bonferroni method (with a type I error equal to 5%) avoids false positives in a multiple-comparison context (Ge et al., 2003).
Reverse transcription reactions and quantitative PCR were performed as described by Chopin et al. (2007) (see Table S2 for primer sets). Results are given as a percentage of the expression of the EF1α gene (At5g60390), which was used as a constitutive reference.
Transgenic plants carrying a pNLP7::GUS construct were grown on horizontal plates (Estelle and Somerville, 1987) for 10 days at 25°C under long-day conditions (16 h light/8 h dark) with 75% relative humidity and with a light intensity of 100 μmol m−2 sec−1. The plantlets were observed under a light microscope (Axioplan 2, Zeiss, http://www.zeiss.com/) after GUS staining (Jefferson, 1987).
Stable expression in Arabidopsis
Transgenic plants carrying a 35S::NLP7cDNA-GFP or 35S::GFP-NLP7cDNA construct (see above) were grown on horizontal plates on standard culture medium (Estelle and Somerville, 1987) for 6 days at 25°C under long-day conditions (16 h light/8 h dark) with 75% relative humidity and a light intensity of 100 μmol m−2 sec−1. The fluorescence was observed using a TCS-SP2-AOBS confocal microscope (Leica, http://www.leica-microsystems.com).
Transient expression in Arabidopsis protoplasts
The pMDC83-NLP7 and pMDC43-NLP7 plasmids used to obtain stable Arabidopsis transformants (see above) were introduced into Arabidopsis cell-suspension protoplasts by polyethylene glycol-mediated transformation (Thomine et al., 2003). Confocal laser-scanning microscopy using a TCS-SP2-AOBS confocal microscope (Leica) was performed 24 h after transformation.
In situ and whole-mount hybridization
A 466 bp 3′NLP7 DNA template for the antisense RNA probe was produced by PCR using the oligonucleotides listed in Table S2. Dioxygenin labelling of the RNA probe, tissue preparation and in situ hybridization were performed as described previously (Smyczynski et al., 2006). Whole-mount hybridization of NLP7 mRNA was performed using the same probe. The coloration was observed under a light microscope (Axioplan 2, Zeiss).
We thank J. Kronenberger, H. Morin and L. Gissot for technical assistance, F. Gosse and J. Talbotec for taking care of the plants, H.-N. Truong and E. Diatloff (Institut des Sciences Végétal, Gif sur Yvettes) for discussions, and D. Tepfer for correcting the use of the English language. This work was supported by grants from Génoplante (to D.P and C.M.) and European Framework 5 (to A.C. and A.K.).