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Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of canonical subunits and various regulators of G-protein signaling (RGS) proteins, plays an important role in transducing signals under environmental stress. Earlier studies demonstrated that Gα and Gβ subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gβ deletion mutant ∆Fvgbb1. Notably, FvFlbA2 has a negative role in FB1 regulation. While many fungi contain a single copy of FlbA, F. verticillioides harbors two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ∆FvflbA1 deletion mutant exhibited no significant defects, ∆FvflbA2 and ∆FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays determined that FvFlbA paralogs interact with key heterotrimeric G protein components, which in turn will lead altered G-protein-mediated signaling pathways that regulate FB1 production and asexual development in F. verticillioides.

Background The heterotrimeric G protein complex, consisting of Gα, Gβ, and Gγ subunits, are conserved signal transduction mechanism in eukaryotes. Recent molecular researches had demonstrated that G protein signaling participates in the regulation of yield related traits. However, the effects of G protein genes on yield components and stress tolerance are not well characterized. Results In this study, we generated heterotrimeric G protein mutants in rice using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology. The effects of heterotrimeric G proteins on the regulation of yield components and stress tolerance were investigated. The mutants of gs3 and dep1 generated preferable agronomic traits compared to the wild-type, whereas the mutants of rga1 showed an extreme dwarf phenotype, which led to a dramatic decrease in grain production. The mutants showed improved stress tolerance, especially under salinity treatment. We found four putative extra-large G proteins (PXLG)1–4 that also participate in the regulation of yield components and stress tolerance. A yeast two hybrid showed that the RGB1 might interact with PXLG2 but not with PXLG1, PXLG3 or PXLG4. Conclusion These findings will not only improve our understanding of the repertoire of heterotrimeric G proteins in rice but also contribute to the application of heterotrimeric G proteins in rice breeding.

SummaryFusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins, on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gβ, and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gβ protein FvGbb1 has direct impact on fumonisin regulation but no other physiological aspects in F. verticillioides. In this study, we identified and characterized a putative receptor for activated C kinase 1 (RACK1) homolog FvGbb2 as a putative Gβ-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may interact with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis, and stress response in F. verticillioides.

Damaged or unwanted cellular proteins are degraded by either autophagy or the ubiquitin/proteasome pathway. In Arabidopsis thaliana, sensing of D-glucose is achieved by the heterotrimeric G protein complex and regulator of G-protein signaling 1 (AtRGS1). Here, we showed that starvation increases proteasome-independent AtRGS1 degradation, and it is correlated with increased autophagic flux. RGS1 promoted the production of autophagosomes and autophagic flux, RGS1-yellow fluorescent protein (YFP) was surrounded by vacuolar dye FM4-64 (red fluorescence). RGS1 and autophagosomes co-localized in the root cells of Arabidopsis and BY-2 cells. We demonstrated that the autophagosome marker ATG8a interacts with AtRGS1 and its shorter form with truncation of the seven transmembrane and RGS1 domains in planta. Altogether, our data indicated the correlation of autophagosome formation with degradation and endocytosis of AtRGS1 through ATG8a.

The nociceptin/orphanin FQ peptide (NOP) opioid receptors regulate neurotransmitter release via inhibition of voltage-gated Ca(2+) channels (CaV2.2) in sympathetic and sensory neurons. Stimulation of NOP receptors by its endogenous agonist, nociception (Noc), leads to membrane-delimited, voltage-dependent (VD) block of CaV2.2 channel currents mediated by Gβγ protein subunits. Previously we reported that the pertussis toxin-sensitive Gαi1 and Gβ2/β4 isoforms mediate the functional coupling of NOP opioid receptors with CaV channels in rat stellate ganglion (SG) sympathetic neurons. In the present report we extended our studies by identifying the Gγ subunit that forms the heterotrimer within this signaling pathway. Small interference RNA (or siRNA) was employed to silence the expression of the natively expressed Gγ subunits. Initial PCR assays indicated that SG neurons expressed seven Gγ subunits. Silencing Gγ3 subunits did not alter signaling between NOP receptors and Ca(2+) channels. However, after Gγ7 isoforms were silenced, the Noc-mediated inhibition of CaV channels was significantly decreased when compared to SG neurons transfected with scrambled siRNA. We observed that Gγ10 and Gγ11 mRNA levels increased 2.5- and 2.7-fold, respectively, after Gγ7 subunits were silenced. However, this compensatory increase in mRNA expression did not appear to fully rescue the NOP receptor coupling efficiency. Additionally, both Gγ2 and Gγ5 levels increased 50 and 75%, respectively, while Gγ3 and Gγ4 expression levels remained relatively unchanged. Taken together, our findings suggest that the Gαi1/Gβ2(β4)/Gγ7 heterotrimeric G protein complex determines the NOP receptor-mediated modulation of CaV channels in SG neurons.

