RESUMO
In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.
Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Arabidopsis/embriologia , Fusão Celular , Endosperma/metabolismo , Mitose , Peptídeos/metabolismo , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismoRESUMO
In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown. Here, we develop a two-photon live imaging method to directly observe pollen tube guidance in the pistil of Arabidopsis thaliana, clarifying signaling and cellular behaviors in the one-to-one guidance. Ovules are suggested to emit multiple signals for pollen tubes, including an integument-dependent directional signal that reaches the inner surface of the septum and adhesion signals for emerged pollen tubes on the septum. Not only FERONIA in the septum but ovular gametophytic FERONIA and LORELEI, as well as FERONIA- and LORELEI-independent repulsion signal, are involved in polytubey blocks on the ovular funiculus. However, these funicular blocks are not strictly maintained in the first 45 min, explaining previous reports of polyspermy in flowering plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Óvulo Vegetal , Tubo Polínico , Transdução de Sinais , Tubo Polínico/crescimento & desenvolvimento , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Óvulo Vegetal/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , FertilizaçãoRESUMO
The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation and final cellularization, it remains unknown when and how the cell fate is determined during development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of individual cell types to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the 1-nucleate stage to the 7-cell stage after cellularization using an in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal (distal-proximal) axis. During cellularization, the polar nuclei migrated while associating with the forming edge of the cell plate, and then, migrated toward each other to fuse linearly. We also tracked the gene expression dynamics and identified that the expression of MYB98pro::GFP-MYB98, a synergid-specific marker, was initiated just after cellularization in the synergid, egg, and central cells and was then restricted to the synergid cells. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild-type and myb98 mutant revealed that the myb98 synergid cells had egg cell-like gene expression profiles. Although in myb98, egg cell-specific gene expression was properly initiated in the egg cells only after cellularization, but subsequently expressed ectopically in one of the 2 synergid cells. These results, together with the various initiation timings of the egg cell-specific genes, suggest complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.
Assuntos
Arabidopsis/metabolismo , Diferenciação Celular/genética , Óvulo Vegetal/metabolismo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Magnoliopsida/metabolismo , Morfogênese , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.
Assuntos
Nicotiana , Plasmodesmos , Microfluídica , Permeabilidade , Plantas , Plasmodesmos/metabolismo , Nicotiana/metabolismoRESUMO
In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.
Assuntos
Arabidopsis/fisiologia , Divisão Celular Assimétrica , Polaridade Celular , Vacúolos/metabolismo , Zigoto/citologia , Zigoto/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Imunofluorescência , MutaçãoRESUMO
In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant-specific nuclear protein that contains the AS2/LOB domain, which includes a zinc-finger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co-localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2-1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Mutação , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Domínios Proteicos , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits-tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Folhas de Planta/anatomia & histologia , Raízes de Plantas/anatomia & histologia , Caules de Planta/anatomia & histologia , Coloração e Rotulagem/métodos , Ureia , XilitolRESUMO
The asymmetric cell division of the zygote is the initial and crucial developmental step in most multicellular organisms. In flowering plants, whether zygote polarity is inherited from the preexisting organization in the egg cell or reestablished after fertilization has remained elusive. How dynamically the intracellular organization is generated during zygote polarization is also unknown. Here, we used a live-cell imaging system with Arabidopsis zygotes to visualize the dynamics of the major elements of the cytoskeleton, microtubules (MTs), and actin filaments (F-actins), during the entire process of zygote polarization. By combining image analysis and pharmacological experiments using specific inhibitors of the cytoskeleton, we found features related to zygote polarization. The preexisting alignment of MTs and F-actin in the egg cell is lost on fertilization. Then, MTs organize into a transverse ring defining the zygote subapical region and driving cell outgrowth in the apical direction. F-actin forms an apical cap and longitudinal arrays and is required to position the nucleus to the apical region of the zygote, setting the plane of the first asymmetrical division. Our findings show that, in flowering plants, the preexisting cytoskeletal patterns in the egg cell are lost on fertilization and that the zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration.
Assuntos
Citoesqueleto de Actina/fisiologia , Arabidopsis/embriologia , Polaridade Celular , Microtúbulos/fisiologia , Sementes/fisiologia , Divisão Celular , FertilizaçãoRESUMO
Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.
