Journal of Experimental Botany

JXB Advance Access published online on May 22, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm066

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FOCUS PAPER

Nitrate transport and signalling

Anthony J. Miller^1,*, Xiaorong Fan^1,2, Mathilde Orsel³, Susan J. Smith¹ and Darren M. Wells¹

¹Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
²College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
³Ameloriation des Plantes et Biotechnologies Végétales, UMR 118, INRA-Agrocampus Rennes, BP 35327, 35653, Le Rheu Cedex, France

^* To whom correspondence should be addressed. E-mail: tony.miller{at}bbsrc.ac.uk

Received 19 December 2005; Revised 2 March 2007 Accepted 6 March 2007

	Abstract

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
Physiological measurements of nitrate () uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity transport system (HATS) and its regulation and yet measurements of soil show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing availability at the surface of the root. Another transporter protein, AtNRT1.1 also has a role in sensing that, like AtNRT2.1, is independent of their transport function. From the range of concentrations present in the soil it is proposed that the -inducible part of HATS functions chiefly as a sensor for root availability. Two other key transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.

Key words: High affinity uptake, nitrate signalling, nitrate transporters, nitrate uptake

	Introduction

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
The first eukaryotic transporter gene was isolated over 15 years ago from the fungus, Aspergillus nidulans (Unkles et al., 1991). A few years later a gene encoding an Arabidopsis transporter was isolated and although this gene also had 12 putative trans-membrane domains it was phylogenetically unrelated to the earlier fungal gene (Tsay et al., 1993). These two types of genes each define the types of transporters that mediate uptake from external sources, and they have become known as the NRT1 and NRT2 families. Both families transport together with a proton (H⁺) in a symport mechanism that is driven by the pH gradients across membranes. Although other membrane proteins that can transport have been identified, these two families are the best characterized and are therefore the main topic of this review. Most of the information for these gene families has been obtained for Arabidopsis and so much of what is discussed refers to the situation in this plant. In this review, consideration is given as to how well our molecular and physiological knowledge can be integrated and what the key questions and opportunities are for the future is discussed. The transporters that might be key targets for future improvement of nitrogen use by crops are discussed along the way, but to understand acquisition by land plants the availability in the soil must be considered and this is where we start.

	Nitrate in the soil

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
Nitrogen (N) can be available to plant roots in several different forms, including , ammonium (), and organic forms chiefly amino acids. Nitrate is usually the most abundant source of N but in this anionic form is readily dissolved in soil water and so is very mobile in the soil profile. Nitrate is generated by the microbial conversion of other soil N forms and this process occurs via intermediates, such as and nitrite, that are usually only found in the soil at very low concentrations. Nitrate is lost from the soil by microbial conversion to N₂ gas or leaching of soil water carrying dissolved . The conversion to gaseous N₂ usually only occurs under oxygen-depleted conditions when soil bacteria can use rather than oxygen for respiration. Nitrate availability to roots is therefore ephemeral and the concentrations of in the soil can rapidly change depending on rainfall and factors influencing microbial activity such as pH, temperature, and oxygen concentrations. Perhaps the single most important soil physical characteristic for determining availability is soil water content. Nonetheless, in models of N uptake by crops, soil water and N supply are usually always considered as independent parameters (SUNDIAL SimUlation of Nitrogen Dynamics In Arable Land http://www.rothamsted.ac.uk/aen/sundial/sundial.htm). There is scope for more research to check if water flux is a good predictor of uptake. When soil is sampled there can be large changes in physical properties over relatively short distances. For example, in a transect across four arable fields with 4 m sampling intervals, soil pH varied by 2 pH units and concentration by almost 100-fold (Fig. 1). Ammonium concentrations were much lower and less variable than with values differing by 10-fold. To some extent, this range of values reflects the soil types and cultivation differences across the transect, but nonetheless the amount of heterogeneity is surprising. At this sampling scale this could be the situation under a single crop of one species and therefore requires considerable root plasticity to achieve maximal yields from each plant. Another interesting aspect of these measurements is the relative ratios of to . Within a field this ratio was typically between 10 and 20:1, but on the margins the ratio could be much higher (Fig. 1). Clearly, plant roots need to be able to adapt to these changing supplies to optimize interception of in the soil. An appreciation of these soil processes is important background information and sets the scene for considering the uptake processes by plant roots.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Variation in soil pH (A) and N concentrations (B, C) along a linear transect sampled at 4 m intervals (redrawn from Lark et al., 2004 and reproduced by kind permission of Blackwell Publishing). Soil cores of 44x150 mm were taken at each sampling site along the transect and standard methods were used to extract and measure the soil parameters including soil water content (see Lark et al., 2004 for details). Transect positions 1–50 were located in a winter barley field, 51–56 grass on waste ground, 57–76 stubble after winter wheat, 77–79 waste ground, 80–152 and 154–256 two drilled winter wheat crops. The dashed line drawn on (B) is the nitrate concentration that indicates the border between HATS and LATS uptake by roots, but HATS may still contribute to N acquisition above this line (see text for details). (D) The calculated ratio of in the soil solution.

