References

13 The Arabidopsis thaliana Glutamate-like Receptor Family(AtGLR) 199
that excess of one subunit could disrupt the formation of normal heteromers
and may have relatively widespread effects, which might be difficult
to interpret but would give crude information about the processes
in which AtGLRs are involved. AtGLRs have been suggested to have wideranging
functions so phenotyping requires testing of plant responses to
a range of different ligands (informed by Sect. 13.4.2), including glutamate,
glycine, GABA, ABA, and other amino acids, as well as agonists and
antagonists of animal iGluRs. Plant responses may involve germination,
root growth, circadian-asssociated phenotypes such as aberrant hypocotyl
angle or length, responses to light, nutrients and toxic ions, solute accumulation,
and electrical and calcium signalling. Phenotyping needs to be
informed to some extent by the reported physiological effects of amino
acids on plant behaviour, but conversely physiological characterisation
will depend partly on mutant characterisation to predict plant responses
to ligands.
13.4.4
Heterologous Expression
Heterologous expression is desirable to demonstrate ion channel function
of AtGLRs. However expression in Xenopus oocytes has been uninformative
(Sect. 13.3.3). Chimaeric iGluRs constructed by transplanting the
pore domain of apparently non-functional iGluRs (such as KA-binding
proteins and C. elegans iGluR) into rat iGluRs have been used to demonstrate
functionality of the pore (Villmann et al. 1997; Strutz-Seebohm et
al. 2003). Although not proof of function in vivo this approach could indicate
whether the pore region is capable of ion conduction and its potential
selectivity. Use of homologous or heterologous plant systems such as cultured
mesophyll cells may result in correct processing. Alternatively the
possible toxicity of some AtGLRs in yeasts and E. coli, whichsuggests
functional expression (Davenport 2002), could be used to advantage by inducible
expression of toxic AtGLRs, which would permit at least ion uptake
studies. In some cases coexpression of subunits in heterologous systems
may be required to produce functional ion channels. iGluRs function as
heteromers and some subunits show no channel activity when expressed
as homomers (Dingledine et al. 1999). The large size of the AtGLR family
and the evidence for simultaneous expression of many different AtGLRs
in single cells (Sect. 13.3.1) makes it difficult to predict likely heteromeric
combinations, and it may be desirable to coexpress large pools of subunits
simultaneously, or to use a yeast two-hybrid approach to identify
interacting subunits.