The short-period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana

Development ◽  
1998 ◽  
Vol 125 (3) ◽  
pp. 485-494 ◽  
Author(s):  
D.E. Somers ◽  
A.A. Webb ◽  
M. Pearson ◽  
S.A. Kay
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Clock ◽  
Red Light ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Environmental Signals ◽  
Developmental States ◽  
Short Period ◽  
Log Linear

The coordination of developmental and physiological events with environmental signals is facilitated by the action of the circadian clock. Here we report a new set of circadian clock-controlled phenotypes for Arabidopsis thaliana. We use these markers together with the short-period mutant, toc1-1, and the clock-controlled cab2::luciferase reporter gene to assess the nature of the circadian clock throughout development and to suggest the position of TOC1 within the circadian clock system. In dark-grown seedlings, the toc1-1 lesion conferred a short period to the cycling of cab2::luciferase luminescence, as previously found in light-grown plants, indicating that the circadian clocks in these two divergent developmental states share at least one component. Stomatal conductance rhythms were similarly approximately 3 hours shorter than wild type in toc1-1, suggesting that a cell-autonomous clockwork may be active in guard cells in 5- to 6-week-old leaves. The effect of daylength on flowering time in the C24 ecotype was diminished by toc1-1, and was nearly eliminated in the Landsberg erecta background where the plants flowered equally early in both short and long days. Throughout a 500-fold range of red light intensities, both the wild type and the mutant showed an inverse log-linear relationship of fluence rate to period, with a 2–3 hour shorter period for the mutant at all intensities. These results indicate that TOC1 acts on or within the clock independently of light input. Temperature entrainment appears normal in toc1-1, and the period-shortening effects of the mutant remain unchanged over a 20 degrees C temperature range. Taken together our results are consistent with the likelihood that TOC1 codes for an oscillator component rather than for an element of an input signaling pathway. In addition, the pervasive effect of toc1-1 on a variety of clock-controlled processes throughout development suggests that a single circadian system is primarily responsible for controlling most, if not all, circadian rhythms in the plant.

scholarly journals Light Gradient-Based Screening of Arabidopsis thaliana on a 384-Well Type Plant Array Chip

Micromachines ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 191
Author(s):  
Youn-Hee Park ◽  
Je-Kyun Park
Keyword(s):  
Arabidopsis Thaliana ◽  
White Light ◽  
Solid Medium ◽  
Red Light ◽  
Phytochrome B ◽  
Wild Type ◽  
Light Gradient ◽  
Gradient Based ◽  
Light Effects ◽  
Germination And Growth

Arabidopsis thaliana (Arabidopsis), as a model for plant research, is widely used for various aspects of plant science. To provide a more sophisticated and microscopic environment for the germination and growth of Arabidopsis, we report a 384-well type plant array chip in which each Arabidopsis seed is independently seeded in a solid medium. The plant array chip is made of a poly(methyl methacrylate) (PMMA) acrylic material and is assembled with a home-made light gradient module to investigate the light effects that significantly affect the germination and growth of Arabidopsis. The light gradient module was used to observe the growth pattern of seedlings according to the intensity of the white light and to efficiently screen for the influence of the white light. To investigate the response to red light (600 nm), which stimulates seed germination, the light gradient module was also applied to the germination test. As a result, the germination results showed that the plant array chip can be used to simultaneously screen wild type seeds and phytochrome B mutant seeds on a single array chip according to the eight red light intensities.

RUNX1/CBFA2 Regulates Myosin Light Chain 9 (MYL9) in Megakaryocytic Cells: Decreased MYL9 Expression in Human RUNX1 Haplodeficiency.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1831-1831
Author(s):  
Gauthami S Jalagadugula ◽  
Gurpreet Kaur ◽  
Guangfen Mao ◽  
Danny Dhanasekaran ◽  
A. Koneti Rao
Keyword(s):  
Transcription Factor ◽  
Protein Binding ◽  
Platelet Function ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Construct ◽  
Hel Cells

