Pascal Tillard

The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches

Localized proliferation of lateral roots in NO3−-rich patches is a striking example of the nutrient-induced plasticity of root development. In Arabidopsis, NO3− stimulation of lateral root elongation is apparently under the control of a NO3−-signaling pathway involving the ANR1 transcription factor. ANR1 is thought to transduce the NO3− signal internally, but the upstream NO3− sensing system is unknown. Here, we show that mutants of the NRT1.1 nitrate transporter display a strongly decreased root colonization of NO3−-rich patches, resulting from reduced lateral root elongation. This phenotype is not due to lower specific NO3− uptake activity in the mutants and is not suppressed when the NO3−-rich patch is supplemented with an alternative N source but is associated with dramatically decreased ANR1 expression. These results show that NRT1.1 promotes localized root proliferation independently of any nutritional effect and indicate a role in the ANR1-dependent NO3− signaling pathway, either as a NO3− sensor or as a facilitator of NO3− influx into NO3−-sensing cells. Consistent with this model, the NRT1.1 and ANR1 promoters both directed reporter gene expression in root primordia and root tips. The inability of NRT1.1-deficient mutants to promote increased lateral root proliferation in the NO3−-rich zone impairs the efficient acquisition of NO3− and leads to slower plant growth. We conclude that NRT1.1, which is localized at the forefront of soil exploration by the roots, is a key component of the NO3−-sensing system that enables the plant to detect and exploit NO3−-rich soil patches.

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.

Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage

AbstractThe NRT2.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-β-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips. Abbreviation: GUS, β-glucuronidase

Regulation of the high-affinity NO3- uptake system by NRT1.1-mediated NO3- demand signaling in arabidopsis

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} transport system (HATS) that plays a crucial role in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} uptake by the plant. Although NRT2.1 was known to be induced by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} concentration decreases to a low level (<0.5 mm) in media containing a high concentration of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document} or Gln (≥1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} is specifically mediated by the NRT1.1 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} in the presence of a reduced N source. Under low \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}/\mathrm{high{\,}NH}_{4}^{{+}}\) \end{document} provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document} toxicity because \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} taken up by the HATS in this situation prevents the detrimental effects of pure \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NH}_{4}^{{+}}\) \end{document} nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} demand of the plant in relation to the various specific roles that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document} plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{{-}}\) \end{document}.

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.

Regulation of Root Nitrate Uptake at the NRT2.1 Protein Level in Arabidopsis thaliana

In Arabidopsis the NRT2.1 gene encodes a main component of the root high-affinity nitrate uptake system (HATS). Its regulation has been thoroughly studied showing a strong correlation between NRT2.1 expression and HATS activity. Despite its central role in plant nutrition, nothing is known concerning localization and regulation of NRT2.1 at the protein level. By combining a green fluorescent protein fusion strategy and an immunological approach, we show that NRT2.1 is mainly localized in the plasma membrane of root cortical and epidermal cells, and that several forms of the protein seems to co-exist in cell membranes (the monomer and at least one higher molecular weight complex). The monomer is the most abundant form of NRT2.1, and seems to be the one involved in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{-}\) \end{document} transport. It strictly requires the NAR2.1 protein to be expressed and addressed at the plasma membrane. No rapid changes in NRT2.1 abundance were observed in response to light, sucrose, or nitrogen treatments that strongly affect both NRT2.1 mRNA level and HATS activity. This suggests the occurrence of post-translational regulatory mechanisms. One such mechanism could correspond to the cleavage of NRT2.1 C terminus, which results in the presence of both intact and truncated proteins in the plasma membrane.

Oxidative Pentose Phosphate Pathway-Dependent Sugar Sensing as a Mechanism for Regulation of Root Ion Transporters by Photosynthesis

Root ion transport systems are regulated by light and/or sugars, but the signaling mechanisms are unknown. We showed previously that induction of the NRT2.1 NO3− transporter gene by sugars was dependent on carbon metabolism downstream hexokinase (HXK) in glycolysis. To gain further insights on this signaling pathway and to explore more systematically the mechanisms coordinating root nutrient uptake with photosynthesis, we studied the regulation of 19 light-/sugar-induced ion transporter genes. A combination of sugar, sugar analogs, light, and CO2 treatments provided evidence that these genes are not regulated by a common mechanism and unraveled at least four different signaling pathways involved: regulation by light per se, by HXK-dependent sugar sensing, and by sugar sensing upstream or downstream HXK, respectively. More specific investigation of sugar-sensing downstream HXK, using NRT2.1 and NRT1.1 NO3− transporter genes as models, highlighted a correlation between expression of these genes and the concentration of glucose-6-P in the roots. Furthermore, the phosphogluconate dehydrogenase inhibitor 6-aminonicotinamide almost completely prevented induction of NRT2.1 and NRT1.1 by sucrose, indicating that glucose-6-P metabolization within the oxidative pentose phosphate pathway is required for generating the sugar signal. Out of the 19 genes investigated, most of those belonging to the NO3−, NH4+, and SO42− transporter families were regulated like NRT2.1 and NRT1.1. These data suggest that a yet-unidentified oxidative pentose phosphate pathway-dependent sugar-sensing pathway governs the regulation of root nitrogen and sulfur acquisition by the carbon status of the plant to coordinate the availability of these three elements for amino acid synthesis.

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.

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.

Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System

In Arabidopsis (Arabidopsis thaliana), the NRT2.1 gene codes for the main component of the root nitrate (NO3−) high-affinity transport system (HATS). Due to the strong correlation generally found between high-affinity root NO3− influx and NRT2.1 mRNA level, it has been postulated that transcriptional regulation of NRT2.1 is a key mechanism for modulation of the HATS activity. However, this hypothesis has never been demonstrated, and is challenged by studies suggesting the occurrence of posttranscriptional regulation at the NRT2.1 protein level. To unambiguously clarify the respective roles of transcriptional and posttranscriptional regulations of NRT2.1, we generated transgenic lines expressing a functional 35S::NRT2.1 transgene in an atnrt2.1 mutant background. Despite a high and constitutive NRT2.1 transcript accumulation in the roots, the HATS activity was still down-regulated in the 35S::NRT2.1 transformants in response to repressive nitrogen or dark treatments that strongly reduce NRT2.1 transcription and NO3− HATS activity in the wild type. In some treatments, this was associated with a decline of NRT2.1 protein abundance, indicating posttranscriptional regulation of NRT2.1. However, in other instances, NRT2.1 protein level remained constant. Changes in abundance of NAR2.1, a partner protein of NRT2.1, closely followed those of NRT2.1, and thus could not explain the close-to-normal regulation of the HATS in the 35S::NRT2.1 transformants. Even if in certain conditions the transcriptional regulation of NRT2.1 contributes to a limited extent to the control of the HATS, we conclude from this study that posttranscriptional regulation of NRT2.1 and/or NAR2.1 plays a predominant role in the control of the NO3− HATS in Arabidopsis.