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Background G protein-coupled receptors (GPCRs) transduce external stimuli into the cell by G proteins via an allosteric mechanism. Agonist binding to the receptor stimulates GDP/GTP exchange within the heterotrimeric G protein complex, whereas recent structures of GPCR–G protein complexes revealed that the H5, S1 and S2 domains of Gα are involved in binding the active receptor, earlier studies showed that a short peptide analog derived from the C-terminus (H5) of the G protein transducin (Gt) is sufficient to stabilize rhodopsin in an active form. Methods We have used Molecular Dynamics simulations along with biological evaluation by means of radio-ligand binding assay to study the interactions between Gαi-derived peptide (G-peptide) and the µ-opioid receptor (µOR). Results Here, we show that a Gαi-derived peptide of 12 amino acids binds the µ-opioid receptor and acts as an allosteric modulator. The Gαi-derived peptide increases µOR affinity for its agonist morphine in a dose-dependent way. Conclusions These results indicate that the GPCR–Gα peptide interaction observed so far for only rhodopsin can be extrapolated to µOR. In addition, we show that the C-terminal peptide of the Gαi subunit is sufficient to stabilize the active conformation of the receptor. Our approach opens the possibility to investigate the GPCR–G protein interface with peptide modification.

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.

In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein-mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In Arabidopsis thaliana, GPCRs are not required for G protein-coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.

A role for the heterotrimeric G protein complex in the induction of a transient burst of reactive oxygen species (ROS) by the Microbial-Associated Molecular Pattern, flg22, a 22-amino acid peptide derived from bacterial flagella, is well established. However, the evidence for a negative or positive role for one component of the Arabidopsis G protein complex, namely, Regulator of G Signaling 1 (AtRGS1) leads to opposing conclusions. We show that the reason for this difference is due to the isolate of Col-0 ecotype used as the wildtype control in flg22-induced ROS and our data further support the idea that AtRGS1 is a negative regulator of the flg22-induced ROS response. Whole-genome genotyping led to the identification and validation of polymorphism in five genes between two Col-0 isolates that are candidates for the different ROS response relative to the rgs1 null mutant.

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 Gα, Gβ, and Gγ subunits, plays an important role in transducing signals under environmental stress. Furthermore, regulators of G-protein signaling (RGS) proteins act as negative regulators in heterotrimeric G protein signaling. 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 ΔFvgbb1. Saccharomyces cerevisiae and Aspergillus nidulans contain a single copy of FlbA, but F. verticillioides has two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. Importantly, FvFlbA2 has a negative role in FB1 regulation. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ΔFvflbA1 deletion mutant exhibited no significant defects, ΔFvflbA2 and ΔFvflbA2/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 suggest that FvFlbA paralogs interact with key heterotrimeric G protein components to impact F. verticillioides FB1 production and asexual development.

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.

Nitrogen is a key nutrient for plant growth and development. Plants regulate nitrogen availability and uptake efficiency through controlling root architecture. While the heterotrimeric G protein complex is an important element to regulate root morphology, it remains unknown whether the G protein regulates the root architecture in response to nitrogen supply. We used rice and Arabidopsis G protein mutants to study the root architecture in response to different nitrogen concentrations. We found that nitrogen inhibits root horizontal projection area (network area), root perimeter, total length, but not root diameter (average root width). Nitrogen influenced bushiness and root spatial distribution by inhibiting horizontal growth and promoting vertical expansion. The dynamic changes of the rice G protein mutant DK22 at different concentrations of nitrogen from day 7 to day 9 were different from the wild type with regard to bushiness and spatial distribution. The agb1-2 mutant in Arabidopsis lacked the inhibitory effect of nitrate on root growth. The heterotrimeric G protein complex regulates the inhibitory effect on root growth caused by high nitrogen supply and root spatial distribution in response to different nitrogen concentrations.

