Extracellular ATP Promotes Stomatal Opening of Arabidopsis thaliana through Heterotrimeric G Protein α Subunit and Reactive Oxygen Species

In recent years, adenosine tri-phosphate （ATP） has been reported to exist in apoplasts of plant cells as a signal molecule. Extracellular ATP （eATP） plays important roles in plant growth, development, and stress tolerance. Here, extra- cellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness. ADP, GTP, and weakly hydrolyzable ATP analogs （ATPγS, Bz-ATP, and 2meATP） showed similar effects, whereas AMP and adenosine did not affect stomatal movement. Apyrase inhibited stomatal opening. ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor （diphenylene iodonium） or deoxidizer （dithiothreitol）, and was impaired in null mutant of NADPH ox- idase （atrbohD/F）. Added ATP triggered ROS generation in guard cells via NADPH oxidase. ATP also induced Ca^2＋ influx and H ＋ efflux in guard cells. In atrbohD/F, ATP-induced ion flux was strongly suppressed. In null mutants of the heterotrimeric G protein α subunit, ATP-promoted stomatal opening, cytoplasmic ROS generation, Ca^2＋ influx, and ^H＋ efflux were all sup- pressed. These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca^2＋, and plasma membrane H＋-ATPase.

Involvement of heterotrimeric G protein in signal transduc-tion of extracellular calmodu-lin in regulating rbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulating rbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhanced rbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibited rbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin on rbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum on rbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely

Activation effect of extracellular calmodulin on heterotrimeric G protein in pollen plasma membrane

IN recent years, calmodulin （CaM）, an important Ca2+ receptor and constituent of cellular signal transduction systems, has been found extracellularly. We have verified that CaM is presented extracellularly in all of plant species we have examined. In addition, we have reported that extracellular CaM has some biological significance, such as stimulation of cell proliferation, cell wall regeneration, initiation of pollen germination and tube growth and inducement of rbcS gene expression. The role of heterotrimeric G proteins in pollen germination, tube growth and signal transduction of extracellular CaM has been examined in Lily pollen, and two kinds of antibodies against animal Gzα internal sequence and N-terminal

G-protein β subunit AGB1 positively regulates salt stress tolerance in Arabidopsis

The heterotrimeric GTP-binding proteins（G-proteins） in eukaryotes consisted of α, β and γ subunits and are important in molecular signaling by interacting with G-protein-coupled receptors（GPCR）, on which to transduce signaling into the cytoplast through appropriate downstream effectors. However, downstream effectors regulated by the G-proteins in plants are currently not well defined. In this study, the transcripts of AGB1, a G protein β subunit gene in Arabidopsis were found to be down-regulated by cold and heat, but up-regulated by high salt stress treatment. AGB1 mutant（agb1-2） was more sensitive to high salt stress than wild-type（WT）. Compared with WT, the cotyledon greening rates, fresh weight, root length, seedling germination rates and survival rates decreased more rapidly in agb1-2 along with increasing concentrations of Na Cl in normal（MS） medium. Physiological characteristic analysis showed that compared to WT, the contents of chlorophyll, relative proline accumulation and peroxidase（POD） were reduced, whereas the malonaldehyde（MDA） content and concentration ratio of Na＋/K＋ were increased in agb1-2 under salt stress condition. Further studies on the expression of several stress inducible genes associated with above physiological processes were investigated, and the results revealed that the expressions of genes related to proline biosynthesis, oxidative stress response, Na＋ homeostasis, stress- and ABAresponses were lower in agb1-2 than in WT, suggesting that those genes are possible downstream genes of AGB1 and that their changed expression plays an important role in determining phenotypic and physiologic traits in agb1-2. Taken together, these findings indicate that AGB1 positively regulates salt tolerance in Arabidopsis through its modulation of genes transcription related to proline biosynthesis, oxidative stress, ion homeostasis, stress- and ABA-responses.

Arabidopsis EXTRA-LARGE G PROTEIN 1(XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity

Pattern-triggered immunity(PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI.Of the three non-canonical EXTRA-LARGE G PROTEINs(XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity,but XLG1 was not considered to function in defense,based on the analysis of a weak xlg1 allele.In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern(PAMP)-triggered activation of mitogen-activated protein kinases(MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.

Photoexcited CRYPTOCHROME 1 Interacts Directly with G-Protein 13 Subunit AGB1 to Regulate the DNA-Binding Activity of HY5 and Photomorphogenesis in Arabidopsis

Light and the heterotrimeric G-protein are known to antagonistically regulate photomorphogenesis in Arabidopsis. However, whether light and G-protein coordinate the regulation of photomorphogenesis is largely unknown. Here we show that the blue light photoreceptor cryptochrome 1 （CRY1） physically inter-acts with the G-protein β subunit, AGB1, in a blue light-dependent manner. We also show that AGB1 directly interacts with HY5, a basic leucine zipper transcriptional factor that acts as a critical positive regulator of photomorphogenesis, to inhibit its DNA-binding activity. Genetic studies suggest that CRY1 acts partially through AGB1, and AGB1 acts partially through HY5 to regulate photomorphogenesis. Moreover, we demonstrate that blue light-triggered interaction of CRY1 with AGB1 promotes the dissociation of HY5 from AGB1. Our results suggest that the CRY1 signaling mechanism involves positive regulation of the DNA-binding activity of HY5 mediated by the CRY1-AGB1 interaction, which inhibits the association of AGB1 with HY5. We propose that the antagonistic regulation of HY5 DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize photomorphogenesis.

