Nitrate regulates primary root growth by two distinct pathways
The generally held view has been that the NO3− supply modifies root architecture predominantly through its effects on lateral root length and numbers, and that primary root growth is largely unresponsive to NO3− (Bloom et al., 2002; Drew and Saker, 1975; Granato and Raper, 1989; Linkohr et al., 2002; Zhang and Forde, 1998). Here we have shown that, in Arabidopsis, NO3− can have a very strong positive effect on primary root growth and that it achieves this in two ways. The first is by antagonising the inhibitory effect of exogenous Glu. This amino acid has previously been shown to be sensed at the primary root tip, resulting in inhibition of meristematic activity and an increase in root branching (Walch-Liu et al., 2006). We found that if an excess of NO3− was present, these effects could be partially or even completely suppressed, depending on the accession and the ratio of NO3− to Glu. The second way in which NO3− stimulates primary root growth is independent of the presence of exogenous Glu. Accessions differed markedly in their sensitivity to this direct effect of NO3−, but primary root growth in the most sensitive line (No-0) was significantly stimulated by an external NO3− concentration of only 50 μm, and a ∼2.5-fold stimulation was achieved by NO3− concentrations above 0.5 mm. The magnitude of these responses is comparable to what was seen when a localised NO3− treatment was applied to lateral roots, although the minimum concentration needed to stimulate lateral root growth was 100 μm (Zhang and Forde, 1998; Zhang et al., 1999).
For both types of primary root response to NO3− we provide evidence that it is the external presence of the NO3− ion itself that is being perceived and that NO3− sensing is taking place at the primary root tip. These conclusions are based on the demonstration that in both cases the primary root tip must be in direct contact with NO3−, that NH4− will not substitute and, in the case of NO3− antagonism of the Glu effect, that a NR-null mutant retains its responsiveness to NO3−. Based on similar evidence, it was previously concluded that stimulation of lateral root growth by NO3− is also a response to the external presence of the NO3− ion, independently of its role as a nutrient (Zhang and Forde, 1998; Zhang et al., 1999).
We considered the possibility that direct stimulation of primary root growth by NO3− might be a manifestation of the same phenomenon as NO3− antagonism of the inhibitory effect of Glu on primary root growth. In this model, primary root growth would be negatively regulated by the endogenous Glu pool, and NO3− would stimulate primary root growth by alleviating this effect. The exceptionally slow growth of primary roots of the Glu-hypersensitive accession, C24 (see Figure 2a), could potentially be explained by a model in which endogenous Glu pools were negatively regulating root growth. Since C24 is also very sensitive to the antagonising effect of NO3−, this model would predict that primary root growth in this accession would also be highly responsive to NO3− in the absence of exogenous Glu. However, C24 was the one accession in which NO3− failed to stimulate primary root growth in the absence of exogenous Glu. Although it is possible that the sensitivity of the root to exogenous Glu is an unreliable indicator of its sensitivity to endogenous Glu pools, we conclude that there is at present no evidence to suggest that the two types of primary root response to NO3− share a common mechanism.
It may be that the signalling pathway by which NO3− stimulates primary root growth directly is the same as the one by which a localised NO3− supply stimulates lateral root growth (Remans et al., 2006b; Zhang and Forde, 1998). However, we were unable to confirm this by demonstrating a role for the NRT1.1 and ANR1 genes because the relevant mutants were in a background (Columbia) whose primary root growth is insufficiently responsive to this NO3− effect.
Nitrate sensing by the NRT1.1 nitrate transporter
In fungi and animals, there are membrane proteins that serve the dual function of transporter and external nutrient sensor (Holsbeeks et al., 2004). Examples include the yeast Pi transporters/receptors (or ‘transceptors’) Pho84p and Pho87p (Giots et al., 2003), the amino acid transceptor Gap1p (Donaton et al., 2003) and the NH4+ transceptor Mep2p (Lorenz and Heitman, 1998). Although there are no known examples of transceptors in plants, a number of studies have suggested that the dual-affinity NRT1.1 NO3− transporter has a direct or indirect role in NO3− sensing (Guo et al., 2001; Muňos et al., 2004; Remans et al., 2006b).
