ABSTRACT
To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3'-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3'-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process.
Long spells of cold winter weather may feel miserable, but they are often necessary for spring to blossom. Indeed, many plants need to face a prolonged period of low temperatures to be able to flower; this process is known as vernalization. While the molecular mechanisms which underpin vernalization are well-known, it is still unclear exactly how plants can 'sense' the difference between short and long periods of cold. Jeon, Jeong et al. set out to explore this question by focusing on COOLAIR, one of the rare genetic sequences identified as potentially being able to trigger vernalization. COOLAIR is a long noncoding RNA, a partial transcript of a gene that will not be 'read' by the cell to produce a protein but which instead regulates how and when certain genes are being switched on. COOLAIR emerges from the locus of the FLC gene, which is one of the main repressors of flowering, and it gradually accumulates in the plant when temperatures remain low for a long period. While some evidence suggests that COOLAIR may help to switch off FLC, other studies have raised some doubts about its involvement in vernalization. In response, Jeon, Jeong et al. examined the FLC gene in a range of plants closely related to A. thaliana, and in which COOLAIR also accumulates upon cold exposure. This helped them identify a class of proteins, known as CBFs, which could bind to sequences near the FLC gene to activate the production of COOLAIR when the plants were kept in cold conditions for a while. CBFs were already known to help plants adapt to short cold snaps, but these experiments confirmed that they could act as both short- and long-term cold sensors. This work allowed Jeon, Jeong et al. to propose a model in which CBF and therefore COOLAIR levels increase as the cold persists, until changes in the structure of the FLC gene prevent CBF from binding to it and COOLAIR production drops. Unexpectedly, examining the fate of mutants which could not produce COOLAIR revealed that these plants could still undergo vernalization, suggesting that the long noncoding RNA is in fact not necessary for this process. These results should prompt other scientists to further investigate the role of COOLAIR in vernalization; they also give insight into how coding and noncoding sequences may have evolved together in various members of the A. thaliana family to adapt to the environment.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , MADS Domain Proteins/genetics , MADS Domain Proteins/metabolism , Flowers/physiology , Arabidopsis/metabolism , Chromatin/metabolism , Gene Expression Regulation, Plant , Cold TemperatureABSTRACT
N 6-methyladenosine (m6A) RNA methylation has been shown to play a crucial role in plant development and floral transition. Two recent studies have identified FIONA1 as an m6A methyltransferase that regulates the floral transition in Arabidopsis through influencing the stability of CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and FLOWERING LOCUS C (FLC). In this study, we confirmed that FIONA1 is an m6A methyltransferase that installs m6A marks in a small group of mRNAs. Furthermore, we show that, in addition to its role in influencing the stability of CO, SOC1, and FLC, FIONA1-mediated m6A methylation influences the splicing of FLC, a key floral repressor, and the stability of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (SPL3) and SEPALLATA3 (SEP3), floral activators, which together play a vital role in floral transition in Arabidopsis. Our study confirms the function of FIONA1 as an m6A methyltransferase and suggests a close molecular link between FIONA1-mediated m6A methylation and the splicing of FLC and the destabilization of SPL3 and SEP3 in flowering time control.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Methyltransferases/genetics , Flowers , MADS Domain Proteins/genetics , MADS Domain Proteins/metabolism , Gene Expression Regulation, PlantABSTRACT
Proper timing of flowering, a phase transition from vegetative to reproductive development, is crucial for plant fitness. The floral repressor FLOWERING LOCUS C (FLC) is the major determinant of flowering in Arabidopsis thaliana. In rapid-cycling A. thaliana accessions, which bloom rapidly, FLC is constitutively repressed by autonomous pathway (AP) genes, regardless of photoperiod. Diverse AP genes have been identified over the past two decades, and most of them repress FLC through histone modifications. However, the detailed mechanism underlying such modifications remains unclear. Several recent studies have revealed novel mechanisms to control FLC repression in concert with histone modifications. This review summarizes the latest advances in understanding the novel mechanisms by which AP proteins regulate FLC repression, including changes in chromatin architecture, RNA polymerase pausing, and liquid-liquid phase separation- and ncRNA-mediated gene silencing. Furthermore, we discuss how each mechanism is coupled with histone modifications in FLC chromatin.
