Abstract

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

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
influx. In addition, efflux analysis using 13N 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
\({\mathrm{NO}_{3}^{{-}}}\)
and one HATS for
\({\mathrm{NH}_{4}^{{+}}}\)
in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for
\({\mathrm{NO}_{3}^{{-}}}\)
and five members of the AMT1 family encoding putative HATS for
\({\mathrm{NH}_{4}^{{+}}}\)
. The induction of high-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of
\({\mathrm{NO}_{3}^{{-}}}\)
, while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity
\({\mathrm{NH}_{4}^{{+}}}\)
transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
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
\({\mathrm{NO}_{3}^{{-}}}\)
transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of
\({\mathrm{NO}_{3}^{{-}}}\)
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.

Introduction

Inorganic ions accumulated in plant cells serve nutritional, osmotic, signalling, and storage functions. Insufficient ion accumulation as well as excess accumulation may therefore compromise these functions. While vacuolar reserves may buffer the cytoplasm against short-term perturbations, in laboratory studies when external sources of ions are removed vacuolar reserves are typically exhausted within a few days (Glass, 1975; Lee et al., 1990; van der Leij et al., 1998). Under field conditions vacuolar reserves may be even more limited. When vacuolar reserves are consumed to sustain cytosolic functions, there is a need to replace their osmotic and charge-balancing function by means of alternative solutes, be they inorganic or organic. Hence vacuolar buffering of cytosolic ion concentrations is not achieved without consequences and, typically, plant roots respond to perturbations of external supply or internal demand long before vacuolar reserves are exhausted. This raises the interesting issue of the signal pathways between vacuole and cytoplasm required to initiate these responses; virtually unexplored territory.

Given that both

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
commonly serve as sources of N for plant growth and that they share some metabolic pathways, it is perhaps not surprising to find that they possess features in common: (1) both ions are actively absorbed into root cells at low external concentrations; (2) influx measurements indicate the presence of two high-affinity transport systems (HATS) for
\({\mathrm{NO}_{3}^{{-}}}\)
(one constitutive and the other inducible) and one HATS for
\({\mathrm{NH}_{4}^{{+}}};\)
(3) influx of both ions is responsive to plant N status and subject to diurnal regulation; (4) molecular studies indicate the presence of seven HATS for
\({\mathrm{NO}_{3}^{{-}}}\)
and five for
\({\mathrm{NH}_{4}^{{+}}}\)
in A. thaliana; and (5) some of the genes encoding
\({\mathrm{NO}_{3}^{{-}}}\)
transporters are subject to transcriptional regulation through inductive effects of
\({\mathrm{NO}_{3}^{{-}}}\)
, while some of those encoding
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
transporters are subject to down-regulating effects of glutamine. Notwithstanding these similarities there are also distinct differences in the characteristics of
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
uptake, as well as differences among species in the extent of their utilization of these different nitrogen sources.

Soil heterogeneity

Heterogeneity of soil nutrient availability is potentially the most important perturbing effect upon plant nutrient status. In addition, seasonal and diurnal changes in growth rates and plant demand for resources are also substantial. In this paper, the main focus will be upon flux regulation in response to perturbations of external supply and, in particular, the responses of the HATS for

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
to these perturbations. In the context of these effects that would displace the plant from steady state, ion fluxes are regulated by feedback from various cellular parameters that serve to counteract such changes.

According to data compiled previously,

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
concentrations of agricultural soils range across three to four orders of magnitude (Wolt, 1994). The situation is even more variable in natural soils (Jackson and Caldwell, 1993). In addition, specific habitats (e.g. mature forests, arctic tundra) may be characterized by nitrogen profiles dominated by ammonium or amino acids, rather than
\({\mathrm{NO}_{3}^{{-}}}\)
. Many species occupying such habitats have become specialists, absorbing
\({\mathrm{NH}_{4}^{{+}}}\)
or amino acids in preference to
\({\mathrm{NO}_{3}^{{-}}}\)
(Kielland, 1994; Kronzucker et al., 1997; Nasholm et al., 1998, 2000). Even when
\({\mathrm{NO}_{3}^{{-}}}\)
exceeds
\({\mathrm{NH}_{4}^{{+}}}\)
by as much as 10-fold,
\({\mathrm{NH}_{4}^{{+}}}\)
uptake may still greatly exceed that of
\({\mathrm{NO}_{3}^{{-}}}\)
in field and laboratory studies (Gessler et al., 1998). In a study of nitrogen absorption by tomato (MY Siddiqi et al., unpublished data), it was demonstrated that 50% of plant N was absorbed as
\({\mathrm{NH}_{4}^{{+}}}\)
, even though this ion represented only 10% of available N, the remaining 90% being
\({\mathrm{NO}_{3}^{{-}}}\)
. In the context of this variability of N supply plants have evolved numerous mechanisms (physiological/biochemical, developmental and life history-based strategies) that enable them to optimize nitrogen acquisition. Included among the physiological adaptations, are the ‘up-regulation’ of nitrogen uptake under conditions of N-limitation, but also the restriction of nitrogen uptake under conditions of N excess. The latter presumably serves to minimize potentially harmful osmotic or specific ion effects.