The evidence presented here indicates that NRT1.1 is a component of at least the signalling pathway that leads to antagonism of the negative effect of Glu on primary root growth. We found that NO3− did not alleviate this effect in the chl1-5 NRT1.1-deficient mutant, but that the NO3− response was fully restored in a chl1-5 line constitutively expressing the NRT1.1 cDNA. However, a major barrier to deciding whether a transporter is directly or indirectly involved in nutrient sensing is eliminating the possibility that it is simply facilitating the uptake of the nutrient for detection by intracellular sensors. Thus although a previous study established that lateral root growth in NRT1.1-deficient mutants was unresponsive to NO3− under conditions where there was no general defect in NO3− uptake (Remans et al., 2006b), a NO3− sensing role for NRT1.1 could not be confirmed because it remained possible that the transporter was necessary for NO3− uptake into specific NO3−-sensing cells in the root tip. The most convincing way to show that a transporter is also a sensor is to be able to uncouple the two functions by mutation (Donaton et al., 2003; Smith et al., 2003). In the case of NRT1.1, we have evidence suggesting that a Thr to Ala substitution at residue 101 may have achieved this effect.
The T101A mutation in NRT1.1 has been shown to inactivate the high-affinity component of this dual-affinity NO3− transporter, while leaving the low-affinity component intact (Liu and Tsay, 2003). We found that in marked contrast to the 35S-CHL1 line, constitutive expression of the mutant form of NRT1.1 in the 35S-CHL1T101A line failed to confer any increase in NO3− sensitivity, even at 5 mm NO3−. To explain this result in terms of the NO3− transport function of NRT1.1 we would have to propose that while constitutive expression of the wild-type (dual-affinity) form of the protein was able to more than fully restore NO3− uptake in the chl1-5 mutant, constitutive expression of the mutant (low-affinity) form made no significant contribution to NO3− uptake in the low-affinity range. This would contradict previous studies showing that the T101A mutant protein expressed in oocytes could catalyse rates of NO3− uptake (from 5 mm NO3−) that were two-thirds those of the wild-type, and experiments showing that the rate of NO3− uptake (again at 5 mm NO3−) by the same 35S-CHL1T101A line as used here was only 35% less than the 35S-CHL1 line (Liu and Tsay, 2003). The presence of alternative N sources such as NH4+ or amino acids at high concentrations (typically 10 mm) can cause feedback inhibition of the high-affinity NO3− transport system, but their effect on uptake in the low-affinity range is much less pronounced (Forde and Clarkson, 1999). l-Glutamate has not been reported to have any effects on NO3− uptake beyond those attributable to its influence on the plant’s N status (Nazoa et al., 2003). Thus there is no reason to expect that the low Glu concentrations used here (0.5–1 mm) would have biased the results, e.g. by differentially inhibiting NO3− uptake in the 35S-CHL1 and 35S-CHL1T101A lines.
The difficulty in explaining the phenotype of the 35S-CHL1T101A line solely in terms of the transport function of NRT1.1 leads us to propose that NRT1.1 has a direct rather than indirect role in NO3− sensing, and that this sensing function is inactivated by the T101A mutation. Since the Thr to Ala substitution blocks phosphorylation at residue 101 (Liu and Tsay, 2003), this would imply that only the phosphorylated protein has NO3−-sensing activity. This would be analogous to the yeast Mep2p NH4+ transceptor, where mutagenesis of a putative phosphorylation site abolished its sensing function while leaving its transport activity intact (Smith et al., 2003).
A regulatory role for NRT1.1 would also help to explain the observation that the 35S-CHL1T101A line was consistently more sensitive to Glu than chl1-5 (or the 35S-CHL1 line), even in the absence of NO3−. This dominant-negative effect of overexpressing the mutant form of the protein suggests that NRT1.1 may be interacting directly with component(s) of the Glu signalling pathway. Thus constitutive expression of NRT1.1 in its unphosphorylated form (but not in its phosphorylatable form) may lead, through protein–protein interactions at the plasma membrane, to conformational changes that make the Glu-sensing system more sensitive to its ligand.
