Biology subjects

Muranaka, T.

Publications and source records attributed to Muranaka, T..

9 recordsLinked to original sources

bioRxiv2026

Nucleotide-driven KaiC dynamics coordinate the core properties of the cyanobacterial circadian clock

Circadian clocks generate stable [~]24-h rhythms with a defined period, temperature compensation, and entrainment to external cues that set phase. However, the molecular reactions that generate these features are not fully understood. In cyanobacteria, timekeeping is driven by the hexameric ATPase KaiC, which consists of two homologous domains, CI and CII, and whose enzymatic turnover underlies rhythmic phosphorylation. Here we identify ADP release as the rate-limiting step in the KaiC ATPase cycle and demonstrate that the KaiC ATPase nucleotide cycle integrates core properties of the circadian clock. Across KaiC period mutants, increased ADP occupancy is associated with longer periods. Elevated temperature shifts KaiC toward an ADP-bound state, offsetting the thermal acceleration of ATP hydrolysis. KaiB reinforces this ADP-bound conformation by inhibiting nucleotide exchange, thereby strengthening inhibition of CI ATPase activity of KaiC and tuning oscillation amplitude in a temperature-dependent manner. In contrast, KaiA accelerates ADP-to-ATP exchange within the KaiB-KaiC complex, thereby stimulating CI ATPase activity and promoting KaiB-KaiC dissociation prior to KaiC phosphorylation. Phosphorylation begins only after this transition, indicating that KaiC nucleotide-bound state sets the phase of the phosphorylation cycle. Collectively, these results establish the nucleotide cycle of KaiC ATPase as a unifying mechanism that connects molecular reactions to the defining properties of the cyanobacterial circadian clock.

biochemistry↗

bioRxiv2026

Circadian entrainment to environmental cues in a natural plant population

Circadian clocks coordinate physiology, development and behaviour with cycles of day and night, which contributes to organismal fitness. For this to occur, the circadian clock must be aligned with the natural daily fluctuations in the environmental conditions. Here, we investigated under natural conditions the role of environmental cues in adjusting the circadian clock, and how this affects clock outputs. By combining novel field-based experimentation in a natural plant population with genome-wide analysis and machine-learning strategies for data interpretation, we inferred that temperature cues change circadian timing under natural conditions. We inferred that low temperature cues can shift circadian phase under naturally fluctuating conditions, and that there are seasonal changes in the effects of temperature upon circadian entrainment. We also identified extensive circadian modulation of temperature responses under field conditions. Therefore, plasticity of circadian timing under natural conditions allows flexible responses of both the clock and its outputs to environmental stimuli.

plant biology↗

bioRxiv2025

Quantitative manipulation of the photoperiodic flowering by a small-molecule clock modulator

Photoperiodism, a seasonal response mechanism that relies on day-length measurement by the circadian clock, is a major regulator of flowering time 1. The external coincidence model proposes that the alignment between the internal circadian phase and external light signals triggers photoperiodic responses 2. It has been challenging to establish robust models for the quantitative relationship between clock and flowering time, due to a lack of tools to modulate clock quantitatively. Here, using a circadian period-lengthening small molecule, we demonstrated the quantitative modulation of the critical day length for flowering in monocots.

plant biology↗

bioRxiv2025

Heritable Large Deletions Using Type I-E CRISPR-Cas3 in Rice

Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 to plants is challenging because 7 CRISPR-Cas3 components (6 Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39-48% and 55-71% of calli transformed with 2 binary vectors carrying 7 expression cassettes of Eco CRISPR-Cas3 components and a compact all-in-one vector carrying 3 expression cassettes of Cas proteins fused to 2A self-cleavage peptide, respectively. The frequency of alleles lacking a region 7.0 kb upstream of the PAM sequence was estimated as 21-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, and genome rearrangement in plants.

plant biology↗

bioRxiv2022

Identification of basic helix-loop-helix transcription factors that activate betulinic acid biosynthesis by RNA-sequencing of hydroponically cultured Lotus japonicus

Although triterpenes are ubiquitous in plant kingdom, their biosynthetic regulatory mechanisms are limitedly understood. Here, we found that hydroponic culture dramatically activated betulinic acid (BA) biosynthesis in the model Fabaceae Lotus japonicus, and investigated its transcriptional regulation. Fabaceae plants develop secondary aerenchyma (SA) on the surface of hypocotyls and roots during flooding for root air diffusion. Hydroponic culture induced SA in L. japonicus and simultaneously increased the accumulation of BA and the transcript levels of its biosynthetic genes. RNA-sequencing of soil-grown and hydroponically cultured plant tissues, including SA collected by laser microdissection, revealed that several transcription factor genes were co-upregulated with BA biosynthetic genes. Overexpression of LjbHLH32 and LjbHLH50 in L. japonicus transgenic hairy roots upregulated the expression of BA biosynthesis genes, resulting in enhanced BA accumulation. However, transient luciferase reporter assays in Arabidopsis mesophyll cell protoplasts showed that LjbHLH32 transactivated promoters of biosynthetic genes in the soyasaponin pathway but not the BA pathway, like its homolog GubHLH3, a soyasaponin biosynthesis regulator in Glycyrrhiza uralensis. This suggested the evolutionary origin and complex regulatory mechanisms of BA biosynthesis in Fabaceae. This study sheds light on the unrevealed biosynthetic regulatory mechanisms of triterpenes in Fabaceae plants. HighlightHydroponic culture enhanced betulinic acid synthesis in Lotus japonicus. RNA-sequencing and functional characterization experiments suggest that LjbHLH32 and LjbHLH50 are the transcription factors activating betulinic acid biosynthesis.