Physiological characterization of
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
uptake

Measurements of 13

\({\mathrm{NO}_{3}^{{-}}}\)
influx and net
\({\mathrm{NO}_{3}^{{-}}}\)
uptake by several groups have revealed the presence of three transport systems for
\({\mathrm{NO}_{3}^{{-}}}\)
and two for
\({\mathrm{NH}_{4}^{{+}}}\)
(reviewed in Glass and Siddiqi, 1995). In roots of species examined for its presence, a low capacity, constitutively expressed, high-affinity transport system (cHATS) allows entry of
\({\mathrm{NO}_{3}^{{-}}}\)
from low external
\({\mathrm{NO}_{3}^{{-}}}\)
. The extent of this flux varies among and within species (Siddiqi et al., 1989; King et al., 1993; Kronzucker et al., 1995; Zhuo et al., 1999). Following first exposure to
\({\mathrm{NO}_{3}^{{-}}}\)
there is a rapid increase of an inducible high-affinity influx (iHATS), which is followed (after several h) by an equally rapid down-regulation of this flux (Siddiqi et al., 1989; Zhuo et al., 1999). There are significant differences in the response time to applied
\({\mathrm{NO}_{3}^{{-}}}\)
among species. For example, in Picea glauca, it was necessary to expose plants to
\({\mathrm{NO}_{3}^{{-}}}\)
for 3 d in order to induce peak 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx (Kronzucker et al., 1995). Both
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NO}_{2}^{{-}}}\)
are capable of inducing this flux (Siddiqi et al., 1992; Aslam et al., 1993).

Several studies have demonstrated that the provision of

\({\mathrm{NH}_{4}^{{+}}}\)
to N-deprived roots may initially increase
\({\mathrm{NH}_{4}^{{+}}}\)
uptake prior to down-regulating the flux, and the term induction has also been applied to this initial increase of influx (see Kronzucker et al., 1998, for references and discussion). However, in these studies high-affinity
\({\mathrm{NH}_{4}^{{+}}}\)
influx was already high (de-repressed) before exposure to
\({\mathrm{NH}_{4}^{{+}}}\)
, and it has been demonstrated that, in rice, the increase of
\({\mathrm{NH}_{4}^{{+}}}\)
influx resulting from
\({\mathrm{NH}_{4}^{{+}}}\)
pretreatment was relatively small (25–40%) (Kronzucker et al., 1998). By comparison, a 30-fold increase of 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx was recorded in Klondike barley following pretreatment with
\({\mathrm{NO}_{3}^{{-}}}\)
(Siddiqi et al., 1990). Kronzucker et al. concluded that the evidence did not support a true inductive effect of
\({\mathrm{NH}_{4}^{{+}}}\)
(Kronzucker et al., 1998).

At nitrate and ammonium concentrations between ∼200 to 500 μM, low-affinity transporter systems (LATS) for these ions become apparent. These were evident in earlier studies (Doddema and Telkamp, 1979; Ullrich et al., 1984), but were largely overlooked, in part because the measurement of

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
uptake at high concentration by depletion methods was typically insufficiently sensitive to characterize these transporters. A perplexing feature of these high capacity low-affinity transporters has been their linear concentration responses (Pace and McClure, 1986; Ullrich et al., 1984), that were earlier suggested to result from diffusive fluxes. However, although
\({\mathrm{NH}_{4}^{{+}}}\)
fluxes via LATS are typically thermodynamically ‘downhill’ (Ullrich et al., 1984; Wang et al., 1993), the LATS for
\({\mathrm{NO}_{3}^{{-}}}\)
was shown to be active even at high external
\({\mathrm{NO}_{3}^{{-}}}\)
concentration and mediated, like the iHATS, by a proton:nitrate symport (Glass et al., 1992).

Homeostatic processes for nitrogen uptake

As outlined above, the uptake of both

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
is subject to down-regulation as tissue N levels approach some upper limit. As early as 1906, Brezeale demonstrated, using hydroponic wheat plants, that withholding K, P, N, Ca or S for 18 h resulted in several-fold increases in rates of absorption of the particular nutrient that had been withheld (Brezeale, 1906). As far as is known, this is the first documented evidence of the physiological regulation of ion uptake by plant roots. Clement et al., using ryegrass as a model system, established that, when available
\({\mathrm{NO}_{3}^{{-}}}\)
concentrations were maintained from 14.3 μM to 14.3 mM, plant growth was only modestly affected and tissue nitrogen concentration remained essentially constant (Clement et al., 1978,a). The up-regulation of nitrate fluxes first observed by Brezeale forms an important component of the processes responsible for achieving nitrogen homeostasis (Brezeale, 1906), while adjustments in growth rate may also be critical under some circumstances (Ingestad and Lund, 1979). While
\({\mathrm{NH}_{4}^{{+}}}\)
transport shows the same general homeostatic propensity (Wang et al., 1993; Rawat et al., 1999), the potential toxicity of elevated ambient
\({\mathrm{NH}_{4}^{{+}}}\)
concentrations severely limits the range of
\({\mathrm{NH}_{4}^{{+}}}\)
concentration over which adaptation is possible. In a study of 13
\({\mathrm{NH}_{4}^{{+}}}\)
fluxes across the plasma membranes of barley roots, Britto et al. showed that at 10 mM external
\({\mathrm{NH}_{4}^{{+}}}\)
, active
\({\mathrm{NH}_{4}^{{+}}}\)
efflux rose to 76% of the value of influx (Britto et al., 2001). Simultaneously, root respiration increased by 40%, and was not diminished by treatment with the GS inhibitor methionine sulphoximine (MSX), indicating that the respiratory increase was not associated with increased assimilation of
\({\mathrm{NH}_{4}^{{+}}}\)
, but with active extrusion. In summary, while high-affinity
\({\mathrm{NH}_{4}^{{+}}}\)
fluxes are effectively regulated, transport via the low-affinity system is poorly regulated, resulting in considerable futile cycling of
\({\mathrm{NH}_{4}^{{+}}}\)
across the plasma membrane as well as toxic effects of excessive
\({\mathrm{NH}_{4}^{{+}}}\)
accumulation (Britto et al., 2001).

