scholarly journals Activation of the Transcription of BrGA20ox3 by a BrTCP21 Transcription Factor Is Associated with Gibberellin-Delayed Leaf Senescence in Chinese Flowering Cabbage during Storage

2019 ◽  
Vol 20 (16) ◽  
pp. 3860
Author(s):  
Xian-mei Xiao ◽  
Yan-mei Xu ◽  
Ze-xiang Zeng ◽  
Xiao-li Tan ◽  
Zong-li Liu ◽  
...  
Keyword(s):  
Transient Expression ◽  
Leaf Senescence ◽  
Biosynthetic Pathway ◽  
Transcript Level ◽  
Mobility Shift ◽  
Transient Expression Assay ◽  
Catabolic Genes ◽  
Mobility Shift Assay ◽  
Psii Quantum Yield ◽  
Senescence Associated Genes

Several lines of evidence have implicated the involvement of the phytohormone gibberellin (GA) in modulating leaf senescence in plants. However, upstream transcription factors (TFs) that regulate GA biosynthesis in association with GA-mediated leaf senescence remain elusive. In the current study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP21 in GA-delayed leaf senescence in Chinese flowering cabbage. Exogenous GA3 treatment maintained a higher value of maximum PSII quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the repression of the expression of senescence-associated genes and chlorophyll catabolic genes, which led to the delay of leaf senescence. A class I member of TCP TFs BrTCP21, was further isolated and characterized. The transcript level of BrTCP21 was low in senescing leaves, and decreased following leaf senescence, while GA3 could keep a higher expression level of BrTCP21. BrTCP21 was further found to be a nuclear protein and exhibit trans-activation ability through transient-expression analysis in tobacco leaves. Intriguingly, the electrophoretic mobility shift assay (EMSA) and transient expression assay illustrated that BrTCP21 bound to the promoter region of a GA biosynthetic gene BrGA20ox3, and activated its transcription. Collectively, these observations reveal that BrTCP21 is associated with GA-delayed leaf senescence, at least partly through the activation of the GA biosynthetic pathway. These findings expand our knowledge on the transcriptional mechanism of GA-mediated leaf senescence.

scholarly journals BrTCP7 Transcription Factor Is Associated with MeJA-Promoted Leaf Senescence by Activating the Expression of BrOPR3 and BrRCCR

2019 ◽  
Vol 20 (16) ◽  
pp. 3963 ◽  
Author(s):  
Yan-mei Xu ◽  
Xian-mei Xiao ◽  
Ze-xiang Zeng ◽  
Xiao-li Tan ◽  
Zong-li Liu ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Transcriptional Activity ◽  
Nuclear Protein ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Transient Expression Assay ◽  
Catabolic Genes ◽  
Meja Treatment ◽  
Mobility Shift Assay ◽  
Chlorophyll Catabolite

The plant hormone jasmonic acid (JA) has been recognized as an important promoter of leaf senescence in plants. However, upstream transcription factors (TFs) that control JA biosynthesis during JA-promoted leaf senescence remain unknown. In this study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP7 in methyl jasmonate (MeJA)-promoted leaf senescence in Chinese flowering cabbage. Exogenous MeJA treatment reduced maximum quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the increased expression of senescence marker and chlorophyll catabolic genes, and accelerated leaf senescence. To further understand the transcriptional regulation of MeJA-promoted leaf senescence, a class I member of TCP TFs BrTCP7 was examined. BrTCP7 is a nuclear protein and possesses trans-activation ability through subcellular localization and transcriptional activity assays. A higher level of BrTCP7 transcript was detected in senescing leaves, and its expression was up-regulated by MeJA. The electrophoretic mobility shift assay and transient expression assay showed that BrTCP7 binds to the promoter regions of a JA biosynthetic gene BrOPR3 encoding OPDA reductase3 (OPR3) and a chlorophyll catabolic gene BrRCCR encoding red chlorophyll catabolite reductase (RCCR), activating their transcriptions. Taken together, these findings reveal that BrTCP7 is associated with MeJA-promoted leaf senescence at least partly by activating JA biosynthesis and chlorophyll catabolism, thus expanding our knowledge of the transcriptional mechanism of JA-mediated leaf senescence.

Characterization of LgnR, an IclR family transcriptional regulator involved in the regulation of l-gluconate catabolic genes in Paracoccus sp. 43P

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 623-634 ◽  
Author(s):  
Tetsu Shimizu ◽  
Akira Nakamura
Keyword(s):  
Constitutive Expression ◽  
Transcriptional Regulator ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Dnase I Footprinting ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Mobility Shift Assay

