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.

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.

Ethnobotanical History: Duckweeds in Different Civilizations.

This presentation examines the history of duckweeds in Chinese, Christian, Greek, Hebrew, Hindu, Japanese, Maya, Muslim, and Roman cultures and details the usage of these diminutive freshwater plants from ancient times through the Middle Ages. We find that duckweeds were widely distributed geographically already in antiquity and were integrated in classical cultures in the Americas, Europe, the Near East, and the Far East 2000 years ago. In ancient medicinal sources, duckweeds are encountered in procedures, concoctions, and incantations involving the reduction of high fever. In this regard, we discuss a potential case of ethnobotanical convergence between the Chinese Han and Classical Maya cultures. Duckweeds played a part in several ancient rituals. In one, the unsuitability of its roots to serve as a wick for Sabbath oil lamps. In another reference to its early use as human food during penitence. In a third, a prominent ingredient in a medicinal incantation, and in a fourth, as a crucial element in ritual body purifications. Unexpectedly, it emerged that in several ancient cultures, the floating duckweed plant featured prominently in the vernacular and religious poetry of the day.

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A.

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore-iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species.

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.

Bioluminescent Monitoring of Circadian Rhythms in Isolated Mesophyll Cells of Arabidopsis at Single-Cell Level.

A bioluminescent monitoring system is used to detect the circadian rhythms of individual plant cells. Transgenic Arabidopsis carrying the firefly luciferase (FLuc) gene driven by a circadian-regulated promoter is used as the material for protoplast isolation. The bioluminescence of these protoplasts in the culture medium is separately captured using a highly sensitive camera system. The time-series data of the bioluminescent imaging reveals the circadian rhythms of these isolated cells, enabling the native properties of the cellular circadian clocks to become elucidated.

Interspecific divergence of circadian properties in duckweed plants.

The circadian clock system is widely conserved in plants; however, divergence in circadian rhythm properties is poorly understood. We conducted a comparative analysis of the circadian properties of closely related duckweed species. Using a particle bombardment method, a circadian bioluminescent reporter was introduced into duckweed plants. We measured bioluminescence circadian rhythms of eight species of the genus Lemna and seven species of the genus Wolffiella at various temperatures (20, 25, and 30°C) and light conditions (constant light or constant dark). Wolffiella species inhabit relatively warm areas and lack some tissues/organs found in Lemna species. Lemna species tended to show robust bioluminescence circadian rhythms under all conditions, while Wolffiella species showed lower rhythm stability, especially at higher temperatures. For Lemna, two species (L. valdiviana and L. minuta) forming a clade showed relatively lower circadian stability. For Wolffiella, two species (W. hyalina and W. repanda) forming a clade showed extremely long period lengths. These analyses reveal that the circadian properties of species primarily reflect their phylogenetic positions. The relationships between geographical and morphological factors and circadian properties are also suggested.

Adaptive Diversification in the Cellular Circadian Behavior of Arabidopsis Leaf- and Root-Derived Cells.

The plant circadian system is based on self-sustained cellular oscillations and is utilized to adapt to daily and seasonal environmental changes. The cellular circadian clocks in the above- and belowground plant organs are subjected to diverse local environments. Individual cellular clocks are affected by other cells/tissues in plants, and the intrinsic circadian properties of individual cells remain to be elucidated. In this study, we monitored bioluminescence circadian rhythms of individual protoplast-derived cells from leaves and roots of a CCA1::LUC Arabidopsis transgenic plant. We analyzed the circadian properties of the leaf- and root-derived cells and demonstrated that the cells with no physical contact with other cells harbor a genuine circadian clock with ∼24-h periodicity, entrainability and temperature compensation of the period. The stability of rhythm was dependent on the cell density. High cell density resulted in an improved circadian rhythm of leaf-derived cells while this effect was observed irrespective of the phase relation between cellular rhythms. Quantitative and statistical analyses for individual cellular bioluminescence rhythms revealed a difference in amplitude and precision of light/dark entrainment between the leaf- and root-derived cells. Circadian systems in the leaves and roots are diversified to adapt to their local environments at the cellular level.

An endogenous basis for synchronisation characteristics of the circadian rhythm in proliferating Lemna minor plants.

