Upregulation of a KN1 homolog by transposon insertion promotes leafy head development in lettuce

Leafy head is a unique type of plant architecture found in some vegetable crops, with leaves bending inward to form a compact head. The genetic and molecular mechanisms underlying leafy head in vegetables remain poorly understood. We genetically fine-mapped and cloned a major quantitative trait locus controlling heading in lettuce. The candidate gene (LsKN1) is a homolog of knotted 1 (KN1) from Zea mays. Complementation and CRISPR/Cas9 knockout experiments confirmed the role of LsKN1 in heading. In heading lettuce, there is a CACTA-like transposon inserted into the first exon of LsKN1 (LsKN1▽). The transposon sequences act as a promoter rather than an enhancer and drive high expression of LsKN1▽. The enhanced expression of LsKN1▽ is necessary but not sufficient for heading in lettuce. Data from ChIP-sequencing, electrophoretic mobility shift assays, and dual luciferase assays indicate that the LsKN1▽ protein binds the promoter of LsAS1 and down-regulates its expression to alter leaf dorsoventrality. This study provides insight into plant leaf development and will be useful for studies on heading in other vegetable crops.

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Functional consensus for mammalian osmotic response elements

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.3.c667 ◽

1999 ◽

Vol 276 (3) ◽

pp. C667-C673 ◽

Cited By ~ 36

Author(s):

Joan D. Ferraris ◽

Chester K. Williams ◽

Akihiko Ohtaka ◽

Arlyn García-Pérez

Keyword(s):

Molecular Mechanisms ◽

Full Range ◽

Renal Medulla ◽

Binding Activity ◽

Organic Osmolytes ◽

Mobility Shift ◽

Osmotic Response ◽

Eukaryotic Gene ◽

Electrophoretic Mobility Shift Assays ◽

Response Region

The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp −1105 to −1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (−1108 to −1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.

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Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

10.1101/230136 ◽

2018 ◽

Author(s):

Ana Rita Borba ◽

Tânia S. Serra ◽

Alicja Górska ◽

Paulo Gouveia ◽

André M. Cordeiro ◽

...

Keyword(s):

Transcription Factors ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Mobility Shift ◽

Helix Loop Helix ◽

Specific Accumulation ◽

Cell Type Specific ◽

Bhlh Transcription Factors ◽

Electrophoretic Mobility Shift Assays ◽

Element Pair

AbstractC4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

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The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16004684 ◽

2016 ◽

Vol 72 (5) ◽

pp. 356-361 ◽

Cited By ~ 4

Author(s):

Catarina S. Silva ◽

Xuelei Lai ◽

Max Nanao ◽

Chloe Zubieta

Keyword(s):

Dna Binding ◽

Molecular Mechanisms ◽

Binding Activity ◽

Mobility Shift ◽

Data Set ◽

Dna Binding Activity ◽

Protein Dna Interactions ◽

Reported Data ◽

Electrophoretic Mobility Shift Assays ◽

Myb Domain

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock.Luxmutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein–DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using theMeshAndCollectworkflow available at the ESRF.

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p24G1 Encoded by Grapevine Leafroll-Associated Virus 1 Suppresses RNA Silencing and Elicits Hypersensitive Response-Like Necrosis in Nicotiana Species

Viruses ◽

10.3390/v12101111 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1111

Author(s):

Chen-Wei Zhang ◽

Qing Liu ◽

Qi Zeng ◽

Wen-Ting Huang ◽

Qi Wang ◽

...

Keyword(s):

Hypersensitive Response ◽

Rna Silencing ◽

Molecular Mechanisms ◽

Potato Virus X ◽

Mobility Shift ◽

Nicotiana Species ◽

P24 Protein ◽

Systemic Rna ◽

Interfering Rna ◽

Electrophoretic Mobility Shift Assays

Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.