Five genes encoding enzymes required for l-gluconate catabolism, together with genes encoding components of putative ABC transporters, are located in a cluster in the genome of Paracoccus sp. 43P. A gene encoding a transcriptional regulator in the IclR family, lgnR, is located in front of the cluster in the opposite direction. Reverse transcription PCR analysis indicated that the cluster was transcribed as an operon, termed the lgn operon. Two promoters, P lgnA and P lgnR , are divergently located in the intergenic region, and transcription from these promoters was induced by addition of l-gluconate or d-idonate, a catabolite of l-gluconate. Deletion of lgnR resulted in constitutive expression of lgnA, lgnH and lgnR, indicating that lgnR encodes a repressor protein for the expression of the lgn operon and lgnR itself. Electrophoretic mobility shift assay and DNase I footprinting analyses revealed that recombinant LgnR binds to both P lgnA and P lgnR , indicating that LgnR represses transcription from these promoters by competing with RNA polymerase for binding to these sequences. d-Idonate was identified as a candidate effector molecule for dissociation of LgnR from these promoters. Phylogenetic analysis revealed that LgnR formed a cluster with putative proteins from other genome sequences, which is distinct from those proteins of known regulatory functions, in the IclR family of transcriptional regulators. Additionally, the phylogeny suggests an evolutionary linkage between the l-gluconate catabolic pathway and d-galactonate catabolic pathways distributed in Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria.

scholarly journals TaWRKY13-A Serves as a Mediator of Jasmonic Acid-Related Leaf Senescence by Modulating Jasmonic Acid Biosynthesis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hualiang Qiao ◽  
Yongwei Liu ◽  
Lingling Cheng ◽  
Xuelin Gu ◽  
Pengcheng Yin ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Leaf Senescence ◽  
Brachypodium Distachyon ◽  
Wrky Transcription Factor ◽  
Luciferase Reporter ◽  
Virus Induced Gene Silencing ◽  
Reporter System ◽  
Mobility Shift ◽  
Yield And Quality ◽  
Mobility Shift Assay

Leaf senescence is crucial for crop yield and quality. Transcriptional regulation is a key step for integrating various senescence-related signals into the nucleus. However, few regulators of senescence implicating transcriptional events have been functionally characterized in wheat. Based on our RNA-seq data, we identified a WRKY transcription factor, TaWRKY13-A, that predominately expresses at senescent stages. By using the virus-induced gene silencing (VIGS) method, we manifested impaired transcription of TaWRKY13-A leading to a delayed leaf senescence phenotype in wheat. Moreover, the overexpression (OE) of TaWRKY13-A accelerated the onset of leaf senescence under both natural growth condition and darkness in Brachypodium distachyon and Arabidopsis thaliana. Furthermore, by physiological and molecular investigations, we verified that TaWRKY13-A participates in the regulation of leaf senescence via jasmonic acid (JA) pathway. The expression of JA biosynthetic genes, including AtLOX6, was altered in TaWRKY13-A-overexpressing Arabidopsis. We also demonstrated that TaWRKY13-A can interact with the promoter of AtLOX6 and TaLOX6 by using the electrophoretic mobility shift assay (EMSA) and luciferase reporter system. Consistently, we detected a higher JA level in TaWRKY13-A-overexpressing lines than that in Col-0. Moreover, our data suggested that TaWRKY13-A is partially functional conserved with AtWRKY53 in age-dependent leaf senescence. Collectively, this study manifests TaWRKY13-A as a positive regulator of JA-related leaf senescence, which could be a new clue for molecular breeding in wheat.

scholarly journals The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Chihiro Oda-Yamamizo ◽  
Nobutaka Mitsuda ◽  
Shingo Sakamoto ◽  
Daisuke Ogawa ◽  
Masaru Ohme-Takagi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transgenic Plants ◽  
Leaf Senescence ◽  
Loss Of Function ◽  
Pheophorbide A ◽  
Delayed Senescence ◽  
Positive Regulator ◽  
Catabolic Genes ◽  
Senescence Associated Genes ◽  
Senescence Phenotype

Abstract Chlorophyll (Chl) degradation occurs during leaf senescence, embryo degreening, bud breaking, and fruit ripening. The Chl catabolic pathway has been intensively studied and nearly all the enzymes involved are identified and characterized; however, the molecular regulatory mechanisms of this pathway are largely unknown. In this study, we performed yeast one-hybrid screening using a transcription factor cDNA library to search for factors controlling the expression of Chl catabolic genes. We identified ANAC046 as a common regulator that directly binds to the promoter regions of NON-YELLOW COLORING1, STAY-GREEN1 (SGR1), SGR2, and PHEOPHORBIDE a OXYGENASE. Transgenic plants overexpressing ANAC046 exhibited an early-senescence phenotype and a lower Chl content in comparison with the wild-type plants, whereas loss-of-function mutants exhibited a delayed-senescence phenotype and a higher Chl content. Microarray analysis of ANAC046 transgenic plants showed that not only Chl catabolic genes but also senescence-associated genes were positively regulated by ANAC046. We conclude that ANAC046 is a positive regulator of Arabidopsis leaf senescence and exerts its effect by controlling the expression of Chl catabolic genes and senescence-associated genes.

scholarly journals LcNAC13 Physically Interacts with LcR1MYB1 to Coregulate Anthocyanin Biosynthesis-Related Genes during Litchi Fruit Ripening

