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• The repertoire of heterotrimeric G-proteins in plant species analyzed thus far is simple, with the presence of only two possible canonical heterotrimers in Arabidopsis and rice vs hundreds in animal systems. We assessed whether genome duplication events have resulted in the multiplicity of G-protein in plant species like soybean that would increase the complexity of G-protein networks. • We identified and amplified four Gα, four Gβ and two Gγ proteins, analyzed their expression profile by quantitative PCR during different developmental stages. We purified the four Gα proteins and analyzed their guanosine-5′-triphosphate (GTP)-binding and GTPase activity. We performed yeast-based interaction analysis to assess the interaction specificity of different G-protein subunits. • Our results show that all 10 G-protein genes are retained in the soybean genome and ubiquitously expressed. The four Gα proteins seem to be plasma membrane-localized. The G-protein genes have interesting expression profiles during seed development and germination. The four Gα proteins form two distinct groups based on their GTPase activity. Yeast-based interaction analyses predict that the proteins interact in most of the possible combinations, with some degree of interaction specificity between duplicated gene pairs. • This research identifies the most elaborate heterotrimeric G-protein network known to date in the plant kingdom.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

The heterotrimeric G‐protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G‐protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G‐protein associates with heptahelical G‐protein‐coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G‐protein effectors and scaffold proteins, we screened a set of proteins from the G‐protein complex using two‐hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G‐protein interactome. Within this core, over half of the interactions comprising two‐thirds of the nodes were retested and validated as genuine in planta . Co‐expression analysis in combination with phenotyping of loss‐of‐function mutations in a set of core interactome genes revealed a novel role for G‐proteins in regulating cell wall modification.