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Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653723 ◽

1995 ◽

Vol 73 (01) ◽

pp. 039-048 ◽

Cited By ~ 12

Author(s):

A Bierhaus ◽

Ch J Hemmer ◽

N Mackman ◽

R Kutob ◽

R Ziegler ◽

...

Keyword(s):

Tissue Factor ◽

Falciparum Malaria ◽

De Novo ◽

Neutralizing Antibody ◽

Mobility Shift ◽

Before And After ◽

Tf Gene ◽

Electrophoretic Mobility Shift Assays ◽

Antiparasitic Treatment ◽

Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

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Transcriptomic Analysis of Short-Term Salt-Stress Response in Mega Hybrid Rice Seedlings

Agronomy ◽

10.3390/agronomy11071328 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1328

Author(s):

Noushin Jahan ◽

Yang Lv ◽

Mengqiu Song ◽

Yu Zhang ◽

Liangguang Shang ◽

...

Keyword(s):

Salt Stress ◽

Molecular Mechanisms ◽

Oryza Sativa L ◽

Transcriptome Profiling ◽

Bhlh Transcription Factor ◽

F1 Hybrid ◽

Salt Stress Response ◽

Productivity Losses ◽

Expression Of Genes ◽

Trait Locus

Salinity is a major abiotic stressor that leads to productivity losses in rice (Oryza sativa L.). In this study, transcriptome profiling and heterosis-related genes were analyzed by ribonucleic acid sequencing (RNA-Seq) in seedlings of a mega rice hybrid, Liang-You-Pei-Jiu (LYP9), and its two parents 93–11 and Pei-ai64s (PA64s), under control and two different salinity levels, where we found 8292, 8037, and 631 salt-induced differentially expressed genes (DEGs), respectively. Heterosis-related DEGs were obtained higher after 14 days of salt treatment than after 7 days. There were 631 and 4237 salt-induced DEGs related to heterosis under 7-day and 14-day salt stresses, respectively. Gene functional classification showed the expression of genes involved in photosynthesis activity after 7-day stress treatment, and in metabolic and catabolic activity after 14 days. In addition, we correlated the concurrence of an expression of DEGs for the bHLH transcription factor and a shoot length/salinity-related quantitative trait locus qSL7 that we fine-mapped previously, providing a confirmed case of heterosis-related genes. This experiment reveals the transcriptomic divergence of the rice F1 hybrid and its parental lines under control and salt stress state, and enlightens about the significant molecular mechanisms developed over time in response to salt stress.

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Arabidopsis MADS-box factor AGL16 negatively regulates drought resistance via stomatal density and stomatal movement

Journal of Experimental Botany ◽

10.1093/jxb/eraa303 ◽

2020 ◽

Vol 71 (19) ◽

pp. 6092-6106 ◽

Cited By ~ 3

Author(s):

Ping-Xia Zhao ◽

Zi-Qing Miao ◽

Jing Zhang ◽

Si-Yan Chen ◽

Qian-Qian Liu ◽

...

Keyword(s):

Drought Stress ◽

Drought Resistance ◽

Molecular Mechanisms ◽

Stomatal Density ◽

Stomatal Closure ◽

Negative Regulator ◽

Stomatal Development ◽

Mads Box ◽

Mobility Shift ◽

Aba Metabolism

Abstract Drought is one of the most important environmental factors limiting plant growth and productivity. The molecular mechanisms underlying plant drought resistance are complex and not yet fully understood. Here, we show that the Arabidopsis MADS-box transcription factor AGL16 acts as a negative regulator in drought resistance by regulating stomatal density and movement. Loss-of-AGL16 mutants were more resistant to drought stress and had higher relative water content, which was attributed to lower leaf stomatal density and more sensitive stomatal closure due to higher leaf ABA levels compared with the wild type. AGL16-overexpressing lines displayed the opposite phenotypes. AGL16 is preferentially expressed in guard cells and down-regulated in response to drought stress. The expression of CYP707A3 and AAO3 in ABA metabolism and SDD1 in stomatal development was altered in agl16 and overexpression lines, making them potential targets of AGL16. Using chromatin immunoprecipitation, transient transactivation, yeast one-hybrid, and electrophoretic mobility shift assays, we demonstrated that AGL16 was able to bind the CArG motifs in the promoters of the CYP707A3, AAO3, and SDD1 and regulate their transcription, leading to altered leaf stomatal density and ABA levels. Taking our findings together, AGL16 acts as a negative regulator of drought resistance by modulating leaf stomatal density and ABA accumulation.