Ras homolog enriched in striatum (Rhes) is predominantly expressed in the corpus striatum. Rhes mRNA is localized in virtually all dopamine D1 and D2 receptor-bearing medium-sized spiny neurons (MSNs), and cholinergic interneurons of striatum. Early studies in rodents showed that Rhes is developmentally regulated by thyroid hormone, as well as by dopamine innervation in adult rat, monkey and human brains. At cellular level, Rhes interferes with adenosine A2A- and dopamine D1 receptor-dependent cAMP/PKA pathway, upstream of the activation of the heterotrimeric G protein complex. Besides its involvement in GPCR-mediated signaling, Rhes modulates Akt pathway activation, acts as E3-ligase of mutant huntingtin, whose sumoylation accounts for neurotoxicity in Huntington's disease, and physically interacts with Beclin-1, suggesting its potential involvement in autophagy-related cellular events. In addition, this protein can also bind to and activate striatal mTORC1, one of the key players in L-DOPA-induced dyskinesia in rodent models of Parkinson's disease. Accordingly, lack of Rhes attenuated such motor disturbances in 6-OHDA-lesioned Rhes knockout mice. In support of its role in MSN-dependent functions, several studies documented that mutant animals displayed alterations in striatum-related phenotypes reminiscent of psychiatric illness in humans, including deficits in prepulse inhibition of startle reflex and, most interestingly, a striking enhancement of behavioral responses elicited by caffeine, phencyclidine or amphetamine. Overall, these data suggest that Rhes modulates molecular and biochemical events underlying striatal functioning, both in physiological and pathological conditions. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Plants tolerate large variations in the intensity of the light environment by controlling the efficiency of solar to chemical energy conversion. To do this, plants have a mechanism to detect the intensity, duration, and change in light as they experience moving shadows, flickering light, and cloud cover. Sugars are the primary products of CO2 fixation, a metabolic pathway that is rate limited by this solar energy conversion. We propose that sugar is a signal encoding information about the intensity, duration and change in the light environment. We previously showed that the Arabidopsis heterotrimeric G protein complex including its receptor-like Regulator of G signaling protein, AtRGS1, detects both the concentration and the exposure time of sugars (Fu et al., 2014. Cell 156: 1084-1095). This unique property, designated dose-duration reciprocity, is a behavior that emerges from the system architecture / system motif. Here, we show that another property of the signaling system is to detect large changes in light while at the same time, filtering types of fluctuation in light that do not affect photosynthesis efficiency. When AtRGS1 is genetically ablated, photosynthesis efficiency is reduced in a changing- but not a constant-light environment. Mathematical modeling revealed that information about changes in the light environment is encoded in the amount of free AtRGS1 that becomes compartmentalized following stimulation. We propose that this property determines when to adjust photosynthetic efficiency in an environment where light intensity changes abruptly caused by moving shadows on top of a background of light changing gradually from sun rise to sun set and fluctuating light such as that caused by fluttering leaves.

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.

SUMMARY Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR desensitizes a cell from its extracellular signal. β-arrestins facilitate GPCR endocytosis but also propagate cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: microdomains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

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.

Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs) because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS) protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS). A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119) were not known previously. AtRGS1 protein interactions were dynamically modulated by d -glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

Skeletal development is exquisitely controlled both spatially and temporally by cell signaling networks. Gαs is the stimulatory α-subunit in a heterotrimeric G protein complex transducing the signaling of G-protein-coupled receptors (GPCRs), responsible for controlling both skeletal development and homeostasis. Gαs, encoded by the GNAS gene in humans, plays critical roles in skeletal development and homeostasis by regulating commitment, differentiation and maturation of skeletal cells. Gαs-mediated signaling interacts with the Wnt and Hedgehog signaling pathways, both crucial regulators of skeletal development, remodeling and injury repair. Genetic mutations that disrupt Gαs functions cause human disorders with severe skeletal defects, such as fibrous dysplasia of bone and heterotopic bone formation. This chapter focuses on the crucial roles of Gαs signaling during skeletal development and homeostasis, and the pathological mechanisms underlying skeletal diseases caused by GNAS mutations.

