Canonical Gα Research Articles

Stomatal responses to VPD utilize guard cell intracellular signaling components.

Stomatal pores, vital for CO2 uptake and water loss regulation in plants, are formed by two specialized guard cells. Despite their importance, there is limited understanding of how guard cells sense and respond to changes in vapor pressure difference (VPD). This study leverages a selection of CO2 hyposensitive and abscisic acid (ABA) signaling mutants in Arabidopsis, including heterotrimeric G protein mutants and RLK (receptor-like kinase) mutants, along with a variety of canola cultivars to delve into the intracellular signaling mechanisms prompting stomatal closure in response to high VPD. Stomatal conductance response to step changes in VPD was measured using the LI-6800F gas exchange system. Our findings highlight that stomatal responses to VPD utilize intracellular signaling components. VPD hyposensitivity was particularly evident in mutants of the ht1 (HIGH LEAF TEMPERATURE1) gene, which encodes a protein kinase expressed mainly in guard cells, and in gpa1-3, a null mutant of the sole canonical heterotrimeric Gα subunit, previously implicated in stomatal signaling. Consequently, this research identifies a nexus in the intricate relationships between guard cell signal perception, stomatal conductance, environmental humidity, and CO2 levels.

An atypical heterotrimeric Gα and its interactome suggest an extra-large role in overcoming abiotic and biotic stress.

Canonical heterotrimeric G-proteins (G-proteins) are comprised of Gα, Gβ, and Gγ subunits. G-proteins regulate multiple crucial plant growth and development processes, incorporating environmental responses. Besides Gα, Gβ and Gγ, the discovery of atypical Gα subunits termed as extra-large G-proteins or extra-large GTP-binding proteins (XLGs) makes G-protein signaling unique in plants. The C-terminus of XLG shares similarities with the canonical Gα subunits; the N-terminus harbors a nuclear localization signal (NLS) and is rich in cysteine. The earlier explorations suggest XLG's role in flowering, the development of embryos and seedlings, root morphogenesis, stamen development, cytokinin-induced development, stomatal opening and regulation of rice grain filling. The XLGs are also known to initiate signaling cascades that prime plants against a variety of abiotic and biotic stresses. They are also engaged in controlling several agronomic parameters such as rice panicle length, grain filling, grain size, and biomass, highlighting their potential contribution to crop improvement. The present review explores the remarkable properties of non-canonical Gα subunits (XLGs) and reflects on the various developmental, abiotic and biotic stress signaling pathways controlled by them. Moreover, the bottleneck dilemma of how a tiny handful of XLGs control a multiplicity of stress-responsive activities is partially resolved in this review by addressing the interaction of XLGs with different interacting proteins. XLG proteins presented in this review can be exploited to gain access to highly productive and stress-tolerant plants.

Extra-large G proteins have extra-large effects on agronomic traits and stress tolerance in maize and rice.

Heterotrimeric G proteins - comprising Gα, Gβ, and Gγ subunits - are ubiquitous elements in eukaryotic cell signaling. Plant genomes contain both canonical Gα subunit genes and a family of plant-specific extra-large G protein genes (XLGs) that encode proteins consisting of a domain with Gα-like features downstream of a long N-terminal domain. In this review we summarize phenotypes modulated by the canonical Gα and XLG proteins of arabidopsis and highlight recent studies in maize and rice that reveal dramatic phenotypic consequences of XLG clustered regularly interspaced short palindromic repeats (CRISPR) mutagenesis in these important crop species. XLGs have both redundant and specific roles in the control of agronomically relevant plant architecture and resistance to both abiotic and biotic stresses. We also point out areas of current controversy, suggest future research directions, and propose a revised, phylogenetically-based nomenclature for XLG protein genes.

Biased agonists of the chemokine receptor CXCR3 differentially signal through Gαi:β-arrestin complexes.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and β-arrestins, to influence nearly every biological process. The G protein and β-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and β-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gαi/o, but not other Gα protein subtypes, directly interacts with β-arrestin, regardless of the canonical Gα protein that is coupled to the GPCR. Here, we report that the endogenous biased chemokine agonists of CXCR3 (CXCL9, CXCL10, and CXCL11), together with two small-molecule biased agonists, differentially formed Gαi:β-arrestin complexes. Formation of the Gαi:β-arrestin complexes did not correlate well with either G protein activation or β-arrestin recruitment. β-arrestin biosensors demonstrated that ligands that promoted Gαi:β-arrestin complex formation generated similar β-arrestin conformations. We also found that Gαi:β-arrestin complexes did not couple to the mitogen-activated protein kinase ERK, as is observed with other receptors such as the V2 vasopressin receptor, but did couple with the clathrin adaptor protein AP-2, which suggests context-dependent signaling by these complexes. These findings reinforce the notion that Gαi:β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.