 

	Nitrate uptake by roots

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
This transport step is the best understood because it is easier to study than other steps. Arabidopsis seedlings can be conveniently grown on agar Petri dishes or in hydroponics and root uptake is easily assayed (Orsel et al., 2006). Physiological investigations of uptake by the roots of many different types of plants have led to the conclusion that plants have developed three types of transport system to cope with the variations in concentrations in cultivated soils (Crawford and Glass, 1998). Two saturable high affinity transport system (HATS) are able to take up at low external concentrations (1 µM to 1 mM). The constitutive system (cHATS) is available even when plants have not been previously supplied with . The inducible system (iHATS) is stimulated by in the external medium. The low affinity transport system (LATS) displays linear kinetics and its contribution to uptake becomes significant at external concentrations above 1 mM (Crawford and Glass, 1998). Although, in principle, both types of HATS can contribute to nitrate uptake at external concentrations above 1 mM, their transport activity is saturated and expression is down-regulated such that they actually contribute marginally to N acquisition. In Arabidopsis, the borders between these various physiological measures of uptake are vague and the explanation for this may be due to experimental design. For example, these parameters can depend on the plant developmental stage, time of day, and the ecotype used but this has yet to be carefully described.

Two gene families have been identified in Arabidopsis that potentially encode transporters responsible for uptake by roots or distribution within the plant; NRT1 with 53 members and NRT2 with seven members. These gene families for transport of have been extensively reviewed and the NRT1 family includes amino acid and peptide transporters and should more correctly be called NRT1/PTR (Forde, 2000; Orsel et al., 2002). The use of Arabidopsis mutants that have disruptions in specific transporter genes has been a powerful tool for the identification of key transporters involved in uptake.