Studies of the many component

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
fluxes that occur in plant cells are severely limited, even in single-celled organisms by cellular compartmentation. In multicellular plants fluxes to and from roots via xylem and phloem further complicate the situation. Therefore, for technical reasons involving the ease of measurement, the emphasis in studies of the mechanisms responsible for ion fluxes and their regulation has been upon the influx step (ϕoc) across the plasma membrane. Nevertheless, there is evidence to suggest that efflux from cytosol to cell wall (ϕco), fluxes across the tonoplast (ϕcv and ϕvc), from cytosol to xylem (ϕcx), as well as fluxes to biochemical pathways appear to be co-ordinated. The use of efflux analysis to estimate the half-lives (t0.5) for 13
\({\mathrm{NO}_{3}^{{-}}}\)
and 13
\({\mathrm{NH}_{4}^{{+}}}\)
residence within the cytosolic compartment, has revealed that t0.5 values are virtually independent of prior nitrogen provision (Siddiqi et al., 1991; Wang et al., 1993; Britto and Kronzucker, 2001). Figure 1 shows data for 13
\({\mathrm{NO}_{3}^{{-}}}\)
efflux from roots of barley grown under steady-state conditions with various concentrations of nitrate for 7 d prior to labelling with 13
\({\mathrm{NO}_{3}^{{-}}}\)
and subsequent measurement of 13
\({\mathrm{NO}_{3}^{{-}}}\)
efflux into non-labelled solutions of the same
\({\mathrm{NO}_{3}^{{-}}}\)
concentration (Britto and Kronzucker, 2001). Despite the wide range of
\({\mathrm{NO}_{3}^{{-}}}\)
concentrations used and the substantial changes of measured fluxes, the rate constants for 13
\({\mathrm{NO}_{3}^{{-}}}\)
efflux were essentially identical (0.0408, 0.0400, 0.0417, 0.0418, and 0.04908 min−1 for plants grown in 10, 1, 0.1, 0.01, and 0 mM
\({\mathrm{NO}_{3}^{{-}}}\)
, respectively). In a study of the effect of perturbing external
\({\mathrm{NH}_{4}^{{+}}}\)
on 13
\({\mathrm{NH}_{4}^{{+}}}\)
efflux from barley roots, Britto and Kronzucker showed that when external
\({\mathrm{NH}_{4}^{{+}}}\)
concentration was increased or decreased, respectively, from 1 mM to either 10 mM or to 100 μM, there was initially a rapid increase or decrease, respectively, of 13
\({\mathrm{NH}_{4}^{{+}}}\)
efflux (Britto and Kronzucker, 2001). Yet, despite this initial perturbation of tracer efflux, rate constants for this flux were restored to their original values within minutes as shown in Fig. 2. Such results point to a precise integration of all component fluxes that impact upon cytosolic ion concentrations.

Several studies using 13

\({\mathrm{NO}_{3}^{{-}}}\)
and 13
\({\mathrm{NH}_{4}^{{+}}}\)
have demonstrated that ϕco increases as external ion concentration increases (Siddiqi et al., 1991; Wang et al., 1993) and that net transfer of nitrogen from vacuole to cytosol (ϕvc–ϕcv) increases (van der Leij et al., 1998), and from cytosol to stele (ϕcx) decreases (Kronzucker et al., 1998), as external ion concentrations decrease. Nevertheless, these fluxes have not been quantified in the same detail that has characterized measurements of ϕoc, nor have genes yet been cloned that encode these transport systems. Likewise there is a lack of detailed studies of the fluxes of
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
into leaf cells. Having noted the paucity of information concerning fluxes other than the root influx step, the remainder of this paper, will focus on the regulation of high-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
influx across the plasma membrane of root cells.