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is an evolutionarily conserved mechanism in diverse eukaryotic organisms. In plants, the repertoire of the heterotrimeric G protein complex, which is composed of the Gα, Gβ, and Gγ subunits, is much simpler than that in metazoans, and the identity of typical G protein-coupled receptors (GPCRs) together with their ligands still remains unclear. Comparative phenotypic analysis in Arabidopsis and rice plants using gain- and loss-of-function mutants of G protein components revealed that heterotrimeric G protein signaling plays important roles in a wide variety of plant growth and developmental processes. Grain yield is a complex trait determined by quantitative trait loci (QTL) and is influenced by soil nitrogen availability and environmental changes. Recent studies have shown that the manipulation of two non-canonical Gγ subunits, GS3 (GRAIN SIZE 3) and DEP1 (DENSE AND ERECT PANICLE 1), represents new strategies to simultaneously increase grain yield and nitrogen use efficiency in rice. This review discusses the latest advances in our understanding of the heterotrimeric G protein signal transduction pathway and its application in improving yield and stress tolerance in crops.

Highlight Maize COMPACT PLANT 2, the alpha subunit of the heterotrimeric G protein complex, is important for some plastic developmental traits: shoot and root system architectures, and ear production., Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. The maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.

SUMMARY Although the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding the intracellular domain of PD-L1. Here, we outline a major role for PD-L1 intracellular signaling in the control of dendritic cell (DC) migration from the skin to the draining lymph node (dLN). Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. The TSS motif proves critical for chemokine-mediated DC migration to the dLN during inflammation. This loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. Finally, the TSS motif is required for chemokine receptor signaling downstream of the Gα subunit of the heterotrimeric G protein complex, ERK phosphorylation, and actin polymerization in DCs., Graphical Abstract, In Brief Lucas et al. define three residues within the cytoplasmic tail of PD-L1 that are required for proper dendritic cell migration from the skin to the lymph node. These three-amino-acid residues promote chemokine signaling in dendritic cells and productive T cell responses to skin infections.

Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Forster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex becomes dissociated thus AGB1 interacts with its effector proteins leading to changes in reactive oxygen species and calcium.

Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization and level was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

Signal transduction typically begins by ligand-dependent activation of a concomitant partner that is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (regulator of G-protein signalling 1), which is the prototype of a seven-transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase-accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required for both G-protein-mediated sugar signalling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis mechanisms.

The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca²⁺ signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein-protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca²⁺ levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca²⁺-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.

Amongst the many stimuli orienting the growth of plant roots, of critical importance are the touch signals generated as roots explore the mechanically complex soil environment. However, the molecular mechanisms behind these sensory events remain poorly defined. We report an impaired obstacle-avoiding response of roots in Arabidopsis lacking a heterotrimeric G-protein. Obstacle avoidance may utilize a touch-induced release of ATP to the extracellular space. While sequential touch stimulation revealed a strong refractory period for ATP release in response to mechano-stimulation in wild-type plants, the refractory period in mutants was attenuated, resulting in extracellular ATP accumulation. We propose that ATP acts as an extracellular signal released by mechano-stimulation and that the G-protein complex is needed for fine-tuning this response.

Plants use sugars as signaling molecules and possess mechanisms to detect and respond to changes in sugar availability, ranging from the level of secondary signaling molecules to altered gene transcription. G-protein-coupled pathways are involved in sugar signaling in plants. The Arabidopsis thaliana regulator of G-protein signaling protein 1 (AtRGS1) combines a receptor-like seven transmembrane domain with an RGS domain, interacts with the Arabidopsis Gα subunit (AtGPA1) in a d -glucose-regulated manner, and stimulates AtGPA1 GTPase activity. We determined that AtRGS1 interacts with additional components, genetically defined here, to serve as a plasma membrane sensor for d -glucose. This interaction between AtRGS1 and AtGPA1 involves, in part, the seven-transmembrane domain of AtRGS1. Structured summary MINT- 6743118 : RGS1 (uniprotkb: Q8H1F2 ) and GPA1 (uniprotkb: P18064 ) physically interact (MI: 0218 ) by bimolecular fluorescence complementation (MI: 0809 )

