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

Abstract Many plant processes occur in the context of and in interaction with a surrounding matrix such as soil (e.g. root growth and root–microbe interactions) or surrounding tissues (e.g. pollen tube growth through the pistil), making it difficult to study them with high-resolution optical microscopy. Over the past decade, microfabrication techniques have been developed to produce experimental systems that allow researchers to examine cell behavior in microstructured environments that mimic geometrical, physical and/or chemical aspects of the natural growth matrices and that cannot be generated using traditional agar plate assays. These microfabricated environments offer considerable design flexibility as well as the transparency required for high-resolution, light-based microscopy. In addition, microfluidic platforms have been used for various types of bioassays, including cellular force assays, chemoattraction assays, and electrotropism assays. Here, we review the recent use of microfluidic devices to study plant cells and organs, including plant roots, root hairs, moss protonemata, and pollen tubes. The increasing adoption of microfabrication techniques by the plant science community may transform our approaches to investigating how individual plant cells sense and respond to changes in the physical and chemical environment.

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Structure and Biomechanics during Xylem Vessel Transdifferentiation in Arabidopsis thaliana

Plants ◽

10.3390/plants9121715 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1715

Author(s):

Eleftheria Roumeli ◽

Leah Ginsberg ◽

Robin McDonald ◽

Giada Spigolon ◽

Rodinde Hendrickx ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Wall ◽

Cell Walls ◽

Turgor Pressure ◽

Plant Cells ◽

Building Blocks ◽

Xylem Vessel ◽

Primary Cell Wall ◽

Individual Plant ◽

Vessel Elements

Individual plant cells are the building blocks for all plantae and artificially constructed plant biomaterials, like biocomposites. Secondary cell walls (SCWs) are a key component for mediating mechanical strength and stiffness in both living vascular plants and biocomposite materials. In this paper, we study the structure and biomechanics of cultured plant cells during the cellular developmental stages associated with SCW formation. We use a model culture system that induces transdifferentiation of Arabidopsis thaliana cells to xylem vessel elements, upon treatment with dexamethasone (DEX). We group the transdifferentiation process into three distinct stages, based on morphological observations of the cell walls. The first stage includes cells with only a primary cell wall (PCW), the second covers cells that have formed a SCW, and the third stage includes cells with a ruptured tonoplast and partially or fully degraded PCW. We adopt a multi-scale approach to study the mechanical properties of cells in these three stages. We perform large-scale indentations with a micro-compression system in three different osmotic conditions. Atomic force microscopy (AFM) nanoscale indentations in water allow us to isolate the cell wall response. We propose a spring-based model to deconvolve the competing stiffness contributions from turgor pressure, PCW, SCW and cytoplasm in the stiffness of differentiating cells. Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. The stiffness of the PCW in all of these conditions is lower than the stiffness of the fully-formed SCW. Our results provide the first experimental characterization of the mechanics of SCW formation at single cell level.

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

10.1101/2020.06.10.143727 ◽

2020 ◽

Author(s):

Emiri Watanabe ◽

Minako Isoda ◽

Tomoaki Muranaka ◽

Shogo Ito ◽

Tokitaka Oyama

Keyword(s):

Circadian Rhythms ◽

Single Cell ◽

Monitoring System ◽

Lemna Minor ◽

Single Cell Level ◽

Cell Level ◽

Dual Color ◽

Stochastic Phenomena ◽

Circadian Behavior ◽

Bioluminescent Reporters

SummaryThe 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 world’s 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.

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Single-nuclei RNA-sequencing of plants

10.1101/2020.11.14.382812 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniele Y. Sunaga-Franze ◽

Jose M. Muino ◽

Caroline Braeuning ◽

Xiaocai Xu ◽

Minglei Zong ◽

...

Keyword(s):

Single Cell ◽

Target Genes ◽

Developmental Stages ◽

Developmental Trajectory ◽

Marker Genes ◽

Individual Plant ◽

Experimental Procedure ◽

Single Cell Genomics ◽

Nuclei Isolation ◽

Environmental Responses

ABSTRACTSingle-cell genomics offers a rich potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nuclei isolation and nanowell-based library preparation for short-read sequencing. This plant-nuclei sequencing (PN-seq) method allows for analyzing the transcriptome in thousands of individual plant cells. We show the applicability of the experimental procedure to seedlings and developing flowers fromArabidopsis thaliana. The developmental trajectory of anther development is reconstructed, and stage-specific master regulators and their target genes are predicted. Novel marker genes for specific anther developmental stages are experimentally verified. The nuclei isolation procedure can be applied in different plant species, thus expanding the toolkit for plant single cell genomics experiments.

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Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2012.03.004 ◽

2012 ◽

Vol 207 (1) ◽

pp. 72-79 ◽

Cited By ~ 14

Author(s):

Ryosuke Enoki ◽

Daisuke Ono ◽

Mazahir T. Hasan ◽

Sato Honma ◽

Ken-ichi Honma

Keyword(s):

Circadian Rhythms ◽

Single Cell ◽

Fluorescence Imaging ◽

Spinning Disk

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Structure and Biomechanics during Xylem Vessel Transdifferentiation in Arabidopsis thaliana

10.20944/preprints202011.0188.v1 ◽

2020 ◽

Author(s):

Eleftheria Roumeli ◽

Leah Ginsberg ◽

Robin McDonald ◽

Giada Spigolon ◽

Rodinde Hendrickx ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Walls ◽

Large Scale ◽

Turgor Pressure ◽

Plant Cells ◽

Building Blocks ◽

Xylem Vessel ◽

Primary Cell Wall ◽

Individual Plant ◽

Vessel Elements

Individual plant cells are the building blocks for all plantae and artificially constructed plant biomaterials, like biocomposites. Secondary cell walls (SCWs) are a key component for mediating mechanical strength and stiffness in both living vascular plants and biocomposite materials. In this paper, we study the structure and biomechanics of cultured plant cells during the cellular developmental stages associated with SCW formation. We use a model culture system that induces transdifferentiation of Arabidopsis thaliana cells to xylem vessel elements, upon treatment with dexamethasone (DEX). We group the transdifferentiation process into three distinct stages, based on morphological observations of the cell walls. The first stage includes cells with only a primary cell wall (PCW), the second covers cells that have formed a SCW, and the third stage includes cells with a ruptured tonoplast and partially or fully degraded PCW. We adopt a multi-scale approach to study the mechanical properties of cells in these three stages. We perform large-scale indentations with a micro-compression system and nanoscale indentations through atomic force microscopy (AFM), in three different osmotic conditions. We introduce a spring-based model to deconvolve the competing stiffness contributions from turgor pressure, PCW, SCW and cytoplasm in the stiffness of differentiating cells. Prior to triggering differentiation, cells in hypotonic pressure conditions are significantly stiffer than cells in isotonic or hypertonic conditions, highlighting the dominant role of turgor pressure. Plasmolyzed cells with a SCW reach similar levels of stiffness as cells with maximum turgor pressure. The stiffness of the PCW in all of these conditions is lower than the stiffness of the fully-formed SCW. Our results provide the first experimental characterization of the mechanics of SCW formation at single cell level.

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