GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response

2021 ◽  
Author(s):  
Zemin Wang ◽  
Darren Chern Jan Wong ◽  
Yi Wang ◽  
Guangzhao Xu ◽  
Chong Ren ◽  
...  
Keyword(s):  
Stress Response ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Cold Treatment ◽  
Ectopic Expression ◽  
Bimolecular Fluorescence Complementation ◽  
Luciferase Reporter ◽  
Vitis Amurensis ◽  
Mobility Shift

Abstract Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

scholarly journals Global identification of full-length cassava lncRNAs unveils the role of CRIR1 in cold stress response

2021 ◽  
Author(s):  
shuxia li ◽  
Zhihao Cheng ◽  
Shiman Dong ◽  
Zhibo Li ◽  
Liangping Zou ◽  
...  
Keyword(s):  
Stress Response ◽  
Cold Stress ◽  
Cold Treatment ◽  
Cold Shock Protein ◽  
Translation Efficiency ◽  
Global Identification ◽  
Non Coding Rnas ◽  
Stress Related Genes ◽  
Rna Chaperones

Long non-coding RNAs (lncRNAs) have been considered to be important regulators of gene expression in a range of biological processes in plants. A large number of lncRNAs have been identified in plants. However, most of their biological functions still remain to be determined. Here, we identified total 3 004 lncRNAs in cassava under normal or cold-treated conditions from Iso-seq data. We further characterized a lincRNA, CRIR1, as a novel positive regulator of the plant response to cold stress. CRIR1 can be significantly induced by cold treatment. Overexpression of CRIR1 in cassava enhanced the cold tolerance of transgenic plants. Transcriptome analysis demonstrated that CRIR1 regulates a range of cold stress-related genes in a CBF-independent pathway. We further found that CRIR1 RNA can interact with MeCSP5, a homolog of the cold shock protein that acts as RNA chaperones, indicating that CRIR1 may recruit MeCSP5 to improve the translation efficiency of mRNA. In summary, our study greatly extends the repertoire of lncRNAs in plants as well as its responding to cold stress. Moreover, it reveals a sophisticated mechanism by which CRIR1 regulates plant cold stress response by modulating the expression of stress-responsive genes and increasing the translational yield.

scholarly journals Characterization of Chromatin Accessibility and Gene Expression Upon Cold Stress Reveals the Transcription Factor RAV1 Functions in Cold Response in Vitis amurensis

2021 ◽  
Author(s):  
Chong Ren ◽  
Li Huayang ◽  
Zemin Wang ◽  
Zhanwu Dai ◽  
Fatma Lecourieux ◽  
...  
Keyword(s):  
Cold Stress ◽  
Cold Tolerance ◽  
Regulatory Networks ◽  
Cold Hardiness ◽  
Target Genes ◽  
Plant Cell Wall ◽  
Cold Treatment ◽  
Vitis Amurensis ◽  
Rna Seq ◽  
Cold Response

Abstract Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. A number of 9 TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde (MDA) content and high peroxidase (POD) activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for rapid identification of TFs in response to cold stress in grapevine. The VaRAV1 may play an important role in adaption to cold stress.

scholarly journals Overexpression of the Apple (Malus× domestica) MdERF100 in Arabidopsis Increases Resistance to Powdery Mildew

2021 ◽  
Vol 22 (11) ◽  
pp. 5713
Author(s):  
Yiping Zhang ◽  
Li Zhang ◽  
Hai Ma ◽  
Yichu Zhang ◽  
Xiuming Zhang ◽  
...  
Keyword(s):  
Stress Response ◽  
Powdery Mildew ◽  
Malus Domestica ◽  
Molecular Mechanisms ◽  
Powdery Mildew Resistance ◽  
Bimolecular Fluorescence Complementation ◽  
Bhlh Protein ◽  
Mildew Resistance ◽  
Sa Signaling

APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors play important roles in plant development and stress response. Although AP2/ERF genes have been extensively investigated in model plants such as Arabidopsis thaliana, little is known about their role in biotic stress response in perennial fruit tree crops such as apple (Malus × domestica). Here, we investigated the role of MdERF100 in powdery mildew resistance in apple. MdERF100 localized to the nucleus but showed no transcriptional activation activity. The heterologous expression of MdERF100 in Arabidopsis not only enhanced powdery mildew resistance but also increased reactive oxygen species (ROS) accumulation and cell death. Furthermore, MdERF100-overexpressing Arabidopsis plants exhibited differential expressions of genes involved in jasmonic acid (JA) and salicylic acid (SA) signaling when infected with the powdery mildew pathogen. Additionally, yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that MdERF100 physically interacts with the basic helix–loop–helix (bHLH) protein MdbHLH92. These results suggest that MdERF100 mediates powdery mildew resistance by regulating the JA and SA signaling pathways, and MdbHLH92 is involved in plant defense against powdery mildew. Overall, this study enhances our understanding of the role of MdERF genes in disease resistance, and provides novel insights into the molecular mechanisms of powdery mildew resistance in apple.

scholarly journals The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta)

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Shihang Sun ◽  
Chungen Hu ◽  
Xiujuan Qi ◽  
Jinyong Chen ◽  
Yunpeng Zhong ◽  
...  
Keyword(s):  
Cold Stress ◽  
Freezing Tolerance ◽  
Luciferase Reporter ◽  
Dna Interactions ◽  
Functional Protein ◽  
Actinidia Arguta ◽  
Protein Dna Interactions ◽  
Similar Phenotype ◽  
Reporter Assays

AbstractBeta-amylase (BAM) plays an important role in plant resistance to cold stress. However, the specific role of the BAM gene in freezing tolerance is poorly understood. In this study, we demonstrated that a cold-responsive gene module was involved in the freezing tolerance of kiwifruit. In this module, the expression of AaBAM3.1, which encodes a functional protein, was induced by cold stress. AaBAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance, and the heterologous overexpression of AaBAM3.1 in Arabidopsis thaliana resulted in a similar phenotype. The results of promoter GUS activity and cis-element analyses predicted AaCBF4 to be an upstream transcription factor that could regulate AaBAM3.1 expression. Further investigation of protein-DNA interactions by using yeast one-hybrid, GUS coexpression, and dual luciferase reporter assays confirmed that AaCBF4 directly regulated AaBAM3.1 expression. In addition, the expression of both AaBAM3.1 and AaCBF4 in kiwifruit responded positively to cold stress. Hence, we conclude that the AaCBF-AaBAM module is involved in the positive regulation of the freezing tolerance of kiwifruit.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

Detection of a Complex That Associates With the Bβ Fibrinogen G−455-A Polymorphism

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3286-3293
Author(s):  
Erika T. Brown ◽  
Gerald M. Fuller
Keyword(s):  
Luciferase Reporter ◽  
Molecular Evidence ◽  
Luciferase Reporter Gene ◽  
Nucleotide Substitutions ◽  
Mobility Shift ◽  
Binding Characteristics ◽  
Base Sequences ◽  
American Society ◽  
Electrophoretic Mobility Shift Assays

The promoter region of the Bβ fibrinogen gene containing the polymorphic site (G−455-A) shows an increase in fibrinogen levels for individuals containing an adenine rather than a guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were performed using specific DNA probes containing base sequences pertinent to the allelic site. Specific DNA binding proteins were detected and their binding characteristics were determined. Secondly, we placed DNA fragments containing different −455 nucleotide substitutions of the Bβ promoter upstream of a luciferase reporter gene and transfected them into HepG2 cells to determine their effect on transactivation. An adenine at position −455 resulted in greater luciferase activity than when a guanine was present. UV cross-linking bound protein to the DNA demonstrated a 47-kD protein binding preferentially to the site when a guanine rather than an adenine was present at −455. We hypothesize that a transactivation protein complex associates with the site, but its association is stronger when guanine is present, thereby slowing downstream Bβ gene transcription. These data provide the first molecular evidence that accounts for the increase in fibrinogen in individuals carrying this allele.© 1998 by The American Society of Hematology.