Induction and down-regulation of influx

It is evident from a number of different studies that only

\({\mathrm{NO}_{3}^{{-}}}\)
or
\({\mathrm{NO}_{2}^{{-}}}\)
among potential products of nitrogen assimilation are capable of inducing
\({\mathrm{NO}_{3}^{{-}}}\)
influx by the iHATS (Tompkins et al., 1978; Behl et al., 1988; Siddiqi et al., 1992; Tischner et al., 1993; Guy and Heimer, 1993; Henriksen and Spanswick, 1993). Nevertheless, as low-N plants accumulate N, the influxes of both
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
are subsequently down-regulated (Lee and Rudge, 1986; Morgan and Jackson, 1988; Siddiqi et al., 1989; Kronzucker et al., 1995; Glass and Siddiqi, 1995; Forde and Clarkson 1999). Prior to the cloning of genes that encoded
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
transporters, two hypotheses emerged to explain this down-regulation. On the one hand it was proposed that accumulated
\({\mathrm{NO}_{3}^{{-}}}\)
or
\({\mathrm{NH}_{4}^{{+}}}\)
themselves, as opposed to their downstream metabolites, were responsible for down-regulation of fluxes. This was based upon inverse correlations between accumulated
\({\mathrm{NO}_{3}^{{-}}}\)
or
\({\mathrm{NH}_{4}^{{+}}}\)
and N fluxes in wild-type plants. This conclusion was supported by the results of experiments in which nitrate reductase (NR) was blocked by tungstate treatment in Lemna gibba and Helianthus annuus (Ingemarsson et al., 1987; De la Haba et al., 1990) or by mutation in barley (Warner and Huffaker, 1989; Siddiqi et al., 1989; King et al., 1993). Incapacitating NR failed to impact upon induction or down-regulation of influx, suggesting that
\({\mathrm{NO}_{3}^{{-}}}\)
itself was responsible for these effects. Likewise effects of MSX application (Ryan and Walker, 1994; King et al., 1993; Feng et al., 1994; Glass et al., 1997) suggested that
\({\mathrm{NH}_{4}^{{+}}}\)
itself was responsible for down-regulating
\({\mathrm{NH}_{4}^{{+}}}\)
influx. On the other hand convincing support for effects of down-stream metabolites has been provided by experiments in which exogenously applied amino acids strongly inhibited both
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
influx, and by several studies in which MSX application blocked down-regulation (Lee and Rudge, 1986; Morgan and Jackson, 1988; Lee et al., 1992; Muller and Touraine, 1992; Rodgers and Barneix, 1993). The contradictory nature of these findings is exemplified by studies on maize and sorghum (Feng et al., 1994). While 15
\({\mathrm{NH}_{4}^{{+}}}\)
influx was stimulated by MSX treatment in maize, in sorghum influx was inhibited. Likewise, Glass et al. observed that, in low-N rice plants, the effects of MSX were consistent with down-regulation of influx by end-products of
\({\mathrm{NH}_{4}^{{+}}}\)
assimilation while in high-N plants
\({\mathrm{NH}_{4}^{{+}}}\)
itself appeared to be involved (Glass et al., 1997). Unfortunately, given that MSX has been used in so many of these studies, it must be acknowledged that cytosolic
\({\mathrm{NH}_{4}^{{+}}}\)
may reach as high as 80 mM when
\({\mathrm{NH}_{4}^{{+}}}\)
assimilation is blocked by this compound (Lee and Ratcliffe, 1991). These are clearly abnormal conditions. As will be evident below, the results of molecular studies has provided some clarification of this question at the transcript level.

Genes encoding putative high-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
transporters

The cloning of genes encoding putative high-affinity

\({\mathrm{NO}_{3}^{{-}}}\)
transporters belonging to the NRT2 family of genes (see Forde, 2000, for a recent review) and putative high-affinity
\({\mathrm{NH}_{4}^{{+}}}\)
transporters of the AMT1 family of genes (see Howitt and Udvardi, 2000, for a recent review), has allowed investigations of the regulation of high-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
influx to proceed to the transcript level. As was the case for induction of
\({\mathrm{NO}_{3}^{{-}}}\)
uptake, only
\({\mathrm{NO}_{3}^{{-}}}\)
or
\({\mathrm{NO}_{2}^{{-}}}\)
were capable of inducing the accumulation of NRT2 transcript. Moreover transcript accumulation followed the same general patterns as had been observed for the induction of
\({\mathrm{NO}_{3}^{{-}}}\)
uptake/influx, namely induction over a period of up to 3 h or more followed by down-regulation (Trueman et al., 1996; Quesada et al., 1997; Amarasinghe et al., 1998; Filleur et al., 1999; Zhuo et al., 1999). In NR mutants, high levels of
\({\mathrm{NO}_{3}^{{-}}}\)
accumulation and increased NRT2 transcript abundance suggested that while
\({\mathrm{NO}_{3}^{{-}}}\)
is responsible for inducing gene expression, it is down-stream metabolites that are responsible for down-regulation (Krapp et al., 1998; Filleur and Daniel-Vedele, 1999; Lejay et al., 1999). Likewise, in barley roots tungstate treatment to block NR caused increased NRT2 transcript abundance (Vidmar et al., 2000). Several reports have documented the down-regulation of NRT2 transcript abundance in response to pretreatment with
\({\mathrm{NH}_{4}^{{+}}}\)
or amino acids (Quesada et al., 1997; Krapp et al., 1998; Zhuo et al., 1999). Unfortunately, exogenous application of amino acids or
\({\mathrm{NH}_{4}^{{+}}}\)
provides little information concerning the N pools that might be responsible for these effects. Differences in uptake or assimilation of applied amino acids, as well as their inter-conversion obscure the sources of observed effects. In addition, exogenous application of various amino acids was shown to increase root [
\({\mathrm{NH}_{4}^{{+}}}\)
] up to 6-fold in rice (Wang, 1994; Kumar et al., unpublished results). Another important consideration is whether or not a particular amino acid is a typical/major component of xylem and phloem-translocated N, since cycling/recycling of amino acids within the vascular system has been proposed as the basis of communicating plant N status to roots so that N uptake may be regulated according to plant N demand (Cooper and Clarkson, 1989; Marschner et al., 1997; Glass et al., 2001). By providing various nitrogen sources (
\({\mathrm{NO}_{3}^{{-}}}\)
,
\({\mathrm{NH}_{4}^{{+}}}\)
, and/or amino acids) in the presence and absence of inhibitors of
\({\mathrm{NO}_{3}^{{-}}}\)
assimilation, for example, tungstate (
\({\mathrm{WO}_{4}^{2{-}}}\)
) to block nitrate reductase, MSX to block glutamine synthetase, and azaserine (AZA) to block glutamate synthase, this confusion can be resolved. In barley, combining results based on the effects of exogenous applications of amino acids with data from inhibitor studies (Fig. 3) demonstrated that NRT2 transcript abundance was most strongly correlated with root glutamine concentrations (Vidmar et al., 2000). Thus, increasing root glutamine by pretreatment with AZA virtually eliminated 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx and NRT2 transcript in both A. thaliana and in H. vulgare (Zhuo et al., 1999; Vidmar et al., 2000).

