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 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 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.     

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 NO(3)(-) and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO(3)(-) 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 NO(3)(-) availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO(3)(-) 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 NO(3)(-)) 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 NO(3)(-)) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO(3)(-) was markedly different. After transfer from 10 to 0.5 mm NO(3)(-), 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 NO(3)(-) uptake measured in the mutant. This suggests that the uptake rate of NO(3)(-), 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 NO(3)(-) uptake by the whole root system and even of the presence of added NO(3)(-) 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 NO(3)(-) availability, both indirectly through its role as a major NO(3)(-) uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.     

Are phloem amino acids involved in the shoot to root control of NO3- uptake in Ricinus communis plants?

The putative role of phloem amino acids as negative feedback signals for root NO3^- uptake was investigated in Ricinus communis L. The NO3^--grown plants were subjected to N-deficiency due either to complete N-deprivation, or to localized N-deprivation on one side of a split-root system. In comparison with controls, complete N-deprivation resulted in a transient increase in ¹⁵NO3^- influx, and in profound changes in downward phloem transport of amino acids. Total amino acid concentration in the phloem sap decreased by 40%, but responses markedly differed between the individual amino acids. Concentrations of Gln and Ser were rapidly lowered by 50%, while those of Val, Phe, Leu, and Ile displayed a marked increase. Localized N-deprivation on one side of the split root system also resulted in the up-regulation of ¹⁵NO3^- influx in the roots still supplied with NO3^-. However, the amino acid composition of the phloem sap directed to these roots was not modified by the treatment, and remained similar to that in N-sufficient control plants. Only amino acid transport to the N-deprived roots was affected as observed in response to complete N-deprivation. The results from split-root plants indicate that the response of root NO3^- influx to N-deficiency is controlled by shoot-borne regulatory signals, and provide a case study where these signals are not related to a qualitative change or a significant decrease in downward phloem transport of amino acids.     

Effects of genetic modification of nitrate reductase expression on 15NO3– uptake and reduction in Nicotiana plants

The physiological consequences for NO₃^– utilization by the plant of underexpression and overexpression of nitrate reductase (NR) were investigated in nine transformants of Nicotiana tabacum and Nicotiana plumbaginifolia. The in vitro NR activities (NRAs) in both roots and leaves of low- and high-NR tobacco transformants ranged from 5–10% to 150–200%, respectively, of those measured in wild-type plants. The level of NR expression markedly affected the NO₃^– reduction efficiency in detached leaves and intact plants. In both species, ¹⁵NO₃^– reduction ranged from 15–45% of ¹⁵NO₃^– uptake in the low-NR plants, to 40–80% in the wild-type, and up to 95% in high-NR plants. In the high-NR genotypes, however, total ¹⁵NO₃^– assimilation was not significantly increased when compared with that in wild-type plants, because the higher ¹⁵NO₃^– reduction efficiency was offset by lower ¹⁵NO₃^– uptake by the roots. The inhibition of NO₃^– uptake appeared to be the result of negative feedback regulation of NO₃^– influx, and is interpreted as an adjustment of NO₃^– uptake to prevent excessive amino acid synthesis. In genotypes underexpressing NR, the low ¹⁵NO₃^– reduction efficiency also was generally associated with a decrease in net ¹⁵NO₃^– uptake as compared with the wild type. Thus, underexpression of NR resulted in an inhibition of reduced ¹⁵N synthesis in the plant, although the effect was much less pronounced than that expected from the very low NRAs. The restricted NO₃^– uptake in low-NR plants emphasizes the point that the products of NO₃^– assimilation are not the only factors responsible for down-regulation of the NO₃^– uptake system.     

Diurnal regulation of NO3-uptake in soybean plants II. Relationship with accumulation of NO and asparagine in the roots

Experiments were performed with soybean plants to test the hypothesisthat the inhibition of NO₃^– uptake in darkness is dueto feedback control by NO₃^– and/or Asn accumulating inthe roots. Xylem export of N compounds was shown to depend onwater flux in both excised root systems and ¹⁵N-labelled intactplants, suggesting that the shortage of transpiration in darknessmay be responsible for the retention of NO₃^– and Asn inthe roots. This was verified in experiments where the light/darkpattern of transpiration was modulated in intact plants by changingthe relative humidity of the atmosphere. Any decrease of transpirationat night was associated with a concurrent stimulation of NO₃^–and Asn accumulations in the roots. However, the light/darkrhythmicity of NO₃^– uptake was only marginally affectedby these treatments, and thusappeared quite independent fromtranspiration and root NO₃^– or Asn levels. Typically,the maintainance of a constant transpiration during the day/nightcycle did not suppress the inhibition of NO₃^– uptake indarkness, whereas it almost prevented the dark increase in rootNO₃^– and Asn contents. These data strongly support theconclusion that the effect of light on NO₃^– uptake isnot mediated by changes in translocation and accumulation ofN compounds. Key words: Glycine max, light/dark, cycles, nitrate uptake, transpiration, transport of N compounds, accumulation of N compounds     

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.