Plants, like mammals, rely on their innate immune system to perceive and discriminate among the majority of their microbial pathogens. Unlike mammals, plants respond to this molecular dialogue by unleashing a complex chemical arsenal of defense metabolites to resist or evade pathogen infection. In basal or non-host resistance, plants utilize signal transduction pathways to detect “non-self”, “damaged-self” and “altered-self”-associated molecular patterns and translate these “danger” signals into largely inducible chemical defenses. The WD40 repeat (WDR)-containing proteins Gβ and TTG1 are constituents of two independent ternary protein complexes functioning at opposite ends of a plant immune signaling pathway. Gβ and TTG1 are also encoded by single-copy genes that are ubiquitous in higher plants, implying the limited diversity and functional conservation of their respective complexes. In this review, we summarize what is currently known about the evolutionary history of these WDR-containing ternary complexes, their repertoire and combinatorial interactions, and their downstream effectors and pathways in plant defense.

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of d-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Gα interaction partner that functions downstream of the plasma membrane–delimited heterotrimeric G-protein (GPA1) in a d-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Förster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous d-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high d-glucose. Additionally, THF1 levels are rapidly degraded by d-glucose but not l-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

Arabidopsis thaliana plants with null mutations in the genes encoding the α and β subunits of the single heterotrimeric G protein are less and more sensitive, respectively, to O3 damage than wild-type Columbia-0 plants. The first peak of the bimodal oxidative burst elicited by O3 in wild-type plants is almost entirely missing in both mutants. The late peak is normal in plants lacking the Gβ protein but missing in plants lacking the Gα protein. Endogenous reactive oxygen species (ROS) are first detectable in chloroplasts of leaf epidermal guard cells. ROS production in adjacent cells is triggered by extracellular ROS signals produced by guard cell membrane-associated NADPH oxidases encoded by the AtrbohD and AtrbohF genes. The late, tissue damage–associated component of the oxidative burst requires only the Gα protein and arises from multiple cellular sources. The early component of the oxidative burst, arising primarily from chloroplasts, requires signaling through the heterotrimer (or the Gβγ complex) and is separable from Gα-mediated activation of membrane-bound NADPH oxidases necessary for both intercellular signaling and cell death.

Heterotrimeric G proteins have been implicated in a wide range of plant processes. These include responses to hormones, drought, and pathogens, and developmental events such as lateral root formation, hypocotyl elongation, hook opening, leaf expansion, and silique development. Results and concepts emerging from recent phenotypic analyses of G-protein component mutants in Arabidopsis and rice are adding to our understanding of G-protein mechanisms and functions in higher plants.

Previous studies implicated the involvement of a heterotrimeric G protein in red (R) and far-red (FR) light signal transduction, but these studies utilized pharmacological or gain-of-function approaches and, therefore, are indirect tests. Here, we reexamine the role of the single canonical heterotrimeric G protein in R and FR control of hypocotyl growth using a loss-of-function approach. Single- and double-null mutants for the GPA1, AGB1genes encoding the alpha and beta subunit of the heterotrimeric G protein, respectively, have wild-type sensitivity to R and FR. Ectopic overexpression of wild type and a constitutive active form of the alpha subunit and of the wild-type beta subunit had no effect that can be unequivocally attributed to altered R and FR responsiveness. These results preclude a direct role for the heterotrimeric G complex in R and FR transduction in Arabidopsis leading to growth control in the hypocotyl.