Abstract RUNX1 (also known as CBFA2 or AML1) is a transcription factor that plays a major role in hematopoiesis. Haplodeficiency of RUNX1 has been associated with familial thrombocytopenia, impaired megakaryopoiesis, impaired platelet function and predisposition to acute myeloid leukemia. We have reported a patient with inherited thrombocytopenia and abnormal platelet function (Gabbeta et al, Blood87:1368–76, 1996). The patient platelets showed impaired phosphorylation of pleckstrin and myosin light chain, diminished GPIIb-IIIa activation and decreased platelet protein kinase C-𝛉. This was associated with a heterozygous nonsense mutation in transcription factor RUNX1 (Sun et al, Blood103: 948–54, 2004). Platelet transcript profiling showed a striking downregulation of myosin light chain 9 (MYL9) by ~77-fold relative to normal platelets (Sun et al, J. Thromb Haemost.5: 146–54, 2007). Myosin light chains (MLCs) play an important role in platelet responses to activation, in platelet biogenesis, and are involved in cellular processes such as cytokinesis, cell adhesion, cell contraction, cell migration. We have addressed the hypothesis that MYL9 is a direct transcriptional target of RUNX1. Studies were performed in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation. To determine endogenous interaction of RUNX1 with MYL9 promoter, we performed chromatin immunoprecipitation (ChIP) assay using anti-RUNX1 antibody. These studies revealed RUNX1 binding to MYL9 chromatin at −742/−529 bp upstream of the ATG codon. TFSEARCH revealed four RUNX1 sites within this region. We performed electrophoretic mobility shift assay (EMSA) using probes containing each of the RUNX1 motifs and PMA-treated nuclear extracts from HEL cells. With each probe, protein binding was observed that was competed by excess unlabelled probe and inhibited by anti-RUNX1 antibody indicating RUNX1 as the protein involved. This protein binding was not competed by oligos containing mutations in the specific RUNX1 sites. No binding was noted directly to the mutant probes. To further corroborate our findings, we performed transient-ChIP analysis where wild type luciferase reporter construct −691/+4 and constructs with each of the RUNX1 sites individually mutated were transiently transfected into HEL cells. ChIP was performed using these cells and anti-RUNX1 antibody, and the expression analyzed by PCR amplification with a forward primer from MYL9 promoter sequence and reverse primer from luciferase vector sequence. Amplification was observed with immunoprecipitated wild type construct but not with any of the mutant constructs. Thus, RUNX1 interacts in vivo with MYL9 promoter, and the multiple RUNX1 sites interact with each other, as also shown for other genes. To test the functional relevance, the wild type construct −691/+4 containing all 4 RUNX1 sites or mutant constructs with each site individually deleted were cloned into firefly luciferase reporter gene vector and transfected into HEL cells. Deletion of RUNX1 site 1, 2, 3 or 4 caused ~60–90% reduction in the activity indicating that each site was functional. Lastly, siRNA mediated knock down of RUNX1 in HEL cells was associated with a decrease in both RUNX1 and MYL9 protein. Conclusions: Our results provide the first evidence that MYL9 gene is transcriptionally regulated by RUNX1. They provide evidence for the presence of multiple RUNX1 sites in MYL9 promoter, as also observed in other genes. Moreover, these studies provide a cogent mechanism for the MYL9 transcript downregulation and the impaired MLC-phosphorylation we have previously described in association with RUNX1 haplodeficiency.

scholarly journals The Coding sequence of firefly luciferase reporter gene affects specific hyperexpressionArabidopsis thaliana cpl1mutant

2017 ◽  
Author(s):  
Hisashi Koiwa ◽  
Akihito Fukudome
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Coding Region ◽  
Luciferase Reporter Gene ◽  
Coding Sequence ◽  
Firefly Luciferase Reporter ◽  
Endogenous Genes ◽  
Firefly Luciferase Reporter Gene

AbstractForward genetic screening of mutants using firefly luciferase (LUC) reporter gene became a standard practice in plant research. Such screenings frequently identified alleles ofCPL1(Carboxyl-terminal Phosphatase-Like 1) regardless of promoters or pathways studied. Expression of the corresponding endogenous genes often shows the minimal difference between wild type andcpl1.Here we show that theLUCcoding sequence is responsible for the high expression incpl1,using a classicalRD29a-LUC. Deletion of theLUC3’-UTR did not change hyperactivation ofLUCincpl1.However, a codon-modifiedLUC(LUC2) produced similar expression levels both in wild type and incpl1. These results indicate that the coding region ofLUCis responsible for thecpl1-specificLUCoverexpression uncoupled with the expression of the endogenous counterpart.

scholarly journals Rhizosphere microbes influence host circadian clock function

2021 ◽  
Author(s):  
Charley Hubbard ◽  
Robby McMinn ◽  
Cynthia Weinig
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Clock ◽  
Microbial Communities ◽  
Abiotic Factors ◽  
Circadian Period ◽  
Local Conditions ◽  
Mutant Genotype ◽  
Short Period ◽  
Clock Mutant ◽  
Boechera Stricta