Krüppel-like factor 4 represses transcription of the survivin gene in esophageal cancer cell lines

2009 ◽  
Vol 390 (5/6) ◽  
Author(s):  
Guo Zhang ◽  
Hongxia Zhu ◽  
Yihua Wang ◽  
Shangbin Yang ◽  
Mei Liu ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Binding Sites ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Aberrant Expression ◽  
Survivin Gene ◽  
Survivin Promoter

Abstract Aberrant expression of survivin has been shown to be regulated at the transcription level in cancer cells. In this study, we demonstrate that there are six putative binding sites of Krüppel-like factor 4 (KLF4) within the 2000-bp region upstream of the transcription start site of the human survivin gene. Luciferase reporter gene assays revealed that survivin promoter activity is repressed upon overexpression of KLF4 in EC9706 cells. A chromatin immunoprecipitation assay indicated that KLF4 indeed binds the survivin promoter in vivo. It specifically binds the site located at position -40 among the six binding sites as determined by electrophoretic mobility shift assay. Ectopic expression of KLF4 decreases the mRNA and protein levels of survivin. Furthermore, overexpression of survivin partially reverses KLF4-induced cell apoptosis. These results indicate that KLF4 is a transcriptional repressor of the human survivin gene in esophageal squamous cancer cells.

scholarly journals Comparative Transcriptomics for Pepper (Capsicum annuum L.) under Cold Stress and after Rewarming

2021 ◽  
Vol 11 (21) ◽  
pp. 10204
Author(s):  
Wu Miao ◽  
Jingshuang Song ◽  
Yanwu Huang ◽  
Rongyun Liu ◽  
Gaofeng Zou ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cold Stress ◽  
Cold Tolerance ◽  
Inbred Line ◽  
Soluble Sugar ◽  
Cold Treatment ◽  
Nac Transcription Factors ◽  
Physiological Indicators ◽  
Damage Repair ◽  
Cytochrome P450 Family

Cold stress has become one of the main abiotic stresses in pepper, which severely limits the growth and development of pepper. In this study, the physiological indicators and transcriptome of a cold-tolerance (CT) inbred line A188 and a cold-sensitive (CS) inbred line A122 under cold–rewarm treatments were studied; the aim of this study was to determine the potential of the key factors in pepper response to cold stress. Compared with CT, CS wilts more seriously after cold stress, with poor resilience, higher content of malondialdehyde, and lower content of soluble sugar and total chlorophyll. Moreover, during cold treatment, 7333 and 5953 differentially expressed genes (DEGs) were observed for CT and CS, respectively. These DEGs were significantly enriched in pathways related to photosynthesis, plant hormone signal transduction, and DNA damage repair. Interestingly, in addition to the widely studied transcription factors related to cold, it was also found that 13 NAC transcription factors increased significantly in the T4 group; meanwhile, the NAC8 (Capana02g003557) and NAC72 (Capana07g002219) in CT were significantly higher than those in CS under rewarming for 1 h after 72 h cold treatment. Notably, weighted gene coexpression network analysis identified four positively correlated modules and eight hub genes, including zinc finger proteins, heat shock 70 kda protein, and cytochrome P450 family, which are related to cold tolerance. All of these pathways and genes may be responsible for the response to cold and even the cold tolerance in pepper.

scholarly journals CDPK6 phosphorylates and stabilizes MYB30 to promote hyperoside biosynthesis that prolongs the duration of full-blooming in okra

2020 ◽  
Vol 71 (14) ◽  
pp. 4042-4056
Author(s):  
Qing Yang ◽  
Biying Dong ◽  
Litao Wang ◽  
Zhihua Song ◽  
Lili Niu ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Fold Increase ◽  
Bimolecular Fluorescence Complementation ◽  
Dependent Manner ◽  
Flavonoid Pathway ◽  
Calcium Dependent ◽  
Key Enzyme ◽  
Dependent Protein ◽  
The Relationship

