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Heterotrimeric G-proteins (G-proteins hereinafter) consisting of α, β and γ subunits are important signal transducers in all eukaryotes (Jones & Assmann, 2004). The Gα subunit of the heterotrimer undergoes a signal-dependent transition between guanosine diphosphate (GDP)- and guanosine-5′-triphosphate (GTP)-bound forms to modulate signal transduction. In the inactive GDP-bound state, Gα interacts with Gβγ and is associated with a cell surface G-protein-coupled receptor (GPCR). Upon signal perception, a change in GPCR conformation facilitates exchange of GTP for GDP on Gα, which results in dissociation of the heterotrimer. The active Gα˙GTP and the released Gβγ interact with different effectors to propagate the signal. Intrinsic GTPase-activity returns Gα to its inactive form (Cabrera-Vera et al., 2003; Offermanns, 2003). These fundamental aspects of G-protein signaling are conserved across phyla (Jones & Assmann, 2004).
In metazoans, G-protein-coupled signaling mediates vision, olfaction, taste, neurotransmission, and hormone responses (Cabrera-Vera et al., 2003; Offermanns, 2003). In these organisms, the multiplicity of G-proteins and GPCRs, in addition to downstream signaling components, provides for complex signal transduction architecture. For example, humans have 23 Gα (including splice variants), five Gβ, 12 Gγ, > 800 GPCRs, and scores of accessory proteins that form an elaborate network of specific GPCR–Gαβγ combinations to transduce a specific signal. Studies in Arabidopsis and rice provide knowledge of plant G-protein signaling and suggest much simpler architecture. The fully sequenced genomes of both these species contain only one conventional Gα, one Gβ and two Gγ subunits and a few GPCRs each (Perfus-Barbeoch et al., 2004), although additional splice variants, or nonconventional genes especially for Gγ subunits may also exist. These limited number of components, however, regulate diverse signaling pathways, including hormone signaling and cross-talk, environment sensing, cell division, ion channel regulation, disease resistance and cell death (Temple & Jones, 2007; Chen, 2008).
Homologs of G-proteins are found in other plant species (Supporting Information, Table S1), but the unavailability of genetic and genomic resources limits insight into their biological function. Moreover, in some cases, the phenotypes observed in Arabidopsis G-protein mutants are not recapitulated in other plant species. For example, in both Arabidopsis and rice, mutation in Gα results in hyposensitivity to gibberellic acid (Ueguchi-Tanaka et al., 2000; Ullah et al., 2001, 2002), but only in rice does this cause plant dwarfism (Ashikari et al., 1999; Fujisawa et al., 1999). Similarly, tobacco plants with suppressed levels of Gβ show defects in anther shape and pollen development, which results in nongerminating pollen grains (Peskan-Berghöfer et al., 2005). These phenotypes are not shared with the corresponding Arabidopsis agb1 mutants (Ullah et al., 2003). Importantly, such examples question whether the relatively simple architecture of the Arabidopsis G-protein signaling pathway is the norm in the plant kingdom, considering that an estimated 30–80% of plant species, including major crops like soybean, have undergone one or more rounds of genome duplication (Wendel, 2000), raising the possibility of expansion of G-protein gene families in such species.
Expanding on studies of model monocots and dicots, growing evidence suggests that G-protein signaling plays a critical role in a range of economically important legume crops during normal growth and development as well as during symbiosis. Some examples include the G-protein-regulated blue and red light signaling pathways in pea (Warpeha et al., 1991), induction of root hair deformation in Vigna, and expression of nodulation-specific genes in Medicago by mastoparan, a known G-protein agonist (Kelly & Irving, 2003; Sun et al., 2007). Additionally, some well-established effectors of G-protein signaling, for example phospholipase C (PLC) and D (PLD) are involved in mastoparan-induced root hair deformation and development of root nodules (Pingret et al., 1998; den Hartog et al., 2001; Charron et al., 2004; Santos-Briones et al., 2009). G-proteins and PLC are also involved in an elicitor-induced oxidative burst in soybean cells (Legendre et al., 1992). Lastly, there is one report of molecular characterization of G-proteins from legumes, where one Gα, one Gβ, and two Gγ cDNAs were isolated from pea and studied for their role in abiotic stress signaling (Misra et al., 2007).
We have designed the present study to identify and characterize soybean heterotrimeric G-proteins. Sequence homology-based screenings and Southern hybridizations identified multiple homologs of G-protein subunits in some plant species, including soybean (Kim et al., 1995; Gotor et al., 1996), but a systematic study to identify and comparatively analyze multiple G-protein subunits with respect to the plant polyploidy has not been performed to date. Soybean was chosen because of the presence of an allopolyploid genome along with the recent availability of suitable genetic and molecular resources (Schmutz et al., 2010) and a lack of any comprehensive data from a legume on the important roles played by G-proteins in regulating fundamental growth and development processes. This study describes multiple G-protein subunits from soybean with distinct biochemical activity, details their expression profiles, and depicts the specificity of interaction between different subunits. This work reveals the most elaborate network of G-proteins in a plant known to date.
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Fig. S1 Nucleotide sequence alignment of the amplified and reported GmGα4 gene along with its duplicated gene pair GmGα1.
Fig. S2 Amino acid sequence alignment of GmGβ proteins.
Fig. S3 Amino acid sequence alignment of GmGγ proteins.
Fig. S4 Evolutionary relationships of Gα proteins.
Fig. S5 Evolutionary relationships of Gβ proteins.
Fig. S6 Syntenic relationship between Gα genes.
Fig. S7 Expression of soybean G-protein genes under various stress conditions.
Fig. S8 Nucleotide binding and competition assay of GmGα1 (A) and GmGα3 (B) proteins.
Fig. S9 Amino acid sequence alignment of Arabidopsis GPA1 (AtGPA1) and moss Gα (PpGα).
Table S1 An inventory of heterotrimeric G-proteins in different plant species
Table S2 Gene-specific primers used for amplification, expression analysis and interaction studies
Table S3 Absolute copy number quantification and primer amplification efficiency test of soybean G-protein genes
Table S4 An analysis of cis-regulatory elements present in the 2 kb upstream region of G-protein genes
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