The circadian clock is an important determinant of fitness that is entrained by local conditions. Aside from abiotic factors, individual pathogenic soil bacteria affect circadian clock function in plant hosts. Yet, in nature, plants interact with diverse microbial communities, and the effect of complex communities on clock function remains unclear. In Arabidopsis thaliana and its wild relative, Boechera stricta, we used diverse rhizosphere inoculates and host genotypes to test the effect of complex rhizosphere microbial communities on the host circadian clock. Arabidopsis thaliana plants with an intact rhizosphere microbiome expressed a circadian period closer to 24h in duration and significantly shorter (by 48 minutes on average) relative to plants grown with a disrupted microbiome. Wild-type host genotypes of A. thaliana differed in clock sensitivity to microbes, with one genotype (Landsberg erecta) expressing a 119-minute difference in circadian period length across rhizosphere microbial treatments. A similar pattern of clock sensitivity to soil microbes was observed in B. stricta. Finally, rhizosphere microbes collected from the mutant genotype toc1-21 of A. thaliana with a short-period phenotype and used as inoculate significantly shortened the long-period phenotype of the clock mutant genotype ztl-1. The results indicate that complex rhizosphere microbial communities affect host clock function.

scholarly journals Time Lag Between Light and Heat Diurnal Cycles Modulates CIRCADIAN CLOCK ASSOCIATION 1 Rhythm and Growth in Arabidopsis thaliana

2021 ◽  
Vol 11 ◽  
Author(s):  
Kosaku Masuda ◽  
Tatsuya Yamada ◽  
Yuya Kagawa ◽  
Hirokazu Fukuda
Keyword(s):  
Arabidopsis Thaliana ◽  
Circadian Rhythms ◽  
Plant Growth ◽  
Circadian Clock ◽  
Time Lag ◽  
Luciferase Reporter ◽  
Time Lags ◽  
Light Cycle ◽  
Growth Responses ◽  
Diurnal Cycles

Plant growth responses to cues such as light, temperature, and humidity enable the entrainment of the circadian rhythms with diurnal cycles. For example, the temperature variations between day and night affect plant growth and accompany the time lag to light cycle. Despite its importance, there has been no systematic investigation into time lags, and the mechanisms behind the entrainment of the circadian rhythms with multiple cycles remain unknown. Here, we investigated systemically the effects of the time lag on the circadian rhythm and growth in Arabidopsis thaliana. To investigate the entrainment status of the circadian clock, the rhythm of the clock gene CIRCADIAN CLOCK ASSOCIATION 1 (CCA1) was measured with a luciferase reporter assay. As a result, the rhythm was significantly modulated by the time lag with +10°C heating for 4 h every day but not −10°C cooling. A model based on coupled cellular oscillators successfully described these rhythm modulations. In addition, seedling growth depended on the time lag of the heating cycle but not that of the cooling cycle. Based on the relationship between the CCA1 rhythms and growth, we established an estimation method for the effects of the time lag. Our results found that plant growth relates to the CCA1 rhythm and provides a method by which to estimate the appropriate combination of light–dark and temperature cycles.

scholarly journals Mutational Analyses of Open Reading Frames within thevraSROperon and Their Roles in the Cell Wall Stress Response ofStaphylococcus aureus

2011 ◽  
Vol 55 (4) ◽  
pp. 1391-1402 ◽  
Author(s):  
N. McCallum ◽  
P. Stutzmann Meier ◽  
R. Heusser ◽  
B. Berger-Bächi
Keyword(s):  
Signal Transduction ◽  
Cell Wall ◽  
Cell Envelope ◽  
Wall Stress ◽  
Integral Membrane Protein ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Cell Wall Stress ◽  
Type Strains

ABSTRACTThe exposure ofStaphylococcus aureusto a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. ThevraSRgenes form part of the four-cistron autoregulatory operonorf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed thatorf1played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF inS. aureusappears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

scholarly journals Rhizosphere microbes influence host circadian clock function

2018 ◽  
Author(s):  
Charley J. Hubbard ◽  
Robby McMinn ◽  
Cynthia Weinig
Keyword(s):  
Circadian Clock ◽  
Microbial Communities ◽  
Circadian Period ◽  
Wild Type ◽  
Host Genotype ◽  
Local Conditions ◽  
Mutant Genotype ◽  
Individual Fitness ◽  
Short Period ◽  
Clock Mutant

