Heterotrimeric G protein α subunit is involved in rice brassinosteroid response

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways.The nullmutation of RGA(rice heterotrimeric G protein α subunit),which encodes the α subunit of heterotrimeric G proteinin rice,causes severe dwarfism and reduced responsiveness to gibberellic acid in rice.However,less is known aboutheterotrimeric G protein in brassinosteroid(BR)signaling,one of the well-understood phytohormone pathways.In thepresent study,we used root elongation inhibition assay,lamina inclination assay and coleoptile elongation analysis todemonstrated reduced sensitivity of dl mutant plants(caused by the null mutation of RGA)to 24-epibrassinolide(24-epiBL),which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development.Moreover,RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner.Our results show thatRGA is involved in rice brassinosteroid response,which may be beneficial to elucidate the molecular mechanisms of Gprotein signaling and provide a novel perspective to understand BR signaling in higher plants.

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.

Heterotrimeric G-protein is involved in phytochrome A-mediated cell death of Arabidopsis hypocotyls

The heterotrimeric guanine nucleotide-binding protein （G-protein） has been demonstrated to mediate various signaling pathways in plants. However, its role in phytochrome A （phyA） signaling remains elusive. In this study, we discover a new phyA-mediated phenotype designated far-red irradiation （FR） preconditioned cell death, which occurs only in the hypocotyls of FR-grown seedlings following exposure to white light （WL）. The cell death is mitigated in the Gα mutant gpal but aggravated in the Gβ mutant agbl in comparison with the wild type （WT）, indicative of antagonistic roles of GPA1 and AGBI in the phyA-mediated cell-death pathway. Further investigation indicates that FR-induced accumulation of nonphotoconvertible protochlorophyllide （Pchlide^633）, which generates reactive oxygen species （ROS） on exposure to WL, is required for FR-preconditioned cell death. Moreover, ROS is mainly detected in chloroplasts using the fluorescent probe. Interestingly, the application of H2O2 to dark-grown seedlings results in a phenotype similar to FR-preconditioned cell death. This reveals that ROS is a critical mediator for the ceil death. In addition, we observe that agb1 is more sensitive to H2O2 than WT seedlings, indicating that the G-protein may also modify the sensitivity of the seedlings to ROS stress. Taking these results together, we infer that the G-protein may be involved in the phyA signaling pathway to regulate FR-preconditioned cell death ofArabidopsis hypocotyls. A possible mechanism underlying the involvement of the G-protein in phyA signaling is discussed in this study.

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

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.

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.