Using A. thaliana as the model system, Rawat et al. demonstrated that up-regulation and down-regulation of 13

\({\mathrm{NH}_{4}^{{+}}}\)
influx (following removal and restoration of exogenous N, respectively) was strongly correlated with AMT1.1 transcript abundance (
Rawat et al., 1999). In the presence of MSX,
\({\mathrm{NH}_{4}^{{+}}}\)
provision caused root [
\({\mathrm{NH}_{4}^{{+}}}\)
] to increase 27-fold, while root glutamine levels remained at the original (N-deprived) level. Concurrent measurements of 13
\({\mathrm{NH}_{4}^{{+}}}\)
influx and Northern analysis revealed that despite this increase of root [
\({\mathrm{NH}_{4}^{{+}}}\)
], transcript abundance and influx remained almost at control (N-starved) levels. These results strongly suggest that glutamine is pivotal in regulating AMT1 transcript abundance.

Fig. 1.

13

\({\mathrm{NO}_{3}^{{-}}}\)
efflux from roots of barley plants grown with different concentrations of
\({\mathrm{NO}_{3}^{{-}}}.\)
Plants were grown for 7 d under steady-state conditions with respect to nitrate provision. Roots were then loaded with 13
\({\mathrm{NO}_{3}^{{-}}}\)
for >5 cytoplasmic half-lives, and subsequently transferred to the same concentration of 14
\({\mathrm{NO}_{3}^{{-}}}\)
for measurement of 13
\({\mathrm{NO}_{3}^{{-}}}\)
efflux. Rate constants for the lines were 0.041 min−1 (10 mM), 0.040 min−1 (1 mM), 0.042 min−1 (0.1 mM), 0.042 min−1 (0.01 mM), and 0.039 min−1 (uninduced plants), respectively (from Britto and Kronzucker, 2001).

Fig. 1.

13

\({\mathrm{NO}_{3}^{{-}}}\)
efflux from roots of barley plants grown with different concentrations of
\({\mathrm{NO}_{3}^{{-}}}.\)
Plants were grown for 7 d under steady-state conditions with respect to nitrate provision. Roots were then loaded with 13
\({\mathrm{NO}_{3}^{{-}}}\)
for >5 cytoplasmic half-lives, and subsequently transferred to the same concentration of 14
\({\mathrm{NO}_{3}^{{-}}}\)
for measurement of 13
\({\mathrm{NO}_{3}^{{-}}}\)
efflux. Rate constants for the lines were 0.041 min−1 (10 mM), 0.040 min−1 (1 mM), 0.042 min−1 (0.1 mM), 0.042 min−1 (0.01 mM), and 0.039 min−1 (uninduced plants), respectively (from Britto and Kronzucker, 2001).

Fig. 2.

Efflux of 13

\({\mathrm{NH}_{4}^{{+}}}\)
efflux from roots of barley plants previously grown on 0.1 mM
\({\mathrm{NH}_{4}^{{+}}}\)
and loaded with 13
\({\mathrm{NH}_{4}^{{+}}}\)
for 1 h, prior to eluting roots with 0.1 mM 14
\({\mathrm{NH}_{4}^{{+}}}\)
for the first 10 min shown. At this time plant roots were subjected to concentration shifts: (A) from 0.1 to 10 mM
\({\mathrm{NH}_{4}^{{+}}}\)
and (B) from 0.1 to 0.01 mM
\({\mathrm{NH}_{4}^{{+}}},\)
during elution (from Britto and Kronzucker, 2001).

Fig. 2.

Efflux of 13

\({\mathrm{NH}_{4}^{{+}}}\)
efflux from roots of barley plants previously grown on 0.1 mM
\({\mathrm{NH}_{4}^{{+}}}\)
and loaded with 13
\({\mathrm{NH}_{4}^{{+}}}\)
for 1 h, prior to eluting roots with 0.1 mM 14
\({\mathrm{NH}_{4}^{{+}}}\)
for the first 10 min shown. At this time plant roots were subjected to concentration shifts: (A) from 0.1 to 10 mM
\({\mathrm{NH}_{4}^{{+}}}\)
and (B) from 0.1 to 0.01 mM
\({\mathrm{NH}_{4}^{{+}}},\)
during elution (from Britto and Kronzucker, 2001).

Fig. 3.

Correlation between transcript abundance of the barley HvNrt2 gene and root glutamine concentrations after exogenous application of different amino acids and various inhibitors of nitrate assimilation (from Vidmar et al., 2000)

Fig. 3.

Correlation between transcript abundance of the barley HvNrt2 gene and root glutamine concentrations after exogenous application of different amino acids and various inhibitors of nitrate assimilation (from Vidmar et al., 2000)

Fig. 4.

