RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Relojes Circadianos/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica de las Plantas , Arabidopsis/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Luz , Fotoperiodo , Flores/genética , Flores/metabolismo , Fitocromo A/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Relojes Circadianos/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ritmo Circadiano/fisiología , Transducción de Señal/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , Análisis de Secuencia de ARN , Factores de TiempoRESUMEN
Like many organisms, plants have evolved a genetic network, the circadian clock, to coordinate processes with day/night cycles. In plants, the clock is a pervasive regulator of development and modulates many aspects of physiology. Clock-regulated processes range from the correct timing of growth and cell division to interactions with the root microbiome. Recently developed techniques, such as single-cell time-lapse microscopy and single-cell RNA-seq, are beginning to revolutionize our understanding of this clock regulation, revealing a surprising degree of organ, tissue, and cell-type specificity. In this review, we highlight recent advances in our spatial view of the clock across the plant, both in terms of how it is regulated and how it regulates a diversity of output processes. We outline how understanding these spatially specific functions will help reveal the range of ways that the clock provides a fitness benefit for the plant.
Asunto(s)
Arabidopsis , Relojes Circadianos , Arabidopsis/genética , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Plantas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Flores , Regulación de la Expresión Génica de las Plantas , Fotoperiodo , Fitomejoramiento , Sulfanilamidas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.
RESUMEN
Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.
Asunto(s)
Proteínas de Arabidopsis , Desarrollo de la Planta , Raíces de Plantas , Plantas , TemperaturaRESUMEN
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.
Asunto(s)
Arabidopsis/citología , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclo Celular/genética , Ciclina B/genética , Ácidos Indolacéticos/farmacología , Mitosis , Ftalimidas/farmacología , Células Vegetales , Raíces de Plantas/citología , Fase S/genéticaRESUMEN
Cell-type-specific transcription factors are key to deducing the distinct functions of specialized cells from gene expression profiles. Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. Palisade and spongy mesophyll cells are sub-tissues that have different morphologies and physiologies. Thus, determining the palisade and spongy mesophyll-specific transcription factors from the respective sub-tissue-specific transcriptomes is vital to understanding or verifying functions of major plant tissues. One way in which gene expression profiles can be addressed is through direct isolation. Here, we present rapid and simple methods to isolate palisade and spongy mesophyll cells mechanically and enzymatically. This method provides a good yield of each isolated cell type, and the isolated cells can be used for various downstream applications.
Asunto(s)
Arabidopsis/citología , Separación Celular/métodos , Células del Mesófilo/citología , Células Vegetales/metabolismo , ADN Complementario/genética , ADN de Plantas/genética , ARN de Planta/aislamiento & purificación , Transcripción GenéticaRESUMEN
In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8-12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport.
Asunto(s)
Florigena/metabolismo , Flores/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/metabolismo , Meristema/metabolismo , Mutación , Floema/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
The article" Circadian clock during plant development".
RESUMEN
Many organisms rely on circadian clocks to synchronize their biological processes with the 24-h rotation of the earth. In mammals, the circadian clock consists of a central clock in the suprachiasmatic nucleus and peripheral clocks in other tissues. The central clock is tightly coupled to synchronize rhythmicity and can organize peripheral clocks through neural and hormonal signals. In contrast to mammals, it has long been assumed that the circadian clocks in each plant cell is able to be entrained by external light, and they are only weakly coupled to each other. Recently, however, several reports have demonstrated that plants have unique oscillator networks with tissue-specific circadian clocks. Here, we introduce our current view regarding tissue-specific properties and oscillator networks of plant circadian clocks. Accumulating evidence suggests that plants have multiple oscillators, which show distinct properties and reside in different tissues. A direct tissue-isolation technique and micrografting have clearly demonstrated that plants have hierarchical oscillator networks consisting of multiple tissue-specific clocks.