Genes responsible for LATS
Figure 1 showed that the concentration of that is available in the soil is chiefly found in the mM range and yet there is much less knowledge of the molecular identity of LATS when compared with HATS. AtNRT1.1 (formerly called CHL1) was originally identified as a contributor to LATS (Tsay et al., 1993), but deletion mutants only showed a deficiency in low affinity uptake when growing on a mixed and supply (Touraine and Glass, 1997). This result led to the suggestion that there may be a second LATS that can only compensate when plants are grown with as the only N source. A later comparison between gene expression levels and LATS activity reckoned that AtNRT1.1 was mainly responsible for influx at 5 mM external concentrations of (Okamoto et al., 2003). However, LATS is assumed to be constitutively present in roots and AtNRT1.1 expression is -inducible. Furthermore, the expression of AtNRT1.1 occurs in very specific tissues at primary and lateral root tips and in stomata where it has a role in water stress responses (Guo et al., 2003). A further complication arises from the fact that the protein was actually described as being dual affinity, with a phosphorylation switch between high and low affinity ranges of nitrate uptake (Liu and Tsay, 2003). By contrast, AtNRT1.2 is constitutively expressed in root epidermal cells and has a K_m for around 6 mM in oocytes (Huang et al., 1999). However, Arabidopsis antisense lines of AtNRT1.2 were characterized by depletion assays and electrophysiology that showed a 50–70% decrease in LATS, but these changes did not correlate with the drop in expression across the lines (Huang et al., 1999). Gene deletion lines of AtNRT1.1 were used for comparison in these experiments and these also showed a 45% decrease in LATS. Better methods for characterizing the influx, such as ¹⁵ influx should be used to check LATS in these phenotypes or gene disrupted lines. Taken together these data seem to suggest a complicated role for both AtNRT1.1 and ATNRT1.2 in LATS, but this result requires re-examination especially as AtNRT1.1 is not strongly expressed in the epidermis and cortex of roots (Guo et al., 2001). AtNRT1.4 has a very specific pattern of expression in the leaf petiole where it has a role in accumulation within these tissues (Chiu et al., 2004). Expression of AtNRT1.3 was -induced in the leaf, but repressed in the root and does not seem to be a significant contributor to LATS (Okamoto et al., 2003). There are many other candidate genes among the NRT1/PTR family. One aspect of the thermodynamics of LATS is the fact that the transport system need not always be coupled to two protons (Miller and Smith, 1996). Therefore the components of LATS may belong to another gene family that are as yet uncharacterized. Clues to identifying the genes responsible for LATS may come from the fact that it is a constitutively expressed system. However, this may also mean that it may be of fundamental importance to plants and gene disrupted mutants may be lethal. The molecular identity of LATS remains a key target for future research especially for uptake by crops.

Genes responsible for HATS
Arabidopsis mutants with impaired expression of AtNRT2.1 and ATNRT2.2 genes are defective in HATS activity (Filleur et al., 2001; Orsel et al., 2004). These two genes lie end to end in the Arabidopsis genome and encode very similar proteins, sharing 90% identity (Orsel et al., 2002). Interestingly, parallel gene pairs of NRT2s that are also located closely within the genome are found in other species, for example, Chlamydomonas (Quesada et al., 1994) and rice (Araki and Hasegawa, 2006) and this may be a more general pattern. In rice, the two sequences appear to be identical, but the UTR flanking sequences result in different patterns of gene expression (Araki and Hasegawa, 2006). This information may suggest that functionally homologous genes encoding major components of HATS may have evolved early in plant development by a gene duplication event. Recently, another Arabidopsis mutant has been described that is uniquely disrupted in expression of AtNRT2.1 and this has been used to dissect the relative contributions of these two genes to iHATS and cHATS and to define AtNRT2.1 as the major contributor to both systems (Li et al., 2007).

	Regulation of transporters

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
At the whole plant level, it has long been recognized that net uptake by roots is regulated by demand and this, in turn, can be measured by the concentrations of N metabolites including in the tissue (Vidmar et al., 2000, and references therein). Feedback from the leaf to the root occurs more specifically via the concentrations in the phloem, frequently amino acids and often glutamine are implicated although this is likely to depend on the species (Tillard et al., 1998). At the cellular level, a number of molecular mechanisms that may be used to regulate the uptake can now be identified.