Heterotrimeric G Proteins in Plants: Canonical and Atypical Gα Subunits.

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors.

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs.

An atypical heterotrimeric Gα protein has substantially reduced nucleotide binding but retains nucleotide-independent interactions with its cognate RGS protein and Gβγ dimer

Plants uniquely have a family of proteins called extra-large G proteins (XLG) that share homology in their C-terminal half with the canonical Gα subunits; we carefully detail here that Arabidopsis XLG2 lacks critical residues requisite for nucleotide binding and hydrolysis which is consistent with our quantitative analyses. Based on microscale thermophoresis, Arabidopsis XLG2 binds GTPγS with an affinity 100 times lower than that to canonical Gα subunits. This means that given the concentration range of guanine nucleotide in plant cells, XLG2 is not likely bound by GTP in vivo. Homology modeling and molecular dynamics simulations provide a plausible mechanism for the poor nucleotide binding affinity of XLG2. Simulations indicate substantially stronger salt bridge networks formed by several key amino-acid residues of AtGPA1 which are either misplaced or missing in XLG2. These residues in AtGPA1 not only maintain the overall shape and integrity of the apoprotein cavity but also increase the frequency of favorable nucleotide-protein interactions in the nucleotide-bound state. Despite this loss of nucleotide dependency, XLG2 binds the RGS domain of AtRGS1 with an affinity similar to the Arabidopsis AtGPA1 in its apo-state and about 2 times lower than AtGPA1 in its transition state. In addition, XLG2 binds the Gβγ dimer with an affinity similar to that of AtGPA1. XLG2 likely acts as a dominant negative Gα protein to block G protein signaling. We propose that XLG2, independent of guanine nucleotide binding, regulates the active state of the canonical G protein pathway directly by sequestering Gβγ and indirectly by promoting heterodimer formation.Communicated by Ramaswamy H. Sarma

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides.

Fusarium 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 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) 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 associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.

BAK1-mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis.

Heterotrimeric G proteins consisting of Gα, Gβ and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein-coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr19 upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1T19A and GPA1T19D , suggesting that the dynamic Thr19 phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22-induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22-induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.

The Arabidopsis heterotrimeric G-protein β subunit, AGB1, is required for guard cell calcium sensing and calcium-induced calcium release.

Cytosolic calcium concentration ([Ca2+ ]cyt ) and heterotrimeric G-proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao ) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Cao responses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+ ]cyt oscillation; and inositol 1,4,5-trisphosphate (InsP3) production. Stomata in wild-type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Cao . By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double-mutants, as well as those of the agg1agg2Gγ double-mutant, were insensitive to Cao . These behaviors contrast with ABA-regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G-protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus-specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6-detected [Ca2+ ]cyt oscillations in response to Cao , initiating only a single [Ca2+ ]cyt spike. Experimentally imposed [Ca2+ ]cyt oscillations restored stomatal closure in agb1. Yeast two-hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Cao induced InsP3 production in Col but not in agb1. In sum, G-protein signaling via AGB1/AGG1/AGG2 is essential for Cao -regulation of stomatal apertures, and stomatal movements in response to Cao apparently require Ca2+ -induced Ca2+ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.

Role of heterotrimeric Gα proteins in maize development and enhancement of agronomic traits.

Plant shoot systems derive from the shoot apical meristems (SAMs), pools of stems cells that are regulated by a feedback between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. The maize heterotrimeric G protein α subunit COMPACT PLANT2 (CT2) functions with CLV receptors to regulate meristem development. In addition to the sole canonical Gα CT2, maize also contains three eXtra Large GTP-binding proteins (XLGs), which have a domain with homology to Gα as well as additional domains. By either forcing CT2 to be constitutively active, or by depleting XLGs using CRISPR-Cas9, here we show that both CT2 and XLGs play important roles in maize meristem regulation, and their manipulation improved agronomic traits. For example, we show that expression of a constitutively active CT2 resulted in higher spikelet density and kernel row number, larger ear inflorescence meristems (IMs) and more upright leaves, all beneficial traits selected during maize improvement. Our findings suggest that both the canonical Gα, CT2 and the non-canonical XLGs play important roles in maize meristem regulation and further demonstrate that weak alleles of plant stem cell regulatory genes have the capacity to improve agronomic traits.

