Characterization of the Arabidopsis Heterotrimeric G Protein

Abstract

We have used fluorescence resonance energy transfer and coimmunoprecipitation to analyze the interactions among the ␣, ␤, and ␥1 subunits of the Arabidopsis heterotrimeric G protein.Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed G␥ 1 localizes to protoplast membranes, but G␤ exhibits membrane localization only when the G␥ 1 protein is co-overexpressed. Overexpressed G␣ shows membrane localization unaccompanied by overexpression of either G␤ or G␥ 1 . We detect fluorescence resonance energy transfer between G␤ and G␥ 1 in the absence of G␣ overexpression and between G␣ and G␥ 1 but only when all three subunits are co-overexpressed. Both G␣ and G␤ are associated with large macromolecular complexes of ϳ700 kDa in the plasma membrane. G␣ is present in both large complexes and as free G␣ in plasma membranes from wild type plants. In plants homozygous for a null allele of the G␤ gene, G␣ is associated with smaller complexes in the 200 -400-kDa range, indicating that its presence in the large complex depends on association with G␤␥. Activation of the G␣ subunit with guanosine 5-3-O-(thio)triphosphate (GTP␥S) results in partial dissociation of G␣ from the complex. Hydrogen peroxide (H 2 O 2 ) promotes extensive dissociation of the G␣ complex but does not interfere with binding of GTP␥S to purified recombinant G␣, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of G␣ itself.The results of pharmacological and genetic studies have provided evidence that the plant heterotrimeric G protein is involved in the transmission of light (1) and hormone signals (2) as well as in the regulation of ion channels (3). Arabidopsis gpa1 mutant plants, which lack the G␣ protein, encoded by the GPA1 gene, exhibit reduced cell division during hypocotyl and leaf formation (4), whereas overexpression of GPA1 causes ectopic cell division, including meristem proliferation (5). Homozygous gpa1 mutant plants are less sensitive to abscisic acid inhibition of stomatal opening and guard cell inward K ϩ currents than wild type plants (6). By contrast, gpa1 mutant seeds exhibit hypersensitivity to abscisic acid in inhibition of germination and in root growth and seedling gene expression. In addition, gpa1 mutant plants are hyposensitive to gibberellic acid and brassinolide (7,8). Arabidopsis agb1-2 mutant plants, which lack the G␤ protein encoded by the AGB1 gene, show alterations in leaf, flower and fruit development, decreased hypocotyl cell division and hypersensitivity to D-glucose (9 -11). There are two G␥ subunit genes in Arabidopsis, AGG1 and AGG2, and mutant analysis indicates that each G␥ subunit participates in a subset of G␤-related developmental processes (12).Evidence is also accumulating that the heterotrimeric G protein mediates plant responses to bacterial and fungal pathogens and abiotic stress. Heterotrimeric G protein signaling to membrane-bound NADPH oxidase has been implicated in the development of disease resistance and in the apoptotic...

Abscisic Acid-Induced Resistance against the Brown Spot Pathogen Cochliobolus miyabeanus in Rice Involves MAP Kinase-Mediated Repression of Ethylene Signaling

“…This view is consistent with the recent discovery of the Arabidopsis Ga-subunit gene GPA1 as a central element in ABAdependent innate immunity in stomatal guard cells . Moreover, there is ample evidence indicating that ABA signaling processes in both seeds and guard cells involve components of the heterotrimeric G-protein complex, further supporting our hypothesis (Pandey and Assmann, 2004;Fan et al, 2008;Wang et al, 2008). According to the G-protein signaling paradigm, the lack of Ga not only abolishes Ga-mediated signaling but also results in free Gbg, thereby possibly enhancing Gbg signal output (Pandey et al, 2006).…”

Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis

“…Interestingly, we observed strong direct binding between XLGs and Gg subunits even in the absence of Gb using protoplasts derived from the agb1-2 mutant. By contrast, interaction between Ga and Gg1 was detected only when Gb was present (Wang et al, 2008). This fact emphasizes the diversification between XLGs and plant Gas but is not unprecedented because it has been demonstrated that the human Ga subunit Ga o directly interacts with the Gg2 subunit (Rahmatullah and Robishaw, 1994).…”

“…Further biochemical studies are required to establish if the XLGGbg interaction is nucleotide dependent (Wall et al, 1995;Digby et al, 2006) or follows the alternative, nondissociable activation mechanism (Bünemann et al, 2003;Galés et al, 2006). Interestingly, while, in rice, the nonhydrolysable nucleotide GTPgS caused full dissociation of Ga from the complex (Kato et al, 2004), only about 30% of Ga was dissociated in Arabidopsis (Wang et al, 2008). Moreover, a mutated GPA1, which is unable to hydrolyze GTP and hence constitutively active, was found in a complex with Gbg1 (Adjobo-Hermans et al, 2006), suggesting that, in Arabidopsis, this canonical heterotrimer does not dissociate upon activation and therefore the Ga/Gbg interaction is not entirely nucleotide dependent.…”

RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants

“…The RNS2 cDNA (6) was obtained from Pamela Green (University of Delaware, Newark, DE). The CFP-coding region was amplified from pAVA321-CFP (37). The cauliflower mosaic virus 35S promoter was obtained from the pGD vector (38).…”