ABSTRACT
KEY MESSAGE: Gene expression analysis coupled with in-planta studies showed that specific Gßγ combination regulates plant growth and defence traits in the allotetraploid Brassica juncea. Plant heterotrimeric G-proteins regulate a wide range of responses despite their limited repertoire of core components. The roles and functional interactions between different G-protein subunits are quite perplexing, which get further complicated with polyploidy. Here, we show that the allotetraploid Brassica juncea comprises multiple homologs of G-protein genes, encoding six BjuGß and ten highly divergent BjuGγ subunit proteins, later being classified into type-A1, type-A2 and type-C Gγ proteins. The encoded BjuGß and BjuGγ proteins shared close evolutionary relationship and have retained distinct spatio-temporal expression patterns during plant developmental stages and in response to the necrotrophic pathogen, Sclerotinia sclerotiorum. RNAi based suppression of BjuGß and BjuGγ genes suggested functional overlap and selectivity of BjuGßs with three distinct BjuGγ type subunits, to regulate plant height (BjuGßγA2 and BjuGßγC), seed weight (BjuGßGγA1 and BjuGßGγC), silique size (BjuGßGγC) and pathogen response (BjuGßGγA1 and BjuGßGγC). Further, the triplicated BjuGß genes, formed due to Brassica specific whole-genome-triplication event, showed differential involvement during pathogen response, wherein overexpression of BjuGß2 displayed higher resistance to Sclerotinia infection. Taken together, our study demonstrates that multiple BjuGß and BjuGγ proteins have retained distinct spatio-temporal expression and functional selectivity to regulate specific plant growth and defence traits in the oilseed B. juncea.
Subject(s)
GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/genetics , Mustard Plant/genetics , Plant Diseases/genetics , Plant Proteins/genetics , Polyploidy , Ascomycota/physiology , Disease Resistance/genetics , GTP-Binding Protein beta Subunits/classification , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/classification , GTP-Binding Protein gamma Subunits/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Host-Pathogen Interactions , Models, Genetic , Mustard Plant/growth & development , Mustard Plant/microbiology , Phylogeny , Plant Diseases/microbiology , Plant Proteins/classification , Plant Proteins/metabolism , Plants, Genetically Modified , Protein Binding , RNA InterferenceABSTRACT
Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gßγ. Since we have shown that signaling efficacy of Gßγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gßγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gßγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization.
Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Receptor, Muscarinic M3/metabolism , Cell Membrane/metabolism , G-Protein-Coupled Receptor Kinase 2/genetics , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/chemistry , GTP-Binding Protein gamma Subunits/classification , HeLa Cells , Humans , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Models, Molecular , Protein Interaction Domains and Motifs , Protein Transport/drug effects , Receptor, Muscarinic M3/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Xanthenes/pharmacologyABSTRACT
In many species, olfactory transduction is triggered by odorant molecules that interact with olfactory receptors coupled to heterotrimeric G-proteins. The role of G-protein-linked transduction in the olfaction of Drosophila is currently under study. Here, we supply a thorough description of the expression in the olfactory receptor organs (antennae and maxillary palps) of all known Drosophila melanogaster genes that encode for G-proteins. Using RT-polymerase chain reaction, we analyzed 6 Galpha (G(s), G(i), G(q), G(o), G(f), and concertina), 3 Gbeta (G(beta5), G(beta13F), and G(beta76C)), and 2 Ggamma genes (G(gamma1) and G(gamma30A)). We found that all Galpha protein-encoding genes showed expression in both olfactory organs, but G(f) mRNA was not detected in palps. Moreover, all the Gbeta and Ggamma genes are expressed in antennae and palps, except for G(beta76C). To gain insight into the hypothesis of different G-protein subunits mediating differential signaling in olfactory receptor neurons (ORNs), we performed immunohistochemical studies to observe the expression of several Galpha and Gbeta proteins. We found that Gs, Gi, Gq, and G(beta13F) subunits displayed generalized expression in the antennal tissue, including ORNs support cells and glial cells. Finally, complete coexpression was found between Gi and Gq, which are mediators of the cyclic adenosine monophosphate and IP3 transduction cascades, respectively.