The G Protein β-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement.

Heterotrimeric guanine nucleotide-binding (G) proteins are composed of Gα, Gβ, and Gγ subunits and function as molecular switches in signal transduction. In Arabidopsis (Arabidopsis thaliana), there are one canonical Gα (GPA1), three extra-large Gα (XLG1, XLG2, and XLG3), one Gβ (AGB1), and three Gγ (AGG1, AGG2, and AGG3) subunits. To elucidate AGB1 molecular signaling, we performed immunoprecipitation using plasma membrane-enriched proteins followed by mass spectrometry to identify the protein interactors of AGB1. After eliminating proteins present in the control immunoprecipitation, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. We identified all of the G protein subunits except XLG1, receptor-like kinases, Ca2+ signaling-related proteins, and 14-3-3-like proteins, all of which may couple with or modulate G protein signaling. We confirmed physical interaction between AGB1 and the receptor-like kinase FERONIA (FER) using bimolecular fluorescence complementation. The Rapid Alkalinization Factor (RALF) family of polypeptides have been shown to be ligands of FER. In this study, we demonstrate that RALF1 regulates stomatal apertures and does so in a G protein-dependent manner, inhibiting stomatal opening and promoting stomatal closure in Columbia but not in agb1 mutants. We further show that AGGs and XLGs, but not GPA1, participate in RALF1-mediated stomatal signaling. Our results suggest that FER acts as a G protein-coupled receptor for plant heterotrimeric G proteins.

Recently duplicated plant heterotrimeric Gα proteins with subtle biochemical differences influence specific outcomes of signal-response coupling

Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, regulate key signaling processes in eukaryotes. The Gα subunit determines the status of signaling by switching between inactive GDP-bound and active GTP-bound forms. Unlike animal systems, in which multiple Gα proteins with variable biochemical properties exist, plants have fewer, highly similar Gα subunits that have resulted from recent genome duplications. These proteins exhibit subtle differences in their GTP-binding, GDP/GTP-exchange, and GTP-hydrolysis activities, but the extent to which these differences contribute to affect plant signaling and development remains unknown. To evaluate this, we expressed native and engineered Gα proteins from soybean in an Arabidopsis Gα-null background and studied their effects on modulating a range of developmental and hormonal signaling phenotypes. Our results indicated that inherent biochemical differences in these highly similar Gα proteins are biologically relevant, and some proteins are more flexible than others in influencing the outcomes of specific signals. These observations suggest that alterations in the rate of the G-protein cycle itself may contribute to the specificity of response regulation in plants by affecting the duration of active signaling and/or by the formation of distinct protein-protein complexes. In species such as Arabidopsis having a single canonical Gα, this rate could be affected by regulatory proteins in the presence of specific signals, whereas in plants with multiple Gα proteins, an even more complex regulation may exist, which likely contributes to the specificity of signal-response coupling.

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein–coupled receptor signaling in platelets

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ1 revealed that Gβ1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ1-GST protein, and PP1c co-immunoprecipitated with Gβ1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ1 complexes with myristoylated Gβ1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ1 protein enlists PP1c to modulate GPCR signaling in platelets.

EXTRA-LARGE G PROTEINs Interact with E3 Ligases PUB4 and PUB2 and Function in Cytokinin and Developmental Processes.

Heterotrimeric GTP-binding proteins (G proteins) composed of Gα, Gβ, and Gγ subunits are conserved signal transduction molecules in animals and plants. In Arabidopsis (Arabidopsis thaliana), there are three Gα-like proteins named EXTRA-LARGE G PROTEINs (XLGs) in addition to the canonical Gα protein GPA1. XLGs have been reported to be implicated in multiple pathways, although the underlying mechanisms of their action remain elusive. Here, we report that all three XLGs interact with two closely related plant U-box (PUB) E3 ligases, PUB2 and PUB4. Three XLGs are predominantly localized at the plasma membrane, whereas XLG2 and XLG3 also show nuclear localization. The interactions between PUB2/4 and XLGs suggest that they might function in the same pathways. Indeed, we found that a newly obtained xlg1/2/3 triple knockout mutant, the pub4 mutant, and the pub2/4 double mutant all exhibited defects in cytokinin responses, stamen development, tapetum development, and male fertility. However, the xlg single mutants and the pub2 mutant did not exhibit an obvious defect in these processes, which suggests functional redundancy among the three XLGs and between PUB2 and PUB4. Overexpressing ARR10 to enhance the cytokinin response in pub4 or in the xlg1/2/3 triple mutant partially restored several phenotypes caused by the pub4 and xlg1/2/3 mutations. Our findings reveal that the XLGs and PUB2/4 are components in the complex cytokinin signaling networks regulating many developmental and physiological processes.