Genetic ablation of the β subunit of the heterotrimeric G protein complex in agb1-2 confers defective activation of microbe-associated molecular pattern (MAMP)-triggered immunity, resulting in agb1-2 enhanced susceptibility to pathogens like the fungus Plectosphaerella cucumerina BMM. A mutant screen for suppressors of agb1-2 susceptibility (sgb) to P. cucumerina BMM identified sgb10, a new null allele (mkp1-2) of the mitogen-activated protein kinase phosphatase 1 (MKP1). The enhanced susceptibility of agb1-2 to the bacterium Pseudomonas syringae pv. tomato DC3000 and the oomycete Hyaloperonospora arabidopsidis is also abrogated by mkp1-2. MKP1 negatively balances production of reactive oxygen species (ROS) triggered by MAMPs, since ROS levels are enhanced in mkp1. The expression of RBOHD, encoding a NADPH oxidase–producing ROS, is upregulated in mkp1 upon MAMP treatment or pathogen infection. Moreover, MKP1 negatively regulates RBOHD activity, because ROS levels upon MAMP treatment are increased in mkp1 plants constitutively overexpressing RBOHD (35S::RBOHD mkp1). A significant reprograming of mkp1 metabolic profile occurs with more than 170 metabolites, including antimicrobial compounds, showing differential accumulation in comparison with wild-type plants. These results suggest that MKP1 functions downstream of the heterotrimeric G protein during MAMP-triggered immunity, directly regulating the activity of RBOHD and ROS production as well as other immune responses.

FLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

Heterotrimeric G proteins are the molecule switch that transmits information from external signals to intracellular target proteins in mammals and yeast cells. In higher plants, heterotrimeric G proteins regulate plant architecture. Rice harbors one canonical &alpha, subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical &beta, subunit gene (RGB1), and five &gamma, subunit genes (tentatively designated RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/qPE9-1/OsGGC3, and RGG5/OsGGC2) as components of the heterotrimeric G protein complex. Among the five &gamma, subunit genes, RGG1 encodes the canonical &gamma, subunit, RGG2 encodes a plant-specific type of &gamma, subunit with additional amino acid residues at the N-terminus, and the remaining three &gamma, subunit genes encode atypical &gamma, subunits with cysteine-rich C-termini. We characterized the RGG4/DEP1/DN1/qPE9-1/OsGGC3 gene product G&gamma, 4 in the wild type (WT) and truncated protein G&gamma, 4∆Cys in the RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutant, Dn1-1, as littele information regarding the native G&gamma, 4 and G&gamma, 4∆Cys proteins is currently available. Based on liquid chromatography-tandem mass spectrometry analysis, immunoprecipitated G&gamma, 4 candidates were confirmed as actual G&gamma, 4. Similar to &alpha, (G&alpha, ) and &beta, subunits (G&beta, ), G&gamma, 4 was enriched in the plasma membrane fraction and accumulated in the developing leaf sheath. As RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants exhibited dwarfism, tissues that accumulated G&gamma, 4 corresponded to the abnormal tissues observed in RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants.

The heterotrimeric G protein complex, composed of Gα, Gβ, and Gγ subunits, plays some role in structural development in plants but this role could be indirect because loss-of-function mutations do not alter the body plan and post-embryonic organs differ only morphologically and not in their identity. This uncertainty has been compounded by the fact that loss of the Gβ subunit in cereals, but not Arabidopsis, is seedling lethal and that loss of maize Gα subunit confers prolificacy of a reproductive organ. In this study, we comprehensively profiled the root and shoot structural traits of rice Gα-null and viable Gβ-RNAi "knockdown" mutants, and found anomalous morphologies caused by Gβ-RNAi that are distinct from the Arabidopsis orthologue. The rice Gβ-RNAi mutant exhibited reduced radial growth of aerial parts as well as a more compact root architecture, among which smaller root mass seems mainly due to increased necrosis when grown on soil. In addition, three dimensional analyses of rice root system architecture revealed that the smaller root architecture of Gβ-RNAi plant is also due to both reduced root elongation and adventitious root formation. This contrasts to the Arabidopsis Gβ-null mutation that promotes cell proliferation. There is elevated cell senescence activity both visualized by Evans Blue staining and inferred from an expression analysis of cell-death marker genes. We propose that the morphological phenotypes of rice Gβ-RNAi plants are predominantly associated with the mediation of various stresses and cell senescence, consistent with an indirect role for Arabidopsis Gβ in development where the orthologous gene ablation mainly confers altered cell proliferation. We also elaborate our speculative working hypothesis that cell division is a type of stress and as such due to impairment in responding to stress in the G protein mutants, manifests as altered morphology and architecture but not an altered body plan or organ identities.

