RESUMO
In order to flower in the appropriate season, plants monitor light and temperature changes and alter downstream pathways that regulate florigen genes such as Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT). In Arabidopsis, FT messenger RNA levels peak in the morning and evening under natural long-day conditions (LDs). However, the regulatory mechanisms governing morning FT induction remain poorly understood. The morning FT peak is absent in typical laboratory LDs characterized by high red:far-red light (R:FR) ratios and constant temperatures. Here, we demonstrate that ZEITLUPE (ZTL) interacts with the FT repressors TARGET OF EATs (TOEs), thereby repressing morning FT expression in natural environments. Under LDs with simulated sunlight (R:FR = 1.0) and daily temperature cycles, which are natural LD-mimicking environmental conditions, FT transcript levels in the ztl mutant were high specifically in the morning, a pattern that was mirrored in the toe1 toe2 double mutant. Low night-to-morning temperatures increased the inhibitory effect of ZTL on morning FT expression by increasing ZTL protein levels early in the morning. Far-red light counteracted ZTL activity by decreasing its abundance (possibly via phytochrome A (phyA)) while increasing GIGANTEA (GI) levels and negatively affecting the formation of the ZTL-GI complex in the morning. Therefore, the phyA-mediated high-irradiance response and GI play pivotal roles in morning FT induction. Our findings suggest that the delicate balance between low temperature-mediated ZTL activity and the far-red light-mediated functions of phyA and GI offers plants flexibility in fine-tuning their flowering time by controlling FT expression in the morning.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Temperatura , Luz Vermelha , Fatores de Transcrição/metabolismo , Flores/fisiologia , Fitocromo A/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , MutaçãoRESUMO
Plants adapt to periodic environmental changes, such as day and night, by using circadian clocks. Cell division and elongation are primary steps to adjust plant development according to their environments. In Arabidopsis, hypocotyl elongation has been studied as a representative model to understand how the circadian clock regulates cell elongation. However, it remains unknown whether similar phenomena exist in other organs, such as roots, where circadian clocks regulate physiological responses. Here, we show that root hair elongation is controlled by both light and the circadian clock. By developing machine-learning models to automatically analyze the images of root hairs, we found that genes encoding major components of the central oscillator, such as TIMING OF CAB EXPRESSION1 (TOC1) or CIRCADIAN CLOCK ASSOCIATED1 (CCA1), regulate the rhythmicity of root hair length. The partial illumination of light to either shoots or roots suggested that light received in shoots is mainly responsible for the generation of root hair rhythmicity. Furthermore, grafting experiments between wild-type (WT) and toc1 plants demonstrated that TOC1 in shoots is responsible for the generation of root hair rhythmicity. Our results illustrate the combinational effects of long-distance signaling and the circadian clock on the regulation of root hair length.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Relógios Circadianos/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Arabidopsis/fisiologiaRESUMO
In natural long days, the florigen gene FLOWERING LOCUS T (FT) shows a bimodal expression pattern with morning and dusk peaks in Arabidopsis. This pattern differs from the one observed in the laboratory, and little is known about underlying mechanisms. A red : far-red (R : FR) ratio difference between sunlight and fluorescent light causes this FT pattern mismatch. We showed that bimodal FT expression patterns were induced in a day longer than 14 h with sunlight R : FR (= c. 1) conditions. By circadian gating experiments, we found that cumulative exposure of R : FR-adjusted light (R : FR ratio was adjusted to 1 with FR supplement) spanning from the afternoon to the next morning required full induction of FT in the morning. Conversely, only 2 h of R : FR adjustment in the late afternoon was sufficient for FT induction at dusk. We identified that phytochrome A (phyA) is required for the morning FT expression in response to the R : FR adjustment on the previous day. As a part of this mechanism, we showed that PHYTOCHROME-INTERACTING FACTOR 7 contributes to FT regulation. Our results suggest that phyA-mediated high-irradiance response and the external coincidence mechanism contribute to morning FT induction under natural long-day conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luz , Fotoperíodo , Flores/genética , Flores/metabolismo , Fitocromo A/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de PlantasRESUMO
