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The Plant Journal (2003) 35, 800±810 doi: 10.1046/j.1365-313X.2003.01849.

A role for glycine in the gating of plant NMDA-like receptors


Christian Dubos1,y, David Huggins2,y, Guy H. Grant2, Marc R. Knight1 and Malcolm M. Campbell1,
1
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK, and
2
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road,
Oxford OX1 3QZ, UK

Received 2 May 2003; revised 25 June 2003; accepted 4 July 2003.



For correspondence (fax ‡44 1865 274074; e-mail malcolm.campbell@plants.ox.ac.uk).
y
These authors contributed equally to this work.

Summary
The amino acid glycine has a well-established role in signalling in the mammalian central nervous system.
For example, glycine acts synergistically with the major excitatory neurotransmitter, glutamate, to regulate
the in¯ux of ions such as calcium, through N-methyl-D-aspartate (NMDA) receptors. Plants possess NMDA-
like receptors, generically referred to as glutamate receptors (GLRs), named on the basis of their presumed
ligand, glutamate. Previously, glycine has not been implicated in plant GLR activity or any other aspect of
plant signalling. Using transgenic Arabidopsis seedlings expressing aequorin to monitor ligand-mediated
changes in the cytosolic concentration of Ca2‡ ([Ca2‡]cyt), the data presented herein show that glutamate
and glycine act synergistically to control ligand-mediated gating of calcium in plants. Glutamate and gly-
cine synergism also regulates hypocotyl elongation. Transient increases in [Ca2‡]cyt mediated by glutamate
and glycine, as well as hypocotyl elongation, were inhibited by 6,7-dinitroquinoxaline-2,3 dione (DNQX), a
competitive inhibitor of animal GLRs. Using a multiscale docking algorithm in combination with a mole-
cular model of the ligand-binding domain of plant GLRs, evidence is provided indicating that glycine, and
not glutamate, is likely to be the natural ligand for most plant GLR subunits. These ®ndings uncover a
hitherto unconsidered role for glycine signalling in plants, and suggest that the synergistic action of glu-
tamate and glycine at NMDA-like receptors predates the divergence of plants and animals.

Keywords: glycine, calcium, ionotropic glutamate receptors, NMDA.

Introduction
Mammalian ionotropic glutamate receptors (GLRs) are trans-membrane regions, one membrane-embedded
multimeric, ligand-gated channels, comprised of four or domain and two domains that form the ligand-binding site
®ve subunits that function to perceive glutamate during (Figure 1). This structure is also predicted for the plant
neurotransmission (Armstrong et al., 1998). Binding of receptors (Chiu et al., 1999). Based on sequence similarity,
glutamate to the ligand-binding domain of these receptors it has been assumed that plant GLRs may function in a
results in gating of the transmembrane channel, allowing manner analogous to mammalian GLRs.
cations to enter the cell. There are three different groups of Mammalian ionotropic GLRs, such as NMDA receptors,
mammalian ionotropic GLRs (AMPA, N-methyl-D-aspartate function as ligand-gated channels for the transport of ions
(NMDA) and Kainate receptors), which are based on their such as Ca2‡ or Na‡ (Scatton, 1993). Similarly, plant GLRs
pharmacological properties and structural similarities have been predicted to function as Ca2‡ channels that are
(Armstrong et al., 1998). gated by glutamate (Davenport, 2002; White et al., 2002).
Twenty putative GLR subunits are encoded in the Arabi- Several lines of evidence suggested that genes encoding
dopsis thaliana genome, and these show the greatest GLR subunits might be involved in regulating cellular and
sequence similarity to the mammalian NMDA receptor developmental phenomena in plants. Treatment of
subunits (Chiu et al., 1999, 2002; Davenport, 2002; Lacombe Arabidopsis seedlings with the GLR antagonist 6,7-dinitro-
et al., 2001; Lam et al., 1998). NMDA receptors are com- quinoxaline-2,3 dione (DNQX) provided preliminary, sug-
posed of assemblies of three different classes of subunits, gestive evidence for the involvement of GLRs in the control
NR1, NR2 and NR3 (Das et al., 1998). Each subunit has three of hypocotyl elongation (Lam et al., 1998). Treatment of
800 ß 2003 Blackwell Publishing Ltd
Unsuspected glycine signalling in plants 801

