Hirokazu Fukuda

Quantitative Analysis of Phase Wave of Gene Expression in the Mammalian Central Circadian Clock Network

Background The suprachiasmatic nucleus (SCN), the master circadian clock, is a heterogeneous oscillator network, yet displays a robust synchronization dynamics. Recent single-cell bioluminescent imaging revealed temporal gradients in circadian clock gene expression in the SCN ex vivo. However, due to technical difficulty in biological approaches to elucidate the entire network structure of the SCN, characteristics of the gradient, which we refer to as phase wave, remain unknown. Methodology/Principal Findings We implemented new approaches, i.e., quantitative analysis and model simulation to characterize the phase waves in Per2::Luciferase clock reporter gene expression of the rat SCN slice. Our quantitative study demonstrated not only a high degree of synchronization between the neurons and regular occurrence of the phase wave propagation, but also a significant amount of phase fluctuations contained in the wave. In addition, our simulations based on local coupling model suggest that the intercellular coupling strength estimated by the model simulations is significantly higher than the critical value for generating the phase waves. Model simulations also suggest that heterogeneity of the SCN neurons is one of the main factors causing the phase wave fluctuations. Furthermore, robustness of the SCN network against dynamical noise and variation of the natural frequencies inherent in these neurons was quantitatively assessed. Conclusions/Significance To our knowledge, this is the first quantitative evaluation of the phase wave and further characterization of the SCN neuronal network features generating the wave i.e., intercellular synchrony, phase fluctuation, strong local coupling, heterogeneous periodicity and robustness. Our present study provides an approach, which will lead to a comprehensive understanding of mechanistic and/or biological significance of the phase wave in the central circadian oscillatory system.

High-Throughput Growth Prediction for Lactuca sativa L. Seedlings Using Chlorophyll Fluorescence in a Plant Factory with Artificial Lighting

Poorly grown plants that result from differences in individuals lead to large profit losses for plant factories that use large electric power sources for cultivation. Thus, identifying and culling the low-grade plants at an early stage, using so-called seedlings diagnosis technology, plays an important role in avoiding large losses in plant factories. In this study, we developed a high-throughput diagnosis system using the measurement of chlorophyll fluorescence (CF) in a commercial large-scale plant factory, which produces about 5000 lettuce plants every day. At an early stage (6 days after sowing), a CF image of 7200 seedlings was captured every 4 h on the final greening day by a high-sensitivity CCD camera and an automatic transferring machine, and biological indices were extracted. Using machine learning, plant growth can be predicted with a high degree of accuracy based on biological indices including leaf size, amount of CF, and circadian rhythms in CF. Growth prediction was improved by addition of temporal information on CF. The present data also provide new insights into the relationships between growth and temporal information regulated by the inherent biological clock.

Effects of Airflow for Lettuce Growth in the Plant Factory with an Electric Turntable

Abstract To promote cultivation performance by controlling the air flow in plant factory, we introduced a new rotation mechanism for the alleviation of the inhomogeneous of airflow by the rotation of plant. The combination of air flow control and rotation mechanism makes about 20% large lettuce compared with the normal condition.

The System of Automatic Seedling Selection by the Early Molecular Diagnosis of Circadian Rhythms

Abstract We have developed the system of automatic seedling selection using an early molecular diagnosis of circadian rhythms. To measure the rhythms of gene expression by bioluminescence with in non-destructive and in real time, we used a measurement system for bioluminescence, called Kondotron. Moreover, to improve the efficiency of seedling selection, a manipulator for automatic seedling selection using the decision for the circadian rhythms in bioluminescence was introduced. Using this manipulator, it became possible to select automatically seedlings in the early stage based on the result of bioluminescent measurement. This system can measure 40 samples simultaneously more than one week in real time. Moreover, it is possible to measure seedlings continuously because new seedlings are supplied to measurement sample holders when they are picked out from the measurement holder. Therefore, it is possible to research in the flexible and wide experiment of gene expression since it can analysis the circadian rhythms under various purposes (automatic selection based on statistical processing, selection by threshold, and so on) and environment (light conditions) by this system.