Control of grain size by G protein signaling in rice

Heterotrimeric G proteins are involved in multiple cellular processes in eukaryotes by sensing and transducing various signals. G protein signaling in plants is quite different from that in animals, and the mechanisms of plant G protein signaling are still largely unknown. Several recent studies have provided new insights into the mechanisms of G protein signaling in rice grain size and yield control. In this review,we summarize recent advances on the function of G proteins in rice grain size control and discuss the potential genetic and molecular mechanisms of plant G protein signaling.

Phytochrome B and AGB1 Coordinately Regulate Photomorphogenesis by Antagonistically Modulating PIF3 Stability in Arabidopsis

Phytochrome B (phyB), the primary red light photoreceptor, promotes photomorphogenesis in Arabidopsis by interacting with the basic helix-loop-helix transcriptional factor PIF3 and inducing its phosphorylation and degradation. Heterotrimeric G proteins are known to regulate various developmental processes in plants and animals. In Arabidopsis, the G-protein β subunit AGB1 is known to repress photomorphogenesis. However, whether and how phyB and AGB1 coordinately regulate photomorphogenesis are largely unknown. Here we show that phyB physically interacts with AGB1 in a red light-dependent manner and that AGB1 interacts directly with PIF3. Moreover, we demonstrate that the AGB1-PIF3 interaction inhibits the association of PIF3 with phyB, leading to reduced phosphorylation and degradation of PIF3, whereas the phyB-AGB1 interaction represses the association of PIF3 with AGB1, resulting in enhaneed phosphorylation and degradation of PIF3. Our results suggest that phyB and AGB1 antagonistically regulate PIF3 stability by dynamically interacting with each other and PIF3. This dynamic mechanism may allow plants to balanee phyB and G-protein signaling to optimize photomorphogenesis.

Arabidopsis cryptochrome 1 promotes stomatal development through repression of AGB1 inhibition of SPEECHLESS DNA-binding activity

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. The heterotrimeric G-protein is known to regulate various physiological processes in plants and mammals. In Arabidopsis, cryptochrome 1(CRY1) and the G-protein β subunit AGB1 act antagonistically to regulate stomatal development.The molecular mechanism by which CRY1 and AGB1 regulate this process remains unknown.Here, we show that Arabidopsis CRY1 acts partially through AGB1, and AGB1 acts through SPEECHLESS(SPCH), a master transcription factor that drives stomatal initiation and proliferation, to regulate stomatal development. We demonstrate that AGB1 physically interacts with SPCH to block the b HLH DNA-binding domain of SPCH and inhibit its DNA-binding activity. Moreover, we demonstrate that photoexcited CRY1 represses the interaction of AGB1 with SPCH to release AGB1 inhibition of SPCH DNA-binding activity, leading to the expression of SPCH-target genes promoting stomatal development. Taken together, our results suggest that the mechanism by which CRY1 promotes stomatal development involves positive regulation of the DNA-binding activity of SPCH mediated by CRY1 inhibition of the AGB1-SPCH interaction. We propose that the antagonistic regulation of SPCH DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize stomatal density and pattern.

G protein controls stress readiness by modulating transcriptional and metabolic homeostasis in Arabidopsis thaliana and Marchantia polymorpha

The core G protein signaling module,which consists of Gαand extra-large Gα(XLG)subunits coupled with the Gβγdimer,is a master regulator of various stress responses.In this study,we compared the basal and salt stress-induced transcriptomic,metabolomic and phenotypic profiles in Gα,Gβ,and XLG-null mutants of two plant species,Arabidopsis thaliana and Marchantia polymorpha,and showed that G protein mediates the shift of transcriptional and metabolic homeostasis to stress readiness status.We demonstrated that such stress readiness serves as an intrinsic protection mechanism against further stressors through enhancing the phenylpropanoid pathway and abscisic acid responses.Furthermore,WRKY transcription factors were identified as key intermediates of G protein-mediated homeostatic shifts.Statistical and mathematical model comparisons between A.thaliana and M.polymorpha revealed evolutionary conservation of transcriptional and metabolic networks over land plant evolution,whereas divergence has occurred in the function of plant-specific atypical XLG subunit.Taken together,our results indicate that the shifts in transcriptional and metabolic homeostasis at least partially act as the mechanisms of G protein-coupled stress responses that are conserved between two distantly related plants.