Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein γ subunit results in a phenotype similar to α and β subunit knockouts

Abstract

Heterotrimeric G proteins (G-proteins) are a diverse class of signal transducing proteins which have been implicated in a variety of important roles in plants. When G-proteins are activated, they dissociate into two functional subunits (α and the βγ dimer) that effectively relay the signal to a multitude of effectors. In animal systems, the βγ dimer is anchored to the plasma membrane by a prenyl group present in the γ subunit and membrane localization has proven vital for heterotrimer function. A semi-dominant negative strategy was designed aiming to disrupt heterotrimer function in Arabidopsis thaliana (ecotype Columbia) plants by over-expressing a truncated γ subunit lacking the isoprenylation motif (γ ⁎). Northern analysis shows that the levels of expression of the mutant γ subunit in several transgenic lines (35S-γ ⁎) are orders of magnitude higher than that of the native subunits. In-depth characterization of the 35S-γ ⁎ lines has been carried out, specifically focusing on a number of developmental characteristics and responses to several stimuli previously shown to be affected in α- and β-deficient mutants. In all cases, the transgenic lines expressing the mutant gamma subunit behave in the same way as the α- and/or the β-deficient mutants, albeit with reduced severity of the phenotype. Our data indicates that signaling from both functional subunits, α and the β/γ dimer, is disrupted in the transgenic plants. Even though physical association of the subunits has been previously reported, our research provides evidence of the functional association of α and β with the γ subunits in Arabidopsis, while also suggesting that plasma membrane localization may be critical for function of plant heterotrimeric G proteins.