Proteins that interact coevolve their structures. When mutation disrupts the interaction, compensation by the partner occurs to restore interaction otherwise counterselection occurs. We show in this study how a destabilizing mutation in one protein is compensated by a stabilizing mutation in its protein partner and their coevolving path. The pathway in this case and likely a general principle of coevolution is that the compensatory change must tolerate both the original and derived structures with equivalence in function and activity. Evolution of the structure of signaling elements in a network is constrained by specific protein pair interactions, by requisite conformational changes, and by catalytic activity. The heterotrimeric G protein-coupled signaling is a paragon of this protein interaction/function complexity and our deep understanding of this pathway in diverse organisms lends itself to evolutionary study. Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTP hydrolysis rate of the Gα subunit of the heterotrimeric G protein complex. An important RGS-contact site is a hydroxyl-bearing residue on the switch I region of Gα subunits in animals and most plants, such as Arabidopsis. The exception is the grasses (e.g., rice, maize, sugarcane, millets); these plants have Gα subunits that replaced the critical hydroxyl-bearing threonine with a destabilizing asparagine shown to disrupt interaction between Arabidopsis RGS protein (AtRGS1) and the grass Gα subunit. With one known exception (Setaria italica), grasses do not encode RGS genes. One parsimonious deduction is that the RGS gene was lost in the ancestor to the grasses and then recently acquired horizontally in the lineage S. italica from a nongrass monocot. Like all investigated grasses, S. italica has the Gα subunit with the destabilizing asparagine residue in the protein interface but, unlike other known grass genomes, still encodes an expressed RGS gene, SiRGS1. SiRGS1 accelerates GTP hydrolysis at similar concentration of both Gα subunits containing either the stabilizing (AtGPA1) or destabilizing (RGA1) interface residue. SiRGS1 does not use the hydroxyl-bearing residue on Gα to promote GAP activity and has a larger Gα-interface pocket fitting to the destabilizing Gα. These findings indicate that SiRGS1 adapted to a deleterious mutation on Gα using existing polymorphism in the RGS protein population.

Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins' regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases.

The serine/threonine protein kinase YpkA is an essential virulence factor produced by pathogenic Yersinia species. YpkA is delivered into host mammalian cells via a type III secretion system and localizes to the inner side of the plasma membrane. We have previously shown that YpkA binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling. To identify residues in YpkA involved in substrate binding activity we generated GFP-YpkA N-terminal deletion mutants and performed coimmunoprecipitation experiments. We located a substrate-binding domain on amino acids 40–49 of YpkA, which lies within the previously identified membrane localization domain on YpkA. Deletion of amino acids 40–49 on YpkA interfered with substrate binding, substrate phosphorylation and substrate inhibition. Autophosphorylation regulates the kinase activity of YpkA. To dissect the mechanism by which YpkA transmits signals, we performed nano liquid chromatography coupled to tandem mass spectrometry to map in vivo phosphorylation sites. Multiple serine phosphorylation sites were identified in the secretion/translocation region, kinase domain, and C-terminal region of YpkA. Using site-directed mutagenesis we generated multiple YpkA constructs harboring specific serine to alanine point mutations. Our results demonstrate that multiple autophosphorylation sites within the N terminus regulate YpkA kinase activation, whereas mutation of serine to alanine within the C terminus of YpkA had no effect on kinase activity. YpkA autophosphorylation on multiple sites may be a strategy used by pathogenic Yersinia to prevent inactivation of this important virulence protein by host proteins.

Background Plant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. Due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. It is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. Less is known about pathways connecting NaCl and cell proliferation and cell death. Plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric G-protein complex with glucose. Prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the Arabidopsis heterotrimeric G protein complex (AtRGS1, AtGPA1, AGB1, and AGG), we tested the hypothesis that G proteins modulate plant growth under salt stress. Results Na+ activates G signaling as quantitated by internalization of Arabidopsis Regulator of G Signaling protein 1 (AtRGS1). Despite being components of a singular signaling complex loss of the Gβ subunit (agb1-2 mutant) conferred accelerated senescence and aborted development in the presence of Na+, whereas loss of AtRGS1 (rgs1-2 mutant) conferred Na+ tolerance evident as less attenuated shoot growth and senescence. Site-directed changes in the Gα and Gβγ protein-protein interface were made to disrupt the interaction between the Gα and Gβγ subunits in order to elevate free activated Gα subunit and free Gβγ dimer at the plasma membrane. These mutations conferred sodium tolerance. Glucose in the growth media improved the survival under salt stress in Col but not in agb1-2 or rgs1-2 mutants. Conclusions These results demonstrate a direct role for G-protein signaling in the plant growth response to salt stress. The contrasting phenotypes of agb1-2 and rgs1-2 mutants suggest that G-proteins balance growth and death under salt stress. The phenotypes of the loss-of-function mutations prompted the model that during salt stress, G activation promotes growth and attenuates senescence probably by releasing ER stress.