Data from the middle of the last century suggested that


glycine might function as a signalling molecule in plants. In
1939, White found that glycine favoured the growth of
excised tomato roots in the presence of appropriate com-
binations of carbohydrates, thiamine and inorganic salts
(White, 1939). Further work based on White's early obser-
vations suggested that cotyledon-derived glycine modu-
lated root development in concert with other amino acids in
a synergistic fashion (Fries, 1953; Skinner and Street, 1953).
In all these instances, glycine was thought to function
Figure 1. Schematic diagram of GLR structure. purely as a nutrient, and the potential involvement of
The regions of the ligand-binding domain that form the focus of these
studies are highlighted in grey. glycine as a signal in plants was not considered. The recent
discovery of NMDA-like GLRs in plants and the role that
glycine plays in gating these receptors in animals suggest
plants with a putative GLR agonist also suggested an that it may be worthwhile to consider the potential involve-
involvement of GLRs in hypocotyl elongation (Brenner ment of glycine signalling in plants.
et al., 2000). Unrelated studies predicted that glutamate- In order to test the hypothesis that glycine may function
gated channels may be involved in regulating changes in to gate plant GLRs, we have examined glycine-mediated
the cytosolic concentration of Ca2‡, [Ca2‡]cyt (Dennison and changes in [Ca2‡]cyt in transgenic Arabidopsis seedlings
Spalding, 2000). Overexpression of one member of the GLR expressing aequorin, a Ca2‡-sensitive luminescent protein
family in Arabidopsis implicated at least one GLR subunit in (Knight et al., 1991, 1996, 1999). In addition, the effects of
the control of calcium homeostasis and related develop- glutamate and glycine on hypocotyl elongation were inves-
mental phenomena (Kim et al., 2001). tigated. In order to determine if any glutamate- or glycine-
To date, studies on plant GLRs have focused on the mediated changes in [Ca2‡]cyt or in hypocotyl elongation
interaction of glutamate with these receptors (Chiu et al., were dependent on GLRs, the plant material was also
1999; Davenport, 2002; Dennison and Spalding, 2000; Kim analysed after treatment with DNQX. Finally, the interaction
et al., 2001; Lacombe et al., 2001; Lam et al., 1998), and of glutamate and glycine, as well as DNQX, with plant GLRs
have not addressed the hypothesis that glutamate may not was examined using a molecular modelling approach. The
be the sole natural ligand for these receptors in plants. This latter approach was based on a multiscale docking algo-
is surprising, as mammalian NMDA receptor subunits, rithm that has been used successfully to examine ligand
which share the highest degree of sequence similarity with binding to proteins such as the anthrax toxin (Glick et al.,
the plant GLR subunits, are well known to bind to glycine. In 2002a,b,c). Taken together, the ®ndings of these analyses
fact, NMDA receptors must bind not only glutamate, but open the door to the possibility that glycine may function as
also the co-agonist, glycine, in order to function (Ivanovic a signalling molecule in plants.
et al., 1998). Furthermore, the NR3 family of NMDA receptor
subunits are not responsive to glutamate, and can be gated
by glycine alone (Chatterton et al., 2002). In animals, the Results and discussion
ratio of glutamate-binding subunits to glycine-binding sub-
units is thought to determine the relative responsiveness of
Glycine mediates changes in [Ca2‡]cyt
the multisubunit NMDA receptor to both glutamate and
glycine (Chatterton et al., 2002). Previous studies have shown that glutamate can induce
Glycine has recently been shown to be a key modulator of changes in the cytosolic concentration of [Ca2‡]cyt, and
cell-to-cell signalling in the mammalian central nervous it was suggested that this occurred through GLRs
system. In addition to the role that glycine plays in stimu- (Dennison and Spalding, 2000). If plant GLRs function like
lating NMDA receptors to initiate calcium signalling, gly- their mammalian counterparts, glycine would also be
cine stimulation of NMDA receptors also primes the predicted to gate GLRs and thereby change [Ca2‡]cyt. This
receptors for clathrin-mediated endocytosis (Nong et al., hypothesis was tested by measuring [Ca2‡]cyt in trans-
2003). Thus, glycine functions to modulate not only the genic Arabidopsis seedlings expressing aequorin (Knight
immediate response of neurons to neurotransmitters, but it et al., 1991, 1996, 1999). Consistent with the previous
also conditions future responsiveness by altering the occur- ®ndings (Dennison and Spalding, 2000), glutamate induced
rence of receptors on the cell surface. Recognition of the an increase in [Ca2‡]cyt in a concentration-dependent
roles played by glycine has profoundly changed the under- fashion (Figure 2a). However, glycine also induced an
standing of synaptic signalling in the central nervous sys- increase in [Ca2‡]cyt in a concentration-dependent manner
tem (Nong et al., 2003). (Figure 2a). Amino acids with similar structures, aspartate
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810
802 Christian Dubos et al.

