Alain Gojon

Nitrate-Regulated Auxin Transport by NRT1.1 Defines a Mechanism for Nutrient Sensing in Plants

Nitrate is both a nitrogen source for higher plants and a signal molecule regulating their development. In Arabidopsis, the NRT1.1 nitrate transporter is crucial for nitrate signaling governing root growth, and has been proposed to act as a nitrate sensor. However, the sensing mechanism is unknown. Herein we show that NRT1.1 not only transports nitrate but also facilitates uptake of the phytohormone auxin. Moreover, nitrate inhibits NRT1.1-dependent auxin uptake, suggesting that transduction of nitrate signal by NRT1.1 is associated with a modification of auxin transport. Among other effects, auxin stimulates lateral root development. Mutation of NRT1.1 enhances both auxin accumulation in lateral roots and growth of these roots at low, but not high, nitrate concentration. Thus, we propose that NRT1.1 represses lateral root growth at low nitrate availability by promoting basipetal auxin transport out of these roots. This defines a mechanism connecting nutrient and hormone signaling during organ development.

Three Functional Transporters for Constitutive, Diurnally Regulated, and Starvation-Induced Uptake of Ammonium into Arabidopsis Roots

Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake–deficient yeast mutant. Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between ≤0.5 and 40 μM. Correlation of gene expression with 15NH4+ uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers. Transcript levels of AtAMT1;1, which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed. All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots. Our results show that high-affinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family.

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.

A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species.

An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high‐affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock‐out mutant where the T‐DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3′ region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low‐affinity system. Moreover, the de‐regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild‐type. These results demonstrate that plant NRT2 proteins do have a role in HATS.

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.

Regulation of Root Ion Transporters by Photosynthesis: Functional Importance and Relation with Hexokinase

Coordination between the activity of ion transport systems in the root and photosynthesis in the shoot is a main feature of the integration of ion uptake in the whole plant. However, the mechanisms that ensure this coordination are largely unknown at the molecular level. Here, we show that the expression of five genes that encode root NO3−, NH4+, and SO42− transporters in Arabidopsis is regulated diurnally and stimulated by sugar supply. We also provide evidence that one Pi and one K+ transporter also are sugar inducible. Sucrose, glucose, and fructose are able to induce expression of the ion transporter genes but not of the carboxylic acids malate and 2-oxoglutarate. For most genes investigated, induction by light and induction by sucrose are strongly correlated, indicating that they reflect the same regulatory mechanism (i.e., stimulation by photosynthates). The functional importance of this control is highlighted by the phenotype of the atnrt2 mutant of Arabidopsis. In this mutant, the deletion of the sugar-inducible NO3− transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO3− influx by light and sugar. None of the sugar analogs used (3-O-methylglucose, 2-deoxyglucose, and mannose) is able to mimic the inducing effect of sugars. In addition, none of the sugar-sensing mutants investigated (rsr1-1, sun6, and gin1-1) is altered in the regulation of AtNrt2.1 expression. These results indicate that the induction of AtNrt2.1 expression by sugars is unrelated to the main signaling mechanisms documented for sugar sensing in plants, such as regulation by sucrose, hexose transport, and hexokinase (HXK) sensing activity. However, the stimulation of AtNrt2.1 transcript accumulation by sucrose and glucose is abolished in an antisense AtHXK1 line, suggesting that HXK catalytic activity and carbon metabolism downstream of the HXK step are crucial for the sugar regulation of AtNrt2.1 expression.

A framework integrating plant growth with hormones and nutrients

It is well known that nutrient availability controls plant development. Moreover, plant development is finely tuned by a myriad of hormonal signals. Thus, it is not surprising to see increasing evidence of coordination between nutritional and hormonal signaling. In this opinion article, we discuss how nitrogen signals control the hormonal status of plants and how hormonal signals interplay with nitrogen nutrition. We further expand the discussion to include other nutrient-hormone pairs. We propose that nutrition and growth are linked by a multi-level, feed-forward cycle that regulates plant growth, development and metabolism via dedicated signaling pathways that mediate nutrient and hormonal regulation. We believe this model will provide a useful concept for past and future research in this field.

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.