We have previously shown that activation of G i2, an subunit of the heterotrimeric G protein complex, induces skeletal mus-cle hypertrophy and myoblast differentiation. To determine whether G i2 is required for skeletal muscle growth or regenera-tion, G i2-null mice were analyzed. G i2 knockout mice display decreased lean body mass, reduced muscle size, and impairedskeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of G i2 insatellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiationex vivo. The impaired differentiationis consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown ofG i2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown toregulate SC proliferation and differentiation, is increased by a constitutively active mutant of G i2 [G i2(Q205L)] and counter-regulated by G i2 knockdown. As for the mechanism, this study demonstrates that G i2(Q205L) regulates satellite cell differen-tiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner. Fil: Minetti, Giulia C.. Novartis Campus. Basilea; Suiza Fil: Feige, Jerome N.. Campus EPFL. Lausania; Suiza Fil: Bombard, Florian. Novartis Campus. Basilea; Suiza Fil: Heier, Annabelle. Novartis Campus. Basilea; Suiza Fil: Morvan, Fredric. Novartis Campus. Basilea; Suiza Fil: Nürnberg , Bernd. University of Tübingen. Tübingen; Alemania Fil: Leiss, Veronika. University of Tübingen. Tübingen; Alemania Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Research Triangle Park. North Carolina; Estados Unidos Fil: Glass, David J. Novartis Institutes for Biomedical Research. Cambridge; Estados Unidos Fil: Fornaro, Mara. Novartis Campus. Basilea; Suiza

The G-protein-coupled receptor (GPCR) signaling system is one of themain signaling pathways in eukaryotes. Here, we analyze the evolutionary history of all its components, from receptors to regulators, to gain a broad picture of its system-level evolution. Using eukaryotic genomes covering most lineages sampled to date, we find that the various components of the GPCR signaling pathway evolved independently, highlighting the modular nature of this system. Our data show that some GPCR families, G proteins, and regulators of G proteins diversified through lineage-specific diversifications and recurrent domain shuffling. Moreover, most of the gene families involved in the GPCR signaling system were already present in the last common ancestor of eukaryotes. Furthermore, we show that the unicellular ancestor of Metazoa already had most of the cytoplasmic components of the GPCR signaling system, including, remarkably, all the G protein alpha subunits, which are typical of metazoans. Thus, we show how the transition to multicellularity involved conservation of the signaling transduction machinery, as well as a burst of receptor diversification to cope with the new multicellular necessities. © The Author(s) 2014., This work was supported by a contract from the Institució Catalana de Recerca i Estudis Avançats, a European Research Council Starting Grant (ERC-2007-StG-206883), a grant (BFU2011-23434) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T, and a pregraduate Formación del Personal Investigador and a pregraduate Formación Profesorado Universitario grant from MINECO to A.d.M. and A.S.-P.

The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gβ, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGβ, CbGβ and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

In animals, heterotrimeric G proteins, comprising α-, β-and γ-subunits, perceive extracellular stimuli through cell surface receptors, and transmit signals to ion channels, enzymes and other effector proteins to affect numerous cellular behaviours. In plants, G proteins have structural similarities to the corresponding molecules in animals but transmit signals by atypical mechanisms and effector proteins to control growth, cell proliferation, defence, stomate movements, channel regulation, sugar sensing and some hormonal responses. In this review, we summarize the current knowledge on the molecular regulation of plant G proteins, their effectors and the physiological functions studied mainly in two model organisms: Arabidopsis thaliana and rice ( Oryza sativa ). We also look at recent progress on structural analyses, systems biology and evolutionary studies.

Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the “self-activating” trait, indicate the existence of divergent Gα regulatory mechanisms in the plant kingdom. In the grasses, purifying selection on the regulatory gene was lost after the physical decoupling of the RGS protein and its cognate Gα partner. More broadly these findings show extreme divergence in Gα activation and regulation that played a critical role in the evolution of G protein signaling pathways., Author Summary Extracellular signals activate intracellular changes that lead to cell behaviors. This spatial coupling is mediated by cell-surface receptor activation of the heterotrimeric G protein complex located on the cytoplasmic side of the plasma membrane. Unlike the case for metazoans, plant G proteins are constitutively active. Plants use multiple mechanisms to keep the G protein complex in its resting state, and activation occurs by inhibition of this property. One mechanism involves a cell surface receptor that accelerates the return to the resting state through direct interaction with the G protein at a specific protein interface. This unique protein, AtRGS1, has both an animal like receptor domain and a domain (RGS box) responsible for accelerating deactivation. One group of plants (cereals) lost this protein through, first, a mutation in the protein interface that reduces the affinity for the RGS box to the G protein, followed by gene loss.