Figure 2. [Ca2‡]cyt changes in plants in response to application of glutamate (Glu), glycine (Gly), aspartate (Asp), alanine (Ala), DNQX or combinations thereof.
The dotted horizontal lines indicate the standard deviation around the control treatment, which was an injection of MS medium, with no effector, as a control for
the touch response.
(a) Arabidopsis seedlings were treated with the effectors at the concentrations indicated, and changes in [Ca2‡]cyt were determined as described previously by
Knight et al. (1996). An analysis of variance was used to compare the mean of each effector treatments relative to the control, which was MS medium alone; $:
signi®cant effect relative to control (injection of MS alone); P < 0.0005. Error bars show the SE of the mean.
(b) As per above, but a cold treatment was used to induce the change in [Ca2‡]cyt as described previously by Knight et al. (1996).

and alanine, did not induce any change in [Ca2‡]cyt


Glycine and glutamate modulation of [Ca2‡]cyt is
(Figure 2a).
DNQX-sensitive

In order to test the hypothesis that glutamate and glycine


Glycine and glutamate act synergistically to modulate
were eliciting their gating effect at GLRs, plants were pre-
[Ca2‡]cyt
treated with a compound that inhibits mammalian iono-
Seedlings were treated with a mixture of glutamate and tropic GLRs, DNQX (Armstrong and Gouaux, 2000), prior to
glycine to determine if the compounds acted synergisti- treatment with the agonists. DNQX pre-treatment comple-
cally. The combination of glutamate and glycine was effec- tely abolished the increase in [Ca2‡]cyt normally induced
tive at inducing an increase in [Ca2‡]cyt at concentrations by the addition of glutamate, or glycine, or both in com-
that were two orders of magnitude less than those required bination (Figure 2a). DNQX pre-treatment did not abolish
to give an equivalent increase when each of the compounds an increase in [Ca2‡]cyt brought about by cold treatment
was added separately (Figure 2a). Addition of a combina- (Figure 2b), showing that the effect of DNQX was neither
tion of aspartate and alanine had no effect, even at con- toxic nor generic, and that it was related to the perception of
centrations that were 10-fold greater than the optimum for glutamate and glycine. The ability of DNQX to block the
glutamate and glycine (Figure 2a). effect of these ligands suggests that they bind to the same
The increase in [Ca2‡]cyt in response to the combination receptor as DNQX, an ionotropic GLR.
of glutamate and glycine did not appear to be dose-depen-
dent at concentrations above 0.01 mM. At even these low
DNQX-sensitive glycine and glutamate modulation of
concentrations, the increase in [Ca2‡]cyt was as great as that
[Ca2‡]cyt occurs in cotyledons, but not in roots
observed when each amino acid was used on its own at the
maximum concentration of 10 mM. This was in contrast to Ligand-induced increases in [Ca2‡]cyt in whole plants were
the increase in [Ca2‡]cyt observed when each amino acid monitored by visualisation of [Ca2‡]-dependent changes in
was added on its own, which was dose-dependent up to aequorin luminescence using a photon-counting camera
1 mM. This suggests that the gating of Ca2‡ by the combi- (Knight et al., 1999). This revealed that the glutamate- and
nation of glutamate and glycine was a saturable response, glycine-gated changes in [Ca2‡]cyt completely overlapped
and that saturation of the channel occurred at very low in both time (Figure 3a) and location (Figure 3b). The
concentrations of the amino acids. Given that 10 mM con- increase in [Ca2‡]cyt took place throughout the plant body,
centrations of each individual amino acid also did not but was strongest in the cotyledons and the root, regardless
induce a statistically signi®cant increase in [Ca2‡]cyt relative of whether the ligand used was glutamate, or glycine, or a
to that observed at 1 mM (Figure 2a) suggests that this combination of both (Figure 3b). DNQX completely inhi-
response is also saturable. bited ligand-gated changes in [Ca2‡]cyt in aerial tissue, but
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810
Unsuspected glycine signalling in plants 803