Asunto(s)
Relojes Circadianos/fisiología , Plantas/genéticaRESUMEN
To determine flowering time, plants perceive multiple environmental stimuli and integrate these signals in the regulation of a florigen gene, FLOWERING LOCUS T (FT). It has been known that nutrient availability affects flowering time in both laboratories and fields. Nitrogen (N), phosphorus (P) and potassium (K) are the three major macronutrients which are important for plant growth and development. Although N and P stimuli can alter the expression of regulators of FT including microRNA156 (miR156) and miR156-targeted transcription factors of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family, how K+ conditions affect flowering is still unclear. We focused on SODIUM POTASSUIM ROOT DEFECTIVE1 (NaKR1) whose mutant plants showed Na+ and K+ overaccumulation and late flowering. It was reported that NaKR1 is involved in the phloem transport of FT protein. Here we report that NaKR1 is also required for the promotion of FT expression in long-day conditions. NaKR1 affects the accumulation of miR156 and SPL3 expression, suggesting that NaKR1 regulates FT expression in part through the miR156-SPL3 module. The late-flowering phenotype of the nakr1-1 mutant was partially suppressed under low K+ conditions, and miR156 abundance and SPL3 expression in the nakr1-1 mutant and, to a lesser extent, in wild-type plants responded to K+ conditions. Taken together, our findings suggest that the miR156-SPL3 module mediates regulation of FT expression by NaKR1 in response to K+ conditions. Finally, we propose a model in which NaKR1 plays dual roles in regulation of flowering, one in the regulation of florigen production, the other in that of florigen transport.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , MicroARNs/metabolismo , Potasio/farmacología , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Flores/efectos de los fármacos , Flores/genética , Flores/fisiología , MicroARNs/genética , Modelos Biológicos , Fenotipo , Transcripción Genética/efectos de los fármacosRESUMEN
Plants have endogenous biological clocks that allow organisms to anticipate and prepare for daily and seasonal environmental changes and increase their fitness in changing environments. The circadian clock in plants, as in animals and insects, mainly consists of multiple interlocking transcriptional/translational feedback loops. The circadian clock can be entrained by environmental cues such as light, temperature and nutrient status to synchronize internal biological rhythms with surrounding environments. Output pathways link the circadian oscillator to various physiological, developmental, and reproductive processes for adjusting the timing of these biological processes to an appropriate time of day or a suitable season. Recent genomic studies have demonstrated that polymorphism in circadian clock genes may contribute to local adaptations over a wide range of latitudes in many plant species. In the present review, we summarize the circadian regulation of biological processes throughout the life cycle of plants, and describe the contribution of the circadian clock to local adaptation.
Asunto(s)
Adaptación Biológica/fisiología , Relojes Circadianos/fisiología , Desarrollo de la Planta/fisiologíaRESUMEN
To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays.
Asunto(s)
Arabidopsis/citología , Separación Celular/métodos , Células del Mesófilo/citología , Epidermis de la Planta/citología , Control de Calidad , Factores de TiempoRESUMEN
Circadian clocks adjust an organism's environmentally relevant physiological responses.. In plants, a decentralized circadian clock system has recently been proposed. Epidermal clock function is crucial for cell elongation; thus, epidermis-specific overexpression of CCA1 caused smaller cotyledons and longer hypocotyls under 27°C, concomitant with elevated night time levels of PIF4 mRNA. However, which tissue's clock regulates PIF4 expression is still an open question. Here we tested spatial expression patterns of PIF4 and its downstream target IAA29 with or without epidermal clock perturbation. Using an epidermal-specific expression system, we revealed that epidermal clock perturbation increases PIF4 expression in both epidermis and mesophyll. However, IAA29 expression is mainly regulated in the epidermis, implying the potential importance of epidermis for regulation of cell elongation through PIF4 and IAA29. We conclude that the circadian clock in epidermis regulates cell elongation mainly in epidermis, and there is also another inter-tissue signaling pathway from epidermis to mesophyll.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Relojes Circadianos , Factores de Transcripción/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica de las Plantas , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In land plants, there are two types of male gametes: one is a non-motile sperm cell which is delivered to the egg cell by a pollen tube, and the other is a motile sperm cell with flagella. The molecular mechanism underlying the sexual reproduction with the egg and pollen-delivered sperm cell is well understood from studies using model plants such as Arabidopsis and rice. On the other hand, the sexual reproduction with motile sperm has remained poorly characterized, due to the lack of suitable models. Marchantia polymorpha L. is a model basal land plant with sexual reproduction involving an egg cell and bi-flagellated motile sperm. To understand the differentiation process of plant motile sperm, we analyzed the gene expression profile of developing antheridia of M. polymorpha. We performed RNA-sequencing experiments and compared transcript profiles of the male sexual organ (antheridiophore and antheridium contained therein), female sexual organ (archegoniophore) and a vegetative organ (thallus). Transcriptome analysis showed that the antheridium expresses nearly half of the protein-coding genes predicted in the genome, but it also has unique features. The antheridium transcriptome shares some common features with male gamete transcriptomes of angiosperms and animals, and homologs of genes involved in male gamete formation and function in angiosperms and animals were identified. In addition, we showed that some of them had distinct expression patterns in the spermatogenous tissue of developing antheridia. This study provides a transcriptional framework on which to study the molecular mechanism of plant motile sperm development in M. polymorpha as a model.
Asunto(s)
Gametogénesis en la Planta/genética , Marchantia/genética , Transcripción Genética , Transcriptoma/genética , Cromosomas de las Plantas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Histonas/metabolismo , Marchantia/anatomía & histología , Marchantia/metabolismo , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Circadian clocks affect a large proportion of differentially expressed genes in many organisms. Tissue-specific hierarchies in circadian networks in mammals have been contentiously debated, whereas little attention has been devoted to the concept in plants, owing to technical difficulties. Recently, several studies have demonstrated tissue-specific circadian clocks and their coupling in plants, suggesting that plants possess a hierarchical network of circadian clocks. The following review summarizes recent studies describing the tissue-specific functions and properties of these circadian clocks and discusses the network structure and potential messengers that might share temporal information on such a network.