The two component NRT2/NAR2.1 system
Unlike the fungal transporters nrtA/B or YNT1, but similar to some Chlamydomonas NRT2s, AtNRT2.1 requires a second protein to mediate transport (Orsel et al., 2006). In Xenopus oocytes, AtNRT2.1 and NRT2 homologues from Chlamydomonas and barley all need to be co-expressed with a NAR2 protein, to yield transport (Zhou et al., 2000a; Tong et al., 2005; Orsel et al., 2006). This has been confirmed by in planta studies that have used mutant plants disrupted in the expression of the second component AtNAR2.1 (Okamoto et al., 2006; Orsel et al., 2006). One of these mutants (atnar2.1-1) shows a stronger phenotype (greater deficiency in HATS) than the NRT2.1 mutants in Arabidopsis (Orsel et al., 2006). In wild-type plants the expression pattern of AtNAR2.1 appears to parallel that of AtNRT2.1 exactly in the results from RT-PCR and microarray experiments and interaction occurs between the two proteins in yeast split ubiquitin assays (Orsel et al., 2006). However, the expression of AtNAR2.1 is not essential for that of AtNRT2.1, but the level of expression is modulated by the presence of the gene, probably as a consequence of the N status of the plant. The AtNAR2.1 protein is predicted to be much smaller than AtNRT2.1, and analysis of the sequence predicts an N-terminal ‘secretory pathway signal’ (Orsel et al., 2006). Taken together this information suggested that NAR2s were involved in targeting NRT2s to the plasma membrane, rather like a reported phosphate transporter traffic facilitator (Gonzalez et al., 2005); but unlike the phosphate model the NAR2s share no sequence similarities to SEC12 proteins. Tagging the AtNRT2.1 protein with GFP suggest that in the atnar2.1-1 mutant background the transporter protein is not correctly targeted to the plasma membrane (Orsel et al., 2007). Taken together these results suggest that the NAR2 proteins are not themselves a new type of transporters (Okamoto et al., 2006), but rather they facilitate targeting of some NRT2s to the plasma membrane.

A mutation in AtNAR2.1 that replaces an aspartate residue with an asparagine in a highly conserved region of the protein greatly decreases uptake (Kawachi et al., 2006). This mutant was called rnc1 and it has a dwarf phenotype when growing on 5 mM , the LATS range of concentration, and this result is different from the other AtNAR2.1 mutants (Okamoto et al., 2006; Orsel et al., 2006). Figure 2 shows wild-type and atnar2.1-1 mutant plants (Orsel et al., 2006) growing in two different hydroponic concentrations, 0.2 mM and 6 mM . The dwarf plant phenotype was evident when the plants were grown on soil that was not supplemented with fertilizer (comparing Figs 2A, B, C). However, when supplied with N fertilizer there was no difference between the growth of wild-type and the atnar2.1-1 mutant plants. The fact that the threshold concentration for the phenotype seems to have been altered by a point mutation in AtNAR2.1 suggests that the gene may alter the affinity of AtNRT2.1 for which is also worthy of further investigation. However, another difference between the plants used in these experiments is the Arabidopsis wild-type background, the atnar2.1-1 mutant was in a Wasselewskija (WS) background while the rnc1 was in Columbia (Col-0). These ecotype backgrounds may be important for the HATS/LATS concentration boundary. The ecotype WS is known to have faster growing roots when compared with other Arabidopsis ecotypes (see Table II in Beemster et al., 2002). These possible differences in the LATS/HATS system of different ecotypes require further investigation and in early papers on transport in Arabidopsis the wild-type background was not always reported.

View larger version (131K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Arabidopsis wild type (WS) and atnar2.1-1 mutant growing at different nitrate supplies on soil and in hydroponics. The missing HATS in the atnar2.1-1 plant (right) produces a dwarf phenotype when compared with wild-type plants (left) grown on an unfertilized soil (A), but the phenotype is less obvious on diluted compost (B) and lost on full nutrient compost (C). The dwarf phenotype of the mutant is seen when plants were grown hydroponically on a full nutrient solution containing 0.5 mM nitrate (D) but not on 6 mM nitrate (E). Four wild-type plants are shown in (D) each labelled W. Alternative WT and mutant plants are shown in (E). All plants were grown for 40 d under the same short-day environmental conditions (Orsel et al., 2006). Scale bar of 1 cm is shown.