Assuntos
Corantes Fluorescentes/química , Indicadores e Reagentes/química , Microscopia de Fluorescência/métodos , Plantas/metabolismo , Ureia/química , Xilitol/química , Arabidopsis , Clorofila/química , Clonagem Molecular , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia de Fluorescência/instrumentação , Floema , Fótons , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
Progression of cell division is controlled by various mitotic kinases. In animal cells, phosphorylation of histone H3 at Thr3 by the kinase Haspin (haploid germ cell-specific nuclear protein kinase) promotes centromeric Aurora B localization to regulate chromosome segregation. However, less is known about the function of Haspin in regulatory networks in plant cells. Here, we show that inhibition of Haspin with 5-iodotubercidin (5-ITu) in Bright Yellow-2 (BY-2) cells delayed chromosome alignment. Haspin inhibition also prevented the centromeric localization of Aurora3 kinase (AUR3) and disrupted its function. This suggested that Haspin plays a role in the specific positioning of AUR3 on chromosomes in plant cells, a function conserved in animals. The results also indicated that Haspin and AUR3 are involved in the same pathway, which regulates chromosome alignment during prometaphase/metaphase. Remarkably, Haspin inhibition by 5-ITu also led to a severe cytokinesis defect, resulting in binuclear cells with a partially formed cell plate. The 5-ITu treatment did not affect microtubules, AUR1/2 or the NACK-PQR pathway; however, it did alter the distribution of actin filaments on the cell plate. Together, these results suggested that Haspin has several functions in regulating cell division in plant cells: in the localization of AUR3 on centromeres and in regulating late cell plate expansion during cytokinesis.
Assuntos
Nicotiana/citologia , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Aurora Quinases/metabolismo , Divisão Celular , Centrômero/metabolismo , Cromossomos de Plantas , Redes e Vias Metabólicas , Metáfase , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Nicotiana/efeitos dos fármacos , Tubercidina/análogos & derivados , Tubercidina/farmacologiaRESUMO
Cell proliferation is crucial to the growth of multicellular organisms, and thus the proper control of cell division is important to prevent developmental arrest or overgrowth. Nevertheless, tools for controlling cell proliferation are still poor in plant. To develop novel tools, we focused on a specific compound family, triarylmethanes, whose members show various antiproliferative activities in animals. By combining organic chemistry to create novel and diverse compounds containing the triarylmethyl moiety and biological screens based on live-cell imaging of a fluorescently labeled tobacco Bright Yellow-2 (BY-2) culture cell line (Nicotiana tabacum), we isolated (3-furyl)diphenylmethane as a strong but partially reversible inhibitor of plant cell division. We also found that this agent had efficient antiproliferative activity in developing organs of Arabidopsis thaliana without causing secondary defects in cell morphology, and induced rapid cell division arrest independent of the cell cycle stage. Given that (3-furyl)diphenylmethane did not affect the growth of a human cell line (HeLa) and a budding yeast (Saccharomyces cerevisiae), it should act specifically on plants. Taking our results together, we propose that the combination of desired chemical synthesis and detailed biological analysis is an effective tool to create novel drugs, and that (3-furyl)diphenylmethane is a specific antiproliferative agent for plants.
Assuntos
Arabidopsis/citologia , Divisão Celular , Técnicas de Química Sintética/métodos , Imagem Molecular/métodos , Nicotiana/citologia , Compostos Benzidrílicos/química , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Especificidade de ÓrgãosRESUMO
Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. In addition to regulating mitotic events, AUR has been shown to regulate protein dynamics during interphase in animal cells. In contrast, there has been no identification and characterization of substrates and/or interacting proteins during interphase in plants. The Arabidopsis thaliana genome encodes three AUR paralogues, AtAUR1, AtAUR2, and AtAUR3. Among them, AtAUR1 and AtAUR2 are considered to function redundantly. Here, we confirmed that both AtAUR1 and AtAUR3 are localized in the nucleus and cytoplasm during interphase, suggesting that they have functions during interphase. To identify novel interacting proteins, we used AlphaScreen to target 580 transcription factors (TFs) that are mainly functional during interphase, using recombinant A. thaliana TFs and AtAUR1 or AtAUR3. We found 133 and 32 TFs had high potential for interaction with AtAUR1 and AtAUR3, respectively. The highly AtAUR-interacting TFs were involved in various biological processes, suggesting the functions of the AtAURs during interphase. We found that AtAUR1 and AtAUR3 showed similar interaction affinity to almost all TFs. However, in some cases, the interaction affinity differed substantially between the two AtAUR homologues. These results suggest that AtAUR1 and AtAUR3 have both redundant and distinct functions through interactions with TFs. In addition, database analysis revealed that most of the highly AtAUR-interacting TFs contained a detectable phosphopeptide that was consistent with the consensus motifs for human AURs, suggesting that these TFs are substrates of the AtAURs. The AtAURs phosphorylated several highly interacting TFs in the AlphaScreen in vitro. Overall, in line with the regulation of TFs through interaction, our results indicate the possibility of phosphoregulation of several TFs by the AtAURs (280/300).