Summary Plant height is among the most important agronomic traits that influence crop yield. However, in addition to the Rht‐1 alleles, the molecular basis of plant height in bread wheat remains largely unclear. Based on wheat gene expression profiling analysis, we identify a light‐regulated gene from bread wheat, designated as TaCOLD1, whose encoding protein is homologous to cold sensor COLD1 in rice. We show that TaCOLD1 protein is localized to the endoplasmic reticulum (ER) and plasma membrane. Phenotypic analyses show that overexpression of a mutated form of TaCOLD1 (M187K) in bread wheat cultivar Kenong199 (Rht‐B1b) background resulted in an obvious reduction in plant height. Further, we demonstrate that the hydrophilic loop of TaCOLD1 (residues 178–296) can interact with TaGα‐7A (the α subunit of heterotrimeric G protein) protein but not TaGα‐1B, and the mutation (M187K) in TaCOLD1 remarkably enhances its interaction with TaGα‐7A. Physical interaction analyses show that the C‐terminal region of TaGα‐7A, which is lacking in the TaGα‐1B protein, is necessary for its interaction with TaCOLD1. Intriguingly, the C‐terminal region of TaGα‐7A is also physically associated with the TaDEP1 protein (an atypical Gγ subunit). Significantly, we discover that TaCOLD1 and mTaCOLD1 (M187K) can interfere with the physical association between TaGα‐7A and TaDEP1. Together, this study reveals that TaCOLD1 acts as a novel regulator of plant height through interfering with the formation of heterotrimeric G protein complex in bread wheat and is a valuable target for the engineering of wheat plant architecture.

The heterotrimeric G protein complex, comprising Gα, Gγ and Gγ subunits, is an evolutionarily conserved signaling molecular machine that transmits signals from transmembrane receptors to downstream target proteins. Plants conserved the core G protein elements, while developing their own regulatory systems differently from animals. Genetic evidence supports the conclusion that the heterotrimeric G proteins regulate shoot, root and epidermis development, as well as sugar sensing, hormone responsiveness and abiotic and biotic stress tolerance. This review is a compendium of the known morphological changes conferred by loss- and gain-of-function mutations of the G protein subunit genes across three higher land plant models, namely Arabidopsis, rice and maize.

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.

The three-member family of Arabidopsis extra-large G proteins (XLG1-3) defines the prototype of an atypical Gα subunit in the heterotrimeric G protein complex. Recent evidence indicate that XLG subunits operate along with its Gβγ dimer in root morphology, stress responsiveness and cytokinin induced development, however downstream targets of activated XLG proteins in the stress pathways are rarely known. To assemble a set of candidate XLG-targeted proteins, a yeast two-hybrid complementation-based screen was performed using XLG protein baits to query interactions between XLG and partner protein found in glucose-treated seedlings, roots, and Arabidopsis cells in culture. Seventy two interactors were identified and greater than 60% of a test set displayed in vivo interaction with XLG proteins. Gene co-expression analysis shows that greater than 70 % of the interactors are positively correlated with the corresponding XLG partners. Gene Ontology enrichment for all the candidates indicates stress responses and posits a molecular mechanism involving a specific set of transcription factor partners to XLG. Genes encoding two of these transcription factors, SZF1 and 2, require XLG proteins for full NaCl-induced expression. The subcellular localization of the XLG proteins in the nucleus, endosome, and plasma membrane is dependent on the specific interacting partner.

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.