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An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽

10.1182/blood.v98.8.2555 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2555-2562 ◽

Cited By ~ 26

Author(s):

Mark Loyevsky ◽

Timothy LaVaute ◽

Charles R. Allerson ◽

Robert Stearman ◽

Olakunle O. Kassim ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Regulatory Protein ◽

Protein S ◽

Fold Increase ◽

Binding Activity ◽

Mobility Shift ◽

Iron Responsive Element ◽

Element Binding Protein ◽

Responsive Element ◽

Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

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Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

Molecular Endocrinology ◽

10.1210/mend.11.11.9971 ◽

1997 ◽

Vol 11 (11) ◽

pp. 1651-1658 ◽

Cited By ~ 1

Author(s):

Limin Liu ◽

Douglas Leaman ◽

Michel Villalta ◽

R. Michael Roberts

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Gene Promoter ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Α Subunit ◽

Choriocarcinoma Cells ◽

Human Choriocarcinoma Cells ◽

Electrophoretic Mobility Shift Assays ◽

Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

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Thyroid hormones regulate phosphate homoeostasis through transcriptional control of the renal type IIa sodium-dependent phosphate co-transporter (Npt2a) gene

Biochemical Journal ◽

10.1042/bj20090671 ◽

2010 ◽

Vol 427 (1) ◽

pp. 161-169 ◽

Cited By ~ 15

Author(s):

Mariko Ishiguro ◽

Hironori Yamamoto ◽

Masashi Masuda ◽

Mina Kozai ◽

Yuichiro Takei ◽

...

Keyword(s):

Thyroid Hormone ◽

Thyroid Hormones ◽

Molecular Mechanisms ◽

Hormone Receptors ◽

Critical Role ◽

Serum Phosphate ◽

Luciferase Assay ◽

Mobility Shift ◽

Intron 1 ◽

Sodium Dependent

The type IIa renal sodium-dependent phosphate (Na/Pi) co-transporter Npt2a is implicated in the control of serum phosphate levels. It has been demonstrated previously that renal Npt2a protein and its mRNA expression are both up-regulated by the thyroid hormone T3 (3,3′,5-tri-iodothyronine) in rats. However, it has never been established whether the induction was mediated by a direct effect of thyroid hormones on the Npt2a promoter. To address the role of Npt2a in T3-dependent regulation of phosphate homoeostasis and to identify the molecular mechanisms by which thyroid hormones modulate Npt2a gene expression, mice were rendered pharmacologically hypo- and hyper-thyroid. Hypothyroid mice showed low levels of serum phosphate and a marked decrease in renal Npt2a protein abundance. Importantly, we also showed that Npt2a-deficient mice had impaired serum phosphate responsiveness to T3 compared with wild-type mice. Promoter analysis with a luciferase assay revealed that the transcriptional activity of a reporter gene containing the Npt2a promoter and intron 1 was dependent upon TRs (thyroid hormone receptors) and specifically increased by T3 in renal cells. Deletion analysis and EMSAs (electrophoretic mobility-shift assays) determined that there were unique TREs (thyroid-hormone-responsive elements) within intron 1 of the Npt2a gene. These results suggest that Npt2a plays a critical role as a T3-target gene, to control phosphate homoeostasis, and that T3 transcriptionally activates the Npt2a gene via TRs in a renal cell-specific manner.

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