Yeast two-hybrid technology is used to build a high-quality protein interaction network centered on Arabidopsis G-protein coupled signaling. The interactions uncovered are without precedent in animals and fungi and help identify new cellular roles for G-protein signaling in plants., 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.

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gβγ dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single β (AGB1) or the two γ (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

Heterotrimeric G protein signaling is important for cell-proliferative and glucose-sensing signal transduction pathways in the model plant organism Arabidopsis thaliana . AtRGS1 is a seven-transmembrane, RGS domain-containing protein that is a putative membrane receptor for d -glucose. Here we show, by using FRET, that d -glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo . AtGPA1 is a unique heterotrimeric G protein α subunit that is constitutively GTP-bound given its high spontaneous nucleotide exchange coupled with slow GTP hydrolysis. Analysis of a point mutation in AtRGS1 that abrogates GTPase-accelerating activity demonstrates that the regulation of AtGPA1 GTP hydrolysis mediates sugar signal transduction during Arabidopsis development, in contrast to animals where nucleotide exchange is the limiting step in the heterotrimeric G protein nucleotide cycle.

Heterotrimeric G-protein complexes couple extracellular signals via cell surface receptors to downstream enzymes called effectors. Heterotrimeric G-protein complexes, together with their cognate receptors and effectors, operate at the apex of signal transduction. In plants, the number of G-protein complex components is dramatically less than in other multicellular eukaryotes. An understanding of how multiple signals propagate transduction through the G-protein node can be found in the unique structural and kinetic properties of the plant heterotrimeric G-protein complex. This review addresses these unique features and speculates on why the repertoire of G-protein signaling elements is dramatically simpler than that in all other multicellular eukaryotes.

Abscisic acid (ABA) plays regulatory roles in a host of physiological processes throughout plant growth and development. Seed germination, early seedling development, stomatal guard cell functions, and acclimation to adverse environmental conditions are key processes regulated by ABA. Recent evidence suggests that signaling processes in both seeds and guard cells involve heterotrimeric G proteins. To assess new roles for the Arabidopsis (Arabidopsis thaliana) Gα subunit (GPA1), the Gβ subunit (AGB1), and the candidate G-protein-coupled receptor (GCR1) in ABA signaling during germination and early seedling development, we utilized knockout mutants lacking one or more of these components. Our data show that GPA1, AGB1, and GCR1 each negatively regulates ABA signaling in seed germination and early seedling development. Plants lacking AGB1 have greater ABA hypersensitivity than plants lacking GPA1, suggesting that AGB1 is the predominant regulator of ABA signaling and that GPA1 affects the efficacy of AGB1 execution. GCR1 acts upstream of GPA1 and AGB1 for ABA signaling pathways during germination and early seedling development: gcr1 gpa1 double mutants exhibit a gpa1 phenotype and agb1 gcr1 and agb1 gcr1 gpa1 mutants exhibit an agb1 phenotype. Contrary to the scenario in guard cells, where GCR1 and GPA1 have opposite effects on ABA signaling during stomatal opening, GCR1 acts in concert with GPA1 and AGB1 in ABA signaling during germination and early seedling development. Thus, cell- and tissue-specific functional interaction in response to a given signal such as ABA may determine the distinct pathways regulated by the individual members of the G-protein complex.

The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTPgammaS has no effect on Gbetagamma interactions, but markedly disrupts binding of the Galphai/o subunits to the receptor tail, suggesting that direct Gbetagamma binding indirectly links Galpha subunits to this region of the receptor. Gbetagamma subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of Galphashis6-beta1gamma2 and Galphaqhis6-beta1gamma2 also interact with the PTH1R tail in vitro. The Gbetagamma interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gbetagamma interactions block PTH signaling via phospholipase Cbeta/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gbetagamma interactions.

Heterotrimeric G proteins composed of alpha, beta, and gamma subunits link ligand perception by G protein-coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Galpha (GPA1) and Gbeta (AGB1) subunits, and two probable Ggamma subunits (AGG1 and AGG2). One Arabidopsis gene, GCR1, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR1 actually interacts with GPA1 was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR1 and GPA1 are indeed physically coupled. GCR1-GPA1 interaction depends on intracellular domains of GCR1. gcr1 T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr1 guard cells are also hypersensitive to the lipid metabolite, sphingosine-1-phosphate (S1P), which is a transducer of the ABA signal upstream of GPA1. Because gpa1 mutants exhibit insensitivity in aspects of guard cell ABA and S1P responses, whereas gcr1 mutants exhibit hypersensitivity, GCR1 may act as a negative regulator of GPA1-mediated ABA responses in guard cells.