Heterotrimeric G‐proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G‐protein subunit mutants of the model plant Arabidopsis thaliana , with or without treatment with the stress hormone, abscisic acid. Although G‐protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G‐protein × hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G‐protein signaling are well represented. Conversely, some theoretical regulatory modes of the G‐protein are not supported; (2) a new mechanism of G‐protein signaling is revealed, in which Gβ regulates gene expression identically in the presence or absence of Gα; (3) guard cells and leaves favor different G‐protein modes in transcriptome regulation, supporting system specificity of G‐protein signaling. Our method holds significant promise for analyzing analogous ‘switch‐like’ signal transduction events in any organism. Synopsis Heterotrimeric G‐proteins, composed of α, β, and γ subunits, participate in a wide range of signaling pathways in eukaryotes (Morris and Malbon, 1999 ). According to the typical, mammalian paradigm, in its inactive state, the G‐protein exists as an associated heterotrimer. G‐protein signaling begins with ligand binding that results in a conformational change in a G‐protein‐coupled receptor (GPCR). Once activated by the GPCR, the Gα separates from the associated Gβγ dimer and the freed Gα and Gβγ proteins can then interact with downstream effector molecules, alone or in combination, to transduce the signal. Subsequent to signal propagation, Gα re‐associates with the Gβγ dimer to reform the G‐protein complex. There are several classical routes for signal propagation through heterotrimeric G‐proteins that have been categorized in mammalian systems (Marrari et al , 2007 ; Dupre et al , 2009 ). One route, which we designate classical I, requires the presence of both subunits, and can invoke one of two distinct mechanisms. In one mechanism, on GPCR activation, freed Gα and Gβγ each interact with downstream effectors to elicit the downstream response. In a related mechanism, Gα but not Gβγ interacts with downstream effectors, but the Gβγ dimer is nevertheless required to facilitate coupling of Gα with the relevant GPCR (Marrari et al , 2007 ). In a second route, which we designate classical II, it is solely the Gβγ dimer that interacts with downstream effectors; in this case, sequestration of Gβγ within the heterotrimer prevents signal propagation. In addition, a few non‐classical G‐protein regulatory modes have also been implicated in some systems, for example signaling by the intact heterotrimer in yeast (Klein et al , 2000 ; Frank et al , 2005 ). Observations such as these lead to a fundamental question, namely, which of all the theoretical regulatory modes of G‐protein signaling are realized biologically. Our study answers this question in the context of the model plant Arabidopsis thaliana , and in addition analyzes the manner in which G‐protein signaling couples with signaling by the plant hormone abscisic acid. The Arabidopsis genome encodes only one canonical Gα subunit, GPA1, and one canonical Gβ subunit, AGB1, and knockout mutants are available for each of these, allowing clear dissection of Gα‐ and Gβ‐related phenotypes. Abscisic acid (ABA) is a major plant hormone, which inhibits growth and promotes tolerance of abiotic stresses such as drought, salinity, and cold. ABA signaling is known to interact with heterotrimeric G‐protein signaling in both developmental and stress responses in a complex manner, causing, for example, ABA hyposensitivity of guard cell stomatal opening in gpa1 and agb1 single mutants as well as agb1 gpa1 double mutants (Fan et al , 2008 ), but ABA hypersensitivity of the inhibition of seed germination and post‐germination seedling development in the same mutants (Pandey et al , 2006 ). These experimental observations implicate G‐proteins as one of the components of ABA signaling, but to date no systematic study has been conducted in either plant or metazoan systems to define the co‐regulatory modes of a G‐protein and a hormone. In this study, we conduct genome‐wide gene expression profiling in G‐protein subunit mutants of A. thaliana guard cells and leaves, with or without treatment with ABA. By introducing one or more mediators acting downstream of the G‐protein and ABA to control transcript levels, we propose nine G‐protein/ABA signaling pathways including ABA‐independent G‐protein signaling pathways, G‐protein‐independent ABA signaling pathways, and seven distinct ABA–G‐protein‐coupled signaling pathways (Figure 1 ). We develop a Boolean modeling framework to systematically enumerate 14 possible theoretical regulatory modes of the G‐protein and 142 co‐regulatory modes of the G‐protein and ABA, and then use a pattern matching approach to associate target genes with theoretical regulatory modes. Our analysis shows that the G‐protein regulatory mode that requires the presence of both Gα and Gβγ subunits (consistent with classical I mechanisms), is well represented in both guard cells and leaves. The G‐protein regulatory mode that requires a freed Gβγ subunit (classical II G‐protein regulatory mechanism) is well supported in guard cells and somewhat less so in leaves. In addition, a G‐protein regulatory mode representing a non‐classical regulatory mechanism is prevalent in guard cells but less so in leaves (Figure 5 ). In this regulatory mode, signaling by Gβ(γ) occurs, and this signaling is not regulated in any way by Gα. By relating the target genes with the nine proposed G‐protein/ABA signaling pathways, we are able to gauge the plausibility of regulatory modes of the G‐protein and ABA at the pathway level. We find that G‐protein‐independent ABA signaling pathways are prevalent in both guard cells and leaves. The existence of an ABA‐independent regulatory activity of the G‐protein is well supported in guard cells, but not supported in leaves. Additive regulation by G‐protein signaling plus G‐protein‐independent ABA signaling is rare in both guard cells and leaves. In addition, combinatorial cross‐talk between G‐protein signaling and ABA signaling and additive cross‐talk between ABA–G‐protein signaling and G‐protein‐independent ABA signaling are observed in both guard cells and leaves. Our transcriptome analysis indicates that in some cases, ABA definitely does not influence G‐protein signaling, though it may do so in some other cases. To investigate whether previously observed hypersensitivity or hyposensitivity of developmental and dynamic transient responses to ABA in G‐protein mutants is recapitulated at the level of transcriptional regulation, we compare gene regulation by ABA in guard cells and leaves of the G‐protein mutants versus wild type. We find that in guard cells, equal ABA hyposensitivity of all mutants combined is significant, although hyposensitivity in individual mutants is not. There is also a separate group of genes in guard cells that show ABA hypersensitivity in the gpa1 mutant, suggesting complex interactions between ABA and G‐protein signaling in gene regulation in this cell type. In leaves, ABA hyposensitivity of gene expression in the three individual mutants and equal hyposensitivity in all mutants are strongly supported. In addition, several of the functional categories identified by our analysis of G‐protein regulatory modes have been implicated in previous physiological analyses of G‐protein mutants, providing validation to the biological interpretation of our results. In summary, by conducting a genome‐wide gene expression profiling study in G‐protein subunit mutants of A. thaliana guard cells and leaves and developing a Boolean modeling framework, we systematically evaluate the biological utilization of mechanisms of G‐protein regulatory action and reveal novel regulatory modes of the G‐protein. The results generate empirical evidence and insights regarding molecular events of G‐protein signaling and response at the physiological level in both plants and mammals. Classical mechanisms of heterotrimeric G‐protein signaling are observed to function in regulation of the transcriptome. Conversely, many theoretical regulatory modes of the G‐protein are not manifested in the transcriptomes we investigate. A new mechanism of G‐protein signaling is revealed, in which the β subunit regulates gene expression identically in the presence or absence of the α subunit. We find evidence of cross‐talk between G‐protein‐mediated and hormone‐mediated transcriptional regulation. We find evidence of system specificity in G‐protein signaling.

Pertussis toxin (PTX) is a typical A-B toxin. The A-protomer (S1 subunit) exhibits ADP-ribosyltransferase activity. The B-oligomer consists of four subunits (S2 to S5) and binds extracellular molecules that allow the toxin to enter the cells. The A-protomer ADP-ribosylates the α subunits of heterotrimeric Gi/o proteins, resulting in the receptors being uncoupled from the Gi/o proteins. The B-oligomer binds proteins expressed on the cell surface, such as Toll-like receptor 4, and activates an intracellular signal transduction cascade. Thus, PTX modifies cellular responses by at least two different signaling pathways; ADP-ribosylation of the Gαi/o proteins by the A-protomer (Gi/o protein-dependent action) and the interaction of the B-oligomer with cell surface proteins (Gi/o protein-independent action).