AbstractThe circadian clock is an important determinant of individual fitness that is entrained by local conditions. In addition to known abiotic inputs that entrain the circadian clock, individual pathogenic soil bacteria affect the circadian period of plant hosts. Yet, in nature, plants interact with diverse microbial communities including hundreds to thousands of microbial taxa, and the effect of these communities on clock function remains unclear. In Arabidopsis thaliana, we used diverse rhizosphere inoculates and both wild-type and clock mutant genotypes to test the effect of complex rhizosphere microbial communities on the host circadian clock. Host plants with an intact rhizosphere microbiome expressed a circadian period that was closer to 24 hrs in duration and significantly shorter (by 60 minutes on average) relative to plants grown with a disrupted microbiome. Wild-type host genotypes differed significantly in clock sensitivity to microbiome treatments, where the effect was most pronounced in the Landsberg erecta genotype and least in the Columbia genotype. Rhizosphere microbes collected from a host genotype with a short-period phenotype (toc1-21) and used as inoculate significantly shortened the long-period phenotype of the ztl-1 clock mutant genotype. The results indicate that complex rhizosphere microbial communities significantly affect host clock function.

scholarly journals Saccharomyces cerevisiae Transcription Elongation Mutants Are Defective in PUR5 Induction in Response to Nucleotide Depletion

2000 ◽  
Vol 20 (20) ◽  
pp. 7427-7437 ◽  
Author(s):  
Randal J. Shaw ◽  
Daniel Reines
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Normal Growth ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Pol Ii

ABSTRACT IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective inPUR5 induction. The inability to fully inducePUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5′ untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.

scholarly journals Changing planetary rotation rescues the biological clock mutantlhy cca1ofArabidopsis thaliana

2015 ◽  
Author(s):  
Andrew J. Millar ◽  
Jamie T. Carrington ◽  
Wei Ven Tee ◽  
Sarah K. Hodge
Keyword(s):  
Gene Expression ◽  
Circadian Clock ◽  
Double Mutant ◽  
Clock Genes ◽  
Biological Clock ◽  
Laboratory Model ◽  
Wild Type ◽  
Planetary Rotation ◽  
Short Period ◽  
Clock Mechanism

Background: Pervasive, 24-hour rhythms from the biological clock affect diverse biological processes in metabolism and behaviour, including the human cell division cycle and sleep-wake cycle, nightly transpiration and energy balance in plants, and seasonal breeding in both plants and animals. The clock mechanism in the laboratory model plant species Arabidopsis thaliana is complex, in part due to the multiple interlocking, negative feedback loops that link the clock genes. Clock gene mutants are powerful tools to manipulate and understand the clock mechanism and its effects on physiology. The LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK ASSOCIATED 1 genes encode dawn-expressed, Myb-related repressor proteins that delay the expression of other clock genes until late in the day. Double mutant plants (lhy cca1) have low-amplitude, short-period rhythms that have been used in multiple studies of the plant circadian clock. Results: We used in vivo imaging of several luciferase (LUC) reporter genes to test how the rhythmic gene expression of wild-type and lhy cca1 mutant plants responded to light:dark cycles. Red, blue and red+blue light were similarly able to entrain these gene expression rhythms. The timing of expression rhythms in double mutant plants showed little or no response to the duration of light under 24h light:dark cycles (dusk sensitivity), in contrast to the wild type. As the period of the mutant clock is about 18h, we tested light:dark cycles of different duration (T cycles), simulating altered rotation of planet Earth. lhy cca1 double mutants regained as much dusk sensitivity in 20h T cycles as the wild type in 24h cycles, though the phase of the rhythm in the mutants was much earlier than wild type. The severe, triple lhy cca1 gi mutants also regained dusk sensitivity in 20h cycles. The double mutant showed some dusk sensitivity under 28h cycles. lhy cca1 double mutants under 28h cycles with short photoperiods, however, had the same apparent phase as wild-type plants. Conclusion: Simulating altered planetary rotation with light:dark cycles can reveal normal circadian performance in clock mutants that have been described as arrhythmic under standard conditions. The features rescued here comprise a dynamic behaviour (apparent phase under 28h cycles) and a dynamic property (dusk sensitivity under 20h cycles). These conditional clock phenotypes indicate that parts of the clock mechanism continue to function independently of LHY and CCA1, despite the major role of these genes in wild-type plants under standard conditions. Accessibility: Most results here will be published only in this format, citable by the DOI. Data and analysis are publicly accessible on the BioDare resource (www.biodare.ed.ac.uk), as detailed in the links below. Transgenic lines are linked to Stock Centre IDs below (Table 7).

scholarly journals Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 309-318 ◽  
Author(s):  
Scott D. Langdon ◽  
Russel E. Kaufman
Keyword(s):  
Binding Sites ◽  
Globin Gene ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Wild Type ◽  
Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

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