scholarly journals Arabidopsis G-Protein β Subunit AGB1 Negatively Regulates DNA Binding of MYB62, a Suppressor in the Gibberellin Pathway

2021 ◽  
Vol 22 (15) ◽  
pp. 8270
Author(s):  
Xin Qi ◽  
Wensi Tang ◽  
Weiwei Li ◽  
Zhang He ◽  
Weiya Xu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transgenic Plants ◽  
G Protein ◽  
G Proteins ◽  
Growth And Development ◽  
Β Subunit ◽  
Wild Type ◽  
Binding Assays ◽  
Plant Growth And Development ◽  
Qrt Pcr

Plant G proteins are versatile components of transmembrane signaling transduction pathways. The deficient mutant of heterotrimeric G protein leads to defects in plant growth and development, suggesting that it regulates the GA pathway in Arabidopsis. However, the molecular mechanism of G protein regulation of the GA pathway is not understood in plants. In this study, two G protein β subunit (AGB1) mutants, agb1-2 and N692967, were dwarfed after exogenous application of GA3. AGB1 interacts with the DNA-binding domain MYB62, a GA pathway suppressor. Transgenic plants were obtained through overexpression of MYB62 in two backgrounds including the wild-type (MYB62/WT Col-0) and agb1 mutants (MYB62/agb1) in Arabidopsis. Genetic analysis showed that under GA3 treatment, the height of the transgenic plants MYB62/WT and MYB62/agb1 was lower than that of WT. The height of MYB62/agb1 plants was closer to MYB62/WT plants and higher than that of mutants agb1-2 and N692967, suggesting that MYB62 is downstream of AGB1 in the GA pathway. qRT-PCR and competitive DNA binding assays indicated that MYB62 can bind MYB elements in the promoter of GA2ox7, a GA degradation gene, to activate GA2ox7 transcription. AGB1 affected binding of MYB62 on the promoter of GA2ox7, thereby negatively regulating th eactivity of MYB62.

scholarly journals A Screen for Genes That Function in Abscisic Acid Signaling in Arabidopsis thaliana

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 1247-1255 ◽  
Author(s):  
Eiji Nambara ◽  
Masaharu Suzuki ◽  
Suzanne Abrams ◽  
Donald R McCarty ◽  
Yuji Kamiya ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Growth And Development ◽  
Plant Hormone ◽  
The Other ◽  
Aba Signaling ◽  
Wild Type ◽  
Plant Growth And Development ◽  
Diverse Range ◽  
Abscisic Acid Signaling ◽  
Aba Insensitive

Abstract The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development under a diverse range of environmental conditions. To identify genes functioning in ABA signaling, we have carried out a screen for mutants that takes advantage of the ability of wild-type Arabidopsis seeds to respond to (−)-(R)-ABA, an enantiomer of the natural (+)-(S)-ABA. The premise of the screen was to identify mutations that preferentially alter their germination response in the presence of one stereoisomer vs. the other. Twenty-six mutants were identified and genetic analysis on 23 lines defines two new loci, designated CHOTTO1 and CHOTTO2, and a collection of new mutant alleles of the ABA-insensitive genes, ABI3, ABI4, and ABI5. The abi5 alleles are less sensitive to (+)-ABA than to (−)-ABA. In contrast, the abi3 alleles exhibit a variety of differences in response to the ABA isomers. Genetic and molecular analysis of these alleles suggests that the ABI3 transcription factor may perceive multiple ABA signals.

scholarly journals G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

1998 ◽  
Vol 141 (7) ◽  
pp. 1529-1537 ◽  
Author(s):  
Barbara Peracino ◽  
Jane Borleis ◽  
Tian Jin ◽  
Monika Westphal ◽  
Jean-Marc Schwartz ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
G Protein ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Fluid Phase ◽  
Β Subunit ◽  
Wild Type ◽  
Fluid Phase Endocytosis ◽  
Green Fluorescent

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

scholarly journals Functional analysis of CgWRKY57 from Cymbidium goeringii in ABA response

