Abstract
Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.
Highlights
Heterotrimeric G-proteins are a class of signal transduction proteins that provide a key mechanism by which extracellular signals are transmitted to cell milieu, across phyla
Identification and sequence analysis of genes encoding heterotrimeric G-protein subunits from B. rapa B. rapa is a mesohexaploid crop with three sub-genomes, sharing the same diploid ancestor with the model plant species A. thaliana [32,33]
Full-length coding DNA sequences (CDS) were isolated for each of the nine G-protein subunit genes using B. rapa cDNA and gene-specific primers; and confirmed with multiple amplifications from different tissue types. We named these sequences as Ga (BraA.Ga1), Gb (BraA.Gb1, BraA.Gb2, and BraA.Gb3), and Gc (BraA.Gc1, BraA.Gc2, BraA.Gc3, BraA.Gc4, and BraA.Gc5), following the universal nomenclature adopted for Brassica genes [39]
Summary
Heterotrimeric G-proteins (hereafter G-proteins) are a class of signal transduction proteins that provide a key mechanism by which extracellular signals are transmitted to cell milieu, across phyla. The three distinct subunits namely, Ga (G-alpha), Gb (Gbeta), and Gc (G-gamma) along with GPCRs (G-protein coupled receptors) are the fundamental components of G-protein signaling complex, wherein, the Ga subunit acts as a switch and found in either GDP bound ‘OFF’ or in GTP bound ‘ON’ form [1]. Signal perception through GPCR causes change in the conformation of Ga subunit leading to exchange of GTP for GDP (i.e. GTP binding). Intrinsic GTPase (i.e. GTP hydrolyzing) activity of Ga causes conversion of GTP bound ‘ON’ form of Ga to GDP bound ‘OFF’ form, leading to re-association of heterotrimer and inhibition of signal perception from the cell surface [1]. Among higher organisms, the plant and animal models seem to have significant quantitative and regulatory differences in G-protein signaling [2]. The plant Ga proteins showed ‘selfactivating’ property, having significantly higher GTP-binding rates and a slower GTPase activity, which is in contrast to the mammalian Ga proteins, thereby indicating that the plant Gproteins remain mostly in constitutively active state [6,7]
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