ABSTRACT
Vernalization, an acceleration of flowering after long-term winter cold, is an intensively studied flowering mechanism in winter annual plants. In Arabidopsis, Polycomb Repressive Complex 2 (PRC2)-mediated suppression of the strong floral repressor, FLOWERING LOCUS C (FLC), is critical for vernalization and a PHD finger domain protein, VERNALIZATION INSENSITIVE 3 (VIN3), recruits PRC2 on FLC chromatin. The level of VIN3 was found to gradually increase in proportion to the length of cold period during vernalization. However, how plants finely regulate VIN3 expression according to the cold environment has not been completely elucidated. As a result, we performed EMS mutagenesis using a transgenic line with a minimal promoter of VIN3 fused to the GUS reporter gene, and isolated a mutant, hyperactivation of VIN3 1 (hov1), which showed increased GUS signal and endogenous VIN3 transcript levels. Using positional cloning combined with whole-genome resequencing, we found that hov1 carries a nonsense mutation, leading to a premature stop codon on the HEAT SHOCK TRANSCRIPTION FACTOR B2b (HsfB2b), which encodes a repressive heat shock transcription factor. HsfB2b directly binds to the VIN3 promoter, and HsfB2b overexpression leads to reduced acceleration of flowering after vernalization. Collectively, our findings reveal a novel fine-tuning mechanism to regulate VIN3 for proper vernalization response.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Chromatin/metabolism , Cold Temperature , DNA-Binding Proteins/genetics , Flowers/physiology , Gene Expression Regulation, Plant , Heat Shock Transcription Factors/metabolism , MADS Domain Proteins/genetics , Polycomb Repressive Complex 2/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Vernalization, a long-term cold-mediated acquisition of flowering competence, is critically regulated by VERNALIZATION INSENSITIVE 3 (VIN3), a gene induced by vernalization in Arabidopsis. Although the function of VIN3 has been extensively studied, how VIN3 expression itself is upregulated by long-term cold is not well understood. In this study, we identified a vernalization-responsive cis-element in the VIN3 promoter, VREVIN3, composed of a G-box and an evening element (EE). Mutations in either the G-box or the EE prevented VIN3 expression from being fully induced upon vernalization, leading to defects in the vernalization response. We determined that the core clock proteins CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE-ELONGATED HYPOCOTYL (LHY) associate with the EE of VREVIN3, both in vitro and in vivo. In a cca1 lhy double mutant background harboring a functional FRIGIDA allele, long-term cold-mediated VIN3 induction and acceleration of flowering were impaired, especially under mild cold conditions such as at 12°C. During prolonged cold exposure, oscillations of CCA1/LHY transcripts were altered, while CCA1 abundance increased at dusk, coinciding with the diurnal peak of VIN3 transcripts. We propose that modulation of the clock proteins CCA1 and LHY participates in the systems involved in sensing long-term cold for the activation of VIN3 transcription.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Circadian Rhythm/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant/genetics , Hypocotyl/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
ABSTRACT
Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.
Subject(s)
Acclimatization/genetics , Ranunculus/growth & development , Ranunculus/genetics , Abscisic Acid/metabolism , Acclimatization/drug effects , Arabidopsis , Ecosystem , Ethylenes/pharmacology , Gene Expression Regulation, Plant/drug effects , Plant Growth Regulators/metabolism , Plant Leaves/drug effects , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Stomata/drug effects , Plant Stomata/growth & development , Plant Stomata/metabolism , Plants, Genetically Modified , Ranunculus/metabolism , Seeds/growth & developmentABSTRACT
Kinetochore, a protein super-complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured-1 (mun-1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun-1 is a weak allele because of the insertion of T-DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9-mediated mutagenesis showed zygotic embryonic lethality similar to nuf2-1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two-hybrid assay and in planta co-immunoprecipitation. MUN is co-localized at the centromere with HTR12/CENH3, which is a centromere-specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Meristem/genetics , Microtubule-Associated Proteins/genetics , Seeds/genetics , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Division/genetics , Chromosome Segregation , Meristem/growth & development , Meristem/metabolism , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Mutation , Plant Shoots/genetics , Plant Shoots/growth & development , Plant Shoots/metabolism , Plants, Genetically Modified , Seeds/growth & development , Seeds/metabolism , Sequence Homology, Amino AcidABSTRACT