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10982
Author(s):  
Huanhuan Liu ◽  
Lianping Wang ◽  
Xijun Jing ◽  
Yue Chen ◽  
Fengrong Hu
Keyword(s):  
Abiotic Stress ◽  
Transgenic Plants ◽  
Real Time ◽  
Growth And Development ◽  
Transmembrane Domain ◽  
Germination Rate ◽  
Wild Type ◽  
Abiotic Stress Response ◽  
Group Iii ◽  
Cymbidium Goeringii

Background The orchid is one of the top ten Chinese flowers and has high ornamental value and elegant color. However, orchids are vulnerable to abiotic stresses during their growth and development, and the molecular mechanism of the abiotic stress response in orchids is unclear. WRKY proteins belong to a transcription factor family that plays important roles in biotic stress, abiotic stress, growth and development in plants, but little is known about the WRKY family in Cymbidium goeringii. Methods The specific fragment of the CgWRKY57 gene of C. goeringii was analyzed by bioinformatics. The expression of the CgWRKY57 gene of C. goeringii under 4 °C, 42 °C water and ABA stress as well as different tissues was detected by real-time fluorescence quantitative PCR. CgWRKY57 gene was overexpressed in wild type Arabidopsis thaliana by inflorescence infection method, and the function of transgenic lines under ABA stress was analyzed. Results CgWRKY57 was cloned from C. goeringii and found to encode 303 amino acids. The CgWRKY57 protein is an acidic, nonsecreted hydrophilic protein without a signal peptide or transmembrane domain. The CgWRKY57 protein is located to the nucleus and may function intracellularly according to its predicted subcellular localization. A domain analysis and homology comparison showed that the CgWRKY57 protein has a “WRKYGQK” domain and belongs to Group III of the WRKY family, and a phylogenetic analysis demonstrated that CgWRKY57 is closely related to OsWRKY47. CgWRKY57 was expressed in the roots, stems, leaves and floral organs of C. goeringii, and its expression level was highest in the roots according to real-time qPCR analysis. There were significant differences in CgWRKY57 expression under 4 °C, 42 °C ABA and water stress treatments, and its expression changed greatly under ABA stress. The expression of CgWRKY57 in transgenic plants was significantly higher than that in wild type plants under ABA stress, and the root length and germination rate were reduced in transgenic plants compared to wild type plants. Conclusions These results indicate that CgWRKY57 overexpression is responsive to ABA stress, and they provide a foundation for future analyses of the biological functions of the WRKY family in C. goeringii.

scholarly journals Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins

1994 ◽  
Vol 297 (1) ◽  
pp. 189-193 ◽  
Author(s):  
A A Bominaar ◽  
P J M Van Haastert
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Wild Type ◽  
Inhibitory Pathway ◽  
Surface Receptors ◽  
G Protein Alpha Subunit ◽  
Camp Receptor ◽  
Protein Alpha Subunit

A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cyclic AMP (cAMP). This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild-type cells both cAMP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein alpha-subunit G alpha 2, showed no stimulation by either cAMP or GTP[S], indicating that G alpha 2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTP[S] was normal, suggesting that the defect in this mutant is upstream of the stimulatory G alpha 2. Inhibition of phospholipase C was achieved in wild-type cells by the partial antagonist 3′-deoxy-3′-aminoadenosine 3′,5′-phosphate (3′NH-cAMP). This inhibition was no longer observed in transformed cell lines lacking either the surface cAMP receptor cAR1 or the G-protein alpha-subunit G alpha 1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2.

scholarly journals Discrimination of DNA binding sites by mutant p53 proteins.