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Interfase , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The female gametophytes of many flowering plants contain one egg cell, one central cell, two synergid cells and three antipodal cells with respective morphological characteristics and functions. These cells are formed by cellularization of a multinuclear female gametophyte. However, the dynamics and mechanisms of female gametophyte development remain largely unknown due to the lack of a system to visualize directly and manipulate female gametophytes in living material. Here, we established an in vitro ovule culture system to examine female gametophyte development in Torenia fournieri, a unique plant species with a protruding female gametophyte. The four-nucleate female gametophyte became eight nucleate by the final (third) mitosis and successively cellularized and matured to attract a pollen tube. The duration of final mitosis was 28 ± 6.5 min, and cellularization was completed in 54 ± 20 min after the end of the third mitosis. Fusion of polar nuclei in the central cell occurred in 13.1 ± 1.1 h, and onset of expression of LURE2, a pollen tube attractant gene, was visualized by a green fluorescent protein reporter 10.7 ± 2.3 h after cellularization. Laser disruption analysis demonstrated that the egg and central cells were required for synergid cells to acquire the pollen tube attraction function. Moreover, aberrant nuclear positioning and down-regulation of LURE2 were observed in one of the two synergid cells after disrupting an immature egg cell, suggesting that cell specification was affected. Our system provides insights into the precise dynamics and mechanisms of female gametophyte development in T. fournieri.
Assuntos
Comunicação Celular , Imageamento Tridimensional/métodos , Lasers , Magnoliopsida/crescimento & desenvolvimento , Óvulo Vegetal/citologia , Óvulo Vegetal/crescimento & desenvolvimento , Diferenciação Celular , Mitose , Modelos Biológicos , Tubo Polínico/citologiaRESUMO
Plant cytokinesis is achieved by formation of cell plates in the phragmoplast, a plant-specific cytokinetic apparatus, which consists of microtubules (MTs) and microfilaments. During cytokinesis, the cell plate is expanded centrifugally outward from the inside of cells in a process that is supported by dynamic turnover of MTs. M-phase-specific kinesin NACK1, which comprises the motor domain at the amino-terminal half to move on MT bundles and the stalk region in the carboxyl-terminal half, is a key player in the process of MT turnover. That is, the specific region in the stalk binds the MAP kinase kinase kinase to activate the whole MAP kinase cascade, which stimulates depolymerization of MTs for the MT turnover. The stalk is also responsible for recruiting the activated kinase cascade to the mid-zone of the phragmoplast, which corresponds to the cell-plate formation site. It should be crucial to uncover roles of the NACK1 kinesin stalk as well as the motor domain in the formation of cell plates in order to understand the mechanisms of cell plate formation. Using dissected Arabidopsis NACK1 (AtNACK1/HINKEL) molecules and AtNACK1-fused GFP, we showed that the C-terminal tail of the stalk in addition to the motor domain is critical for its proper localization to the site of cell plate formation in the phragmoplast, probably by affecting its motility activity.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Citocinese , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Genome integrity is continuously threatened by external stresses and endogenous hazards such as DNA replication errors and reactive oxygen species. The DNA damage checkpoint in metazoans ensures genome integrity by delaying cell-cycle progression to repair damaged DNA or by inducing apoptosis. ATM and ATR (ataxia-telangiectasia-mutated and -Rad3-related) are sensor kinases that relay the damage signal to transducer kinases Chk1 and Chk2 and to downstream cell-cycle regulators. Plants also possess ATM and ATR orthologs but lack obvious counterparts of downstream regulators. Instead, the plant-specific transcription factor SOG1 (suppressor of gamma response 1) plays a central role in the transmission of signals from both ATM and ATR kinases. Here we show that in Arabidopsis, endoreduplication is induced by DNA double-strand breaks (DSBs), but not directly by DNA replication stress. When root or sepal cells, or undifferentiated suspension cells, were treated with DSB inducers, they displayed increased cell size and DNA ploidy. We found that the ATM-SOG1 and ATR-SOG1 pathways both transmit DSB-derived signals and that either one suffices for endocycle induction. These signaling pathways govern the expression of distinct sets of cell-cycle regulators, such as cyclin-dependent kinases and their suppressors. Our results demonstrate that Arabidopsis undergoes a programmed endoreduplicative response to DSBs, suggesting that plants have evolved a distinct strategy to sustain growth under genotoxic stress.
