The Arabidopsis thaliana Glutamate-like Receptor Family (AtGLR) - Communication in Plants: Neuronal Aspects of Plant Life - page 198

2005). It is also desirable to follow changes in abundance at the protein
level (to substantiate, for instance, the possibility of rapid protein turnover
in response to stresses), and to determine the membrane localisation of
At GLR with confidence. GFP tags can interfere with normal protein tar-
geting, especially in the case of endomembrane proteins where both the
N-andtheC-terminiaswellasinternalsignalsmaybeinvolvedintar-
geting, and it is desirable to have a test of retention of functionality or
phenotype of the tagged protein (which we do not have at present for
most At GLRs). Alternatives to large tags such as GFP include antibodies
or smaller tags (which could be incorporated internally in the At GLR pro-
tein to allow in vivo monitoring of membrane protein dynamics as in the
case of iGluR tagged with a toxin binding site; Sekine-Aizawa and Huganir
2004).
13.4.2
Ligand Binding and Regulation
The ligand-binding profile of At GLRneedstobeexaminedexperimentally.
Although glutamate and glycine binding has been modelled, no ligand-
binding assays have been performed in vitro to confirm the prediction of
the model. It is possible that other ligands exist for At GLR and elucidation
of these could inform phenotyping studies (Sect. 13.4.3). In the absence
of a functional expression system, ligand binding could be explored by
constructing chimaeras consisting of only the soluble ligand-binding do-
mains and using these to measure affinity of binding, as has been done
with S1,S2 domains from animal iGluRs (Kuusinen et al. 1995). In addition,
theproposedsignallingroleofglutamateorglycineshouldbesubstanti-
ated by high-resolution quantification of changes in apoplastic concentra-
tion (e.g. by fluorescence-based techniques; Acher et al. 2005; Fehr et al.
2005).
13.4.3
Knockout and Overexpression Phenotyping
As there are 20 AtGLR s, knockout of some members may result in sub-
tle or no phenotypes owing to specificity of function or compensation
by other members, making it necessary to reduce expression of several
members simultaneously to readily identify phenotypes. This could be
achieved by pyramiding knockouts or by employing an RNA interfer-
ence strategy to silence several At GLRs simultaneously (Essah et al. 2005).
Overexpression of At GLRs is also a desirable strategy, since it is possible