plant biology↗

bioRxiv2022

Circadian and environmental signal transduction in a natural population of Arabidopsis

Plants sense and respond to environmental cues during 24 h fluctuations in their environment. This requires the integration of internal cues such as circadian timing with environmental cues such as light and temperature to elicit cellular responses through signal transduction. However, the integration and transduction of circadian and environmental signals within plants growing in natural environments remains poorly understood. To gain insights into the 24 h dynamics of environmental signalling in nature, we performed a field study of signalling from the nucleus to chloroplasts in a natural population of Arabidopsis halleri. Using advanced modelling approaches to interpret the data, we identified that the circadian clock and temperature are key regulators of this pathway under natural conditions. We identified potential time-delay steps between pathway components, and diel fluctuations in the response of the pathway to temperature cues that are reminiscent of the process of circadian gating. This approach of combining studies of gene expression in the field with modelling allowed us to identify the dynamic integration and transduction of environmental cues, in plant cells, under naturally fluctuating diel cycles.

plant biology↗

bioRxiv2022

No circadian clock-gene circuit in the generation of cellular bioluminescence rhythm of CaMV35S::PtRLUC in duckweed

Physiological circadian rhythms are coordinated in the plant body in an orderly manner. The coordination of time information has been studied from the aspects of cell-cell local coupling and long-distance communication between tissues. These studies were based on the idea that the behavior of the clock gene circuit represents the physiological rhythms. Here we report the cellular circadian rhythm of a bioluminescence reporter which is not governed by the clock gene circuit in the expressing cells. Using a dual-color bioluminescence monitoring system in Lemna minor transfected with the AtCCA1::LUC+ and CaMV35S::PtRLUC reporters, cellular bioluminescence rhythms with different free-running periods (FRPs) were detected in the same cells. Co-transfection experiments with the two reporters and a clock gene overexpressing effector revealed that the circadian properties of the AtCCA1::LUC+ rhythm, but not those of the CaMV35S::PtRLUC rhythm, were altered in the cells with a dysfunctional clock gene circuit. This indicates that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian clock while the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared while the AtCCA1::LUC+ rhythm persisted. The plant circadian system consists of both cell-autonomous rhythms and non-cell-autonomous rhythms that are unaffected by the cellular clock.

plant biology↗

bioRxiv2022

A chronotype effect: circadian-period variation underlies the local adaptation of photoperiodism in the short-day plant Lemna aequinoctialis.

Phenotypic variation is the basis for trait adaptation via evolutionary selection.1, 2, 3 However, the driving forces behind the quantitative trait variations remain unclear owing to their complexity at the molecular level.4, 5, 6 This study focused on the natural variation of the free-running period (FRP) of the circadian clock because FRP is a determining factor of the internal clock phase (chronotype), which is responsible for physiological timing during a day.7 Although natural variations in FRP have been widely reported,8, 10, 11 few studies have shown the association between FRP and adaptive temporal traits. As a clock-dependent physiological process, photoperiodism is a typical target of local adaptation.12, 13 Lemna aequinoctialis in Japan is a paddy-field duckweed exhibiting a latitudinal cline of critical day-lengths (CDLs) for short-day flowering.14 To investigate the relationship between FRP and CDL, we collected 72 strains of L. aequinoctialis within a latitudinal range between 31.5{degrees}N to 43.8{degrees}N. We found a significant correlation (P = 7.5E-8) between FRPs and locally adaptive CDLs, confirming that the variation in FRP-dependent chronotypes underlies geographically differentiated photoperiodism. Diel transcriptome analysis revealed that the induction timing of a florigen gene is key for connecting chronotypes to photoperiodism at the molecular level. Based on these results, we propose a fundamental rule concerning the "chronotype effect" in evolution: the variation of FRP functions as a resource for the variation of temporal traits. This study highlights the adaptive significance of FRP variation and provides a reason for the maintenance of FRP variation in natural populations.

ecology↗

bioRxiv2020

Detection of uncoupled circadian rhythms in individual cells of Lemna minor by using a dual-color bioluminescence monitoring system

The plant circadian oscillation system is based on the circadian clock of individual cells and coordinates the circadian behavior of the plant body. To observe the cellular circadian behavior of both the oscillator and its output in plants, 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 to observe the cellular behavior of the oscillator and output, respectively. 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 studies using other plants. In contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. Thus, we clearly demonstrated the uncoupling between the oscillator and output in individual cells. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression. Significance statementWe succeeded in establishing the worlds first dual-color bioluminescence monitoring system at a single-cell level that enables simultaneous measurement of the luminescence activities of two reporter genes in plants. This system is a strong tool to analyze stochastic phenomena, and we clearly demonstrated the uncoupling of rhythmic behavior between two bioluminescent reporters in individual cells that stochastically occurred in the same plant.

plant biology↗