It has been found that phosphorylation/dephosphorylation at Thr101 is nitrogen regulated, being most highly phosphorylated under N-limiting conditions and then rapidly dephosphorylated when N is resupplied (Liu and Tsay, 2003). If, as our results suggest, phosphorylation at Thr101 is required to activate the sensing function of NRT1.1, then reversible phosphorylation/dephosphorylation of the protein would provide a useful mechanism for regulating its NO3− sensitivity according to changes in N availability. Phosphorylation-dependent sensitization/desensitization of receptor proteins is a commonly observed phenomenon in animal systems (e.g. Ribas et al., 2007).
The observation that the NRT2.1 gene remains inducible by NO3− in an NRT1.1-defective mutant (Muňos et al., 2004) indicates that there are NO3− sensors additional to NRT1.1 in the Arabidopsis root. Possible candidates include other NRT1-related proteins with a confirmed NO3− transport function (Tsay et al., 2007), or the NRT2.1 NO3− transporter itself, which is reported to have a sensing or signalling role in the regulation of lateral root initiation (Little et al., 2005; Remans et al., 2006a).
How does nitrate antagonise l-glutamate signalling?
If uptake of Glu by Glu-sensing cells at the root tip were required for perception of the Glu signal, then one straightforward explanation for nitrate’s ability to antagonise the Glu effect would be by blocking Glu uptake. However, we found that excess NO3− had no inhibitory effect on Glu uptake at the root tip. The evidence suggests that exogenous Glu is most likely sensed at the cell surface (Walch-Liu et al., 2006), and although there is a plant family of GLR genes that encode homologues of mammalian ionotropic Glu receptors (Chiu et al., 2002), their involvement in this process has not yet been established. Once the receptor(s) and other components of the Glu signalling pathway at the root tip have been identified, we can begin to address the question of how the NO3− signal, sensed by the NRT1.1 protein, is able so effectively to antagonise the Glu signal. A model for how NO3− and Glu signalling pathways may interact at the primary root tip is presented in Figure 4(a).
Figure 4. Nitrate and l-glutamate (Glu) signalling pathways at the primary root tip and their possible role in modulating root architecture. (a) Model for antagonistic interactions between NO3− and Glu signalling pathways. l-Glutamate sensed at the primary root tip by an unknown receptor triggers the slowing of primary root growth and increased branching behind the root tip (Walch-Liu et al., 2006). Nitrate sensed by NRT1.1 at the primary root tip antagonises the Glu signalling pathway and alleviates the effect of Glu on root architecture. The model suggests that NRT1.1 is only active as a NO3− sensor when phosphorylated at Thr101, phosphorylation being reversible and regulated by the N supply (Liu and Tsay, 2003). (b) A diagram illustrating how two environmental factors (NO3− and Glu concentrations in the soil) could work in opposition to modulate root architecture, with the root’s response being dependent on its genetically determined sensitivity to each signal.
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As previously discussed (Walch-Liu et al., 2006), Glu is one of the most abundant amino acids in soil and the concentrations that affect root growth in Arabidopsis are in the range likely to be encountered in organic N-rich soil patches. Likewise, the NO3− concentrations that were able to alleviate Glu inhibition (0.5–5 mm) and directly stimulate primary root growth (0.05–5 mm) are commonly found in many soil types (Farley and Fitter, 1999; Glass and Siddiqi, 1995). While NO3− is the major form of N available in fertile aerobic soils, the dissolved organic N pool can be the main N source available in nutrient-poor soils (Christou et al., 2005). Thus the antagonistic effects of NO3− and Glu on primary root growth suggest a mechanism that would allow root architecture to be modulated according to differences in soil fertility, or in response to spatial or temporal variations in the relative abundance of organic and inorganic N. Figure 4(b) illustrates how differences in soil N composition and in a plant’s intrinsic sensitivity to the different N signals could combine to influence the development of the architecture of the root system.