TATA-binding protein-associated factors (TAFs) are general transcription factors within the transcription factor IID (TFIID) complex, which recognizes the core promoter of genes. In addition to their biochemical function, it is known that several TAFs are involved in the regulation of developmental processes. In this study, we found that TAF15b affects flowering time, especially through the autonomous pathway (AP) in Arabidopsis. The mutant taf15b shows late flowering compared with the wild type plant during both long and short days, and vernalization accelerates the flowering time of taf15b. In addition, taf15b shows strong upregulation of FLOWERING LOCUS C (FLC), a flowering repressor in Arabidopsis, and the flc taf15b double mutant completely offsets the late flowering of taf15b, indicating that TAF15b is a typical AP gene. The taf15b mutant also shows increased transcript levels of COOLAIR, an antisense transcript of FLC. Consistently, chromatin immunoprecipitation (ChIP) analyses showed that the TAF15b protein is enriched around both sense and antisense transcription start sites of the FLC locus. In addition, co-immunoprecipitation showed that TAF15b interacts with RNA polymerase II (Pol II), while ChIP showed increased enrichment of the phosphorylated forms, both serine 2 (Ser2) and Ser5, of the C-terminal domain of Pol II at the FLC locus, which is indicative of transcriptional elongation. Finally, taf15b showed higher enrichment of the active histone marker, H3K4me3, on FLC chromatin. Taken together, our results suggest that TAF15b affects flowering time through transcriptional repression of FLC in Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , MADS Domain Proteins/metabolism , TATA-Binding Protein Associated Factors/metabolism , Arabidopsis/growth & development , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Chromatin/genetics , Chromatin Immunoprecipitation , Flowers/genetics , Flowers/growth & development , Flowers/physiology , Histones/genetics , MADS Domain Proteins/genetics , Mutation , Promoter Regions, Genetic/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , TATA-Binding Protein Associated Factors/genetics , Time Factors , Up-RegulationABSTRACT
Annual plants complete life cycle in a year while perennial plants maintain growth for several years. Arabis alpina, a polycarpic perennial, is a close relative of monocarpic annual Arabidopsis. Pajares is an accession of A. alpina requiring vernalization, a long-term cold for flowering. Arabidopsis shows holistic flowering whereas Pajares shows idiographic flowering, producing axillary branches under variable developmental phases from juvenile, adult vegetative to reproductive phases. To understand the molecular mechanism behind diverse phases of axillary branches, we analyzed the levels of primary miR156 expressions because miR156-SPL module is a key regulator for developmental phase transition. We found that in Pajares, miR156 levels were highly variable among the axillary branches, which causes differential sensitivity to vernalization. Thus, the axillary branches expressing high levels of miR156 remain in juvenile phase even after vernalization, whereas the axillary branches expressing low levels of miR156 produce flowers after vernalization. In contrast, every axillary branches of Arabidopsis winter annual Sy-0 expressed similar levels of miR156 and synchronously responded to vernalization, which causes holistic flowering. Therefore, we suggest that variable miR156 expression levels and the resulting differential response to vernalization among axillary branches are distinctive features determining polycarpic perenniality of A. alpina Pajares.
ABSTRACT
One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-ß, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (ß-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.
Subject(s)
Gene Expression Regulation , Influenza A Virus, H5N1 Subtype/physiology , Influenza, Human/genetics , Interleukin-6/genetics , Interleukin-8/genetics , Receptors, Purinergic P2/metabolism , Respiratory Mucosa/virology , Animals , Cell Line , Chickens , Humans , Influenza, Human/metabolism , Influenza, Human/virology , RNA, Messenger/genetics , Respiratory Mucosa/metabolism , Signal TransductionABSTRACT
Traditionally, proteins are considered to perform a single role, be it as an enzyme, a channel, a transporter or as a structural scaffold. However, recent studies have described moonlighting proteins that perform distinct and independent functions; for example, TRPM7 is both an ion channel and a kinase. ZnT-1 is a member of the Carrier Diffusion Facilitator family that is expressed throughout the phylogenetic tree from bacteria to humans. Since its cloning in 1995, ZnT-1 is considered a major extruder of Zn2+ based on its capability to protect cells against zinc toxicity. Recently, we reported that ZnT-1 inhibits the L-type calcium channel (LTCC), a major Zn2+ and Ca2+ entry pathway. Here we show that ZnT-1 is a dual-function protein by demonstrating that its abilities to exchange Zn2+/H+ and to inhibit the LTCC are independent of each other and are mediated by different parts of the protein. Specifically, mutations in the membrane-spanning helices that render ZnT-1 unable to transport zinc do not prevent it from inhibiting the LTCC. Moreover, a fragment consisting of the intracellular ZnT-1 C-terminal, which lacks all ion-transfer segments, inhibits the LTCC as efficiently as wild-type ZnT-1. Our data therefore indicates that ZnT-1 performs two structurally independent functions related to zinc homeostasis.