G‐protein‐coupled receptors (GPCRs), also known as seven transmembrane receptors (7‐TMRs), are the largest protein receptor superfamily in the body. These receptors and their ligands direct a diverse array of physiological responses, and hence have broad relevance to numerous diseases. As a result, they have generated considerable interest in the pharmaceutical industry as drug targets. Recently, GPCRs have been demonstrated to elicit signals through interaction with the scaffolding proteins, β‐arrestins‐1 and 2, independent of heterotrimeric G‐protein coupling. This review discusses several known G‐protein‐independent, β‐arrestin‐dependent pathways and their potential physiological and pharmacological significance. The emergence of G‐protein‐independent signalling changes the way in which GPCR signalling is evaluated, from a cell biological to a pharmaceutical perspective and raises the possibility for the development of pathway specific therapeutics. British Journal of Pharmacology (2008) 153, S298–S309; doi:10.1038/sj.bjp.0707508; published online 26 November 2007

The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1. The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2, the Gβ protein AGB1, and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism. Here we show that in the pre-activation state, XLG2 directly interacts with FLS2 and BIK1, and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation. Following the activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD. The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms. Living cells need to be able to detect changes in their environment and respond accordingly. This ability involves signals from outside of the cell triggering changes to the activity inside the cell. Heterotrimeric G proteins are important for this kind of signaling in a wide range of organisms. In animals and fungi, these proteins directly work with a specific class of receptor proteins called G protein-coupled receptors (or GPCRs for short). Plants also have heterotrimeric G proteins, but it remains unclear whether they similiarly work with GPCRs. Plants detect invading microbes by using receptors that are completely different from GPCRs. For example, a receptor called FLS2 from the model plant Arabidopsis senses a telltale protein produced by bacteria, and then passes the signal to another protein called BIK1 to activate the plant’s defenses. Heterotrimeric G proteins are required for this process, but the underlying mechanisms remain unknown. Liang, Ding et al. now show that heterotrimeric G proteins regulate FLS2-controlled defenses by directly interacting with FLS2 and BIK1. Heterotrimeric G proteins also enhance defenses in at least two different ways. Firstly, in the absence of an infection, heterotrimeric G proteins stabilize the BIK1 protein to ensure that it is ready to respond. Secondly, if FLS2 does detect the telltale bacterial protein, BIK1 marks one of the heterotrimeric G proteins with a phosphate group. This then allows the G protein to boost the activity of another plant enzyme that is vital for defense signaling. In the future, it will be important to work out how activation of FLS2 leads to the activation of heterotrimeric G proteins. Furthermore, heterotrimeric G proteins are likely to regulate additional plant proteins when defenses are activated, and further studies are needed to identify these proteins.

Heterotrimeric G-proteins regulate multiple aspects of plant growth, development, and response to biotic and abiotic stresses. While the core components of heterotrimeric G-proteins and their basic biochemistry are similar in plants and metazoans, key differences exist in their regulatory mechanisms. In particular, the activation mechanisms of plant G-proteins appear diverse and may include both canonical and novel modes. Classical G-protein-coupled receptorlike proteins exist in plants and interact with Gα proteins, but their ability to activate Gα by facilitating GDP to GTP exchange has not been demonstrated. Conversely, there is genetic and functional evidence that plant G-proteins interact with the highly prevalent receptor-like kinases (RLKs) and are phosphorylated by them. This suggests the exciting scenario that in plants the G-proteins integrate RLK-dependent signal perception at the plasma membrane with downstream effectors. Because RLKs are active kinases, it is also likely that the activity of plant G-proteins is regulated via phosphorylation/dephosphorylation rather than GTP–GDP exchange as in metazoans. This review discusses our current knowledge of the possible RLK-dependent regulatory mechanisms of plant G-protein signaling in the context of several biological systems and outlines the diversity that might exist in such regulation.

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell’s decision to “go” vs. “grow.” These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.

As sessile organisms, plants are constantly exposed to a variety of environmental stresses that have detrimental effects on their growth and development, leading to major crop yield losses worldwide. To cope with adverse conditions, plants have developed several adaptive mechanisms. A thorough understanding of these mechanisms is critical to generate plants for the future. The heterotrimeric G-protein complex, composed of Gα, Gβ, and Gγ subunits, participates in the regulation of diverse cellular signaling pathways and has multiple roles in regulating plant stress responses. The complex has two functional entities, the GTP-bound Gα subunit and the Gβγ dimer, both of which by interacting with additional proteins can activate various signaling networks. The involvement of G-proteins has been shown in plants’ response to drought, salinity, extreme temperatures, heavy metals, ozone, and UV-B radiation. Due to their versatility and the number of processes modulated by them, G-proteins have emerged as key targets for generating stress-tolerant crops. In this review, we provide an overview of the current knowledge of the roles of G-proteins in abiotic stress tolerance, with examples from the model plant Arabidopsis thaliana , where these processes are most widely studied and from additional agriculturally relevant crops, where their potential is realized for human usage.