Language：English   Publishing type：Research paper (scientific journal)  

The plant circadian oscillation system is based on the circadian clock of individual cells. Circadian behavior of cells has been observed by monitoring the circadian reporter activity such as bioluminescence of AtCCA1::LUC+. To deeply analyze different circadian behaviors in individual cells, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC. CaMV35S::LUC+ was previously reported as a circadian reporter with a low amplitude rhythm. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported. In contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. These results strongly suggested the uncoupling of cellular behavior between these circadian reporters. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression.

DOI： 10.1093/pcp/pcab037

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Timing adjustment is an important ability for living organisms. Wild animals need to act at the right moment to catch prey or escape a predator. Land plants, although limited in their movement, need to decide the right time to grow and bloom. Humans also need to decide the right moment for social actions. Although scientists can pinpoint the timing of such behaviors by observation, we know extremely little about how living organisms as actors or players decide when to act – such as the exact moment to dash or pounce. The time measurements of an outsider-observer and the insider-participants are utterly different. We explain how such essential operations of timing adjustment and temporal spanning, both of which constitute a single regulated set, can be carried out among organisms. For this purpose, we have to reexamine the ordinary conception of time. Our specific explanatory tools include the natural movement known as the upbeat (<italic>anacrusis</italic>) in music, a rhythmic push for the downbeat that follows, which predicts future moves as an anticipatory lead-in. The scheme is situated in and is the extension of our formulation of E-series time, i.e., timing co-adjusted through interaction, which is derived from the semiotic/communicative perspectives. We thereby demonstrate that a prediction-based timing system is not mechanical but communicative and entails meanings for future anticipation.

DOI： 10.1007/s12304-020-09398-5

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Other Link： http://link.springer.com/article/10.1007/s12304-020-09398-5/fulltext.html

Authorship：Lead author   Language：English  

The bioluminescent reporter system is a powerful tool for the long-term monitoring of gene expression because of its noninvasive nature. Furthermore, in combination with high-sensitive imaging technology, spatiotemporal analysis on regulation and heterogeneity in gene expression is possible. We developed a single-cell bioluminescent imaging system for plants through a transient gene transfection by particle bombardment. By applying this system to a duckweed species, we succeeded in monitoring circadian rhythms of individual cells in an intact plant for over a week. Here we describe methods for gene transfection by particle bombardment and single-cell bioluminescence monitoring by a high-sensitive camera. This technique provides a platform for characterizing gene expression patterns of individual cells in the same tissue.

DOI： 10.1007/978-1-4939-9940-8_17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Anticipatory acts or predictive behavior are prerequisites for living organisms to sustain their survival when escaping from a predator, catching prey, or schooling. For example, catching prey requires that the predator perform some procedures that are equivalent to estimating the directional movement of the prey, its speed and its distance relative to the predator. Underlying these procedures is time experience, which does not adhere to man-made mechanical clocks. Living organisms keep time based on the local activities of each participant and form ecological clocks together. The timekeeping of ecological clocks has been called E-series time, which is interactive in character and consists of mutual alignment of timing that is co-adjusted to each other's movements and rhythms. A main objective of our current work is to illustrate how E-series time is used for flows of anticipatory acts. To explain such predictive moves and their efforts based on how the perspective of the immediate future affects the present, we resort to the organismic activity of revising the preceding acts in retrospect and semiotic scaffolding that extends beyond simple linear causality. Special attention is paid to the construction of the notion of retrocausal scaffolding, which is a series of dialogical punctuations or mutual coordination of rhythms for the joint production of the present moment of now. Retrocausal scaffolding is synonymous with negotiated anticipation, which is a semiotic/communicative account of revising the preceding acts in the present moment.

DOI： 10.1007/s12304-019-09363-x

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Language：English  

Circadian rhythms produce a biological measure of the time of day. In plants, circadian regulation forms an essential adaptation to the fluctuating environment. Most of our knowledge of the molecular aspects of circadian regulation in plants is derived from laboratory experiments that are performed under controlled conditions. However, it is emerging that the circadian clock has complex roles in the coordination of the transcriptome under natural conditions, in both naturally occurring populations of plants and in crop species. In this review, we consider recent insights into circadian regulation under natural conditions. We examine how circadian regulation is integrated with the acute responses of plants to the daily and seasonally fluctuating environment that also presents environmental stresses, in order to coordinate the transcriptome and dynamically adapt plants to their continuously changing environment.

DOI： 10.3389/fgene.2019.01239

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

We develop a semiotic scheme of time, in which time precipitates from the repeated succession of punctuating the progressive tense by the perfect tense. The underlying principle is communication among local participants. Time can thus be seen as a meaning-making, semiotic system in which different time codes are delineated, each having its own grammar and timekeeping. The four time codes discussed are the following: the subjective time having tense, the objective time without tense, the static time without timekeeping, and the inter-subjective time of the E-series. Living organisms adopt a time code called the E-series, which emerges through the local synchronization among organisms or parts of organisms. The inter-subjective time is a new theoretical dimension resulting from the time-aligning activities of interacting agents. Such synchronization in natural settings consists of incessant mutual corrections and adjustments to one’s own punctuation, which is then constantly updated. Unlike the third-person observer keeping the objective time while sitting outside a clock, the second-person negotiators participate in forming the E-series time by punctuating and updating the interface through which different tenses meet at the moment of “now.” Although physics allows physicists to be the only interpreters, the semiotic perspective upends the physical perspective by letting local participants be involved in the interpretation of their mutual negotiations to precipitate that which is called time.

