Four low-affinity (NRT1), and seven high-affinity (NRT2) nitrate transporter gene homologues have been identified in Arabidopsis thaliana. We investigated the transcript abundances of all eleven genes in shoot and root tissues in response to the provision of 1 mM NO(3)(-), using relative quantitative RT-PCR. Based upon this criterion, genes were classified as nitrate-inducible, nitrate-repressible, or nitrate-constitutive. AtNRT1.1, 2.1, and 2.2 were strongly induced by NO(3)(-), peaking at 3-12 h and subsequently declining. By contrast AtNRT2.4 showed only modest induction both in shoots and roots. Expression of AtNRT2.5, one of the nitrate-repressible genes, was strongly suppressed by nitrate provision in both roots and shoots. The last group, characterized by a constitutive expression pattern, included AtNRT1.2, 1.4, 2.3, 2.6, and 2.7. Correlation coefficients between (13)NO(3)(-) influx from 100 micro M and 5 mM [NO(3)(-)], suggest that high- and low-affinity transport systems are mediated primarily by AtNRT2.1 and AtNRT1.1, respectively. Functional roles for the other members of these families remain uncertain.

Inorganic nitrogen concentrations in soil solutions vary across several orders of magnitude among different soils and as a result of seasonal changes. In order to respond to this heterogeneity, plants have evolved mechanisms to regulate and influx. In addition, efflux analysis using (13)N has revealed that there is a co-ordinated regulation of all component fluxes within the root, including biochemical fluxes. Physiological studies have demonstrated the presence of two high-affinity transporter systems (HATS) for and one HATS for in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for and five members of the AMT1 family encoding putative HATS for. The induction of high-affinity transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of, while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated and may act post-transcriptionally on transporter function. The present challenge is to resolve the functions of all of these genes. In Aspergillus nidulans and Chlamydomonas reinhardtii there are but two high-affinity transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of absorption over the wide range of concentration normally found in nature. Such kinetic differentiation may also have occurred among higher plant transporters. The characterization of transporter function in higher plants is currently being inferred from patterns of gene expression in roots and shoots, as well as through studies of heterologous expression systems and knockout mutants.

SummaryPutative high-affinity nitrate (NO 3 -) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic highaffinity NO 3 -transporters. Both genes were induced by NO 3 -, but Nrt2;1At gene expression was not apparent in 2-and 5-day-old plants. By 10 days, and thereafter, Nrt2; 1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13 NO 3 -influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH 4 ⍣ , gln and other amino acids.

SummarySulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO 4 2-. Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO 4 2-uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO 4 2-restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO 4 2-transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.

To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.