 
Gene expression
The regulation of NRT2.1 expression has been thoroughly investigated at the mRNA level. NRT2.1 transcript accumulation mainly occurs in epidermis and cortex of the mature root regions (Nazoa et al., 2003), and is strongly influenced by a range of different environmental factors. Expression of NRT2.1 is induced by , repressed by high N status through a negative feedback regulation involving reduced N metabolites such as or amino acids (Zhou et al., 1999; Nazoa et al., 2003), and stimulated by light and sugars (Lejay et al., 2003). The expression pattern of AtNAR2.1 almost exactly parallels that of AtNRT2.1 and is similarly repressed by feedback regulation from N metabolites (Krouk et al., 2006). More recently, AtNRT2.1/NAR2.1 has been shown to be down-regulated by itself, through a mechanism independent of the feedback repression exerted by N metabolites, but specifically triggered by the dual-affinity transporter NRT1.1 (Munos et al., 2004; Krouk et al., 2006). There is generally a strong positive correlation between changes in NRT2.1 transcript level and HATS activity, suggesting that the transcriptional regulation of NRT2.1 expression plays a major role in governing root high-affinity uptake. Over-expression of the endogenous NpNRT2.1 gene driven by either the 35S or RolD promoter did not stimulate HATS influx in N. plumbaginifolia plants (Fraisier et al., 2000). However, when HATS influx was measured in plants supplied with 10 mM there was less feedback inhibition of HATS in the over-expressing lines when compared with the wild type (Fraisier et al., 2000). This work has been cited as an example of the over-expression of a transporter gene resulting in altered uptake by plants, but actually the plants did not accumulate any more than the wild type and no growth differences were reported. These plants may have lacked a necessary second NAR2-type component and over-expression of both components may be more likely to result in increased accumulation.

Post-translational regulation of transporters
The use of chemical inhibitors in physiological studies had long ago suggested that protein synthesis was important for uptake (Agüera et al., 1990) and the transporters may turn over relatively slowly. In barley roots, the application of inhibitors showed that the iHATS had a relatively long half-life when compared with transport to the xylem and with potassium uptake (Behl et al., 1988). Recently, in the yeast Hansenula polymorpha it was shown that degradation of a NRT2-type protein (YNT1) in the vacuole was associated with the removal of this transporter from the plasma membrane (Navarro et al., 2006). The YNT1 protein is ubiquitinylated when glutamine is supplied to the cells, and is transferred to the vacuole where it is rapidly degraded by a specific proteinase A. Recently, a similar mechanism for Arabidopsis AtNRT2.1 degradation has been suggested (J Wirth et al., unpublished data) and this could provide a direct molecular mechanism for feedback by a N metabolite.

The presence of a number of conserved protein kinase C recognition motifs in the N- and C-terminal domains of HvNRT2.1 (Forde, 2000) may suggest that phosphorylation events are involved in regulating AtNRT2.1 activity in response to environmental cues, as was shown to be the case for AtNRT1.1 (Liu and Tsay, 2003). Sequence analysis of the NRT2s has identified some possible 14-3-3 regulatory sites; this is particularly interesting because of the role of these proteins in the regulation of key N assimilatory enzymes. The C-terminus of the tobacco NRT2.1 gene has a perfect 14-3-3 binding consensus (G Moorhead, personal communication). Using this sequence to compare with the Arabidopsis NRT2s identifies four genes with some consensus. In particular, AtNRT2.4 has a perfect 14-3-3 binding motif, consensus sequence RSXSXP, but in contrast to the tobacco gene this region is not present in the C-terminus of the protein. As 14-3-3 binding and regulation involves phosphorylation of the protein, this aspect of the NRT2s is worth further investigation. The 14-3-3 proteins can regulate the activity of both reductase and glutamine synthetase and so it is tempting to speculate that a common regulatory mechanism may exist for the uptake and assimilation of N. It was shown there may be several different forms of the NRT2.1 protein, some that are truncated, phosphorylated, or modified in some way and it is possible that the protein has different functions in each of these states (J Wirth et al., unpublished data).