Abstract The flowers of okra (Abelmoschus esculentus) open and wilt within only a few hours, and this is accompanied by accumulation of hyperoside, a secondary metabolite in the flavonoid pathway. However, little is known about the relationship between flavonoids and flowering. Here, we found that exogenous application of hyperoside extended the duration of the full-blooming period by more than 3-fold, and this was accompanied by a 14.7-fold increase in the expression of CALCIUM-DEPENDENT PROTEIN KINASE6 (AeCDPK6). Gene expression profiling indicated that the transcription factor AeMYB30 was co-expressed with AeCDPK6, and detailed protein interaction and phosphorylation experiments together with yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated an interaction between AeMYB30 and AeCDPK6. AeCDPK6 specifically phosphorylated AeMYB30S191, leading to increased protein stability and prevention of degradation. Furthermore, AeMYB30 directly bound to the promoter of AeUF3GaT1, a key enzyme in the hyperoside biosynthesis pathway. Analysis of transgenic plants showed that AeCDPK6 was required for the hyperoside-induced phosphorylation of AeMYB30 to enhance its stability and transcriptional activity. Ectopic expression of AeCDPK6 promoted hyperoside accumulation and prolonged the full-blooming period in an AeMYB30-dependent manner. Our results indicate the role of AeCDPK6–AeMYB30 in the molecular mechanism by which hyperoside regulates the period of full blooming in okra, a plant with a short duration of flowering.

Genes Identified in a Hemopoietic Lineage Switch Influence Transcription.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1250-1250
Author(s):  
Louise N. Winteringham ◽  
Raelene Endersby ◽  
Ian J. Majewski ◽  
Jennifer Beaumont ◽  
Simon Kobelke ◽  
...  
Keyword(s):  
Myeloid Leukaemia ◽  
Globin Gene ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Phenotypic Change ◽  
Subcellular Localisation ◽  
Erythroid Cells ◽  
Mobility Shift ◽  
Lineage Switch

Abstract The J2E erythroblastoid cell line responds to erythropoietin (Epo) by morphological maturation and hemoglobin synthesis. However, on rare occasions, these cells have undergone a spontaneous lineage switch and display features of monoblastoid cells which do not respond to Epo. Amongst the genes up-regulated in the monoblastoid variants were Hemopoietic lineage switch (Hls) 5 and 7. Hls5 is a recently identified member of the RING finger, B Box, Coiled coil (RBCC) or tripartite motif (TRIM) family, which includes PML, a gene involved in acute promyelocytic leukaemia. Hls7 is the murine orthologue of Myeloid leukaemia factor 1 (Mlf1), a gene involved in a t(3;5), associated with acute myeloid leukaemia. We have shown previously that Hls7/Mlf1 imposes a dramatic phenotypic change upon the erythroid cells, rendering them monoblastoid (Williams, J. et al EMBO 1999). We have studied the role of Hls5 and Mlf1 in erythroid commitment and differentiation. Ectopic expression of Hls5 inhibits globin production in erythroid cells and suppresses development of B-FUE and C-FUE. A yeast-two-hybrid screen identified FOG-1 as a binding partner of Hls5. Significantly, FOG-1 is a transcriptional co-regulator of GATA-1, which controls globin gene expression. While Hls5 is able to enhance the repression of GATA-1 activity imposed by FOG-1, it is also able to repress GATA-1 transcriptional activity in the absence of FOG-1. Using electrophoretic mobility shift assay we have shown that Hls5 is able to reduce GATA-1 binding to DNA in a dose dependant manner. This observation that Hls5 reduces GATA-1 binding to promoter elements is mirrored by chromatin immunoprecipitation assays. Expression of MLF1 is highest in CD34+ cells, but is markedly down regulated during erythroid differentiation. Microarray analysis identified a number of known transcriptional regulators differentially expressed in the presence of Mlf1 including ets1, Myc intron binding protein and Tbr2. Mlf1 is able to bind DNA and luciferase reporter assays demonstrated that Mlf1 is able to affect transcription. In addition, Mlf1 interacts with a novel member of the hnRNP family viz Mlf1 associated nuclear protein (Manp). Manp binds to DNA, is able to influence the subcellular localisation of Mlf1 by translocating Mlf1 from the cytoplasm to the nucleus. Importantly, Manp also has an affect on transcription. These data demonstrate that both Hls5 and Mlf1 affect transcription of genes associated with erythroid differentiation.

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