1995 ◽  
Vol 15 (9) ◽  
pp. 5196-5202 ◽  
Author(s):  
S K Thukral ◽  
Y Lu ◽  
G C Blain ◽  
T S Harvey ◽  
V L Jacobsen
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Base Pair ◽  
Molecular Genetic ◽  
Full Length ◽  
Single Amino Acid ◽  
Wild Type ◽  
Binding Assays ◽  
Wild Type P53

Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.

scholarly journals Identification of Potential Auxin-Responsive Small Signaling Peptides through a Peptidomics Approach in Arabidopsis thaliana

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3146 ◽  
Author(s):  
Weigui Luo ◽  
Yuan Xiao ◽  
Qiwen Liang ◽  
Yi Su ◽  
Langtao Xiao
Keyword(s):  
Arabidopsis Thaliana ◽  
Growth And Development ◽  
Plant Protoplasts ◽  
Wild Type ◽  
Plant Growth And Development ◽  
Technical Challenge ◽  
Low Concentrations ◽  
Defective Mutant ◽  
Optimized Protocol ◽  
Signaling Peptides

Small signaling peptides (SSPs) are a class of short peptides playing critical roles in plant growth and development. SSPs are also involved in the phytohormone signaling pathway. However, identification of mature SSPs is still a technical challenge because of their extremely low concentrations in plant tissue and complicated interference by many other metabolites. Here, we report an optimized protocol to extract SSPs based on protoplast extraction and to analyze SSPs based on tandem mass spectrometry peptidomics. Using plant protoplasts as the material, soluble peptides were directly extracted into phosphate buffer. The interference of non-signaling peptides was significantly decreased. Moreover, we applied the protocol to identify potential SSPs in auxin treated wild type and auxin biosynthesis defective mutant yuc2yuc6. Over 100 potential SSPs showed a response to auxin in Arabidopsis thaliana.

scholarly journals The Effect of the Upregulated Expression of Fe Regulators IMA1 and bHLH104 in Arabidopsis on the Root Length and Fe Homeostasis Under Pi Starvation

2021 ◽  
Author(s):  
Xiangxiang Meng ◽  
Wenfeng Li ◽  
Renfang Shen ◽  
Ping Lan
Keyword(s):  
Growth And Development ◽  
Primary Root ◽  
Mineral Nutrients ◽  
Wild Type ◽  
Plant Growth And Development ◽  
Pi Starvation ◽  
Significant Difference ◽  
Fe Uptake ◽  
Fe Homeostasis ◽  
Total P

Abstract Phosphate (Pi) and iron (Fe) are two essential mineral nutrients for plant growth and development. Pi starvation triggers the Fe local redistribution and over-accumulation, resulting in the reduction of the primary root, while represses the expression of Fe uptake genes. Nevertheless, the antagonistic mechanism between P and Fe nutrition in plant remain not addressed. Here, the effect of the upregulated expression of Fe regulators IMA1 and bHLH104 driven by the different-type promoters (proCaMV 35S, the promoters of Pi-starvation responsive genes proIPS1 and proPHT1;4) in response to Pi starvation was investigated in Arabidopsis. The results showed that the expression of Fe uptake genes IRT1 and FRO2 was successfully upregulated in proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 under Pi starvation while decreased in pro35S::IMA1, pro35S::bHLH104 and proPHT1;4::bHLH104, compared with that in the corresponding plants under Pi sufficiency. Although the length and Fe distribution in roots of them didn’t have significant difference with wild type under Pi starvation, the Fe distribution and total Fe contents were significantly increased in shoots of proIPS1::IMA1, proPHT1;4::IMA1 and proIPS1::bHLH104 while were decreased in proPHT1;4::bHLH104. The higher Fe concentrations in the Pi-starved transgenic plants also conferred the obviously tolerance to Fe deficiency. Their biomasses and total P concentrations showed no difference with wild type, regardless of Pi sufficiency or deficiency. Therefore, this approach would be a novel manipulation to modify Fe nutrient via coupling with Pi starvation in plants.

scholarly journals Suppression of Phytochrome-Interacting Factors Enhances Photoresponses of Seedlings and Delays Flowering With Increased Plant Height in Brachypodium distachyon

2021 ◽  
Vol 12 ◽  
Author(s):  
Quyen T. N. Hoang ◽  
Sharanya Tripathi ◽  
Jae-Yong Cho ◽  
Da-Min Choi ◽  
Ah-Young Shin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transgenic Plants ◽  
Plant Height ◽  
Growth And Development ◽  
Floral Induction ◽  
Leaf Growth ◽  
Chlorophyll Biosynthesis ◽  
Brachypodium Distachyon ◽  
Active Form ◽  
Cell Number