In humans, heterotrimeric G proteins couple stimulus perception by G-protein-coupled receptors (GPCRs) with numerous downstream effectors. By contrast, despite great complexity in their signal-transduction attributes, plants have a simpler repertoire of G-signalling components. Nonetheless, recent studies on Arabidopsis thaliana have shown the importance of plant G-protein signalling in such fundamental processes as cell proliferation, hormone perception and ion-channel regulation.

In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA1 (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.

We used rice dwarf1 ( d1 ) mutants lacking a single-copy Gα gene and addressed Gα's role in disease resistance. d1 mutants exhibited a highly reduced hypersensitive response to infection by an avirulent race of rice blast. Activation of PR gene expression in the leaves of the mutants infected with rice blast was delayed for 24 h relative to the wild type. H 2 O 2 production and PR gene expression induced by sphingolipid elicitors (SE) were strongly suppressed in d1 cell cultures. Expression of the constitutively active OsRac1, a small GTPase Rac of rice, in d1 mutants restored SE-dependent defense signaling and resistance to rice blast. Gα mRNA was induced by an avirulent race of rice blast and SE application on the leaf. These results indicated the role of Gα in R gene-mediated disease resistance of rice. We have proposed a model for the defense signaling of rice in which the heterotrimeric G protein functions upstream of the small GTPase OsRac1 in the early steps of signaling.

Seed germination is regulated by many signals. We investigated the possible involvement of a heterotrimeric G protein complex in this signal regulation. Seeds that carry a protein null mutation in the gene encoding the alpha subunit of the G protein in Arabidopsis (GPA1) are 100-fold less responsive to gibberellic acid (GA), have increased sensitivity to high levels of Glc, and have a near-wild-type germination response to abscisic acid and ethylene, indicating that GPA1 does not directly couple these signals in germination control. Seeds ectopically expressingGPA1 are at least a million-fold more responsive to GA, yet still require GA for germination. We conclude that the GPA1 indirectly operates on the GA pathway to control germination by potentiation. We propose that this potentiation is directly mediated by brassinosteroids (BR) because the BR response and synthesis mutants,bri1-5 and det2-1, respectively, share the same GA sensitivity as gpa1 seeds. Furthermore,gpa1 seeds are completely insensitive to brassinolide rescue of germination when the level of GA in seeds is reduced. A lack of BR responsiveness is also apparent in gpa1 roots and hypocotyls suggesting that BR signal transduction is likely coupled by a heterotrimeric G protein at various points in plant development.

Signal-transducing GTPases in plants include small G proteins, heterotrimeric G proteins, and, potentially, several unique types of GTP binding proteins that are not members of either of the aforementioned classes. This review will focus on recent discoveries concerning the roles of heterotrimeric

The α subunit of a prototypical heterotrimeric GTP-binding protein (G protein), which is encoded by a single gene ( GPA1 ) in Arabidopsis , is a modulator of plant cell proliferation. gpa1 null mutants have reduced cell division in aerial tissues throughout development. Inducible overexpression of GPA1 in Arabidopsis confers inducible ectopic cell division. GPA1 overexpression in synchronized BY-2 cells causes premature advance of the nuclear cycle and the premature appearance of a division wall. Results from loss of function and ectopic expression and activation of GPA1 indicate that this subunit is a positive modulator of cell division in plants.

