Functional characterization of Arabidopsis thaliana glutamate receptors

Nitrogen (N) is a major determinant of plant growth, development and yield. Arabidopsis thaliana glutamate receptors (AtGLRs) are demonstrated here to mediate N sensing. Using a lateral root elongation assay, AtGLR1.1 and AtGLR3.2 are demonstrated to form components of a positive and negative regulatory network respectively that controls N sensing, N assimilation, stomatal conductance and biomass production. Pharmacological analyses using ionotropic glutamate receptor antagonist 6,7 dinitroquinoxaline-2-3-[1H,4H]-dione (DNQX) confirm that AtGLRs mediate lateral root (LR) elongation. It is proposed that AtGLR regulates plant growth and development based on the levels of N sensed and form a major component contributing to traits such as N use efficiency and yield in higher plants. Analyses of signaling proteins interacting with AtGLR3.2 using immuno-precipitation and mass spectroscopy resulted in the identification of ∼300 proteins. Signaling proteins and enzymes localized to the animal post synaptic density complex have been identified as part of the AtGLR3.2 complex indicating shared processes. Two dimensional gel analysis and western blot analyses resulted in validation of 14-3-3s, small GTPases and FLA8 interacting specifically with the AtGLR3.2 complex. Evaluation of T-DNA knockouts for 14-3-3 chi and kappa for their role in carbon (C), N metabolic regulation using germination assays demonstrated modified germination and primary root growth. Probably, 14-3-3 chi and kappa by interacting with AtGLR channels co-ordinate major C/N metabolic pathways based on the level of N sensing. Receptor binding analysis using Arabidopsis homogenate preparation demonstrated specific, reversible, heat labile, saturable binding of L-[3H]-Glu that is linear with the protein concentration. Saturation and homologous competition analysis showed an affinity of 134 nM and 250 nM L-Glu respectively. Pharmacological analyses demonstrated that β-N-methyl-amino-L-alanine (BMAA) competed for 20% of the total L-[3H]-Glu binding. Incubation with kainic acid (KA) resulted in an increase of L-[3H]-Glu binding. Neither DNQX at 100 μM nor amino-5-phosphonobutyric acid (AP5) at 1 mM compete for L-Glu binding. This would indicate broader structural differences between animal versus plant and microbial GLR ligand binding domains. The role of plant GLRs in N sensing opens up targeted approaches to improving traits that are ecologically and economically important such as yield, and resource use efficiency.