Finally, one aspect of regulation that is overlooked is the fact that the physical parameters of the cell can have a major effect on transport. Nitrate transport is driven by co-transport with two protons and is therefore electrically sensitive. Xenopus oocyte expression experiments have shown that membrane voltage can have a direct effect on the activity of the transporter protein. If the electrical potential difference across the cell membrane is decreased there is less energy for transport, but also the protein's affinity for (K_m) can change (Zhou et al., 1998). Indeed membrane-potential dependent changes in K_m can explain why measurements in oocytes may appear to give kinetic parameters that seem at odds with in planta measurements. Trans-membrane gradients of potential and pH can have a major effect on transport into a cell and these may be very different in oocytes and plant cells. The importance of changes in cytosolic pH and membrane potential as regulators of uptake really depends on how much and how fast these parameters might change for cells in planta and it is very difficult to get accurate measurements of these physical parameters for roots growing in soil.

	Nitrate efflux from cells

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
In general, efflux systems have been much less studied than influx systems; however, it is known that efflux is protein-mediated, passive, saturable, and selective for (Aslam et al., 1996). Aquaporins, or more likely, anion channels are an obvious route for efflux because they are thermodynamically downhill. However, it is worth noting that one member of the NRT2 gene family was demonstrated to have both a proton-cotransport and passive mechanism (Zhou et al., 2000b). The efflux system is under a degree of regulation, induced by (Aslam et al., 1996) and it is also proportional to whole tissue concentrations (Teyker et al., 1988; van der Leij et al., 1998). It can be predicted that the membrane protein mediating efflux must be -inducible.

Concentrations of in the xylem sap can be quite high (10–30 mM) especially in plants which transport most of the taken up to the shoot for reduction. Nonetheless the entry of into the xylem can be mediated by anion channels assuming cytosolic concentrations and membrane potentials in xylem parenchyma cells similar to those measured in epidermal and cortical cells (Miller and Smith, 1996). Genome analysis has identified several different anion channel families that may fulfil this function. Candidate quickly-activating anion channels have been characterized for barley roots (Kohler et al., 2002), but their molecular identity has not yet been determined. Nitrate in the xylem exerts positive feedback on its loading through a change in the voltage dependence of the channel. Interestingly, this effect was specific for and was not found for Cl^–. By transport through this channel, efflux into the xylem can be maintained with high concentrations in the xylem sap; a situation which can occur during the night. There is a clear diurnal change in xylem sap concentrations (Siebrecht et al., 2003) of , related to changes in the transpiration rate. In maize xylem parenchyma cells, the activity of a quickly activating anion channel was altered by changes in the water status of plants and the addition of ABA to protoplasts inhibited the channels activity (Gilliham and Tester, 2005).

	Nitrate storage and remobilization from the vacuole

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
Nitrate storage in the vacuole is important for osmotic balance and as an N reserve. Tissue levels are indicators of N status and this is the basis of tissue testing for crops. Interestingly, land plants accumulate in the vacuole but aquatic plants may have either lost this ability or never developed it. Cells of the algae Chara do not accumulate in the vacuole above passive transport levels after growing in high concentrations for many months (Miller and Zhen, 1991). Seven members of the CLC gene family have been identified in the Arabidopsis genome (De Angeli et al., 2006) and disruption of one of these genes was shown to alter the accumulation of in leaf tissues (Geelen et al., 2000). An earlier paper had reported that some members of the CLC family were localized in endomembranes (Heckenberger et al., 1996), but mammalian examples have been localized to the mitochondria (Jentsch et al., 2002). Studying the activity of mammalian CLCs has been made easier by the fact that the proteins have been characterized by expression in Xenopus oocytes (Jentsch et al., 2002), but this has been unsuccessful for plant homologues (Heckenberger et al., 1996). GFP-tagging of the proteins in Arabidopsis and rice has localized them to the tonoplast where they are implicated in the storage and remobilization of and chloride in the vacuole (De Angeli et al., 2006; Nakamura et al., 2006). Much earlier studies with isolated vacuoles (Schumaker and Sze, 1987) and intracellular measurements of pH and gradients had used thermodynamic calculations to predict an antiport mechanism for accumulation in the vacuole (Miller and Smith, 1992). These results are consistent with the new finding that AtCLCa is able specifically to accumulate nitrate in the vacuole and behaves as a 2 antiporter (De Angeli et al., 2006). This family of transporters are important for further studies to understand and manipulate N use efficiency in crops. Nitrate storage pools in the leaves and stems of crop plants are important for several quite different reasons; firstly, increased storage in tissue enables over-wintered crops in the mopping up of soil that would otherwise cause environmental damage through leaching in rainwater. Secondly, when leafy vegetables are eaten there is medical evidence to suggest that it is beneficial to health to decrease tissue concentrations. Finally, this family of transporters may have a role in salt tolerance as these genes can also transport chloride, resulting in accumulation in the vacuole to avoid any possible harmful effects of the anion in the cytoplasm.