The circadian clock allows plants to anticipate and adapt to periodic environmental changes. Organ- and tissue-specific properties of the circadian clock and shoot-to-root circadian signaling have been reported. While this long-distance signaling is thought to coordinate physiological functions across tissues, little is known about the feedback regulation of the root clock on the shoot clock in the hierarchical circadian network. Here, we show that the plant circadian clock conveys circadian information between shoots and roots through sucrose and K+. We also demonstrate that K+ transport from roots suppresses the variance of period length in shoots and then improves the accuracy of the shoot circadian clock. Sucrose measurements and qPCR showed that root sucrose accumulation was regulated by the circadian clock. Furthermore, root circadian clock genes, including PSEUDO-RESPONSE REGULATOR7 (PRR7), were regulated by sucrose, suggesting the involvement of sucrose from the shoot in the regulation of root clock gene expression. Therefore, we performed time-series measurements of xylem sap and micrografting experiments using prr7 mutants and showed that root PRR7 regulates K+ transport and suppresses variance of period length in the shoot. Our modeling analysis supports the idea that root-to-shoot signaling contributes to the precision of the shoot circadian clock. We performed micrografting experiments that illustrated how root PRR7 plays key roles in maintaining the accuracy of shoot circadian rhythms. We thus present a novel directional signaling pathway for circadian information from roots to shoots and propose that plants modulate physiological events in a timely manner through various timekeeping mechanisms.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Relógios Circadianos/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ritmo Circadiano/fisiologia , Transdução de Sinais/genética , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/metabolismoRESUMO
Circadian rhythms and progression of cell differentiation are closely coupled in multicellular organisms. However, whether establishment of circadian rhythms regulates cell differentiation or vice versa has not been elucidated due to technical limitations. Here, we exploit high cell fate plasticity of plant cells to perform single-cell RNA sequencing during the entire process of cell differentiation. By analyzing reconstructed actual time series of the differentiation processes at single-cell resolution using a method we developed (PeakMatch), we find that the expression profile of clock genes is changed prior to cell differentiation, including induction of the clock gene LUX ARRYTHMO (LUX). ChIP sequencing analysis reveals that LUX induction in early differentiating cells directly targets genes involved in cell-cycle progression to regulate cell differentiation. Taken together, these results not only reveal a guiding role of the plant circadian clock in cell differentiation but also provide an approach for time-series analysis at single-cell resolution.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas , Análise de Sequência de RNA , Fatores de TempoRESUMO
Flowering time is an agriculturally important trait that can be manipulated by various approaches such as breeding, growth control and genetic modifications. Despite its potential advantages, including fine-tuning the regulation of flowering time, few reports have explored the use of chemical compounds to manipulate flowering. Here, we report that sulfanilamide, an inhibitor of folate biosynthesis, delays flowering by repressing the expression of florigen FLOWERING LOCUS T (FT) in Arabidopsis thaliana. Transcriptome deep sequencing and quantitative polymerase chain reaction analyses showed that the expression of the circadian clock gene LUX ARRYTHMO/PHYTOCLOCK1 (LUX/PCL1) is altered by sulfanilamide treatment. Furthermore, in the lux nox mutant harboring loss of function in both LUX and its homolog BROTHER OF LUX ARRHYTHMO (BOA, also named NOX), the inhibitory effect of sulfanilamide treatment on FT expression was weak and the flowering time was similar to that of the wild type, suggesting that the circadian clock may contribute to the FT-mediated regulation of flowering by sulfanilamide. Sulfanilamide also delayed flowering time in arugula (Eruca sativa), suggesting that it is involved in the regulation of flowering across Brassicaceae. We propose that sulfanilamide is a novel modulator of flowering.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Flores , Regulação da Expressão Gênica de Plantas , Fotoperíodo , Melhoramento Vegetal , Sulfanilamidas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The plant circadian clock regulates multiple developmental and physiological events that occur at specific times and seasons. As many of the currently known clock proteins and clock-associated regulators are transcription factors, analyzing molecular events in the nuclei is crucial. In addition, long-time course analyses of protein abundance and interactions are often required to assess the role of the circadian clock on clock-regulated phenomena. Here we introduce a simple procedure to prepare nuclear-enriched tissues, which we routinely use to study time-resolved accumulation changes in low-abundance nuclear proteins (i.e., transcription factors). In addition to measuring changes in abundance, investigating the protein-protein interaction dynamics at specific times of day or under certain environmental conditions is needed for plant chronobiology studies. Therefore, we also present our co-immunoprecipitation method for studying diurnal/circadian protein-protein interactions, tailored to nuclear-localized proteins in Arabidopsis and tobacco.