G proteins diverge and conquer

Evolution In plants, signaling through G (heterotrimeric GTP-binding) proteins involves either canonical Gα proteins, which are structurally and functionally similar to animal Gα proteins, or extra-large Gα (XLG) proteins. Urano et al. found that plant canonical Gα proteins evolved slowly, like their animal counterparts, and were involved in developmental processes. In contrast, XLG proteins rapidly diversified early in the land plant lineage and mediated stress responses. Thus, diversification of plant XLG proteins coincided with the colonization of land and may have helped plants to withstand the new stresses encountered in this environment. Sci. Signal. 9 , ra93 (2016).

Sporophyte formation and life cycle completion in moss requires heterotrimeric G-proteins

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.

The GCR1 and GPA1 Participate in Promotion of Arabidopsis Primary Root Elongation Induced by N-Acyl-Homoserine Lactones, the Bacterial Quorum-Sensing Signals

Many gram-negative bacteria use N-acyl-homoserine lactones (AHL) as quorum-sensing signals to coordinate their collective behaviors. Accumulating evidence indicates that plants can respond to AHL. However, little is known about the molecular mechanism of plants reacting to these bacterial signals. In this study, we show that the treatment of Arabidopsis roots with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) and N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) resulted in significant root elongation. The genetic analysis revealed that the T-DNA insertional mutants of gcr1, encoding a G-protein-coupled receptor GCR1, were insensitive to 3OC6-HSL or 3OC8-HSL in assays of root growth. The loss-of-function mutants of the sole canonical Gα subunit GPA1 showed no response to AHL promotion of root elongation whereas Gα gain-of-function plants overexpressing either the wild type or a constitutively active version of Arabidopsis Gα exhibited the exaggerated effect on root elongation caused by AHL. Furthermore, the expression of GCR1 and GPA1 were significantly upregulated after plants were contacted with both AHL. Taken together, our results suggest that GCR1 and GPA1 are involved in AHL-mediated elongation of Arabidopsis roots. This provides insight into the mechanism of plant responses to bacterial quorum-sensing signals.

Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1

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.

Plant volatiles regulate the activities of Ca2+-permeable channels and promote cytoplasmic calcium transients in Arabidopsis leaf cells

A variety of plant species emit volatile compounds in response to mechanical stresses such as herbivore attack. Although these volatile compounds promote gene expression leading to anti-herbivore responses, the underlying transduction mechanisms are largely unknown. While indirect evidence suggests that the cytoplasmic free Ca2+ concentration ([Ca2+]c) plays a crucial role in the volatile-sensing mechanisms in plants, these roles have not been directly demonstrated. In the present study, we used Arabidopsis leaves expressing apoaequorin, a Ca2+-sensitive luminescent protein, in combination with a luminometer, to monitor [Ca2+]c transients that occur in response to a variety of volatile compounds and to characterized the pharmacological properties of the increase in [Ca2+]c. When leaves were exposed to volatiles, [Ca2+]c was transiently raised. The [Ca2+]c increases induced by acyclic compounds were disrupted by Ruthenium Red, a potential plasma-membrane and endo-membrane Ca2+-permeable channel inhibitor, but not by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular Ca2+-chelator, suggesting that acyclic compounds promote Ca2+-release from intracellular stores. On the other hand, the electrophilic compound (E)-2-hexenal promoted Ca2+-influx via ROS production by natural oxidation at the aquarius phase. In a gpa1-2 mutant lacking a canonical Gα subunit, the [Ca2+]c transients induced by all tested volatiles were not attenuated, suggesting that G-protein coupled receptors are not involved in the volatile-induced [Ca2+]c transients in Arabidopsis leaves.