	Nitrate transporters, root morphology, and signalling

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
As is not only a major N source for nutrition of plants, but also acts as a signal to modulate plant metabolism and development (Crawford and Glass, 1998) this had led to the hypothesis that plant cells must have a sensor for availability. One way that this hypothesis can be demonstrated is through the use of Arabidopsis root growth assays on Petri dishes. These experiments have compared the root growth pattern of wild type (WT) with mutant plants that have a gene disruption in specific transporters. The initiation and elongation of lateral root (LR) development is stimulated by local availability of and it has been proposed in Arabidopsis roots that NRT2.1 may itself be a signal transducer or sensor (Little et al., 2005). This function of the transporter is reckoned to be independent from influx (Little et al., 2005; Remans et al., 2006a). The many regulatory processes that might change the activity of this protein (e.g. phosphorylation, the two components system etc) described previously seems to fit with the idea that the protein may have multiple functions. Furthermore, AtNRT1.1 has been implicated in the signalling pathway triggering root colonization of -rich patches and this has been linked to changes in the expression of a putative transcription factor MADS box gene (Remans et al., 2006b).

A key aspect of these signalling functions for transporters is the assumption that this effect is independent of the proteins transport function (Little et al., 2005; Remans et al., 2006a, b). This result is fundamental to the proposed separation of the signalling and transport functions of these proteins and so this aspect of the experimental system needs careful examination. For AtNRT2.1, the case rests on the fact that a point mutation in the protein results in a loss of the normal LR repression that occurs in WT growing on high sucrose and low nitrate (Little et al., 2005). The mutant phenotype is actually independent of exogenous and is even seen in -free conditions when obviously no influx is occurring. As AtNAR2.1 interacts with AtNRT2.1 at the protein level and it is required for membrane targeting, it would be important to know if the protein–protein interaction can still occur with the mutated AtNRT2.1. In another paper for AtNRT2.1, measurements of cumulative uptake were used to show that the LR phenotype was independent of the total amount of nitrate taken up (Remans et al., 2006a). Both types of experiments actually measure whole root nitrate influx, but LR initiation occurs at very specific discrete regions of root and there may be subtle changes at localized regions that get hidden within measures of whole root influx. Of course, there are important practical reasons why these assays are performed on whole roots: in order to obtain sufficient ¹⁵N enrichment to detect the influx in the tissue samples. These Petri dish experiments provide a good assay tool and the concentrations of nitrate used are appropriate for the soil ranges (Fig. 1). However, when interpreting the experiments it is necessary to remember how this environment may differ from that experienced by a root growing in soil. For example, patchiness may be much greater in the soil. Recent experiments to investigate the signalling role of AtNRT1.1 have recognized this problem and used split root treatments to offer localized -rich patches (Remans et al., 2006b). Clearly, under low levels of supply Arabidopsis roots are very good at growing to forage for N, and this is relevant to breeding new varieties adapted to lower fertilizer inputs. For example, wheat QTL analysis has identified that root architecture is important for N foraging under deficiency (Laperche et al., 2006).

