Marc Lepetit

A Central Role for the Nitrate Transporter NRT2.1 in the Integrated Morphological and Physiological Responses of the Root System to Nitrogen Limitation in Arabidopsis

Up-regulation of the high-affinity transport system (HATS) for NO3− and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO3− HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis (Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture (RSA) to low NO3− availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO3− medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation (transfer from 10 mm to 1 or 0.5 mm NO3−) mostly led to an increase in the number of visible laterals, while severe nitrogen stress (transfer from 10 mm to 0.1 or 0.05 mm NO3−) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO3− was markedly different. After transfer from 10 to 0.5 mm NO3−, the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO3− uptake measured in the mutant. This suggests that the uptake rate of NO3−, rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO3− uptake by the whole root system and even of the presence of added NO3− in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO3− availability, both indirectly through its role as a major NO3− uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.

Mutation of the Arabidopsis NRT1.5 Nitrate Transporter Causes Defective Root-to-Shoot Nitrate Transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

Transcript Profiling in the chl1-5 Mutant of Arabidopsis Reveals a Role of the Nitrate Transporter NRT1.1 in the Regulation of Another Nitrate Transporter, NRT2.1

Arabidopsis thaliana mutants deficient for the NRT1.1 NO3− transporter display complex phenotypes, including lowered NO3− uptake, altered development of nascent organs, and reduced stomatal opening. To obtain further insight at the molecular level on the multiple physiological functions of NRT1.1, we performed large-scale transcript profiling by serial analysis of gene expression in the roots of the chl1-5 deletion mutant of NRT1.1 and of the Columbia wild type. Several hundred genes were differentially expressed between the two genotypes, when plants were grown on NH4NO3 as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO3− transport system (HATS), was found to be strongly derepressed in the chl1-5 mutant (as well as in other NRT1.1 mutants). This was associated with a marked stimulation of the NO3− HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH4NO3-fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2.1 expression is strongly altered in the chl1-5 mutant, where this gene is no more repressible by high N provision to the plant. This indicates that NRT1.1 plays an unexpected but important role in the regulation of both NRT2.1 expression and NO3− HATS activity. Overexpression of NRT2.1 was also found in wild-type plants supplied with 1 mM NH4+ plus 0.1 mM NO3−, a situation where NRT1.1 is likely to mediate very low NO3− transport. Thus, we suggest that it is the lack of NRT1.1 activity, rather than the absence of this transporter, that derepresses NRT2.1 expression in the presence of NH4+. Two hypotheses are discussed to explain these results: (1) NRT2.1 is upregulated by a NO3− demand signaling, indirectly triggered by lack of NRT1.1-mediated uptake, which overrides feedback repression by N metabolites, and (2) NRT1.1 plays a more direct signaling role, and its transport activity generates an unknown signal required for NRT2.1 repression by N metabolites. Both mechanisms would warrant that either NRT1.1 or NRT2.1 ensure significant NO3− uptake in the presence of NH4+ in the external medium, which is crucial to prevent the detrimental effects of pure NH4+ nutrition.

Systemic Signaling of the Plant Nitrogen Status Triggers Specific Transcriptome Responses Depending on the Nitrogen Source in Medicago truncatula

Legumes can acquire nitrogen (N) from NO3−, NH4+, and N2 (through symbiosis with Rhizobium bacteria); however, the mechanisms by which uptake and assimilation of these N forms are coordinately regulated to match the N demand of the plant are currently unknown. Here, we find by use of the split-root approach in Medicago truncatula plants that NO3− uptake, NH4+ uptake, and N2 fixation are under general control by systemic signaling of plant N status. Indeed, irrespective of the nature of the N source, N acquisition by one side of the root system is repressed by high N supply to the other side. Transcriptome analysis facilitated the identification of over 3,000 genes that were regulated by systemic signaling of the plant N status. However, detailed scrutiny of the data revealed that the observation of differential gene expression was highly dependent on the N source. Localized N starvation results, in the unstarved roots of the same plant, in a strong compensatory up-regulation of NO3− uptake but not of either NH4+ uptake or N2 fixation. This indicates that the three N acquisition pathways do not always respond similarly to a change in plant N status. When taken together, these data indicate that although systemic signals of N status control root N acquisition, the regulatory gene networks targeted by these signals, as well as the functional response of the N acquisition systems, are predominantly determined by the nature of the N source.

Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses.

Adaptation of Medicago truncatula to local nitrogen (N) limitation was investigated to provide new insights into local and systemic N signaling. The split-root technique allowed a characterization of the local and systemic responses of NO(3)(-) or N(2)-fed plants to localized N limitation. (15)N and (13)C labeling were used to monitor plant nutrition. Plants expressing pMtENOD11-GUS and the sunn-2 hypernodulating mutant were used to unravel mechanisms involved in these responses. Unlike NO(3)(-)-fed plants, N(2)-fixing plants lacked the ability to compensate rapidly for a localized N limitation by up-regulating the N(2)-fixation activity of roots supplied elsewhere with N. However they displayed a long-term response via a growth stimulation of pre-existing nodules, and the generation of new nodules, likely through a decreased abortion rate of early nodulation events. Both these responses involve systemic signaling. The latter response is abolished in the sunn mutant, but the mutation does not prevent the first response. Local but also systemic regulatory mechanisms related to plant N status regulate de novo nodule development in Mt, and SUNN is required for this systemic regulation. By contrast, the stimulation of nodule growth triggered by systemic N signaling does not involve SUNN, indicating SUNN-independent signaling.

Analysis of a sunflower polyubiquitin promoter by transient expression

Abstract We have recently isolated two sunflower polyubiquitin genes (UbB1 and UbB2) which are induced by heat-stress and show a highly conserved region of about 250 bp upstream of the transcription initiation site. The promoter activity of this region has been analyzed by transient expression using the β-glucoronidase (GUS) gene as reporter in tobacco protoplasts. The GUS expression in absence of any external inducer is five times higher than that observed under the control of the CaMV 35S promoter. Analysis of different deletions within the 250-bp region showed that a 65-bp sequence is responsible for most of the promoter activity in tobacco protoplasts. This activity is intron-independent and is not enhanced when protoplasts are heat-stressed. Sequence homologies strongly suggest that UbB1 plays a major role in the stress response in sunflower.

HIGH NITROGEN INSENSITIVE 9 (HNI9)-mediated systemic repression of root NO3− uptake is associated with changes in histone methylation

In plants, root nitrate uptake systems are under systemic feedback repression by the N satiety of the whole organism, thus adjusting the N acquisition capacity to the N demand for growth; however, the underlying molecular mechanisms are largely unknown. We previously isolated the Arabidopsis high nitrogen-insensitive 9-1 (hni9-1) mutant, impaired in the systemic feedback repression of the root nitrate transporter NRT2.1 by high N supply. Here, we show that HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex. HNI9/AtIWS1 acts in roots to repress NRT2.1 transcription in response to high N supply. At a genomic level, HNI9/AtIWS1 is shown to play a broader role in N signaling by regulating several hundred N-responsive genes in roots. Repression of NRT2.1 transcription by high N supply is associated with an HNI9/AtIWS1-dependent increase in histone H3 lysine 27 trimethylation at the NRT2.1 locus. Our findings highlight the hypothesis that posttranslational chromatin modifications control nutrient acquisition in plants.