Assuntos
Arabidopsis/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , DNA de Plantas/genética , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia , Bleomicina/toxicidade , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cisplatino/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Replicação do DNA/efeitos da radiação , Raios gama , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Ploidias , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Raios UltravioletaRESUMO
OBJECTIVE: This study investigated the strain surround implants and the pressure distribution of the alveolar ridge related to implant overdentures with stress-breaking ball (SBB) attachments. METHODS: Simulation models of edentulous mandibles were embedded with 1 and 2 implants to simulate a single implant-retained overdenture (S-IOD) and a two-implant-retained overdenture (T-IOD), respectively. The implants were connected to the denture base with the following conditions: complete separation between the denture base and implant with cover screws as a conventional complete denture (CD), 3 types of settlements with an SBB attachment between them, and a conventional ball attachment (BALL). Strain surround the implant, pressure at 5 different soft tissue areas, and displacement of the denture base were measured with loading up to 50 N. RESULTS: In S-IOD and T-IOD, the strain of each SBB attachment was less than the strain shown by the BALL. The pressure at each region of the SBB attachment was less than the pressure shown with the CD and BALL. CONCLUSIONS: SBB attachments prevented strain surround the implants in loading and provided optimal stress distribution.
Assuntos
Implantes Dentários , Análise do Estresse Dentário , Revestimento de Dentadura , Humanos , Técnicas In VitroRESUMO
Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.
RESUMO
Sexual reproduction ensures propagation of species and enhances genetic diversity within populations. In flowering plants, sexual reproduction requires complicated and multi-step cell-to-cell communications among male and female cells. However, the confined nature of plant reproduction processes, which occur in the female reproductive organs and several cell layers of the pistil, limits our ability to observe these events in vivo. In this review, we discuss recent live-cell imaging in in vitro systems and the optical manipulation techniques that are used to capture the dynamic mechanisms representing molecular and cellular communications in sexual plant reproduction.
Assuntos
Arabidopsis/genética , Microscopia/métodos , Tubo Polínico/metabolismo , Arabidopsis/fisiologia , Comunicação Celular , Fertilização , Flores , Genes de Plantas , Variação Genética , Magnoliopsida/genética , Magnoliopsida/fisiologia , Óptica e Fotônica , Plantas , Transdução de SinaisRESUMO
After double fertilization, the endosperm in the seeds of many flowering plants undergoes repeated mitotic nuclear divisions without cytokinesis, resulting in a large coenocytic endosperm that then cellularizes. Growth during the coenocytic phase is strongly associated with the final seed size; however, a detailed description of the cellular dynamics controlling the unique coenocytic development in flowering plants has remained elusive. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm of Arabidopsis thaliana including nuclear divisions, their timing intervals, nuclear movement and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive actin filament (F-actin) organization. Microtubules promote nuclear movement after division, while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and the mature seed. The characterization of cytoskeleton dynamics in real time throughout the entire coenocyte endosperm period provides foundational knowledge of plant coenocytic development, insights into the coordination of F-actin and microtubules in nuclear dynamics, and new opportunities to increase seed size and our food security.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Endosperma , Actinas , Sementes , CitoesqueletoRESUMO
Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division-affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.