Previously, we reported that the rice dwarf mutant, d1 , is defective in the α subunit of the heterotrimeric G protein (Gα). In the present study, gibberellin (GA) signaling in d1 and the role of the Gα protein in the GA-signaling pathway were investigated. Compared with the wild type, GA induction of α-amylase activity in aleurone cells of d1 was greatly reduced. Relative to the wild type, the GA 3 -treated aleurone layer of d1 had lower expression of Ramy1A , which encodes α-amylase, and OsGAMYB , which encodes a GA-inducible transcriptional factor, and no increase in expression of Ca 2 + -ATPase . However, in the presence of high GA concentrations, α-amylase induction occurred even in d1 . The GA sensitivity of second leaf sheath elongation in d1 was similar to that of the wild type in terms of dose responsiveness, but the response of internode elongation to GA was much lower in d1 . Furthermore, Os20ox expression was up-regulated, and the GA content was elevated in the stunted internodes of d1 . All these results suggest that d1 affects a part of the GA-signaling pathway, namely the induction of α-amylase in the aleurone layer and internode elongation. In addition, a double mutant between d1 and another GA-signaling mutant, slr , revealed that SLR is epistatic to the D1 , supporting that the Gα protein is involved in GA signaling. However, the data also provide evidence for the presence of an alternative GA-signaling pathway that does not involve the Gα protein. It is proposed that GA signaling via the Gα protein may be more sensitive than that of the alternative pathway, as indicated by the low GA responsiveness of this Gα-independent pathway.

Background: All organisms perceive and respond to a profusion of environmental and endogenous signals that influence growth, development and behaviour. The G-protein signalling pathway is a highly conserved mechanism for transducing extracellular signals, and the superfamily of receptors that have seven transmembrane (7TM) domains is a primary element of this pathway. Evidence that heterotrimeric G proteins are involved in signal transduction in plants is accumulating, prompting speculation that plant 7TM receptors might exist. Results: Using information in the dbEST database of expressed sequence tags, we isolated an Arabidopsis thaliana gene ( GCR1 ) that encodes a protein with seven predicted membrane-spanning domains and other features characteristic of 7TM receptors. The protein shows 18–23% amino-acid identity (46–53% similarity) to, and good colinear alignment with, 7TM receptors from three different families. Its highest sequence identity is with the Dictyostelium cAMP receptors. GCR1 is expressed at very low levels in the roots, stems and leaves of Arabidopsis; it is a single-copy gene which maps close to the restriction fragment length polymorphism marker m291 on chromosome 5. Transgenic Arabidopsis expressing antisense GCR1 under the control of the constitutive cauliflower mosaic virus 35S promoter have reduced sensitivity to cytokinins in roots and shoots, yet respond normally to all other plant hormones. This suggests a functional role for GCR1 in cytokinin signal transduction. Conclusions: GCR1 encodes the first 7TM receptor homologue identified in higher plants and is involved in cytokinin signal transduction. This discovery suggests that 7TM receptors are ancient and predate the divergence of plants and animals.

Regulatory guanine nucleotide-binding proteins (G proteins) have been studied extensively in animal and microbial organisms, and they are divided into the heterotrimeric and the small (monomeric) classes. Heterotrimeric G proteins are known to mediate signal responses in a variety of pathways in animals and simple eukaryotes, while small G proteins perform diverse functions including signal transduction, secretion, and regulation of cytoskeleton. In recent years, biochemical analyses have produced a large amount of information on the presence and possible functions of G proteins in plants. Further, molecular cloning has clearly demonstrated that plants have both heterotrimeric and small G proteins. Although the functions of the plant heterotrimeric G proteins are yet to be determined, expression analysis of an Arabidopsis G alpha protein suggests that it may be involved in the regulation of cell division and differentiation. In contrast to the very few genes cloned thus far that encode heterotrimeric G proteins in plants, a large number of small G proteins have been identified by molecular cloning from various plants. In addition, several plant small G proteins have been shown to be functional homologues of their counterparts in animals and yeasts. Future studies using a number of approaches are likely to yield insights into the role plant G proteins play.

Plant heterotrimeric G-proteins have been implicated in a number of signaling processes. However, most of these studies are based on biochemical or pharmacological approaches. To examine the role of heterotrimeric G-proteins in plant development, we generated transgenic Arabidopsis expressing the Galpha subunit of the heterotrimeric G-protein under the control of a glucocorticoid-inducible promoter. With the conditional overexpression of either the wild type or a constitutively active version of Arabidopsis Galpha, transgenic seedlings exhibited a hypersensitive response to light. This enhanced light sensitivity was more exaggerated in a relatively lower intensity of light and was observed in white light as well as far-red, red, and blue light conditions. The enhanced responses in far-red and red light required functional phytochrome A and phytochrome B, respectively. Furthermore, the response to far-red light depended on functional FHY1 but not on FIN219 and FHY3. This dependence on FHY1 indicates that the Arabidopsis Galpha protein may act only on a discrete branch of the phytochrome A signaling pathway. Thus, our results support the involvement of a heterotrimeric G-protein in the light regulation of Arabidopsis seedling development.