Brian G. Forde

Nitrate transporters in plants : structure, function and regulation.

Physiological studies have established that plants acquire their NO(-3) from the soil through the combined activities of a set of high- and low-affinity NO(-3) transport systems, with the influx of NO(-3) being driven by the H(+) gradient across the plasma membrane. Some of these NO(-3) transport systems are constitutively expressed, while others are NO(-3)-inducible and subject to negative feedback regulation by the products of NO(-3) assimilation. Here we review recent progress in the characterisation of the two families of NO(-3) transporters that have so far been identified in plants, their structure and their regulation, and consider the evidence for their roles in NO(-3) acquisition. We also discuss what is currently known about the genetic basis of NO(-3) induction and feedback repression of the NO(-3) transport and assimilatory pathway in higher plants.

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.

Nitrate and ammonium nutrition of plants : physiological and molecular perspectives.

Nitrogen is the mineral nutrient that plants need in the greatest quantities and the one that most frequently limits plant growth and crop yields. Most plants get their nitrogen (N) from the soil as either nitrate or ammonium, with some species showing a strong preference for one ionic form over the other. The uptake of nitrate and ammonium ions by roots involves a complex set of membrane transport systems that includes both high- and low-affinity transporters; net uptake rates can also be strongly influenced by the rate at which these ions efflux from root cells. Here we review our current picture of the mechanisms responsible for the uptake and efflux of nitrate and ammonium, attempting to integrate the large body of physiological data with the recent advances in the molecular biology of nitrate and ammonium transporters in bacteria and algae as well as in higher plants. We also review what is known at the physiological and molecular levels about the regulation of the N uptake systems, a process which involves both positive signals from soil nitrate or ammonium and feedback inhibitory signals that are generated by the plants internal N status

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.

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.

Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans

The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane transporters. Degenerate oligonucleotides corresponding to the crnA sequences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related sequences from barley root poly(A)+ RNA. A 130 bp cDNA fragment with sequence similarities to crnA was amplified and used as a probe to screen a barley root cDNA library. Two full-length clones (pBCH1 and pBCH2) were isolated. The nt sequences of pBHC1 and pBCH2 are closely related (80% identical) and potentially encode hydrophobic polypeptides of 54.7 and 55.0 kDa respectively, with twelve predicted transmembrane domains. The encoded polypeptides are 41-43% identical to the A. nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate transporter from the eukaryotic alga Chlamydomonas reinhardtii. Phylogenetic analysis indicated that crnA, nar-3 and the barley homologues belong to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli. In northern blots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly induced in barley roots by NO3-, but not by NH4+, and genomic Southern blots indicated that there may be seven to ten BCH1-related genes in the barley genome.

Nitrate signalling mediated by the NRT1.1 nitrate transporter antagonises l‐glutamate‐induced changes in root architecture

Arabidopsis root architecture is highly responsive to changes in the nitrogen supply. External NO(3)(-) stimulates lateral root growth via a signalling pathway involving the ANR1 MADS box transcription factor, while the presence of exogenous l-glutamate (Glu) at the primary root tip slows primary root growth and stimulates root branching. We have found that NO(3)(-), in conjunction with Glu, has a hitherto unrecognized role in regulating the growth of primary roots. Nitrate was able to stimulate primary root growth, both directly and by antagonising the inhibitory effect of Glu. Each response depended on direct contact between the primary root tip and the NO(3)(-), and was not elicited by an alternative N source (NH(4)(+)). The chl1-5 mutant, which is defective in the NRT1.1 (CHL1) NO(3)(-) transporter, was insensitive to NO(3)(-) antagonism of Glu signalling, while an anr1 mutant retained its sensitivity. Sensitivity to NO(3)(-) was restored in a chl1-5 mutant constitutively expressing NRT1.1. However, expression in chl1-5 of a transport-competent but non-phosphorylatable form of NRT1.1 not only failed to restore NO(3)(-) sensitivity but also had a dominant-negative effect on Glu sensitivity. Our results indicate the existence of a NO(3)(-) signalling pathway at the primary root tip that can antagonise the roots response to Glu, and they further suggest that NRT1.1 has a direct NO(3)(-) sensing role in this pathway. We discuss how the observed signalling interactions between NO(3)(-) and Glu could provide a mechanism for modulating root architecture in response to changes in the relative abundance of organic and inorganic N.

Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris

In plants, glutamine synthetase (GS) is the enzyme primarily responsible for the assimilation of ammonia into organic nitrogen. In Phaseolus vulgaris a number of isoenzymic forms of GS are found, each of which consists of eight subunits of mol. wt 41 000‐45 000. The GS subunits of P. vulgaris have previously been shown to be encoded by a small multigene family and a partial cDNA clone for a nodule‐specific GS subunit has been obtained. We report here the isolation and nucleotide sequencing of two essentially full‐length GS cDNA clones (pR‐1 and pR‐2) from a root cDNA library and the deduced amino acid sequences of the corresponding GS subunits (355 amino acid residues each). The coding sequences of pR‐1 and pR‐2 are closely related (80% nucleotide homology, 88% amino acid homology), but their 5′‐ and 3′‐untranslated regions have diverged almost completely. Both pR‐1 and pR‐2 are related to, but distinct from, the nodule GS clone, pcPvNGS‐01 (or pN‐1). Hybridization to genomic Southern blots showed that the three GS mRNAs are encoded by three seperate genes and indicated the existence of a fourth class of GS gene. An S1 nuclease protection assay demonstrated the presence of R‐1 and R‐2 mRNA in both roots and leaves and confirmed that expression of the N‐1 gene is nodule‐specific. Expression of the R‐1 and R‐2 genes in the roots did not change significantly during nodulation. However, only the R‐1 gene is expressed in the nodules themselves, indicating that the R‐2 gene is specifically repressed during nodule development.

Molecular and Developmental Biology of Inorganic Nitrogen Nutrition

Unique among the major mineral nutrients, inorganic N is available to plants in both anionic and cationic forms (NO3− and NH4+, respectively). The relative abundance of these two ions in natural soils is highly variable and to a large degree depends on the relative rates of two microbial processes: mineralisation (the release of NH4+ from organic N) and nitrification (the conversion of NH4+ to NO3−) (Marschner, 1995). In well-aerated soils nitrification is rapid, so that NH4+ concentrations are low and NO3− is the main N source, but in waterlogged or acidic soils nitrification is inhibited and NH4+ accumulates. Most plants (including Arabidopsis) seem to be able to use either form of N, although exceptions to this rule are known (e.g. Kronzucker et al., 1997). Nitrogens importance in plant biology extends far beyond its role as a nutrient. It is now clear that several different N compounds, including NO3−, NH4+ and some of the products of their assimilation, exert strong regulatory effects on both metabolic and developmental pathways (Redinbaugh and Campbell, 1991; Crawford, 1995; Forde and Clarkson, 1999; Stitt, 1999; Zhang and Forde, 2000; Coruzzi and Bush, 2001; Coruzzi and Zhou, 2001). Both the biochemical and the regulatory aspects of inorganic N nutrition with emphasis on Arabidopsis will be considered in this chapter.

PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.