A model representing proposed feedback processes involved in regulating the abundances of root Nrt2 and Amt1 transcripts by root glutamine (- - -), and in direct effects upon the transporters by root cytosolic

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
concentrations (……). Induction of NRT2 expression by
\({\mathrm{NO}_{3}^{{-}}}\)
is also indicated. Solid lines from NRT2 and Amt1.1 indicate the pathways of transcription and translation leading to high-affinity nitrate and ammonium transporters (circles) in the plasma membrane (outer rectangle). For purposes of simplicity, the diagram makes no attempt to distinguish between plastidic and cytosolic nitrogen pools (from Glass et al., 2001).

Fig. 4.

A model representing proposed feedback processes involved in regulating the abundances of root Nrt2 and Amt1 transcripts by root glutamine (- - -), and in direct effects upon the transporters by root cytosolic

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
concentrations (……). Induction of NRT2 expression by
\({\mathrm{NO}_{3}^{{-}}}\)
is also indicated. Solid lines from NRT2 and Amt1.1 indicate the pathways of transcription and translation leading to high-affinity nitrate and ammonium transporters (circles) in the plasma membrane (outer rectangle). For purposes of simplicity, the diagram makes no attempt to distinguish between plastidic and cytosolic nitrogen pools (from Glass et al., 2001).

Multiple members of the Nrt2 and Amt1 families

In the study of barley NRT2 genes by Trueman et al., it was suggested that there might be as many as 8–10 homologues in this species (Trueman et al., 1996). Following completion of the Arabidopsis genome sequencing project, it is now apparent that there are seven homologues in A. thaliana. A major task to be resolved is the individual functions of these genes. Work in the senior author's laboratory has been directed toward this goal, using A. thaliana as a model system. Under the conditions of this growth system, in which plants are grown hydroponically in open vessels, it has been possible to detect expression of all seven NRT2 homologues in roots and shoots using RT-PCR (Okamoto et al., unpublished data). Based upon the number of PCR cycles required and quantities of template RNA provided, it appears that AtNRT2.1 and AtNRT2.2 are the most abundantly expressed genes. In roots these genes are expressed at roughly 10 times the levels of all other genes whether in roots or shoots. The seven genes have been grouped into three categories according to their responses to nitrate feeding in plants previously deprived of

\({\mathrm{NO}_{3}^{{-}}}\)
for a period of 7 d before resupplying this ion. Category No. 1 includes AtNRT2.1 and AtNRT2.2, genes whose expression in roots increased 3–5-fold following provision of 1 mM
\({\mathrm{NO}_{3}^{{-}}}\)
. Both genes are subsequently down-regulated, presumably by a gradual increase of tissue glutamine. In shoots expression levels of these genes increased by less than 50% in response to
\({\mathrm{NO}_{3}^{{-}}}\)
provision, but, as in roots, this increase was followed by substantial down-regulation. Category No. 2 contains genes that are constitutively expressed, showing virtually no response to provision of
\({\mathrm{NO}_{3}^{{-}}}\)
. In both roots and shoots AtNRT2.5 and AtNRT2.6 show this pattern while for AtNRT2.3 this pattern was restricted to roots. In shoots, AtNRT2.3 expression levels doubled by 48 h. Category No. 3 contains AtNRT2.4 and AtNRT2.7, genes that are immediately down-regulated following exposure to
\({\mathrm{NO}_{3}^{{-}}}\)
(Okamoto et al., unpublished results). Interestingly, when AtNRT2.1 and AtNRT2.2 were first cloned from plants grown for several days with 1 mM KNO3 (Zhuo et al., 1999), it was stated that AtNRT2.2 was expressed at substantially lower levels than AtNRT2.1. However, it is apparent from these time-course studies (Okamoto et al., unpublished data) that, following initial exposure to
\({\mathrm{NO}_{3}^{{-}}}\)
, AtNRT2.2 transcript abundance is roughly equivalent to that of AtNRT2.1, however, by 12 h AtNRT2.2 transcript abundance is substantially reduced compared to AtNRT2.1. Based on the high levels of AtNRT2.1 and AtNRT2.2 transcript abundance in roots and the correspondence between the patterns of changes in transcript abundance and high-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
influx, these genes are good candidates for encoding iHATS. Recently, Filleur et al. have isolated a T-DNA insertional mutant of A. thaliana disrupted in adjoining AtNRT2.1 and AtNRT2.2 genes (Filleur et al., 2001). High-affinity
\({\mathrm{NO}_{3}^{{-}}}\)
transport in this mutant was reduced to 27% of wild-type rates. Thus it can be concluded that AtNRT2.1 and AtNRT2.2 make major contributions to the iHATS. The extent to which the remaining transport is due to other NRT2 genes or to NRT1 (low-affinity transport) is presently unknown (Wang et al., 1998).

If both AtNRT2.1 and AtNRT2.2 genes encode iHATS in roots, an important question is what (if any) differential roles these transporters might serve. Some suggestive answers to this question may be provided by comparisons with NRT2 genes of other organisms. In Aspergillus nidulans only two functional NRT2 genes appear to exist, and all four genotypes (wild type, double mutant and two single mutants) have been characterized with respect to 13