Another example of nitrate signalling has been demonstrated in the breaking of seed dormancy. In Arabidopsis, a role for AtNRT1.1 has been specifically implicated in this response (Alboresi et al., 2005). Like root development, seed dormancy can provide another useful model system for studying nitrate signalling in plants.

	Conclusions

Top Abstract Introduction Nitrate in the soil Nitrate uptake by roots Regulation of transporters Nitrate efflux from cells Nitrate storage and... Nitrate transporters, root... Conclusions References

 
In Arabidopsis roots, the AtNRT2.1, AtNRT2.2, and AtNAR2.1 genes are all important contributors to HATS. The transporter function of two of the seven AtNRT2 family members has been clearly identified, but care should be taken in assigning this transport function to all members of the NRT2 family. Although reassuringly, this number of NRT2s is a similar figure to that for other nutrient transporter families, for example, sulphate and phosphate (ARAMEMNON database, http://aramemnon.botanik.uni-koeln.de/). Perhaps too much emphasis is given to phylogeny analysis. It is known that changing just a few amino acid residues can alter the transported ion so why do we believe that all related proteins must have the same function? The AtNRT1 family is a large family with members transporting many different N-containing substrates including a dicarboxylate (Jeong et al., 2004). Family members may have evolved alternative functions in different plant species. Heterologous expression systems such as yeast and oocytes, together with gene disruption mutants will continue to be essential tools in unravelling the functions of these family members.

The different regulatory mechanisms that have already been identified for controlling the known transporters fit with our knowledge of the transient availability of in the soil. These mechanisms might also fit with a dual function role for some of these transporters. It may be questioned why it has taken 10–15 years to identify some of these genes as not only transporters but also sensors. Should a sensor have fundamental importance in plants and, therefore, a deletion mutant is lethal? Not necessarily, because plants can use other N sources and there may be a gene family of sensors. Indeed iHATS may primarily be a sensing system as we know it is a low capacity system operating at low availability and soil concentrations rarely occur in this range (Fig. 1). Associating nitrate sensing with HATS does not exclude a role for AtNRT1.1 because the protein can function in both LATS and HATS (Liu and Tsay, 2003). It is generally assumed that in natural ecosystems the soil nitrate availability is lower than shown in Fig. 1 and therefore wild plants use HATS for nitrate uptake. The transect data in Fig. 1 include natural ground and these regions do not show particularly low nitrate concentrations (51–56 and 77–79), but these are field margins and may be influenced by agricultural inputs. There are few measurements of soil nitrate availability and often when they are available they are not expressed in soil water concentration units; more data are needed. Both and are available in all soils (Fig. 1), but the relative amounts of each can be very different and this ratio must be important for the function of a nitrate sensor. The polarity of nitrate fluxes through a cell may have an important role in sensing and an interesting analogy here may be with auxin transport (Teale et al., 2006). Localized gradients of nitrate in the rhizosphere may be detected by differing nitrate influx at points along the root. Measurements of cellular responses have shown that there are differences between outer and inner cell layers and along the length of roots (Radcliffe et al., 2005). The homeostasis of cytosolic is controversial, but recent measurements have shown that there can be transient changes in response to external stimuli such as light in leaf cells (Cookson et al., 2005) and glutamine in barley root cells (Fan et al., 2006) that fit with a signalling role for . Such transient signalling events may also be part of the localized sensing mechanism needed for LR initiation and elongation. The sensor role has been ascribed to transporters after the transport function was identified and we should now be asking if these same genes would be detected in a screen to find a root sensor if it can be designed.

	Acknowledgements

 
This work was funded by EU grants BIO4CT972231, Research Training Network ‘Plant use of nitrate’ HPRN-CT-2002–00247. Rothamsted Research is grant-aided by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. Thanks to Murray Lark (Rothamsted Research) and Greg Moorhead (University of Calgary) for their help and access to their data.

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