Summary Cells continuously adjust their behavior in response to changing environmental conditions. Both intensity and duration of external signals are critical factors in determining what response is initiated. To understand how intracellular signaling networks process such multidimensional information, we studied the AtRGS1-mediated glucose response system of Arabidopsis . By combining experiments with mathematical modeling, we discovered a reciprocal dose and duration response relying on the orchestrated action of three kinases (AtWNK1, AtWNK8, and AtWNK10) acting on distinct timescales and activation thresholds. Specifically, we find that high concentrations of D-glucose rapidly signal through AtWNK8 and AtWNK10, whereas low, sustained sugar concentration slowly activate the pathway through AtWNK1, allowing the cells to respond similarly to transient, high-intensity signals and sustained, low-intensity signals. This "dose-duration reciprocity" allows encoding of both the intensity and persistence of glucose as an important energy resource and signaling molecule.

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.

Currently, there are strong inconsistencies in our knowledge of plant heterotrimeric G-proteins that suggest the existence of additional members of the family. We have identified a new Arabidopsis G-protein γ-subunit (AGG3) that modulates morphological development and ABA-regulation of stomatal aperture. AGG3 strongly interacts with the Arabidopsis G-protein β-subunit in vivo and in vitro. Most importantly, AGG3-deficient mutants account for all but one of the 'orphan' phenotypes previously unexplained by the two known γ-subunits in Arabidopsis. AGG3 has unique characteristics never before observed in plant or animal systems, such as its size (more than twice that of canonical γ-subunits) and the presence of a C-terminal Cys-rich domain. AGG3 thus represent a novel class of G-protein γ-subunits, widely spread throughout the plant kingdom but not present in animals. Homologues of AGG3 in rice have been identified as important quantitative trait loci for grain size and yield, but due to the atypical nature of the proteins their identity as G-protein subunits was thus far unknown. Our work demonstrates a similar trend in seeds of Arabidopsis agg3 mutants, and implicates G-proteins in such a crucial agronomic trait. The discovery of this highly atypical subunit reinforces the emerging notion that plant and animal G-proteins have distinct as well as shared evolutionary pathways.

Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different functional properties, suitable for their role as dim and bright light receptors, respectively. For example, rod and cone visual pigments couple to distinct variants of the heterotrimeric G protein transducin. However, the role of the structural differences between rod and cone transducin α subunits (Tα) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod Tα expressed in cones and cone Tα expressed in rods in three mouse strains: rod Tα knockout, cone Tα GNAT2 cpfl3 mutant, and rod and cone Tα double mutant rd17 mouse. Surprisingly, although the rod/cone Tα are only 79% identical, exogenously expressed rod or cone Tα localized and translocated identically to endogenous Tα in each photoreceptor type. Moreover, exogenously expressed rod or cone Tα rescued electroretinogram responses (ERGs) in mice lacking functional cone or rod Tα, respectively. Ex vivo transretinal ERG and single-cell recordings from rd17 retinas treated with rod or cone Tα showed comparable rod sensitivity and response kinetics. These results demonstrate that cone Tα forms a functional heterotrimeric G protein complex in rods and that rod and cone Tα couple equally well to the rod phototransduction cascade. Thus, rod and cone transducin α-subunits are functionally interchangeable and their signaling properties do not contribute to the intrinsic light sensitivity differences between rods and cones. Additionally, the technology used here could be adapted for any such homologue swap desired.

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.