DOI： 10.1007/s12304-018-9316-0

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Tokyo  

The circadian clock is an endogenous timing system based on the self-sustained oscillation in individual cells. These cellular circadian clocks compose a multicellular circadian system working at respective levels of tissue, organ, plant body. However, how numerous cellular clocks are coordinated within a plant has been unclear. There was little information about behavior of circadian clocks at a single-cell level due to the difficulties in monitoring circadian rhythms of individual cells in an intact plant. We developed a single-cell bioluminescence imaging system using duckweed as the plant material and succeeded in observing behavior of cellular clocks in intact plants for over a week. This imaging technique quantitatively revealed heterogeneous and independent manners of cellular clock behaviors. Furthermore, these quantitative analyses uncovered the local synchronization of cellular circadian rhythms that implied phase-attractive interactions between cellular clocks. The cell-to-cell interaction looked to be too weak to coordinate cellular clocks against their heterogeneity under constant conditions. On the other hand, under light–dark conditions, the heterogeneity of cellular clocks seemed to be corrected by cell-to-cell interactions so that cellular clocks showed a clear spatial pattern of phases at a whole plant level. Thus, it was suggested that the interactions between cellular clocks was an adaptive trait working under day–night cycles to coordinate cellular clocks in a plant body. These findings provide a novel perspective for understanding spatio-temporal architectures in the plant circadian system.

DOI： 10.1007/s10265-017-1001-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

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.

DOI： 10.1111/nph.14487

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Individual cells in a plant can work independently as circadian clocks, and their properties are the basis of various circadian phenomena. The behaviour of individual cellular clocks in Lemna gibba was orderly under 24-h light/dark cycles despite their heterogeneous free-running periods (FRPs). Here, we reveal the entrainment habits of heterogeneous cellular clocks using non-24-h light/dark cycles (T-cycles). The cellular rhythms of AtCCA1:: LUC under T = 16 h cycles showed heterogeneous entrainment that was associated with their heterogeneous FRPs. Under T = 12 h cycles, most cells showed rhythms having similar to 24-h periods. This suggested that the lower limit of entrainment to the light/dark cycles of heterogeneous cellular circadian clocks is set to a period longer than 12 h, which enables them to be synchronous under similar to 24-h daily cycles without being perturbed by short light/dark cycles. The entrainment habits of individual cellular clocks are likely to be the basis of the circadian behaviour of plant under the natural day-night cycle with noisy environmental fluctuations. We further suggest that modifications of EARLY FLOWERING3 (ELF3) in individual cells deviate the entrainability to shorter T-cycles possibly by altering both the FRPs and light responsiveness.

DOI： 10.1038/s41598-017-00454-8

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Recent advances in single-cell analysis have revealed the stochasticity and nongenetic heterogeneity inherent to cellular processes. However, our knowledge of the actual cellular behaviors in a living multicellular organism is still limited. By using a single-cell bioluminescence imaging technique on duckweed, Lemna gibba, we demonstrate that, under constant conditions, cells in the intact plant work as individual circadian clocks that oscillate with their own frequencies and respond independently to external stimuli. Quantitative analysis uncovered the heterogeneity and instability of cellular clocks and partial synchronization between neighboring cells. Furthermore, we found that cellular clocks in the plant body under light-dark cycles showed a centrifugal phase pattern in which the effect of cell-to-cell heterogeneity in period lengths was almost masked. The inherent heterogeneity in the properties of cellular clocks observed under constant conditions is corrected under light-dark cycles to coordinate the daily rhythms of the plant body. These findings provide a novel perspective of spatiotemporal architectures in the plant circadian system.

DOI： 10.1126/sciadv.1600500

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The plant circadian clock controls various physiological phenomena that are important for adaptation to natural day-night cycles. Many components of the circadian clock have been identified in Arabidopsis thaliana, the model plant for molecular genetic studies. Recent studies revealed evolutionary conservation of clock components in green plants. Homologues of clock-related genes have been isolated from Lemna gibba and Lemna aequinoctialis, and it has been demonstrated that these homologues function in the clock system in a manner similar to their functioning in Arabidopsis. While clock components are widely conserved, circadian phenomena display diversity even within the Lemna genus. In order to survey the full extent of diversity in circadian rhythms among duckweed plants, we characterised the circadian rhythms of duckweed by employing a semi-transient bioluminescent reporter system. Using a particle bombardment method, circadian bioluminescent reporters were introduced into nine strains representing five duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna gibba, L.aequinoctialis and Wolffia columbiana. We then monitored luciferase (luc+) reporter activities driven by AtCCA1, ZmUBQ1 or CaMV35S promoters under entrainment and free-running conditions. Under entrainment, AtCCA1::luc+ showed similar diurnal rhythms in all strains. This suggests that the mechanism of biological timing under day-night cycles is conserved throughout the evolution of duckweeds. Under free-running conditions, we observed circadian rhythms of AtCCA1::luc+, ZmUBQ1::luc+ and CaMV35S::luc+. These circadian rhythms showed diversity in period length and sustainability, suggesting that circadian clock mechanisms are somewhat diversified among duckweeds.

DOI： 10.1111/plb.12202

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of &gt;= 50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

DOI： 10.1093/pcp/pct131

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