effect. Glycine and glutamate functioned synergistically


when applied together with DNQX to seedlings, function-
ing at signi®cantly lower concentrations than when they
were used alone (Figure 4b).
While DNQX pre-treatment of seedlings completely
inhibited ligand-gated changes in [Ca2‡]cyt in aerial tissues,
as monitored by aequorin (Figure 3b), it is clear that growth
of seedlings in medium containing a mixture of DNQX and
amino acids allows for competition to occur between the
agonists and the antagonist, so that the effect is not abso-
lute but concentration-dependent. It is important to note
that the experiments with aequorin document the ability of
DNQX pre-treatment to block the immediate change in
[Ca2‡]cyt, and do not indicate the long-term effect on
[Ca2‡]cyt, gating by the three ligands acting together. The
growth of plants in a combination of DNQX with the amino
acids would almost certainly result in a dynamic competi-
tion for the binding site by the inhibitor and the two
agonists. Furthermore, long-term exposure of plants to
DNQX may alter the abundance of GLRs, and thereby alter
plant responsiveness to the agonists. In animals, the abun-
dance of GLRs is affected by the extent to which a neuron is
stimulated by neurotransmitters. The greater the stimula-
Figure 3. [Ca2‡]cyt changes in plants in response to application of glutamate
(Glu), glycine (Gly), DNQX or combinations thereof. tion, the lower the abundance of GLRs and vice versa
Arabidopsis seedlings were treated with the effectors at the concentrations (Platenik et al., 2000; Scannevin and Huganir, 2000; Sheng
indicated, and changes in [Ca2‡]cyt were determined as described previously and Kim, 2002). Similarly, continual exposure of plants to
by Knight et al. (1996). Ligand-based changes in [Ca2‡]cyt were (a) examined
luminometrically or (b) visualised using a photon-counting camera as DNQX and concomitant decreases in GLR stimulation may
described previously by Knight et al. (1999). lead to an increase in the abundance of GLRs, which would,
in turn, enhance plant responsiveness to any glutamate and
glycine that is present. This may account for the ability of
only slightly changed the activity in the root. These results glutamate and glycine to reverse the effects of DNQX when
reveal that GLRs found in aerial tissues of Arabidopsis plants are grown in a mixture of these ligands. Finally, it
behave in a manner analogous to their mammalian coun- may also be that the combined action of glutamate and
terparts in that they are DNQX-sensitive. These results also glycine reverses the inhibitory effects of DNQX through an
suggest that either DNQX does not penetrate the root to alternative signalling mechanism, which may be calcium-
inhibit GLR activity there, or, that at least two types of independent and which could account for the apparent
receptors function to perceive glutamate and glycine in differences between the calcium in¯ux data and the hypo-
Arabidopsis: a DNQX-sensitive form in aerial tissues and cotyl elongation data.
another that is DNQX-insensitive found in the roots.
Molecular modelling shows binding of plant GLRs to
DNQX-mediated hypocotyl elongation is competitively glutamate, glycine and DNQX
inhibited by glycine and glutamate
As was the case in previous studies, the results described
Plant GLRs have been implicated in the control of an herein provide circumstantial evidence for the interaction
important component of light-mediated development ± between plant GLRs and their putative ligand, glutamate.
hypocotyl elongation (Brenner et al., 2000; Lam et al., Beyond this, however, the current results provide more
1998). As has been shown previously (Lam et al., 1998), comprehensive evidence in support of the hypothesis that
plants grown in the presence of DNQX have elongated plant GLRs function like their mammalian counterparts in
hypocotyls (Figure 4a). If DNQX elicits this response by that the gating of [Ca2‡]cyt is modulated by both glutamate
antagonising plant GLRs, then simultaneous application and glycine, and that this is inhibited by DNQX. Further-
of GLR agonists should reverse this effect. Both glutamate more, the results presented herein show that this signalling
and glycine reversed DNQX-induced hypocotyl elongation mechanism may be involved in the control of hypocotyl
in a concentration-dependent fashion (Figure 4). Glycine elongation. Nevertheless, it remains to be determined if any
was at least as effective as glutamate at reversing the DNQX of the compounds in question actually interact with plant
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810
804 Christian Dubos et al.