Study of the Applicability of CFD Simulation for Analysis of the Transpiration Properties in Sunagoke Moss

Abstract Urban heat island (UHIP) is one of the environmental problems in the urban and is a feature commonly observed in many cities. The implementation of roof top greening is one of effective options to increase the amount of well-watered vegetation. Sunagoke moss have attracted attention as cover plants recently because they have desiccation-tolerance and are easy to manage. In this study, we obtained knowledge of transpiration properties in Sunagoke moss to optimal control the irrigation system of moss roof.

Theoretical Control of the Plant Biological Clock by the Periodic Dark Pulses

Abstract The endogenous circadian rhythms close to a 24-h period are observed by nearly all living organisms including bacteria, plants, animals, and humans. Plants use the circadian clocks to coordinate gene expression, metabolism, physiology and growth. The phase of circadian rhythm can be changed (advanced or delayed) by some stimuli. We used a dark pulse in continuous light pulse (LL) in the stimuli. During cultivation of plants, the use of dark pulses in LL is more suitable than that of light pulse in DD, because the light energy for photosynthesis is sufficient in LL with short dark pulses. By application of a 2-h dark pulse at subjective dusk, a small phase advance was induced (about 2-h phase advance). In contrast, by the application of a 2-h dark pulse at subjective dawn, a phase was induced (about 4-h phase delay).With knowledge of behavior of rhythms for dark pulse, we can control circadian clock and promote productivity. We described the phase response curve (PRC) of 2-h dark pulse by a sinusoidal curve with second harmonic component. By application of the PRC, we evaluated the relationship of plant growth and the rhythm of the circadian clock control with dark pulses.

Local Control of Circadian Rhythms in Lettuce Leaves Using Laser

Abstract The circadian rhythm is a biological clock with an approximately 24 hour period. This rhythm is observed in many living organisms. In plant, the cells are known to act as coupled oscillators by clock gene expression and cell-cell interaction, resulting that the phase wave and spiral wave can be observed in leaf as a two-dimensional self-oscillatory system. In this study, we experiment on local control of circadian rhythms in leaves of genetically modified plants using LCD projector and laser projector. Our results recognized that the circadian rhythm can be controlled by spatiotemporal illumination using low-power laser projector.

Simultaneous Measurement Device of the Luciferase Bioluminescence and the Delayed Fluorescence in Lettuce

Abstract In this study, we developed the devise named “Kondotron-lettuce” that is possible to measure luciferase bioluminescence and delayed fluorescence intensity of plants which are in the state that nearer to plant factory than the conventional circadian rhythm measurement. The main refinements are two points: One is that the devise became able to measure bioluminescence from plants which are cultured under hydroponic culturing for a long term. The another is that the devise became able to measure the delayed fluorescence. This devise is useful for measuring of luciferase bioluminescence and delayed fluorescence intensity under the controlled light and hydroponic culturing conditions in the long term.

Optimal Arrangement of the Dry-Heat Sterilizer in a Plant Factory

Abstract In this research, we propose a dry-heat sterilizer as substitute for the HEPA filter, which is the sterile filtration method of the conventional plant factory. It is important to determine the optimal placement in the dry-heat sterilizer for its practical use. There, in this research, we examined the evaluation methods aiming at the determination of the optimal placement of the dry-heat sterilizer in the chamber using a thermal flow simulation. We proposed the residence time and diffusive concentration as an evaluation index. As a result, it turned out that optimal placement can be determined by two indexes. Moreover, it was revealed that arrangement of the dry-heat sterilizer to near the wall of the opposite side of air-conditioning is an optimal placement of the dry-heat sterilizer.

Estimation of Bacterial Count by Electrochemical Method with Small Potentiostat

Abstract In plant factory, creation of additional values for products is very important. Cleanliness is one of the most important additional values to appeal to consumers. In particular, rapid measuring of bacteria is a significant method that keeps cleanliness of all products. Electrochemical measuring is useful to measure the number of bacteria rapidly and to estimate the number of E.coli with small potentiostat and Hoechst33258. In this study, we created a calibration curve and used it for the measuring of bacterial count on the surface of Lactuca sattiva. In our measurement, estimated count and actual count showed a similar value. Our study is clarified the possibility of electrochemical quantification of bacteria with small potentiostat.