Function of α subunit of heterotrimeric G protein in brassinosteroid response of rice plants

The α subunit of heterotrimeric G-proteins (Gα) is involved in a broad range of aspects of the brassinosteroid (BR) response, such as the enhancement of lamina bending. However, it has been suggested from epistatic analysis of d1 and d61, which are mutants deficient for Gα and the BR receptor BRI1, that Gα and BRI1 may function via distinct pathways in many cases. In this study, we investigated further the genetic interaction between Gα and BRI1. We report the analysis of transformants of T65d1 and T65d1/d61-7 into which were introduced a constitutively active form of Gα, Q223L. The application of 24-epi-brassinolide (24-epiBL) to T65d1 expressing Q223L still resulted in elongation of the coleoptile and, in fact, it was enhanced over the wild-type plant (WT) level in a concentration dependent manner. In T65d1/d61-7 expressing Q223L, the seed size was enlarged over that of d61-7 due to activation of Gα. These results suggest that Q223L is able to augment the BR response in response to 24-epiBL and also that Q223L functions independently of BRI1 in the process of determining seed morphology, given that Q223L was functional in the BRI1-deficient mutant, d61-7.Key words: brassinosteroid, BRASSINOSTEROID INSENSITIVE1 (BRI1), genetic interaction, G-protein α subunit, rice plants, seed morphology, transgenic plants     

Characterization of the putative α subunit of a heterotrimeric G protein in rice

A recombinant protein with a cDNA that encodes the putative subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice ) with an apparent molecular mass of 45 kDa was bound with guanosine 5-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its Kcat was 0.44. rGrice was ADP-ribosylated by activated cholera toxin.Monoclonal antibodies raised against rGrice reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice . A 45 kDa polypeptide in the plasma membrane, as well as rGrice , was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the subunits of heterotrimeric G proteins in animals, suggesting that the putative subunit is truly the subunit itself.     

Expression profile of the α subunit of the heterotrimeric G protein in rice

Previous studies on the activity of the rice Gα promoter using a β-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.Key words: expression pattern, Gα subunit, GUS staining pattern, heterotrimeric G protein, riceThe rice mutant d1 is deficient in the heterotrimeric G protein α subunit (Gα). Recently it was found that the dwarfism phenotype of d1 is due to a reduction in cell numbers.¹ This discovery has led to new questions regarding how rice Gα regulates cell number, and which other signaling molecules are involved in this process in various tissues and at different development stages. Studies of d1 suggest that rice Gα participates in both gibberellin signaling^2–4 and brassinosteroid signaling.^5–8 Promoter studies using the β-Glucuronidase (GUS) reporter indicate that Gα expression is highest in developing organs.¹ In this paper, we report on the expression pattern of a Gα promoter::GUS construct in seeds and developing leaves of rice.     

Tryptophan207 is involved in the GTP-dependent conformational switch in the α subunit of the G protein transducin: Chymotryptic digestion patterns of the GTPγS and GDP-bound forms

The limited proteolytic pattern of transducin,G _t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the α _t subunit were identified. The α _t subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas α _t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγ _t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γ _t subunit was not cleaved and only a small portion of β _t was digested into several fragments. In order to determine which proteolytic fragment of α _t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG _t previously³²P-labeled at Cys³⁴⁷ by petrussis toxin-catalyzed ADP-ribosylation. The³²P-label was mainly associated with the α _t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not³²P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of α _t , at Leu¹⁵ and Leu¹⁹. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of α _t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp²⁰⁷ in a conformation-dependent manner. Trp²⁰⁷ of α _t -GTPγS was resistant to proteolysis but α _t -GDP and the 38 kD fragments of α _t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp²⁰⁷ changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.     

Heterotrimeric G protein Gi is involved in a signal transduction pathway for ATP release from erythrocytes

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.     

Heterotrimeric G-protein subunit function in Candida albicans: both the alpha and beta subunits of the pheromone response G protein are required for mating

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Galpha subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Galpha subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gbeta subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Galpha and Gbeta subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.     

A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation

A Galpha subunit-encoding gene (CGA1) was cloned from Cochliobolus heterostrophus, a heterothallic foliar pathogen of corn. The deduced amino acid sequence showed similarity to Galpha proteins from other filamentous fungi and suggested that CGA1 is a member of the Galphai class. cga1 mutants had reduced ability to form appressoria on glass surfaces and on corn leaves; mutants nevertheless caused lesions on corn plants like those of wild type. cga1 mutants were female sterile; sexual development was completely abolished when the mutant allele was homozygous in a cross. Ascospores produced in crosses heterozygous at Cga1 were all wild type. The signal transduction pathway represented by CGA1 appears to be involved in developmental pathways leading to either appressorium formation or mating; in sexual development CGA1 is required for both fertility and ascospore viability.     

The heterotrimeric [corrected] G protein subunit G alpha i is present on mitochondria

Receptors that signal through heterotrimeric [corrected] GTP binding (G) proteins mediate the majority of intercellular communication. Recent evidence suggests that receptors acting through G proteins also transfer signals across the nuclear membrane. Here we present cell fractionation and immunolabeling data showing that the heterotrimeric [corrected] G protein subunit Galphai is associated with mitochondria. This finding suggests that G protein receptor signaling may be a feature common to all membranes.     

Heterotrimeric G protein alpha and beta subunits antagonistically modulate stomatal density in Arabidopsis thaliana

Stomata are essential for efficient gas and water-vapor exchange between the atmosphere and plants. Stomatal density and movement are controlled by a series of signal molecules including phytohormones and peptides as well as by environmental stimuli. It is known that heterotrimeric G-proteins play an important role in the ABA-inhibited stomatal opening. In this study, the G-protein signaling pathway was also found to regulate stomatal density on the lower epidermis of Arabidopsis cotyledons. The loss-of-function mutation of the G-protein α-subunit (GPA1) showed a reduction in stomatal density, while overexpression of the constitutively active form of GPA1^QL increased stomatal density, indicating a positive role of the active form of GPA1 in stomatal development. In contrast, stomatal density increased in the null mutant of the G-protein β-subunit (AGB1) but decreased in transgenic lines that overexpressed AGB1. Stomatal analysis of the gpa1 agb1 double mutants displayed an average value of stomatal density compared to the single mutants. Taken together, these results suggest that the stomatal density in Arabidopsis is modulated by GPA1 and AGB1 in an antagonistic manner.     

Heterotrimeric G protein signalling in the plant kingdom

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

Heterotrimeric G protein activation by G-protein-coupled receptors

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP-GDP exchange on the G protein alpha-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.