Figure 4. Variation in hypocotyl elongation in


response to application of glutamate (Glu), gly-
cine (Gly), DNQX or combinations thereof.
(a) Arabidopsis seedlings were grown in med-
ium containing the effectors at the concentra-
tions indicated, and changes in hypocotyl
elongation were visualised.
(b) The quantitative response to the effectors
was determined. Values represent the mean of a
minimum of 15 seedlings. Pairwise t-tests were
used to compare the mean of each treatment
against the mean for the seedlings grown in MS
plus DNQX ($: signi®cant difference; P <
0.0001) or MS alone (*: no signi®cant differ-
ence; P < 0.0001). Error bars show the SE of the
mean.

GLRs. As an important ®rst step in determining if gluta- between the protein and the ligand, the iterations reveal
mate, glycine and DNQX are able to bind to plant GLRs, a that only one or several overlapping protein sites are opti-
molecular modelling approach was employed. mal for that ligand.
The structure of the extracellular ligand-binding region of Using this approach, the docking algorithm con®rmed
an A. thaliana GLR subunit (AtGLR) was modelled on the that glutamate bound to the R. norvegicus GLR at the site
crystal structure of the analogous region of a Rattus nor- predicted by the crystal structure (Figures 7a±c and 8a,b). In
vegicus GLR (PDB ID 1FTJ; Armstrong and Gouaux, 2000). contrast, attempts to dock glutamate to the AtGLR2.9
The structure of the rat GLR and its interaction with its model failed. The ®nal iteration revealed that there was a
ligands was empirically determined by crystallography as high likelihood that glutamate would only associate with
well as detailed functional analyses of GLR-ligand interac- the surface of the protein, and not in the predicted ligand-
tions (Armstrong and Gouaux, 2000; Armstrong et al., 1998; binding site (Figure 7d±f). Furthermore, attempts to `force'
Yoneda and Ogita, 1991). Using a multiscale docking algo- glutamate into the predicted ligand-binding site in silico
rithm that provides accurate predictions of protein±ligand showed that this could not occur naturally because of
interactions (Glick et al., 2002a,b,c), the nature of the inter- signi®cant steric hindrance (Figure 8c,d). This is attributa-
action of the plant GLRs with glutamate and glycine was ble to the replacement of one of the key residues that are
explored. The A. thaliana GLR, AtGLR2.9, was chosen for required for binding of the side chain carboxylate group of
the modelling study as it exhibited the greatest sequence glutamate (Figure 8d). The residue in question, Thr655 of
similarity to the R. norvegicus GLR, particularly in those the R. norvegicus GLR, is conserved amongst all known
regions implicated in ligand binding (Figures 5 and 6). mammalian GLRs (Figure 5). In AtGLR2.9, Thr655 is
The multiscale docking algorithm runs a series of pro- replaced by a bulky, hydrophobic Phe residue, which blocks
gressive iterations to explore the three-dimensional space the ligand-binding pocket where glutamate would dock
of a protein molecule for the most likely binding sites of a (Figures 6 and 8d). In fact, Thr655 is replaced either by
chosen ligand. In early iterations, many possible binding Phe or the bulky, hydrophobic branch-chain amino acids,
sites are found, as all surfaces of the protein are available Leu or Ile, in 18 of the 20 AtGLR subunits, and is missing
for ligand binding. As the algorithm proceeds, preferred altogether from one other (Figure 5). This suggests that
binding sites are identi®ed as they are `discovered' iteration glutamate is not the natural ligand for the majority of AtGLR
upon iteration. For those proteins where there is a good ®t subunits.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810
Unsuspected glycine signalling in plants 805

