Heterotrimeric G-proteins are composed of three different subunits (α, β and γ) and mediate cellular signalling in many physiological processes. In humans, the required functional specificity is possible through the generation of multiple heterotrimer variants from a pool of 23 Gα, 5 Gβ and 12 Gγ subunits. Comparatively, most plants have only one Gα, one Gβ and several Gγ subunits, hence rising questions regarding how the seemingly simple heterotrimeric G-protein system controls the various cellular processes in plants where there is demonstrated involvement. It was speculated that the ability of G-protein to regulate numerous signaling pathways in plants is largely attributed to the most divergent subunit, the Gγ subunit. The research conducted in this thesis endeavors to shed light on the functional aspects of plant G-proteins and the roles of the multiple Gγ isoforms.
In Arabidopsis, heterotrimeric G-proteins consist of one Gα (GPA1), one Gβ (AGB1) and three Gγ (AGG1, AGG2 and AGG3) subunits. Gβ and Gγ subunits function as obligate heterodimers, hence any phenotypes evident in Gβ-deficient mutants should be apparent in Gγ-deficient mutants. Nevertheless, the first two Gγ subunits discovered failed to explain many of the phenotypes observed in the agb1 mutants in Arabidopsis, prompting the search for additional Gγ subunits. The recent discovery of an additional though atypical Gγ subunit in Arabidopsis (AGG3) has helped to complete the picture and explains almost all of the missing agb1 ‘orphan’ phenotypes. However, there is still one unexplained phenotype, the reduction in rosette size reported for agb1, that has not been observed in any of the individual agg mutants or the double agg1agg2 mutant. Described in this thesis is the creation of a triple gamma mutant (agg1agg2agg3) in Arabidopsis which recapitulates the remaining ‘orphan’ agb1 phenotypes. Triple agg1agg2agg3 mutants display the reduction in rosette size previously observed in agb1 mutants. In addition, small differences in flower and silique size observed between agb1 and agg3 mutants are also accounted for by the triple agg1agg2agg3 mutant. The results presented here strongly suggest that different Gβγ dimers may play synergistic roles resulting in additive effects in the modulation of plant morphology. In addition, the fact that the triple agg1agg2agg3 mutants recapture all the agb1‘orphan’ phenotypes implies that there are no additional members of the G-protein family remaining to be discovered in Arabidopsis.
Unlike the recently identified AGG3, which bears unusual features never before seen in animal G-protein systems, the other two Gγ subunits, AGG1 and AGG2, resemble the canonical mammalian Gγ subunits. AGG1 and AGG2 have been shown to provide functional selectivity to the Gβγ dimer in Arabidopsis. However, it is unclear if such selectivity is embedded in their molecular structures or conferred by the different expression patterns observed in both subunits. In order to study the molecular basis for such selectivity, complementation studies in AGG1- and AGG2-deficient mutants were performed. The results presented in the later part of this thesis show that, if expressed in the correct tissues, AGG2 can rescue agg1 mutant phenotypes such as the hypersensitivity to Fusarium oxysporum and D-mannitol as well as the levels of lateral roots observed in agg1 mutant, but not the early flowering phenotype. Similarly, AGG1 can complement the osmotic stress and lateral root phenotypes observed in agg2 mutants but failed to complement the heat-stress induction of flowering. The fact that AGG1 and AGG2 are functionally interchangeable in a number of pathways implies that, at least for those pathways, signaling specificity resides in the distinctive spatiotemporal expression patterns exhibited by each Gγ subunit. Nevertheless the lack of complementation for some phenotypes indicates that there are some pathways in which signaling specificity is provided by non-conserved amino acid residues in AGG1 and AGG2.
Overall, the research detailed in this thesis has contributed to insights on how the simple plant heterotrimeric G-protein system generates functional differences through its Gβγ dimers to modulate numerous cellular processes. These include 1) diverging amino acid sequences of Gγ isoforms which lead to protein structural differences of each Gβγ dimer, allowing specific effector interactions; 2) genetic interactions or cross-talk in downstream pathways controlled by different Gβγs resulting in additive and/or synergistic effects; 3) unique expression pattern and spatiotemporal separation of each Gγ which allows modulation of processes in different tissues.