Seasonal switching of integrated leaf senescence controls in an evergreen perennial Arabidopsis.

Evergreeness is a substantial strategy for temperate and boreal plants and is as common as deciduousness. However, whether evergreen plants switch foliage functions between seasons remains unknown. We conduct an in natura study of leaf senescence control in the evergreen perennial, Arabidopsis halleri. A four-year census of leaf longevity of 102 biweekly cohorts allows us to identify growth season (GS) and overwintering (OW) cohorts characterised by short and extended longevity, respectively, and to recognise three distinct periods in foliage functions, i.e., the growth, overwintering, and reproductive seasons. Photoperiods during leaf expansion separate the GS and OW cohorts, providing primal control of leaf senescence depending on the season, with leaf senescence being shut down during winter. Phenotypic and transcriptomic responses in field experiments indicate that shade-induced and reproductive-sink-triggered senescence are active during the growth and reproductive seasons, respectively. These secondary controls of leaf senescence cause desynchronised and synchronised leaf senescence during growth and reproduction, respectively. Conclusively, seasonal switching of leaf senescence optimises resource production, storage, and translocation for the season, making the evergreen strategy adaptively relevant.

A non-cell-autonomous circadian rhythm of bioluminescence reporter activities in individual duckweed cells.

The circadian clock is responsible for the temporal regulation of various physiological processes in plants. Individual cells contain a circadian oscillator consisting of a clock gene circuit that coordinates physiological rhythms within the plant body in an orderly manner. The coordination of time information has been studied from the perspective of cell-cell local coupling and long-distance communication between tissues based on the view that the behavior of circadian oscillators represents physiological rhythms. Here, we report the cellular circadian rhythm of bioluminescence reporters that are not governed by the clock gene circuit in expressing cells. We detected cellular bioluminescence rhythms with different free-running periods in the same cells using a dual-color bioluminescence monitoring system in duckweed (Lemna minor) transfected with Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1::luciferace+ (AtCCA1::LUC+) and Cauliflower mosaic virus 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC) reporters. Co-transfection experiments with the two reporters and a clock gene-overexpressing effector revealed that the AtCCA1::LUC+ rhythm, but not the CaMV35S::PtRLUC rhythm, was altered in cells with a dysfunctional clock gene circuit. This indicated that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian oscillator, whereas the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared, whereas the AtCCA1::LUC+ rhythm persisted. This suggests that the CaMV35S::PtRLUC bioluminescence has a symplast/apoplast-mediated circadian rhythm generated at the organismal level. The CaMV35S::PtRLUC-type bioluminescence rhythm was also observed when other bioluminescence reporters were expressed. These results reveal that the plant circadian system consists of both cell-autonomous and noncell-autonomous rhythms that are unaffected by cellular oscillators.

Effects of fruit dimorphism on genetic structure and gene flow in the coastal shrub Scaevola taccada.

BACKGROUND AND AIMS: Plant propagules often possess specialized morphologies that facilitate dispersal across specific landscapes. In the fruit dimorphism of a coastal shrub, Scaevola taccada, individual plants produce either cork-morph or pulp-morph fruits. The former is buoyant and common on sandy beaches, whereas the latter does not float, is bird-dispersed, and is common on elevated sites such as slopes on sea cliffs and behind rocky shores. We hypothesized that beach populations bridge the heterogeneous landscapes by serving as a source of both fruit types, while dispersal is biased for the pulp morph on elevated sites within the islands and for the cork morph between beaches of different islands. Based on this hypothesis, we predicted that populations in elevated sites would diverge genetically over time due to isolation by distance, whereas beach populations would maintain high genetic similarity via current gene flow. METHODS: The genetic structure and gene flow in S. taccada were evaluated by investigating genome-wide single nucleotide polymorphisms in plants from 17 sampling sites on six islands (belonging to the Ryukyu, Daito and Ogasawara Islands) in Japan. KEY RESULTS: Geographical isolation was detected among the three distant island groups. Analyses within the Ryukyu Islands suggested that sandy beach populations were characterized by genetic admixture, whereas populations in elevated sites were relatively isolated between the islands. Pairwise FST values between islands were lowest between sandy beaches, intermediate between sandy beaches and elevated sites, and highest between elevated sites. CONCLUSIONS: Dispersal across the ocean by cork morphs is sufficiently frequent to prevent genetic divergence between beaches of different islands. Stronger genetic isolation of elevated sites between islands suggests that bird dispersal by pulp morphs is restricted mainly within islands. These contrasting patterns of gene flow realized by fruit dimorphism provide evidence that fruit characteristics can strongly mediate genetic structure.

Multifunctional chemical inhibitors of the florigen activation complex discovered by structure-based high-throughput screening.

Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.

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. However, the driving forces behind quantitative trait variations remain unclear owing to their complexity at the molecular level. 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 phase phenotype of clock-dependent physiology. Lemna aequinoctialis in Japan is a paddy field duckweed that exhibits a latitudinal cline of critical day length (CDL) for short-day flowering. We collected 72 strains of L. aequinoctialis and found a significant correlation between FRPs and locally adaptive CDLs, confirming that variation in the FRP-dependent phase phenotype underlies photoperiodic adaptation. Diel transcriptome analysis revealed that the induction timing of an FT gene is key to connecting the clock phase to photoperiodism at the molecular level. This study highlights the importance of FRP as a variation resource for evolutionary adaptation.

Characterization of Frond and Flower Development and Identification of FT and FD Genes From Duckweed Lemna aequinoctialis Nd.

Duckweeds (Araceae: Lemnoideae) are aquatic monocotyledonous plants that are characterized by their small size, rapid growth, and wide distribution. Developmental processes regulating the formation of their small leaf-like structures, called fronds, and tiny flowers are not well characterized. In many plant species, flowering is promoted by the florigen activation complex, whose major components are florigen FLOWERING LOCUS T (FT) protein and transcription factor FD protein. How this complex is regulated at the molecular level during duckweed flowering is also not well understood. In this study, we characterized the course of developmental changes during frond development and flower formation in Lemna aequinoctialis Nd, a short-day plant. Detailed observations of frond and flower development revealed that cell proliferation in the early stages of frond development is active as can be seen in the separate regions corresponding to two budding pouches in the proximal region of the mother frond. L. aequinoctialis produces two stamens of different lengths with the longer stamen growing more rapidly. Using high-throughput RNA sequencing (RNA-seq) and de novo assembly of transcripts from plants induced to flower, we identified the L. aequinoctialis FT and FD genes, whose products in other angiosperms form a transcriptional complex to promote flowering. We characterized the protein-protein interaction of duckweed FT and FD in yeast and examined the functions of the two gene products by overexpression in Arabidopsis. We found that L. aequinoctialis FTL1 promotes flowering, whereas FTL2 suppresses flowering.

Detection of Uncoupled Circadian Rhythms in Individual Cells of Lemna minor using a Dual-Color Bioluminescence Monitoring System.

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. By 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.

Application of Single-Cell Bioluminescent Imaging to Monitor Circadian Rhythms of Individual Plant Cells.

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.

Circadian Regulation of the Plant Transcriptome Under Natural Conditions.

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.

Time from Semiosis: E-series Time for Living Systems.

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.

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.

Early evolution of the land plant circadian clock.

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.

Synchrony of plant cellular circadian clocks with heterogeneous properties under light/dark cycles.

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 ~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 ~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.

Heterogeneity of cellular circadian clocks in intact plants and its correction under light-dark cycles.

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.

A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

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 ≥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.