Subject(s)
Calcium Channels, L-Type/chemistry , Cation Transport Proteins/metabolism , Xenopus/physiology , Zinc/pharmacology , Amino Acid Sequence , Animals , CHO Cells , Calcium Channels, L-Type/metabolism , Cation Transport Proteins/genetics , Cricetinae , Cricetulus , Cytoplasm/drug effects , Cytoplasm/metabolism , HEK293 Cells , Homeostasis , Humans , Ion Transport , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Sequence AlignmentABSTRACT
The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , DNA Methylation/genetics , Gene Expression Regulation, Plant , N-Glycosyl Hydrolases/genetics , Ovule/genetics , Pollen/genetics , Trans-Activators/genetics , DNA Glycosylases , DNA Transposable Elements , Endosperm/genetics , Genomic Imprinting , Germ Cells , Mutation , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
The ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) exchanges the histone H2A-H2B dimer with the H2A.Z-H2B dimer, producing variant nucleosomes. Arabidopsis thaliana SWR1-C contributes to the active transcription of many genes, but also to the repression of genes that respond to environmental and developmental stimuli. Unlike other higher eukaryotic H2A.Z deposition mutants (which are embryonically lethal), Arabidopsis SWR1-C component mutants, including arp6, survive and display a pleiotropic developmental phenotype. However, the molecular mechanisms of early flowering, leaf serration, and the production of extra petals in arp6 have not been completely elucidated. We report here that SWR1-C is required for miRNA-mediated developmental control via transcriptional regulation. In the mutants of the components of SWR1-C such as arp6, sef, and pie1, miR156 and miR164 levels are reduced at the transcriptional level, which results in the accumulation of target mRNAs and associated morphological changes. Sequencing of small RNA libraries confirmed that many miRNAs including miR156 decreased in arp6, though some miRNAs increased. The arp6 mutation suppresses the accumulation of not only unprocessed primary miRNAs, but also miRNA-regulated mRNAs in miRNA processing mutants, hyl1 and serrate, which suggests that arp6 has a transcriptional effect on both miRNAs and their targets. We consistently detected that the arp6 mutant exhibits increased nucleosome occupancy at the tested MIR gene promoters, indicating that SWR1-C contributes to transcriptional activation via nucleosome dynamics. Our findings suggest that SWR1-C contributes to the fine control of plant development by generating a balance between miRNAs and target mRNAs at the transcriptional level.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/genetics , Chromatin Assembly and Disassembly/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , MicroRNAs/metabolism , Arabidopsis/drug effects , Arabidopsis Proteins/genetics , Body Patterning/drug effects , Body Patterning/genetics , Chromatin Assembly and Disassembly/drug effects , Cyclopentanes/pharmacology , Environment , Flowers/drug effects , Flowers/genetics , Flowers/physiology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Plant , MicroRNAs/genetics , Mutation/genetics , Nucleosomes/drug effects , Nucleosomes/metabolism , Oxylipins/pharmacology , Plant Roots/drug effects , Plant Roots/genetics , Plant Roots/growth & development , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Initiation Site , Transcriptional Activation/drug effects , Transcriptional Activation/geneticsABSTRACT
BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI) and its three homologs (BOIs) are RING domain-containing proteins that repress flowering. Here, we investigated how BOIs repress flowering. Genetic analysis of the boiQ quadruple mutant indicates that BOIs repress flowering mainly through FLOWERING LOCUS T (FT). BOIs repress the expression of FT by CONSTANS (CO)-dependent and -independent mechanisms: in the CO-dependent mechanism, BOIs bind to CO, inhibit the targeting of CO to the FT locus, and thus repress the expression of FT; in the CO-independent mechanism, BOIs target the FT locus via a mechanism that requires DELLAs but not CO. This dual repression of FT makes BOIs strong repressors of flowering in both CO-dependent and CO-independent pathways in Arabidopsis thaliana. Our finding that BOIs inhibit CO targeting further suggests that, in addition to modulating CO mRNA expression and CO protein stability, flowering regulation can also modulate the targeting of CO to FT.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , DNA-Binding Proteins/metabolism , Flowers/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , Flowers/chemistry , Flowers/genetics , Flowers/growth & development , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Protein Structure, Tertiary , Repressor Proteins/chemistry , Repressor Proteins/genetics , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.