The circadian clock is a cell-autonomous system that functions through the coordination of time information in the plant body. Synchronisation of cellular clocks is based on coordination mechanisms; the synchronisation characteristics of proliferating plants remain unclear. The bioluminescence circadian rhythms of fronds (leaf-like plant units) of proliferating Lemna minor plants carrying a circadian bioluminescence reporter, AtCCA1:LUC, were spatiotemporally analysed at a cell-level resolution. We focused on spontaneous circadian organisation under constant light conditions for plants with light : dark treatment (LD grown) or without it (LL grown). Fronds developing even from an LL-grown parental frond showed coherent circadian rhythms among them. This allowed the maintenance of circadian rhythmicity in proliferating plants. Inside a frond, a centrifugal phase/period pattern was observed in LD-grown plants, whereas various phase patterns with travelling waves were formed in LL-grown plants. These patterns were model simulated by local coupling of heterogeneous cellular circadian oscillators with different initial synchronous states in fronds. Spatiotemporal analysis of the circadian rhythms in proliferating plants reveals spontaneous synchronisation manners that are associated with local cell-cell coupling, spatial phase patterns and developmental stages.

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.

Return of the Lemnaceae: duckweed as a model plant system in the genomics and postgenomics era.

The aquatic Lemnaceae family, commonly called duckweed, comprises some of the smallest and fastest growing angiosperms known on Earth. Their tiny size, rapid growth by clonal propagation, and facile uptake of labeled compounds from the media were attractive features that made them a well-known model for plant biology from 1950 to 1990. Interest in duckweed has steadily regained momentum over the past decade, driven in part by the growing need to identify alternative plants from traditional agricultural crops that can help tackle urgent societal challenges, such as climate change and rapid population expansion. Propelled by rapid advances in genomic technologies, recent studies with duckweed again highlight the potential of these small plants to enable discoveries in diverse fields from ecology to chronobiology. Building on established community resources, duckweed is reemerging as a platform to study plant processes at the systems level and to translate knowledge gained for field deployment to address some of society's pressing needs. This review details the anatomy, development, physiology, and molecular characteristics of the Lemnaceae to introduce them to the broader plant research community. We highlight recent research enabled by Lemnaceae to demonstrate how these plants can be used for quantitative studies of complex processes and for revealing potentially novel strategies in plant defense and genome maintenance.

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.

Indigenous bacteria, an excellent reservoir of functional plant growth promoters for enhancing duckweed biomass yield on site.

The advantages of aquatic biomass production using wastewater as a cost-free fertilizer have recently been highlighted. Here, we report a successful study in which duckweed, Lemna gibba, biomass production in a food factory effluent containing low nitrogen and high salts was enhanced by employing customized plant growth-promoting bacteria (PGPB). Two common PGPB strains previously obtained from natural pond water, Acinetobacter calcoaceticus P23 and Pseudomonas fulva Ps6, hardly promoted the growth of duckweed; on the contrary, they inhibited its growth in treated factory wastewater, far different water conditions. Then, we asked if some indigenous wastewater bacteria could promote the growth of duckweed. We found that Chryseobacterium strains, a group of bacteria with limited nitrogen metabolism, were dominantly selected as effective PGPB. Moreover, we demonstrated that nitrogen limitation is the crucial environmental factor that induces the plant growth-inhibiting behavior of A. calcoaceticus P23 through competition for mineral nutrients with the host duckweed. This study uncovered points to be considered in PGPB technology to achieve efficient production of duckweed biomass in a factory effluent with unbalanced content of mineral nutrients.

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.

Monitoring single-cell bioluminescence of Arabidopsis leaves to quantitatively evaluate the efficiency of a transiently introduced CRISPR/Cas9 system targeting the circadian clock gene ELF3.

The rapid assessment of gene function is crucial in biological research. The CRISPR/Cas9 system is widely used as a tool for targeted gene editing in many organisms including plants. Previously, we established a transient gene expression system for investigating cellular circadian rhythms in duckweed. In this system, circadian reporters and clock gene effectors-such as overexpressors, RNA interference (RNAi), and CRISPR/Cas9-were introduced into duckweed cells using a particle bombardment method. In the present study, we applied the CRISPR/Cas9 system at a single cell level to Arabidopsis thaliana, a model organism in plant biology. To evaluate the mutation induction efficiency of the system, we monitored single-cell bioluminescence after application of the CRISPR/Cas9 system targeting the ELF3 gene, which is essential for robust circadian rhythmicity. We evaluated the mutation induction efficiency by determining the proportion of cells with impaired circadian rhythms. Three single guide RNAs (sgRNAs) were designed, and the proportion of arrhythmic cells following their use ranged from 32 to 91%. A comparison of the mutation induction efficiencies of diploid and tetraploid Arabidopsis suggested that endoreduplication had a slight effect on efficiency. Taken together, our results demonstrate that the transiently introduced CRISPR/Cas9 system is useful for rapidly assessing the physiological function of target genes in Arabidopsis cells.