Assuntos
Arabidopsis , Relógios Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano , Regulação da Expressão Gênica de Plantas , Imunoprecipitação , Fatores de Transcrição/metabolismoAssuntos
Proteínas de Arabidopsis , Desenvolvimento Vegetal , Raízes de Plantas , Plantas , TemperaturaRESUMO
Appropriate cell cycle regulation is crucial for achieving coordinated development and cell differentiation in multicellular organisms. In Arabidopsis, endoreduplication is often observed in terminally differentiated cells and several reports have shown its molecular mechanisms. Auxin is a key factor for the mode transition from mitotic cell cycle to endocycle; however, it remains unclear if and how auxin maintains the endocycle mode. In this study, we reanalyzed root single-cell transcriptome data and reconstructed cell cycle trajectories of the mitotic cell cycle and endocycle. With progression of the endocycle, genes involved in auxin synthesis, influx and efflux were induced at the specific cell phase, suggesting that auxin concentration fluctuated dynamically. Such induction of auxin-related genes was not observed in the mitotic cell cycle, suggesting that the auxin fluctuation plays some roles in maintaining the endocycle stage. In addition, the expression level of CYCB1;1, which is required for cell division in the M phase, coincided with the expected amount of auxin and cell division. Our analysis also provided a set of genes expressed in specific phases of the cell cycle. Taking these findings together, reconstruction of single-cell transcriptome data enables us to identify properties of the cell cycle more accurately.
Assuntos
Arabidopsis/citologia , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclo Celular/genética , Ciclina B/genética , Ácidos Indolacéticos/farmacologia , Mitose , Ftalimidas/farmacologia , Células Vegetais , Raízes de Plantas/citologia , Fase S/genéticaRESUMO
The plant-specific protein GIGANTEA (GI) controls many developmental and physiological processes, mediating rhythmic post-translational regulation. GI physically binds several proteins implicated in the circadian clock, photoperiodic flowering, and abiotic stress responses. To understand GI's multifaceted function, we aimed to comprehensively and quantitatively identify potential interactors of GI in a time-specific manner, using proteomics on Arabidopsis plants expressing epitope-tagged GI. We detected previously identified (in)direct interactors of GI, as well as proteins implicated in protein folding, or degradation, and a previously uncharacterized transcription factor, CYCLING DOF FACTOR6 (CDF6). We verified CDF6's direct interaction with GI, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LIGHT KELCH PROTEIN 2 proteins, and demonstrated its involvement in photoperiodic flowering. Extending interaction proteomics to time series provides a data resource of candidate protein targets for GI's post-translational control.
Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Fotoperíodo , Proteômica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genéticaRESUMO
In plants, light receptors play a pivotal role in photoperiod sensing, enabling them to track seasonal progression. Photoperiod sensing arises from an interaction between the plant's endogenous circadian oscillator and external light cues. Here, we characterize the role of phytochrome A (phyA) in photoperiod sensing. Our metaanalysis of functional genomic datasets identified phyA as a principal regulator of morning-activated genes, specifically in short photoperiods. We demonstrate that PHYA expression is under the direct control of the PHYTOCHROME INTERACTING FACTOR transcription factors, PIF4 and PIF5. As a result, phyA protein accumulates during the night, especially in short photoperiods. At dawn, phyA activation by light results in a burst of gene expression, with consequences for physiological processes such as anthocyanin accumulation. The combination of complex regulation of PHYA transcript and the unique molecular properties of phyA protein make this pathway a sensitive detector of both dawn and photoperiod.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ritmo Circadiano , Fotoperíodo , Fitocromo A/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Luz , Fitocromo A/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
Plants sense light and temperature changes to regulate flowering time. Here, we show that expression of the Arabidopsis florigen gene, FLOWERING LOCUS T (FT), peaks in the morning during spring, a different pattern than we observe in the laboratory. Providing our laboratory growth conditions with a red/far-red light ratio similar to open-field conditions and daily temperature oscillation is sufficient to mimic the FT expression and flowering time in natural long days. Under the adjusted growth conditions, key light signalling components, such as phytochrome A and EARLY FLOWERING 3, play important roles in morning FT expression. These conditions stabilize CONSTANS protein, a major FT activator, in the morning, which is probably a critical mechanism for photoperiodic flowering in nature. Refining the parameters of our standard growth conditions to more precisely mimic plant responses in nature can provide a powerful method for improving our understanding of seasonal response.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Fotoperíodo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Fitocromo A/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Ritmo Circadiano/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação , Fotoperíodo , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras GenéticasRESUMO
While angiosperm clocks can be described as an intricate network of interlocked transcriptional feedback loops, clocks of green algae have been modelled as a loop of only two genes. To investigate the transition from a simple clock in algae to a complex one in angiosperms, we performed an inventory of circadian clock genes in bryophytes and charophytes. Additionally, we performed functional characterization of putative core clock genes in the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis. Phylogenetic construction was combined with studies of spatiotemporal expression patterns and analysis of M. polymorpha clock gene mutants. Homologues to core clock genes identified in Arabidopsis were found not only in bryophytes but also in charophytes, albeit in fewer copies. Circadian rhythms were detected for most identified genes in M. polymorpha and A. agrestis, and mutant analysis supports a role for putative clock genes in M. polymorpha. Our data are in line with a recent hypothesis that adaptation to terrestrial life occurred earlier than previously expected in the evolutionary history of charophyte algae. Both gene duplication and acquisition of new genes was important in the evolution of the plant circadian clock, but gene loss has also contributed to shaping the clock of bryophytes.