BIM-46187 (7-[2-amino-1-oxo-3-thio-propyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo-[1,2a]-pyrazine dimer, hydrochloride) is an inhibitor of the heterotrimeric G-protein complex signalling. Since many mediators of pain act through G-protein coupled receptors, the anti-hyperalgesic effects of BIM-46187 were assessed on experimental models of pain. In addition since opioids are widely used in pain management and act through specific G-protein-coupled receptors, the effects of BIM-46187 on the analgesic properties of morphine have also been investigated. BIM-46187 elicited a dose dependent analgesic effect in the models of carrageenan-induced hyperalgesia (0.1-1 mg/kg; i.v.) and chronic constriction injury (0.3-3 mg/kg; i.v.) in rats. BIM-46187, however, up to 10 mg/kg did not modify the paw oedema induced by carrageenan excluding an anti-inflammatory effect. In addition, at these doses, the compound was not sedative as shown by the lack of effect on the motor performance in the rotarod test. The combination of BIM-46187 and morphine (ratio 1/1) resulted in an unexpected synergistic effect in the model of carrageenan-induced hyperalgesia and in the chronic constriction injury model in rats when evaluated by isobolographic analysis. This synergy allowed a reduction of at least 20 fold in the dose of each compound. Conversely, the drug combination did not increase the side effects of morphine as assessed in the rotarod test. In conclusion, BIM-46187 elicits a potent anti-hyperalgesic effect and strongly synergizes with morphine. This work highlights the role of heterotrimeric G-protein complexes in pain and supports further investigations of the use of BIM-46187 alone, or in combination with low doses of morphine, in the management of pain.

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 )

In mammals, basal currents through G protein-coupled inwardly rectifying K + (GIRK) channels are repressed by Gα i/o GDP, and the channels are activated by direct binding of free Gβγ subunits released upon stimulation of Gα i/o -coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana , is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis Gα (At GPA1 ), Gβ ( AGB1 ), and Regulator of G protein Signaling (At RGS1 ) genes for impacts on ion channel regulation. In guard cells, where K + fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K + (K in ) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in At gpa1 and agb1 single mutants and the At gpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The At rgs1 mutation did not affect K in current magnitude or ABA sensitivity, but K in voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the Gα subunit or regulation of the heterotrimer to mediate K in channel modulation after ligand perception. In contrast, outwardly rectifying (K out ) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.

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.

Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.

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.

Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

Signaling through heterotrimeric G proteins is conserved in diverse eukaryotes. Compared to vertebrates, the simpler repertoire of G-protein complex and accessory components in Arabidopsis (Arabidopsis thaliana) offers a unique advantage over all other multicellular, genetic-model systems for dissecting the mechanism of G-protein signal transduction. One of several biological processes that the G-protein complex regulates in Arabidopsis is cell division. We determined cell production rate in the primary root and the formation of lateral roots in Arabidopsis to define individually the types of modulatory roles of the respective G-protein α- and β-subunits, as well as the heterotrimer in cell division. The growth rate of the root is in part a consequence of cell cycle maintenance in the root apical meristem (RAM), while lateral root production requires meristem formation by founder pericycle cells. Thus, a comparison of these two parameters in various genetic backgrounds enabled dissection of the role of the G-protein subunits in modulation of cell division, both in maintenance and initiation. Cell production rates were determined for the RAM and lateral root formation in gpa1 (Arabidopsis G-protein α-subunit) and agb1 (Arabidopsis G-protein β-subunit) single and double mutants, and in transgenic lines overexpressing GPA1 or AGB1 in agb1 or gpa1 mutant backgrounds, respectively. We found in the RAM that the heterotrimeric complex acts as an attenuator of cell proliferation, whereas the GTP-bound form of the Gα-subunit's role is a positive modulator. In contrast, for the formation of lateral roots, the Gβγ-dimer acts largely independently of the Gα-subunit to attenuate cell division. These results suggest that Arabidopsis heterotrimeric G-protein subunits have differential and opposing roles in the modulation of cell division in roots.

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.

The dimorphic fungus Candida albicans secretes farnesol, which acts as a quorum-sensing molecule and prevents the yeast to mycelium conversion. In this study we examined the effect of farnesol in the filamentous fungus Aspergillus nidulans. We show that externally added farnesol has no effect on hyphal morphogenesis; instead, it triggers morphological features characteristic of apoptosis. Additional experiments suggest that mitochondria and reactive oxygen species (ROS) participate in farnesol-induced apoptosis. Moreover, the effects of farnesol appear to be mediated by the FadA heterotrimeric G protein complex. Because A. nidulans does not secrete detectable amounts of farnesol, we propose that it responds to farnesol produced by other fungi. In agreement with this notion, growth and development were impaired in a farnesol-dependent manner when A. nidulans was co-cultivated with C. albicans. Taken together, our data suggest that farnesol, in addition to its quorum-sensing function that regulates morphogenesis, is also employed by C. albicans to reduce competition from other microbes.

Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina is complex and depends on the ethylene, jasmonic acid and salicylic acid signaling pathways. A quantitative trait loci (QTL) analysis of resistance to this fungus was performed using two populations of recombinant inbred lines. Three loci QRP1-QRP3 (for Quantitative Resistance to Plectosphaerella) were identified and mapped on chromosome 2 (QRP1 and QRP2) and 5 (QRP3). QRP1, the locus showing the strongest effect, was found to correspond to the ERECTA (ER) gene that encodes a receptor-like-kinase (RLK), which has been previously implicated in plant development, and resistance to the bacterium Ralstonia solanacearum. The leucine-rich repeat and the kinase domains of ERECTA were specifically required for resistance to P. cucumerina, as er mutant alleles impaired in any of these domains showed enhanced susceptibility to this fungus, but not to other virulent pathogens. The involvement of the ER-signaling pathway in resistance to P. cucumerina was supported by the fact that three mutants defective in this pathway, elk2, elk5 and elk4 (agb1-1), which encodes the beta-subunit of Arabidopsis heterotrimeric G protein, were also impaired in their resistance to this fungus. The putative function of the Arabidopsis heterotrimeric G protein in resistance to P. cucumerina suggested by the enhanced susceptibility of agb1-1 was corroborated by the demonstration that a null allele (gpa1-4) of the G protein alpha-subunit showed enhanced resistance to this pathogen. Deposition of beta-1,3-glucan callose at infection sites was specifically impaired in er-1 and agb1-1 mutants upon P. cucumerina inoculation. Taken together, these data suggest a putative function of ERECTA and heterotrimeric G protein in P. cucumerina perception.

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.

Signal recognition by seven-transmembrane (7TM) cell-surface receptors is typically coupled by heterotrimeric G-proteins to downstream effectors in metazoan, fungal, and amoeboid cells. Some responses perceived by 7TM receptors in amoeboid cells and possibly in human cells can initiate downstream action independently of heterotrimeric G-proteins. Plants use heterotrimeric G-protein signaling in the regulation of growth and development, particularly in hormonal control of seed germination, but it is not yet clear which of these responses utilize a 7TM receptor. Arabidopsis GCR1 has a predicted 7TM-spanning domain and other features characteristic of 7TM receptors. Loss-of-function gcr1 mutants indicate that GCR1 plays a positive role in gibberellin- (GA) and brassinosteroid- (BR) regulated seed germination. The null mutants of GCR1 are less sensitive to GA and BR in seed germination. This phenotype is similar to that previously observed for transcript null mutants in the Gα-subunit, gpa1. However, the reduced sensitivities toward GA and BR in the single gcr1, gpa1, and agb1 (heterotrimeric G-protein β-subunit) mutants are additive or synergistic in the double and triple mutants. Thus, GCR1, unlike a typical 7TM receptor, apparently acts independently of the heterotrimeric G-protein in at least some aspects of seed germination, suggesting that this alternative mode of 7TM receptor action also functions in the plant kingdom.

Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells. Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit. When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical alpha-subunit (Galpha) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLDalpha1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with Galpha. PLDalpha1 contains a sequence motif analogous to the G alpha-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD(K564A) of this motif abolished the PLDalpha1-Galpha binding, whereas mutation of the two flanking residues PLD(E563A) and PLD(F565A) decreased the binding. Addition of Galpha to PLDalpha1 inhibited PLDalpha1 activity, whereas the PLD(K564A) mutation that disrupted the Galpha-PLDalpha1 binding abolished the inhibition. GTP relieved the Galpha inhibition of PLDalpha1 activity and also inhibited the binding between PLDalpha1 and Galpha. Meanwhile, the PLDalpha1-Galpha interaction stimulated the intrinsic GTPase activity of Galpha. Therefore, these results have demonstrated the direct binding between Galpha and PLDalpha1, identified the DRY motif on PLDalpha1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLDalpha1 and Galpha.