Subject(s)
Epidermal Growth Factor/pharmacology , Epithelial Sodium Channel Blockers/pharmacology , Epithelial Sodium Channels/metabolism , Oncogene Proteins/metabolism , ras Proteins/metabolism , Animals , Down-Regulation/drug effects , HEK293 Cells , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Signaling System/drug effects , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mutation , Oncogene Proteins/genetics , Proto-Oncogene Proteins c-raf/metabolism , Rats , ras Proteins/geneticsABSTRACT
ZnT-1 is a Cation Diffusion Facilitator (CDF) family protein, and is present throughout the phylogenetic tree from bacteria to humans. Since its original cloning in 1995, ZnT-1 has been considered to be the major Zn(2+) extruding transporter, based on its ability to protect cells against zinc toxicity. However, experimental evidence for ZnT-1 induced Zn(2+) extrusion was not convincing. In the present study, based on the 3D crystal structure of the ZnT-1 homologue, YiiP, that predicts a homodimer that utilizes the H(+) electrochemical gradient to facilitate Zn(2+) efflux, we demonstrate ZnT-1 dependent Zn(2+) efflux from HEK 293T cells using FluoZin-3 and Fura 2 by single cell microscope based fluorescent imaging. ZnT-1 facilitates zinc efflux in a sodium-independent, pH-driven and calcium-sensitive manner. Moreover, substitution of two amino acids in the putative zinc binding domain of ZnT-1 led to nullification of Zn(2+) efflux and rendered the mutated protein incapable of protecting cells against Zn(2+) toxicity. Our results demonstrate that ZnT-1 extrudes zinc from mammalian cells by functioning as a Zn(2+)/H(+) exchanger.
Subject(s)
Cation Transport Proteins/metabolism , Hydrogen/metabolism , Mammals/metabolism , Zinc/metabolism , Animals , Biological Transport/drug effects , Calcium/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cytoprotection/drug effects , Electrochemistry , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Mutant Proteins/metabolism , Sodium/metabolism , Zinc/toxicityABSTRACT
THz metamaterials are employed to examine changes in the meta-resonances when two anisotropic organic materials, liquid crystal and carbon nanotubes, are placed on top of metamaterials. In both anisotropic double split-ring resonators and isotropic four-fold symmetric split-ring resonators, anisotropic interactions between the electric field and organic materials are enhanced in the vicinity of meta-resonances. In liquid crystal, meta-resonance frequency shift is observed with the magneto-optical coupling giving rise to the largest anisotropic shift. In carbon nanotube, meta-resonance absorptions, parallel and perpendicular to nanotube direction, experience different amount of broadening of Lorentzian oscillator of meta-resonance. Investigation reported here opens the application of metamaterials as a sensor for anisotropic materials.
ABSTRACT
Plants monitor changes in day length to coordinate flowering with favorable seasons to increase their fitness. The day-length specific induction of FLOWERING LOCUS T (FT) regulated by CONSTANS (CO) is the crucial aspect of photoperiodic flowering in Arabidopsis thaliana. Recent studies have elucidated some mechanisms of CO-dependent FT induction. Here, we demonstrate another mechanism of CO-dependent FT regulation. Our results indicate that CO protein partially regulates FT transcription by forming a complex with ASYMMETRIC LEAVES 1 (AS1) protein, which regulates leaf development partly by controlling gibberellin (GA) levels. We identified AS1 as a CO-interacting protein in yeast and verified their interaction in vitro and in planta. We also showed that the temporal and spatial expression pattern of AS1 overlapped with that of CO. In addition, as1 mutants showed GA-independent delayed flowering under different light/dark conditions. FT expression levels in the as1 mutants and the SUC2:CO-HA/as1 line under long-day and 12-h light/12-h dark conditions were reduced compared with wild-type plants and the SUC2:HA-CO line, respectively. Moreover, AS1 bound directly to the specific regions of the FT promoter in vivo. These results indicate that CO forms a functional complex with AS1 to regulate FT expression and that AS1 plays different roles in two regulatory pathways, both of which concomitantly regulate the precise timing of flowering.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Flowers/physiology , Gene Expression Regulation, Plant/genetics , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flowers/genetics , Flowers/growth & development , Mutation , Phenotype , Photoperiod , Plant Leaves/genetics , Plant Leaves/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Promoter Regions, Genetic , Protein Interaction Mapping , Signal Transduction/genetics , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Two-Hybrid System TechniquesABSTRACT
A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.