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 135 ◽  
Author(s):  
Guoxiang Jiang ◽  
Zhiwei Li ◽  
Yunbo Song ◽  
Hong Zhu ◽  
Sen Lin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Fruit Ripening ◽  
Regulatory Networks ◽  
Anthocyanin Biosynthesis ◽  
Anthocyanin Accumulation ◽  
Mobility Shift ◽  
Transient Expression Assay ◽  
Mobility Shift Assay ◽  
Litchi Fruit ◽  
Tf Gene

Anthocyanin accumulation is crucial for the development of quality for most fruit. The mechanism underlying the regulation of anthocyanin biosynthesis by transcription factors in litchi fruit remains largely unknown. In this study, we isolated one NAC (NAM, ATAF1/2 and CUC2) TF gene, LcNAC13. Expression of LcNAC13 was upregulated as ripening proceeded, followed by the accumulation of anthocyanins. Electrophoretic mobility shift assay (EMSA) and transient expression assay showed that LcNAC13 could negatively regulate the expression of anthocyanin biosynthesis-related genes, including LcCHS1/2, LcCHI, LcF3H, LcF3’H, LcDFR, and LcMYB1. Furthermore, LcR1MYB1, as one R1-MYB type MYB, was identified to physically interact with LcNAC13 and reverse the effect of LcNAC13. Taken together, these results suggested that LcNAC13 and LcR1MYB1 may act together to antagonistically regulate anthocyanin biosynthesis during litchi fruit ripening, which helps to provide new insights into the regulatory networks of anthocyanin biosynthesis.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot

2021 ◽  
Author(s):  
Weiguo Dong ◽  
Wenqing Ren ◽  
Xuan Wang ◽  
He Yuke
Keyword(s):  
Plant Resistance ◽  
Host Susceptibility ◽  
Stem Rot ◽  
Sclerotinia Stem Rot ◽  
Sequencing Data ◽  
Mobility Shift ◽  
Expression Levels ◽  
Affected Plant ◽  
Mobility Shift Assay ◽  
Rot Disease

Abstract MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infections by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affected plant resistance to stem rot disease of Sclerotinia sclerotiorum. In the plants of Brassica rapa infected with S. sclerotiorum, miR319a levels increased while expression levels of several BraTCP genes significantly decreased compared with those of the uninfected plants. The overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas the overexpression of BraTCP4-1 promoted the plant resistance. Our RNA-sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation (ChIP) assay, electrophoretic mobility shift assay (EMSA) and reporter transaction assay showed that BraTCP4-1 was bound to the promoters of WRKY75, WRKY70, and WRKY33 genes and directly activated these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in the plants overexpressing BraMIR319a declined significantly whereas those of the plants overexpressing BraTCP4-1 increased significantly. These results suggest that miR319a and its targeted gene BraTCP4 regulate stem rot resistance through pathways of WRKY genes.

Amifostine Inhibits Hematopoietic Progenitor Cell Apoptosis by Activating NF-κB/Rel Transcription Factors

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4060-4066 ◽  
Author(s):  
Maria Fiammetta Romano ◽  
Annalisa Lamberti ◽  
Rita Bisogni ◽  
Corrado Garbi ◽  
Antonio M. Pagnano ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Apoptosis ◽  
Progenitor Cell ◽  
Cell Types ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Mobility Shift ◽  
Human Cord Blood ◽  
Mobility Shift Assay ◽  
Induced Apoptosis

Abstract We investigated the involvement of NF-κB/Rel transcription factors that reportedly can inhibit apoptosis in various cell types in the antiapoptotic mechanism of the cytoprotectant amifostine. In the nontumorigenic murine myeloid progenitor 32D cells incubated with amifostine, we detected a reduction of the IκB cytoplasmic levels by Western blotting and a raising of nuclear NF-κB/Rel complexes by electrophoretic mobility shift assay. Amifostine inhibited by more than 30% the growth factor deprivation-induced apoptosis, whereas its effect failed when we blocked the NF-κB/Rel activity with an NF-κB/Rel-binding phosphorothioate decoy oligodeoxynucleotide. In human cord blood CD34+ cells, the NF-κB/Rel p65 subunit was detectable (using immunofluorescence analysis) mainly in the cytoplasm in the absence of amifostine, whereas its presence was appreciable in the nuclei of cells incubated with the cytoprotectant. In 4 CD34+ samples incubated for 3 days in cytokine-deficient conditions, cell apoptosis was reduced by more than 30% in the presence of amifostine (or amifostine plus a control oligo); the effect of amifostine was abolished in cultures with the decoy oligo. These findings indicate that the inhibition of hematopoietic progenitor cell apoptosis by amifostine requires the induction of NF-κB/Rel factors and that the latter can therefore exert an antiapoptotic activity in the hematopoietic progenitor cell compartment. Furthermore, the identification of this specific mechanism underlying the survival-promoting activity of amifostine lends support to the possible use of this agent in apoptosis-related pathologies, such as myelodysplasias.

scholarly journals DNA-binding specificity of GATA family transcription factors.

1993 ◽  
Vol 13 (7) ◽  
pp. 3999-4010 ◽  
Author(s):  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Binding Specificity ◽  
Mobility Shift ◽  
Binding Domains ◽  
Dna Binding Specificity ◽  
Mobility Shift Assay ◽  
Gata Family

GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.

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