Figure 5. Multiple sequence alignment of GLR


sequences.
The peptide sequences (and their correspond-
ing accession numbers) that were used in the
alignment included: R. norvegicus GLRs, Rat-
GlurB (CAA38465), RatKain1 (AAA02873), NR2a
(AAC03565),NR2b(AAA41714),NR2c(AAA41713),
NR2d (AAC37647), NR1a (AAA16366) and
NR3b (AAL69893); Homo sapiens GLR, NR3a
(AAL40734) and A. thaliana GLRs, AtGLR1.1
(AAF26802.1), AtGLR1.2 (BAA96960.1), AtGLR1.3
(BAA96961.2), AtGLR1.4 (AAF02156.1), AtGLR2.1
(AAB61068.1), AtGLR2.2 (AAD26895.1), AtGLR2.3
(AAD26894.1), AtGLR2.4 (CAA19752.1), AtGLR2.5
(CAB96656.1), AtGLR2.6 (CAB96653.1), AtGLR2.7
(AAC33239.1),AtGLR2.8(AAC33237.1),AtGLR2.9
(AAC33236.1), AtGLR3.1 (AAF63223.1), AtGLR3.2
(CAA18740.1), AtGLR3.3 (AAG51316.1), AtGLR3.4
(AAB71458.1),AtGLR3.5(AAC69939.1),AtGLR3.6
(CAB63012.1) and AtGLR3.7 (AAC69938.1). Resi-
duesimportantfor ligandbinding arehighlighted
in grey in domain one (a) and domain two (b).

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810


806 Christian Dubos et al.

There are two types of mammalian NMDA receptor sub-


units: glutamate and glycine receptors (Ivanovic et al.,
1998). Glutamate is bound by NR2 subunits, whereas gly-
cine is bound by NR1 and NR3 subunits (Ivanovic et al.,
1998). All NR2 subunits possess a conserved glutamate-
binding GST motif, corresponding to Gly653, Ser654 and
Thr655 in the glutamate-binding site of the R. norvegicus
GLR (Figure 5). In contrast, most of the AtGLR sequences
are similar to the glycine-binding subunits NR1 and NR3,
where Thr655 is replaced by amino acids with large, hydro-
Figure 6. A multiple sequence alignment of the R. norvegicus GLR phobic side chains (Figure 5).
(AAA41240) with the A. thaliana GLRs, AtGLR2.9 (AAC33236.1) and
AtGLR1.1 (AAF26802.1) shows locations of residues that have been impli- To determine if glycine could function as a ligand for
cated in ligand binding highlighted in grey. plant GLR subunits, glycine was docked to the AtGLR2.9
The two regions that bind glutamate are shown. In domain one, relatively model. Glycine preferentially docks with the proposed glu-
highly conserved residues at Pro478, Thr480 and Arg485 bind the backbone
of glutamate ($). In domain two, residues corresponding to Ser654, Thr655 tamate-binding site (Figures 7g±i and 8e,f). The data
and Glu705 from the R. norvegicus GLR also bind to glutamate (^). strongly suggest that glycine is the natural agonist for
the majority of AtGLRs. The one exception to this is

Figure 7. `Snapshots' of the cumulative iterations from the multiscale docking algorithm, for the interactions of GLR subunits and putative ligands.
The full ligand-binding domain, showing the amino-acid residues important in binding in the ligand-binding pocket (in yellow), is shown with the potential
binding sites (green dots).
(a) An early iteration of the R. norvegicus GLR docking with glutamate.
(b) A middle iteration of the R. norvegicus GLR docking with glutamate.
(c) The last cumulative iteration of the R. norvegicus GLR docking with glutamate. Note that all potential binding sites cluster in the ligand-binding pocket.
(d) An early iteration of AtGLR2.9 docking with glutamate.
(e) A middle iteration of AtGLR2.9 docking with glutamate.
(f) The last cumulative iteration of AtGLR2.9 docking with glutamate. Arrows denote the fact that no speci®c binding site has been identi®ed.
(g) An early iteration of AtGLR2.9 docking with glycine.
(h) A middle iteration of AtGLR2.9 docking with glycine.
(i) The last cumulative iteration of AtGLR2.9 docking with glycine. Note that all potential binding sites coalesce to unify in a single site in the ligand-binding
pocket.