Gene Expression of the NO3– Transporter NRT1.1 and the Nitrate Reductase NIA1 Is Repressed in Arabidopsis Roots by NO2–, the Product of NO3– Reduction

NRT1.1 and NIA1 genes, which encode a nitrate (NO3–) transporter and the minor isoform of NO3– reductase (NR), respectively, are overexpressed in roots of NR-deficient mutants of Arabidopsis grown on nutrient solution containing NO3– and reduced N. The overexpression is found only in mutants with reduced NIA2 activity, and disruption of the NIA1 gene alone has no effect on NRT1.1 expression. Because the up-regulation of NRT1.1 and NIA1 is observed in N-sufficient NR mutant plants, it cannot be related to a release of the general feedback repression exerted by the N status of the plant. Our data do not support the hypothesis of overinduction of these genes by an increased concentration of NO3– in tissues. Furthermore, although a control by external pH might contribute to the regulation of NRT1.1, changes in external pH due to lack of NR activity cannot alone explain the up-regulation of both genes. The stimulation of NRT1.1 and NIA1 in NR mutants in these conditions suggests that NR activity is able to repress directly the expression of both genes independently of the availability of reduced N metabolites in wild-type plants. Accordingly, nitrite (NO2–) strongly represses NRT1.1 and NIA1 transcript accumulation in the roots. This effect is rapid, specific, and reversible. Furthermore, transport studies on plants exposed to NO2– show that down-regulation of the NRT1.1 gene is associated with a decrease in NO3– influx. These results indicate that feedback regulation of genes of NO3– assimilation relies not only on the repression exerted by reduced N metabolites, such as NH4+ or amino acids, but may also involve the action of NO2– as a regulatory signal.

Organization and expression of the gene coding for the potassium transport system AKT1 of Arabidopsis thaliana

We have isolated and sequenced the genomic clone coding for the potassium transport system AKT1 of Arabidopsis thaliana. Southern blot analysis indicated that the gene is present in one copy in the Arabidopsis genome. The coding sequence is interrupted by ten introns. Sequence comparisons of AKT1 polypeptide with the voltage-gated inward rectifying Arabidopsis K+ channel KAT1, and with voltageor cyclic nucleotide-gated channels from insects and mammals, revealed a highly conserved domain found specifically in both plant polypeptides, and correponding to about the last 50 amino acids of their C-terminal region. Northern blot analysis of AKT1 expression in Arabidopsis seedlings indicated that AKT1 is preferentially expressed in roots. No transcript was detected in extracts from heterotrophic suspension culture cells. Depleting K+ in the Arabidopsis seedling culture medium for 4 days led to a strong decrease in K+ tissue content (ca. 50%), but did not affect AKT1 transcript level.

Identification of Arabidopsis Mutants Impaired in the Systemic Regulation of Root Nitrate Uptake by the Nitrogen Status of the Plant

Nitrate uptake by the roots is under systemic feedback repression by high nitrogen (N) status of the whole plant. The NRT2.1 gene, which encodes a NO3− transporter involved in high-affinity root uptake, is a major target of this N signaling mechanism. Using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the pNRT2.1::LUC reporter gene (NL line), we performed a genetic screen to isolate mutants altered in the NRT2.1 response to high N provision. Three hni (for high nitrogen insensitive) mutants belonging to three genetic loci and related to single and recessive mutations were selected. Compared to NL plants, these mutants display reduced down-regulation of both NRT2.1 expression and high-affinity NO3− influx under repressive conditions. Split-root experiments demonstrated that this is associated with an almost complete suppression of systemic repression of pNRT2.1 activity by high N status of the whole plant. Other mechanisms related to N and carbon nutrition regulating NRT2.1 or involved in the control of root SO4− uptake by the plant sulfur status are not or are slightly affected. The hni mutations did not lead to significant changes in total N and NO3− contents of the tissues, indicating that hni mutants are more likely regulatory mutants rather than assimilatory mutants. Nevertheless, hni mutations induce changes in amino acid, organic acid, and sugars pools, suggesting a possible role of these metabolites in the control of NO3− uptake by the plant N status. Altogether, our data indicate that the three hni mutants define a new class of N signaling mutants specifically impaired in the systemic feedback repression of root NO3− uptake.