\({\mathrm{NO}_{3}^{{-}}}\)
influx kinetics (Unkles et al., 2001). Hoffstee plots of 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx indicate that both transporters contribute to
\({\mathrm{NO}_{3}^{{-}}}\)
influx in wild-type strains, although the transporters show distinct kinetic differentiation. The NrtA (originally crnA) transporter has a high Vmax and high Km (564 nmol mg−1 DW h−1 and 96.3 μM, respectively) while the second transporter (NrtB) has a low Vmax and low Km (141 nmol mg−1 DW h−1 and 11 μM, respectively). Interestingly the corresponding transporters in Chlamydomonas reinhardtii also possess widely different Km values for
\({\mathrm{NO}_{3}^{{-}}}\)
uptake (1.6 and 11 μM, respectively), but differ only slightly in Vmax values (9.0 and 5.6 μmol h−1 mg−1 chlorophyll, respectively (Galvan et al., 1996). This kinetic differentiation presumably enables the organism to access
\({\mathrm{NO}_{3}^{{-}}}\)
efficiently over a much wider range of concentration than would be possible by means of a single transporter. The A. nidulans double mutant is incapable of using
\({\mathrm{NO}_{3}^{{-}}}\)
as sole source of N at concentrations up to 250 mM
\({\mathrm{NO}_{3}^{{-}}}\)
or of absorbing 13
\({\mathrm{NO}_{3}^{{-}}}\)
at concentrations up to 500 μM. Continued exposure to
\({\mathrm{NO}_{3}^{{-}}}\)
leads to down-regulation of 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx in wild-type strains. This is due to down-regulation of NrtA, activity (Vmax values were 564±67 and 300±71 nmol mg−1 DW h−1 at 6 h and 16 h, respectively). By contrast, 13
\({\mathrm{NO}_{3}^{{-}}}\)
influx via the NrtB protein was unaffected by duration of exposure to
\({\mathrm{NO}_{3}^{{-}}}\)
(Vmax values were 141±6 and 162±26 nmol mg−1 DW h−1 at 6 and 16 h, respectively). This difference in response to duration of
\({\mathrm{NO}_{3}^{{-}}}\)
exposure among the strains may be due to slower accumulation of
\({\mathrm{NO}_{3}^{{-}}}\)
and products of
\({\mathrm{NO}_{3}^{{-}}}\)
assimilation that would normally down-regulate gene expression in mutant strains expressing only the NrtB protein. Thus, by default, gene mutation is partially compensated for.

The AMT1 family of high-affinity

\({\mathrm{NH}_{4}^{{+}}}\)
transporters contains five members, of which AtAMT1.1, AtAMT1.2 and AtAMT1.3 have been studied in detail (Gazzarini et al., 1999). All three genes are expressed in roots, while only AMT1.1 is expressed in significant amounts in leaves. By measuring 14C-methylamine uptake by Saccharomyces cerevisiae mutants expressing these genes individually, it was possible to estimate Km values of ∼0.5 μM for the AtAMT1.1, transporter and ∼40 μM for the AtAMT1.2 and AtAMT1.3 transporters. During N starvation, transcript abundance of AtAMT1.1 increased 7-fold during 24 h (Rawat et al., 1999). In a comparative study of root AtAMT1.1, AtAMT1.2 and AtAMT1.3 expression in response to N deprivation, it was shown that AtAMT1.1 increased 5-fold within 72 h, compared to a 2-fold increase in AtAMT1.3 and no change in AtAMT1.2 transcript abundance (Gazzarini et al., 1999). In tomato, LeAMT1.1 and LeAMT 1.2 transporters are expressed in roots, while LeAMT1.3 is preferentially expressed in shoots (von Wiren et al., 2000). Levels of LeAMT1.1 transcript in tomato roots also increased over time under conditions of N-deprivation and this was associated with a decline of glutamine and
\({\mathrm{NH}_{4}^{{+}}}\)
pool sizes (von Wiren et al., 2000). By contrast, and perhaps contrary to expectation, LeAMT1.2 transcript abundance increased following re-supply of
\({\mathrm{NH}_{4}^{{+}}}\)
or
\({\mathrm{NO}_{3}^{{-}}}\)
. This response may account for the initial stimulation of
\({\mathrm{NH}_{4}^{{+}}}\)
influx that was discussed above following resupply of N to N-starved plants (Kronzucker et al., 1998). LeAMT1.3 was not detected in roots.

A T-DNA insertional mutant has recently been isolated from Arabidopsis that fails to express AtAMT1.1 mRNA (Glass et al., 2001). Surprisingly, since AMT1.1 shows the strongest response to N-deprivation and also had the highest affinity for

\({\mathrm{NH}_{4}^{{+}}}\)
(at least when expressed heterologously in S. cerevisiae) disruption of this gene function reduced 13
\({\mathrm{NH}_{4}^{{+}}}\)
influx by only 20–30% (Glass et al., 2001). It is possible that, because of reduced
\({\mathrm{NH}_{4}^{{+}}}\)
uptake and thereby reduced negative feedback effects on transcript abundance of other AMT genes, there was compensation for the disruption of AtAMT1.1. This isssue is currently being explored.