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810


Unsuspected glycine signalling in plants 807

Figure 8. Molecular models of ligands binding


to GLR subunits.
The full ligand-binding domains showing the
amino-acid residues important in binding (in
green) are shown with the ligands (in red) in
panels (a,c,e,g,i), whereas the detailed ligand-
binding pockets with the amino acids impli-
cated in binding are shown with the ligands
in panels (b,d,f,h,j).
(a) Model of the ligand-binding site of the
R. norvegicus GLR docked with glutamate.
(b) Detailed model of the ligand-binding pocket
of the R. norvegicus GLR docked with gluta-
mate.
(c) Model of the ligand-binding site of AtGLR2.9
docked with glutamate, showing the regions of
steric hindrance as coloured spheres.
(d) Detailed model of the ligand-binding pocket
of AtGLR2.9 docked with glutamate, showing
the regions of steric hindrance as coloured
spheres.
(e) Model of the ligand-binding site of AtGLR2.9
binding to glycine.
(f) Detailed model of the ligand-binding pocket
of AtGLR2.9 binding to glycine.
(g) Model of the ligand-binding site of AtGLR1.1
binding to glutamate.
(h) Detailed model of the ligand-binding pocket
of AtGLR1.1 binding to glutamate.
(i) Model of the ligand-binding site of AtGLR2.9
binding to DNQX.
(j) Detailed model of the ligand-binding pocket
of AtGLR2.9 binding to DNQX.

AtGLR1.1, which contains the glutamate-binding GST motif Analysis of the modelling results highlights residues
in the correct region of domain 2 (Figures 5 and 6). When implicated in glutamate binding in mammalian GLRs that
the ligand-binding domain of AtGLR1.1 was modelled on also appear important for glycine binding in the AtGLRs. For
the R. norvegicus receptor, glutamate bound to the same example, the Glu and Arg residues in the binding site appear
residues as in the mammalian GLRs (Figure 8g,h). Thus, to be important for ligand binding in both the R. norvegicus
while the majority of AtGLR subunits should bind glycine, GLR (Glu705, Arg485) and the AtGLR (Glu724, Arg529).
we predict that plant GLR channels may function with both Both are perfectly conserved over all receptors investigated
glycine and glutamate. This prediction is entirely consistent (Figure 5). Similarly, other amino acids implicated in estab-
with the observations that we made with respect to both the lishing the three-dimensional structure of the ligand-bind-
gating of [Ca2‡]cyt and the changes in hypocotyl elongation. ing region of domain 1, such as Thr480 and Ile481, are
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810
808 Christian Dubos et al.