Assuntos
Evolução Biológica , Relógios Circadianos , Embriófitas/fisiologia , Relógios Circadianos/genética , Ritmo Circadiano/genética , Embriófitas/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Genes Reporter , Luciferases/metabolismo , Medições Luminescentes , Família Multigênica , Mutação/genética , Filogenia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de TempoAssuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Proteínas de Ligação a DNA/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Fotoperíodo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Modelos Biológicos , Especificidade de Órgãos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genéticaRESUMO
Light regulates various aspects of development throughout the life cycle of sessile land plants. Photoreceptors, such as the red (R) and far-red (FR) light receptors phytochromes, play pivotal roles in modulating developmental programs. Reflecting high developmental plasticity, plants can regenerate tissues, organs, and whole bodies from varieties of cells. Among land plants, bryophytes exhibit extraordinary competency of regeneration under hormone-free conditions. As an environmental factor, light plays critical roles in regeneration of bryophytes. However, how light regulates regeneration remains unknown. Here we show that using the liverwort Marchantia polymorpha, which contains a single phytochrome gene, the phytochrome regulates re-entry into the cell cycle and cell shape in newly regenerating tissues. Our morphological and cytological observations revealed that S-phase entry of G1-arrested epidermal cells around the midrib on the ventral surface of thallus explants was greatly retarded in the dark or under phytochrome-inactive R/FR cycle irradiation conditions, where, nevertheless, small, laterally narrow regenerants were eventually formed. Thus, consistent with earlier descriptions published over a century ago, light is not essential for, but exerts profound effects on regeneration in M. polymorpha. Ventral cells in regenerants grown under R/FR cycle conditions were longer and narrower than those under R cycle. Expression of a constitutively active mutant of M. polymorpha phytochrome allowed regeneration of well grown, widely expanded thalli even in the dark when sugar was supplied, further demonstrating that the phytochrome signal promotes cell proliferation, which is rate-limited by sucrose availability. Similar effects of R and FR irradiation on cell division and elongation were observed in sporelings as well. Thus, besides activation of photosynthesis, major roles of R in regeneration of M. polymorpha are to facilitate proliferation of rounder cells through the phytochrome by mechanisms that are likely to operate in the sporeling.
Assuntos
Ciclo Celular/fisiologia , Desdiferenciação Celular/fisiologia , Marchantia/fisiologia , Fitocromo/fisiologia , Transdução de Sinais/fisiologia , Carboidratos/fisiologia , Forma Celular , Luz , Marchantia/efeitos da radiação , Marchantia/ultraestrutura , Modelos Biológicos , Fotossíntese/fisiologia , RegeneraçãoRESUMO
Recent studies have demonstrated that allelic variation in daily expression profiles of the GIGANTEA gene may account for the variance in plant growth in different accessions. Studying natural variation in daily transcriptional patterns of circadian-clock regulated genes provides new insights into plant adaptive strategies to different geographical regions.
Assuntos
Fotoperíodo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica de Plantas , Locos de Características Quantitativas/genética , TranscriptomaRESUMO
Photoperiodic control of the phase transition from vegetative to reproductive growth is critical for land plants. The GIGANTEA (GI) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) protein complex controls this process in angiosperms. However, little is known about how plants evolved this regulatory system. Here, we report that orthologues of GI and FKF1 are present in a basal plant, the liverwort Marchantia polymorpha, and describe the molecular interaction between their products. Knockout of either the GI or FKF1 orthologue completely abolishes the long-day-dependent growth-phase transition in M. polymorpha. Overexpression of either gene promotes growth-phase transition, even under short-day conditions. Introduction of the GI orthologue partially rescues the late-flowering phenotype of the Arabidopsis thaliana gi mutant. Our findings suggest that plants had already acquired the GI-FKF1 system to regulate growth-phase transition when they colonized land, and that this system was co-opted from gametophyte to sporophyte generation during evolution.
Assuntos
Regulação da Expressão Gênica de Plantas , Marchantia/genética , Fotoperíodo , Proteínas de Plantas/genética , Reprodução/genética , Arabidopsis , Proteínas de Arabidopsis/genética , Sequência de Bases , Evolução Biológica , Dados de Sequência MolecularRESUMO
The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Marchantia/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/genética , Cinamatos/farmacologia , DNA Bacteriano/genética , DNA de Cadeia Simples , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Marchantia/efeitos dos fármacos , Marchantia/crescimento & desenvolvimento , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regeneração , Fatores de Tempo , Transformação GenéticaRESUMO
We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.