Community composition and methane oxidation activity of methanotrophs associated with duckweeds in a fresh water lake.

Methanotrophs are the only biological sink of the greenhouse gas methane. To understand the ecological features of methanotrophs in association with plants in the methane emitting environments, we investigated the community composition and methane oxidation of methanotrophs associated with duckweeds in a fresh water lake. Duckweeds collected from Lake Biwa, Japan over three summers showed methane consumption activity between 0.0067 and 0.89 µmol h-1 g-1 (wet weight), with the highest values occurring from the end of July to August. The methanotrophic community on duckweeds consisted primarily of γ-proteobacterial groups including the genera Methylomonas and Methylocaldum. Further analysis of co-cultures of a methanotroph isolate with sterilized duckweed revealed that the duckweed plant as well as the duckweed spent culture supernatant exerted an enhancing effect on methane oxidation. These results indicate that duckweeds not only provide a habitat for methanotrophs but also stimulate methanotrophic growth.

KaiC family proteins integratively control temperature-dependent UV resistance in Methylobacterium extorquens AM1.

KaiC protein is the pivotal component of the circadian clock in cyanobacteria. While KaiC family proteins are well-conserved throughout divergent phylogenetic lineages, studies of the physiological roles of KaiC proteins from other microorganisms have been limited. We examined the role of the KaiC proteins, KaiC1 and KaiC2, in the methanol-utilizing bacterium Methylobacterium extorquens AM1. Wild-type M. extorquens AM1 cells exhibited temperature-dependent UV resistance (TDR) under permissive growth temperatures (24 °C -32 °C). Both the phosphorylation of KaiC2 and the intracellular levels of KaiC1 were temperature-dependent, and the TDR phenotype was positively regulated by KaiC1 and negatively regulated by KaiC2. Taken together with biochemical and functional analogies to the KaiC protein of cyanobacteria, our present results suggest that KaiC family proteins function to integrate environmental cues, that is, temperature and UV light, and output appropriate cellular responses to allow cells to adapt to changing environmental conditions.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Long-term monitoring of bioluminescence circadian rhythms of cells in a transgenic Arabidopsis mesophyll protoplast culture.

The circadian system of plants is based on the cell-autonomously oscillating circadian clock. In the plant body, these cellular clocks are associated with each other, but their basic and intrinsic properties are still largely unknown. Here we report a method that enables long-term monitoring of bioluminescence circadian rhythms of a protoplast culture in a complete synthetic medium. From the leaves of Arabidopsis transgenic plants carrying the luciferase gene under a clock-gene promoter, mesophyll protoplasts were isolated and their bioluminescence was automatically measured every 20 min for more than one week. Decreasing luminescence intensities were observed in protoplasts when they were cultured in a Murashige and Skoog-based medium and also in W5 solution. This decrease was dramatically improved by adding the phytohormones auxin and cytokinin to the MS-based medium; robust circadian rhythms were successfully monitored. Interestingly, the period lengths of bioluminescence circadian rhythms of protoplasts under constant conditions were larger than those of detached leaves, suggesting that the period lengths of mesophyll cells in leaves were modulated from their intrinsic properties by the influence of other tissues/cells. The entrainability of protoplasts to light/dark signals was clearly demonstrated by using this monitoring system. By analyzing the circadian behavior of isolated protoplasts, the basic circadian system of plant cells may be better understood.

Use of a duckweed species, Wolffiella hyalina, for whole-plant observation of physiological behavior at the single-cell level.

We developed a new model system to analyze physiological behavior at the single-cell level in whole plants. Wolffiella hyalina is a species of rootless duckweed, which has a thin and very small structure and can grow rapidly on the surface of culture medium. Epidermal and mesophyll cells were transfected with a reporter gene using particle bombardment and were observed at the single-cell level in the whole living plant. An EM-CCD camera system with a macro zoom microscope was used to capture time-lapse images of bioluminescence, and we successfully detected circadian rhythms in individual cells that expressed a luciferase gene under the control of a circadian promoter. We also detected individual S-phase cells in meristematic tissues of intact W. hyalina plants by using a 5-ethynyl-2'-deoxyuridine (EdU)-labeling assay. Our observations indicated that low-molecular-weight compounds could access the inside of the plant body. Thus, W. hyalina showed the experimental characteristics suitable for single-cell analyses that could be combined with whole-plant observations and/or pharmacological analyses/chemical biology.