conserved between species (Figure 5). In contrast, the bind- glutamate and glycine is an ancient mechanism, which
ing-site Pro residue at position 478 in the R. norvegicus GLR predates the split between plants and animals some
is replaced by Asp522 in AtGLR2.9, and appears to bind to 1000 million years ago. Furthermore, the preponderance
the glycine via a side chain interaction. The replacement of of plant GLRs that can bind glycine, relative to those that
Pro with Asp would anchor glycine more strongly within bind glutamate, suggests that glycine receptor subunits
the binding site. In domain 2, Ser654 and Thr655 of the may have diversi®ed to function in different developmental
R. norvegicus GLR are replaced by non-polar Ala and Phe contexts. As is the case in animals, where timing and locali-
residues, respectively, in the AtGLR. The Ala681 residue in sation of the expression of different NMDA receptor sub-
the AtGLR is predicted to bind glycine via its backbone units can alter cell responsiveness to agonists (Chatterton
nitrogen (much as Ser 654 binds glutamate in mammalian et al., 2002), plants may also alter their responsiveness to
GLRs). Crucially, the Phe682 residue in AtGLR is not pre- glycine by altering the expression of different GLR subunits.
dicted to interact with glycine; rather, the presence of this Indeed, the subunit encoded by AtGLR1.1, which is the only
amino acid hinders binding of other amino acids, such as subunit that the modelling results predict to bind glutamate,
glutamate, because of the presence of their side chains. may be amongst those subunits that is the most important
DNQX appears to have the capacity to bind to all plant in altering GLR speci®city. Recent evidence suggests that
GLR subunits. This ®nding supports the hypothesis that AtGLR1.1 does play a role in perceiving C:N balance in
DNQX interacts with plant GLRs (Lam et al., 1998). Model- Arabidopsis (Kang and Turano, 2003), but it remains to be
ling with AtGLR2.9 showed that DNQX bound in the pre- determined if this relates to the perception of the glycine.
dicted ligand-binding pocket (Figure 8i,j). Thus, DNQX While earlier studies suggested that glycine might func-
would be predicted to compete for the plant GLR ligand- tion as a signal in plants (Fries, 1953; Skinner and Street,
binding pocket with other compounds, such as GLR ago- 1953; White, 1939), this possibility was not explored. It is
nists. This is entirely consistent with the predicted interac- tempting to speculate that glycine may function as a sig-
tion of DNQX with animal GLRs. Importantly, these ®ndings nalling molecule in plants. Diurnal changes in glycine levels
are also consistent with the observation that glutamate and have been documented in tobacco. In fact, in tobacco, the
glycine compete with DNQX in the gating of [Ca2‡]cyt levels of glycine in the leaves increased by a factor of almost
(Figure 2) and in the regulation of hypocotyl elongation sixfold from the end of the night until the plants were
(Figure 4), and substantiate the contention that glutamate exposed to 9 h of daylight (Geiger et al., 1998). This was
and glycine mediate their effect at plant GLRs. in contrast to the concentration of glutamate in the leaves,
Future studies should aim to obtain empirical evidence where the levels remained almost constant over the same
for ligand binding to plant GLR subunits. The ®ndings time period (Geiger et al., 1998). Furthermore, growth of
presented here suggest that this may not be a trivial task. tobacco plants in elevated carbon dioxide had very little
Given the fact that the animal receptors function as hetero- effect on the normal diurnal changes in glutamate levels, but
meric channels (Armstrong et al., 1998) and given the pre- dampened the extent of the ¯uctuations in glycine concen-
ponderance of plant genes encoding the different subunits, tration in the leaves (Geiger et al., 1998). These changes in
it may prove dif®cult to devise experimental systems that glycine levels and the responsiveness of the levels to altered
allow the re-constitution of the plant channels to provide an carbon dioxide suggest that glycine could function as a sig-
accurate indication of the in vivo ligands. Indeed, animal nalling molecule, at least in tobacco. It is notable that glu-
GLRs are unable to bind to their ligands when expressed as tamate remained virtually unchanged over the time period
homomeric channels. However, the modelling work pre- that was monitored, while glycine levels increased during
sented here should provide guidance for future studies the day. This observation is consistent with glycine func-
aimed at determining the ligands for these receptors, at tioning as the signalling molecule, as opposed to glutamate.
least in vitro. Where glycine might not have previously been These observations, coupled with the ®ndings presented
considered as a ligand for these receptors, or where dif®- herein, open the door to future studies aimed at investigating
culties may have been encountered when using glutamate the hitherto undiscovered role of glycine in plant signalling
as a ligand, new alternatives can be considered. Further- and to explore the role of glycine signalling in organism
more, the contention that these receptors may not function development that extends beyond the nervous system.
as amino acid receptors, based on lack of glutamate bind-
ing (Davenport, 2002), may have to be re-assessed.
Experimental procedures

Conclusion
Growth of Arabidopsis plants
These studies provide evidence that glycine signalling may Seeds from A. thaliana wild-type plants (Col-0 ecotype) were
be widespread, to include the plant kingdom. The results sterilised by sequential treatment with 70% ethanol, satura-
show that the activation of GLRs by the synergistic action of ted calcium hypochlorite and sterile water, and then germinated

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 35, 800±810


Unsuspected glycine signalling in plants 809

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We are most grateful to Drs Mark Fricker and Heather Knight for Kang, J. and Turano, F.J. (2003) The putative glutamate receptor
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useful comments on the manuscript. D.H. was supported by a Kim, S.A., Kwak, J.M., Jae, S.K., Wang, M.H. and Nam, H.G. (2001)
studentship from the Engineering and Physical Sciences Research Overexpression of the AtGluR2 gene encoding an Arabidopsis
Council, UK. This work was supported by grants from the Biotech- homolog of mammalian glutamate receptors impairs calcium
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