SUMMARYCurrently, there are strong inconsistencies in our knowledge of plant heterotrimeric G-proteins that suggest the existence of additional members of the family. We have identified a new Arabidopsis G-protein c-subunit (AGG3) that modulates morphological development and ABA-regulation of stomatal aperture. AGG3 strongly interacts with the Arabidopsis G-protein b-subunit in vivo and in vitro. Most importantly, AGG3-deficient mutants account for all but one of the 'orphan' phenotypes previously unexplained by the two known c-subunits in Arabidopsis. AGG3 has unique characteristics never before observed in plant or animal systems, such as its size (more than twice that of canonical c-subunits) and the presence of a C-terminal Cys-rich domain. AGG3 thus represent a novel class of G-protein c-subunits, widely spread throughout the plant kingdom but not present in animals. Homologues of AGG3 in rice have been identified as important quantitative trait loci for grain size and yield, but due to the atypical nature of the proteins their identity as G-protein subunits was thus far unknown. Our work demonstrates a similar trend in seeds of Arabidopsis agg3 mutants, and implicates G-proteins in such a crucial agronomic trait. The discovery of this highly atypical subunit reinforces the emerging notion that plant and animal G-proteins have distinct as well as shared evolutionary pathways.

Heterotrimeric G proteins have been previously linked to plant defense; however a role for the Gbetagamma dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Galpha or Gbeta subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gbeta-deficient mutants while Galpha-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gbeta-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gbeta-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Galpha-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbetagamma functional subunit but not by Galpha. We hypothesize that Gbetagamma acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Ga and Gb subunit genes and two Gg subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gg genes revealed specialized roles for each Gg subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1-and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbg1 and Gbg2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gg subunit in this root response differs, with Gbg1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbg2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbg signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gg subunits form functional Gbg dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

Yeast two-hybrid technology is used to build a high-quality protein interaction network centered on Arabidopsis G-protein coupled signaling. The interactions uncovered are without precedent in animals and fungi and help identify new cellular roles for G-protein signaling in plants.