The molecular genetics of circadian rhythms in Arabidopsis.

While a number of physiological and biochemical processes in plants have been found to be regulated in a circadian manner, the mechanism underlying the circadian oscillator remains to be elucidated. Advances in the identification and characterization of components of the plant circadian system have been made largely through the use of genetics in Arabidopsis thaliana. Results so far indicate that the generation of rhythmicity by the Arabidopsis clock relies on molecular mechanisms that are similar to those described for other organisms, but that a totally different set of molecular components has been recruited to perform these functions.     

Monitoring circadian rhythms of individual cells in plants

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.     

RAP, an integrated program for monitoring bioluminescence and analyzing circadian rhythms in real time

To process the large number of circadian rhythmic bioluminescence data that we generated with specially developed, high-throughput monitoring apparatuses, we developed an integrated rhythm-analyzing program (RAP) with a user-friendly graphical interface. RAP does all of the following in real time: (i) displays the bioluminescence time course, (ii) records time-series data, (iii) analyzes the data, (iv) displays the analyzed results, (v) statistically evaluates the analyzed results, and (vi) provides printouts. Because RAP can import files, it can analyze not only bioluminescence data but also other data such as those obtained by DNA array experiments and by Northern and Western blot analyses. The program is a powerful tool for large-scale investigations of biological rhythmic phenomena in general.     

Electrifying rhythms in plant cells

Physiological oscillations (or rhythms) pervade all spatiotemporal scales of biological organization, either because they perform critical functions or simply because they can arise spontaneously and may be difficult to prevent. Regardless of the case, they reflect regulatory relationships between control points of a given system and offer insights as read-outs of the concerted regulation of a myriad of biological processes. Here we review recent advances in understanding ultradian oscillations (period < 24h) in plant cells, with a special focus on single-cell oscillations. Ion channels are at the center stage due to their involvement in electrical/excitabile phenomena associated with oscillations and cell-cell communication. We highlight the importance of quantitative approaches to measure oscillations in appropriate physiological conditions, which are essential strategies to deal with the complexity of biological rhythms. Future development of optogenetics techniques in plants will further boost research on the role of membrane potential in oscillations and waves across multiple cell types.     

Glycosylation of sesamol by cultured plant cells

The glycosylation of sesamol was investigated using cultured cells of Nicotiana tabacum and Eucalyptus perriniana. The cultured suspension cells of N. tabacum converted sesamol into its β-glucoside (7%) as well as the disaccharide, sesamyl 6-O-(β-D-glucopyranosyl)-β-D-glucopyranoside (β-gentiobioside, 30%). On the other hand, sesamyl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside (β-rutinoside, 56%), together with the β-glucoside (3%), was produced when sesamol was incubated with suspension cells of E. perriniana.     

Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.     

A method to record circadian plant movements, with application to Oxalis leaf rhythms

A transducer was developed to record the circadian movement of the individual leaflets in Oxalis regnellii Mig. The method can easily be adapted to measure other kinds of plant movements as well. It is based on the detection of the shadow each leaflet casts on the small side of a specially formed Perspex plate. The light is guided through the Perspex and collected by a phototransistor, which provides an electrical signal that is proportional to the light intensity falling onto it. The output signal can be made a linear function of the leaf angle. This equipment was used in experiments to study the coupling between the 3 leaflets in Oxalis . Pulses of 4 h of red light were given to one of the leaflets, the two others were shielded from the light. A phase response curve was determined for each leaflet, but there was no significant difference in the phase response between the 3 leaflets. Experiments were also made in which the 3 leaflets were separated physically by cuts along the petiole between the pulvini. In this case ultradian oscillations were observed.     

Calcium and rhythms in plant cells

Plants cells, like any other living organism, experience the daily rotation of the Earth. They also depend strikingly on light, as a result of which much of the plant's biochemistry, physiology, and behaviour are temporally organised with respect to the environmental oscillation of day and night. Here we review the most recent findings on plants rhythms and how they seem to be so tightly connected to calcium-signalling aspects. We also try to establish parallels between different cell types, such as pollen tubes and fungal hyphae, where the existence and function of rhythms and oscillations is not obvious. Additionally, we discuss new methodologies and how these are shaping our current working hypothesis to study Ca²⁺ rhythms in plant cells.     

Entrainment of Drosophila circadian rhythms by temperature cycles

Sleep and Biological Rhythms - The fruit fly, Drosophila melanogaster, has been a good organism for elucidating the molecular and cellular bases of circadian behavioral rhythms. The fly shows a...     

Beyond Arabidopsis: The circadian clock in non-model plant species

Circadian clocks allow plants to temporally coordinate many aspects of their biology with the diurnal cycle derived from the rotation of Earth on its axis. Although there is a rich history of the study of clocks in many plant species, in recent years much progress in elucidating the architecture and function of the plant clock has emerged from studies of the model plant, Arabidopsis thaliana. There is considerable interest in extending this knowledge of the circadian clock into diverse plant species in order to address its role in topics as varied as agricultural productivity and the responses of individual species and plant communities to global climate change and environmental degradation. The analysis of circadian clocks in the green lineage provides insight into evolutionary processes in plants and throughout the eukaryotes.     

Exploring the signaling pathway of circadian bioluminescence

Bioluminescence in the unicellular dinoflagellate Gonyaulax polyedra represents an excellent model for studying a circadian controlled process at the biochemical and molecular levels. There are three key components involved in the bioluminescence reaction: the enzyme, luciferase, its substrate, luciferin, and a luciferin-binding protein (LBP), which sequesters the substrate at p^H 7.5 and thus prevents it from reacting with the enzyme. All components are tightly packed together in organdies, designated scintillons. The entire bioluminescent system is under circadian control with maximum amounts in the night. For both proteins circadian control is exerted at the translational level. In case of Ibp mRNA a small interval in its 3'untranslated region serves as a cis -acting element to which a trans -factor binds in a circadian manner. The binding activity of this factor decreases at the beginning of the night phase, when synthesis of LBP starts, and it increases al the end of the night, when synthesis of LBP stops indicating that it functions as a clock-controlled represser.     

Masking of circadian activity rhythms in hamsters by darkness

Summary Wheel-running activity was recorded in male golden hamsters (Mesocricetus auratus) kept in constant dim illumination. For periods of several weeks the lights in the cabinet were turned off daily at the same time of day, either for 1 h or 2 h. Despite these periodically recurring dark pulses, the circadian activity rhythms continued to free-run, and consequently crossed through the pulses at a more or less regular speed. During a dark pulse, the activity was usually enhanced. The amount of these masking effects varied with the phase of the circadian cycle at which the pulse occurred. The responses were maximal a few hours after the onset of spontaneous activity, and minimal during the rest-time of the animal.     

Mutagenesis of cultured plant cells

Experiments were designed to study the effectiveness of the chemical mutagens ethylmethane sulfonate and nitrosoguanidine on plant cells growing in liquid suspensions. Mutation frequency was defined as the number of colonies appearing on selective plates divided by the number of colonies growing on non-selective plates. The compounds tested usually increased mutation frequency by one order of magnitude over the spontaneously occurring rate, although the increase ranged from one to 140-fold. Cell killing was found to be directly correlated with mutation frequency.