Phytochromes are red and far-red photoreceptors that regulate plant growth and development under ambient light conditions. During phytochrome-mediated photomorphogenesis, phytochrome-interacting factors (PIFs) are the most important signaling partners that regulate the expression of light-responsive genes. However, the function of PIFs in monocots has not been studied well. In this study, using RNA interference (RNAi), we investigated the functions of BdPIL1 and BdPIL3, two PIF-like genes identified in Brachypodium distachyon, which are closely related to Arabidopsis PIF1 and PIF3. The expression of their genes is light-inducible, and both BdPIL1 and BdPIL3 proteins interact with phytochromes in an active form-specific manner. Transgenic Brachypodium seedlings with the RNAi constructs of BdPIL1 and BdPIL3 showed decreased coleoptile lengths and increased leaf growth when exposed to both red and far-red light. In addition, the transgenic plants were taller with elongated internodes than wild-type Bd21-3 plant, exhibiting late flowering. Moreover, RNA-seq analysis revealed downregulation of many genes in the transgenic plants, especially those related to the regulation of cell number, floral induction, and chlorophyll biosynthesis, which were consistent with the phenotypes of increased plant height, delayed flowering, and pale green leaves. Furthermore, we demonstrated the DNA-binding ability of BdPIL1 and BdPIL3 to the putative target promoters and that the DNA-binding was inhibited in the presence of phytochromes. Therefore, this study determines a molecular mechanism underlying phytochrome-mediated PIF regulation in Brachypodium, i.e., sequestration, and also elucidates the functions of BdPIL1 and BdPIL3 in the growth and development of the monocot plant.

scholarly journals G protein- and β-arrestin Signaling Profiles of Endothelin Derivatives at the Type A Endothelin Receptor

Kidney360 ◽  
2021 ◽  
pp. 10.34067/KID.0005462020
Author(s):  
Xinyu Xiong ◽  
Nour Nazo ◽  
Ritika Revoori ◽  
Sudarshan Rajagopal ◽  
Matthew A. Sparks
Keyword(s):  
Cardiovascular System ◽  
G Protein ◽  
G Proteins ◽  
Knockout Mice ◽  
Type A ◽  
Physiological Effects ◽  
Endothelin Receptor ◽  
Wild Type ◽  
Biased Agonism ◽  
Wide Range

Background: Endothelin-1 (ET-1) is a potent vasoconstrictor in the cardiovascular system, an effect mediated through the type A endothelin receptor (ETAR), a G protein-coupled receptor (GPCR). Antagonist of the ETAR have shown promising results in randomized clinical trials. However, side effects limit widespread use. Biased agonists have been developed to mitigate untoward effects of a number of GPCR antagonist. These agents block deleterious G-coupled pathways while stimulating protective β-arrestin pathways. The goal of this study was to test whether there was any significant ligand bias between endothelin derivatives, and whether this could have any physiological effects in the cardiovascular system. Methods: A panel of endothelin derivatives were tested in assays of G protein signaling and β-arrestin 2 recruitment at the ETAR. We then tested the effects of ET-1 on the vasopressor response in wild type and β-arrestin 1 and 2 KO mice. Results: We found that the endothelins activated a wide range of G proteins at the ETAR, but none of the endothelin derivatives demonstrated significant biased agonism. Endothelin derivatives did display structure-activity relationships with regards to their degrees of agonism. β-arrestin 1 and 2 knockout mice did not display any differences to wild type mice in the acute pressor response to ET-1 and β-arrestin 2 knockout mice did not display any blood pressure differences to wild type mice in the chronic responses to ET-1. Conclusions: Our findings are consistent with vasoconstriction being mediated by G proteins with a lack of significant desensitization by β-arrestins at the ETAR. These findings suggest that G protein- and β-arrestin-biased ETAR agonists could have distinct physiological effects from balanced agonists, although the endothelin peptide scaffold does not appear suitable for designing such ligands.

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