Diurnal effects on
\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
uptake

There is now abundant evidence to confirm that

\({\mathrm{NO}_{3}^{{-}}}\)
and
\({\mathrm{NH}_{4}^{{+}}}\)
uptake display characteristic diurnal patterns (Clement et al., 1978,b; Macduff et al., 1997; Peuke and Jeschke, 1998; Gazzarini et al., 1999; Tischner, 2000). In the study by Clement et al., peak
\({\mathrm{NO}_{3}^{{-}}}\)
uptake occurred in the late afternoon while minimum uptake rates occurred at the end of the dark period or even in the first hours of daylight (Clement et al., 1978,b). It is notable that the amplitude of the diurnal pattern and the absolute values of the
\({\mathrm{NO}_{3}^{{-}}}\)
flux declined substantially during the course of the greenhouse study (Clement et al., 1978,b). This was associated with the onset of poor weather and a 75% reduction of irradiance. This may account for the low amplitude of the diurnal pattern reported in many growth chamber studies where plants are generally maintained under low irradiance. For example, in soybeans maintained on a 9/15 h light/dark regimen, uptake of 15
\({\mathrm{NO}_{3}^{{-}}}\)
was reduced by only 6% in the dark compared to the light period (Rufty et al., 1984). It has been suggested that reduced
\({\mathrm{NO}_{3}^{{-}}}\)
uptake associated with darkness may be countered by exogenously applied carbohydrates (Sehtiya and Goyal, 2000). Thus, in barley and maize, 1% sucrose additions caused 31% and 70% increases of
\({\mathrm{NO}_{3}^{{-}}}\)
uptake, respectively, in the light, while in dark-grown plants the values were 38% for both barley and maize. Nevertheless, given that dark-grown seedlings should have been substantially more carbohydrate-depleted than light-grown plants, it is surprising that the sucrose effect was actually less (maize) or similar (barley) in dark-grown plants.

\({\mathrm{NH}_{4}^{{+}}}\)
uptake in Phleum, Festuca and Arabidopsis also exhibits a diurnal periodicity, gradually increasing to a peak level toward the end of daylight hours (Macduff et al., 1997; Gazzarini et al., 1999), and the amplitude of the diurnal pattern of
\({\mathrm{NO}_{3}^{{-}}}\)
,
\({\mathrm{NH}_{4}^{{+}}}\)
and K+ uptake was highest on high irradiance days (Macduff et al., 1997).

Molecular studies have demonstrated that diurnal patterns of N uptake are correlated with diurnal patterns of transcript abundance for the high-affinity NRT2 and AMT1 genes (Lejay et al., 1999; Ono et al., 2000; von Wiren et al., 2000; Matt et al., 2001). In A. thaliana, NRT2.1 expression in roots increased in daylight hours and declined in the first hours of the dark period, this night-time reduction being prevented by additions of sucrose (Lejay et al., 1999). In roots of A. thaliana, all three members of the AMT1 family exhibited diurnal variation, with AtAMT1.3 expression showing the strongest correlation with diurnal patterns of 15

\({\mathrm{NH}_{4}^{{+}}}\)
uptake. In leaves of tomato, LeAMT1.2 and LeAMT1.3 showed a reciprocal diurnal pattern of expression with LeAMT1.3 transcript being highest in darkness.

The conclusion that C and N metabolism are tightly linked is inescapable (Coruzzi and Bush, 2001). In the study by Matt et al., the activities of various enzymes involved in nitrogen metabolism and their transcript abundances, including the high-affinity nitrate transporter, as well as concentrations of various metabolites (

\({\mathrm{NO}_{3}^{{-}}}\)
, amino acids, sugars and 2-oxoglutarate) were measured during a diurnal cycle in tobacco (Matt et al., 2001). Based upon the correspondence between root sugar levels and NRT2 transcript abundance (and a lack of correspondence with other metabolites) the authors concluded that root sugars were responsible for the diurnal pattern of NRT2 expression. It is intriguing to consider whether the effects of carbohydrate supply might act directly or indirectly on nitrogen pools and/or transcript abundances. For example, when carbohydrate supply to the root limits N assimilation and/or growth, accumulation of N metabolites might reduce expression of transporter genes or even act directly upon the transporters. Furthermore, the study by Matt et al. acknowledged that the observed correlations between NRT2 expression and root sugar levels were based upon whole root analyses (Matt et al., 2001). Clearly, cytosolic metabolite concentrations might have provided a different conclusion.

In summary, a high degree of heterogeneity with respect to soil N availability and diurnal and seasonal variation in plant requirements for N impose a need to regulate N fluxes across the plasma membrane of plant roots in order to optimize plant N capture. The need to integrate/co-ordinate N acquisition from several potential soil N sources (

\({\mathrm{NO}_{3}^{{-}}}\)
,
\({\mathrm{NH}_{4}^{{+}}}\)
and amino acids) suggests that regulation might be most effective if a common end-product of
\({\mathrm{NO}_{3}^{{-}}}\)
assimilation such as glutamine were to serve as the source of negative feedback. Experiments listed above indicate that this may be the case. Nevertheless, there is no reason to assume that, in addition to the clearly demonstrated regulation by transcript abundance, there will not be post-transcriptional regulation by other nitrogen sources. Indeed preliminary evidence for such effects has already been presented (Fraisier et al., 2000; Vidmar et al., 2000; Rawat et al., 1999).

In addition to regulating influx across root plasma membranes, internal redistributions to vacuole and to xylem suggest that there is a need for integration of all component fluxes as well as for the integration of amino acid fluxes involved in nutrient cycling within plants. Thus far, the focus of attention in studies of inorganic N uptake at the physiological and molecular levels has been upon the regulation of root plasma membrane transporters. It is to be anticipated that future physiological and molecular studies will include fluxes to subcellular compartments and between major organs of the plant (such as fluxes from root to xylem, xylem to shoot) and leaf uptake of inorganic N.

Acknowledgments

The work undertaken by the authors was financed by grants from NSERC to ADM Glass, who gratefully acknowledges this support. In addition we gratefully acknowledge the provision of 13N by the UBC TRIUMF cyclotron.

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Author notes

5
Present address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.

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