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.

Localized proliferation of lateral roots in NO 3؊ -rich patches is a adaptive root development ͉ ANR1 signaling pathway ͉ plant growth ͉ nitrate sensing ͉ nitrogen nutrition

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-b-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 rootto-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. INTRODUCTIONNitrate and ammonium ions are the two major nitrogen sources for higher plants. Due to its toxicity, ammonium is preferentially assimilated in the root and then transported in an organic form to the aerial parts. By contrast, nitrate can be assimilated into ammonium and then amino acids in the root or shoot. Partitioning of nitrate assimilation between the root and shoot depends on the plant species, external nitrate concentration, temperature, and light intensity (reviewed in Smirnoff and Stewart, 1985). If there is sufficient light, nitrate assimilation in the leaf has a lower energy cost than in the root. However, some disadvantages of leaf nitrate assimilation include (1) if light is limited, nitrate assimilation and carbon dioxide fixation will compete directly for the reductants and ATP generated by photosynthetic electron transport (Canvin and Atkins, 1974), and (2) hydroxyl ions generated in the leaf need to be neutralized by the synthesis of organic acids (in the root, the pH balance may possibly be maintained by reducing proton excretion or increasing bicarbonate excretion). Due to these factors, the partition of nitrate assimilation between the root and shoot shows both seasonal and diurnal fluctuations, allowing the plant to sustain maximal growth. In turn, the partition of nitrate assimilation depends on the partition of nitrate between the root and shoot.To transport nitrate to the aerial parts of the plant, nitrate has to be loaded into the xylem vessels of the root vascular stele. In Arabidopsis thaliana roots, four layers of cells are found surrounding the xylem, these being the epidermis, cortex, endodermis, and pericycle (in the order external to ...

SummaryRoot NO 3 ± uptake and expression of two root NO 3 ± transporter genes (Nrt2;1 and Nrt1) were investigated in response to changes in the N-or C-status of hydroponically grown Arabidopsis thaliana plants.

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.

Arabidopsis thaliana mutants deficient for the NRT1.1 NO 3 ÿ transporter display complex phenotypes, including lowered NO 3 ÿ 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 NH 4 NO 3 as N source. Among these genes, the N satiety-repressed NRT2.1 gene, encoding a major component of the root high-affinity NO 3 ÿ 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 NO 3 ÿ HATS activity in the mutant, suggesting adaptive response to a possible N limitation resulting from NRT1.1 mutation. However, derepression of NRT2.1 in NH 4 NO 3 -fed chl1-5 plants could not be attributed to lowered production of N metabolites. Rather, the results show that normal regulation of NRT2

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 NO 3 ؊ , NH 4 ؉ , and SO 4 2 ؊ 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 NO 3 ؊ transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO 3 ؊ 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.

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 NO 3 ؊ 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 posttranslational 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.The NRT2.1 gene of Arabidopsis thaliana is part of a small multigene family comprising 7 members, which with the exception of NRT2.7, are predominantly expressed in the roots (1, 2). NRT2 genes are found in a large variety of organisms (fungi, certain yeasts, green algae, and plants) and belong to the nitrate nitrite porter family of transporter genes (3).It is generally assumed that NRT2 genes encode high-affinity nitrate (NO 3 Ϫ ) or nitrite transporters (4 -11), and that in higher plants, they play a key role in the root high-affinity transport system (HATS), 3 which ensures uptake of NO 3 Ϫ from the soil solution (3,(12)(13)(14). In all plant species investigated to date, the NO 3 Ϫ HATS displays a saturable activity, with a V max generally reached for NO 3 Ϫ concentrations comprised between 0.2 and 0.5 mM (3,12,14). Although the functional characterization of almost all higher plant NRT2 transporters remains to be done, it is now well documented that NRT2.1 is a major component of the HATS in A. thaliana as shown by the fact that (i) of the seven NRT2 members, only NRT2.1 transcript abundance showed significant correlation (r 2 ϭ 0.74) with HATS activity (2, 11) and (ii) several mutants disrupted for the NRT2.1 gene (15, 16) or for both NRT2.1-NRT2.2 genes (17, 18) have lost up to 75% of the high-affinity NO 3 Ϫ uptake activity. As a consequence, growth of these mutants is severely impaired at low NO 3 Ϫ concentration (15,19,20), but not at high NO 3 Ϫ concentration when low-affinity transporters, most probably those of the NRT1 family (3,12,14), are active.Despite its firmly established role in root NO 3 Ϫ uptake, several aspects of NRT2.1 function remain enigmatic. First, unlike the Aspergillus nidulans or Chlorella sorokiniana NRT2 trans...