Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of &lt;i&gt;Arabidopsis&lt;/i&gt; cotyledon pavement cells

Higaki, Takumi; Mizuno, H.

doi:10.5511/plantbiotechnology.20.0605a

Cited by 5 publications

(4 citation statements)

References 33 publications

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“…Here, we presented a confocal imaging procedure and image processing pipeline for morphometric analysis and mechanical simulation of A. thaliana cotyledon epidermal cells. We focused on two-dimensional approximation analysis; however, it is possible to acquire three-dimensional structural information of these cells and their changes over time (i.e., four dimensions) (Higaki and Mizuno 2020). It is better to obtain three-or four-dimensional information, but there are many cases where two-dimensional approximations are sufficient.…”

Section: Discussionmentioning

confidence: 99%

“…Fluorescent labeling of plasma membranes or cell walls is a standard method for visualizing leaf epidermal cell contours. We prefer using transgenic A. thaliana plants that stably express the plasma membrane marker GFP-PIP2a (Cutler et al 2000, Higaki and Mizuno 2020. Figure 2A shows an example of the single optical section of GFP-PIP2alabeled plasma membranes in the cotyledon surface of a 15-day-old A. thaliana seedling that was captured by our confocal microscope [a fluorescence microscope (IX-70; Olympus, Tokyo, Japan) equipped with a low magnification (10 ) objective lens (UPLXAPO10X, NA= 0.4; Olympus), a spinning disk confocal laser scanning unit (CSU-W1; Yokogawa, Tokyo, Japan), a laser illumination homogenization unit (Uniformizer; Yokogawa), and a complementary metal-oxide-semiconductor camera (Zyla; Andor, Belfast, UK)].…”

Section: Confocal Imaging and Image Processing To Extract Pavement Cell Shapesmentioning

confidence: 99%

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Wide-Range Segmentation of Cotyledon Epidermal Cells for Morphometrical Analysis and Mechanical Simulation

et al. 2021

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Cell segmentation from microscopic images is conventionally used to investigate cell morphology. However, the time expense for manual segmentation becomes extreme with increasing numbers of cells to be analyzed. Recent progress in automated image analysis techniques can facilitate efficient and accurate cell segmentation in wide-range confocal images. Pavement cells, which mainly comprise the epidermal tissue of plant leaves, show jigsaw puzzle-like shapes and provide a model for elucidating the mechanisms underlying the complex morphology of plant cells. This mini-review demonstrates the effectiveness of using a confocal image processing pipeline for morphometric analysis and mechanical simulation using Arabidopsis thaliana cotyledon pavement cells as an example. We examined A. thaliana cotyledon surfaces using wide-range confocal images and used an image processing pipeline in ImageJ software to extract epidermal cell contours. We then used the segmented epidermal cell images to provide examples of how this information can be used for morphometry and mechanical simulation. The use of this high-throughput segmentation method is not limited to plant epidermal tissue and can be applied to various biological materials. Therefore, our approach to microscopic image analysis will hopefully contribute to the advancement of quantitative cell morphology research.

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Section: Discussionmentioning

confidence: 99%

Section: Confocal Imaging and Image Processing To Extract Pavement Cell Shapesmentioning

confidence: 99%

Wide-Range Segmentation of Cotyledon Epidermal Cells for Morphometrical Analysis and Mechanical Simulation

et al. 2021

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“…A limitation of this method is that it cannot provide three-dimensional shape information for cells because it captures only the cotyledon surface and is based on a two-dimensional approximation. To obtain three-dimensional structures of epidermal cells and their temporal changes (i.e., four-dimensional information), conventional confocal microscopy-based methods can be used ( Wong et al, 2019 ; Haas et al, 2020 ; Higaki and Mizuno, 2020 ; Eng et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…It is relatively easy to observe cell shapes over time with this method, but it possibly causes contact damage to the cotyledon with each observation. The most common technique is fluorescent labeling, in which fluorescent proteins or dyes are used to label cell walls or plasma membranes ( Zhang et al, 2011 ; Armour et al, 2015 ; Kierzkowski et al, 2019 ; Higaki and Mizuno, 2020 ; Seerangan et al, 2020 ; Eng et al, 2021 ). However, transformation is required when using fluorescent proteins, and therefore the plant species that can be studied with this method are limited.…”

Section: Introductionmentioning

confidence: 99%

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis

et al. 2021

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During cotyledon growth, the pavement cells, which make up most of the epidermal layer, undergo dynamic morphological changes from simple to jigsaw puzzle-like shapes in most dicotyledonous plants. Morphological analysis of cell shapes generally involves the segmentation of cells from input images followed by the extraction of shape descriptors that can be used to assess cell shape. Traditionally, replica and fluorescent labeling methods have been used for time-lapse observation of cotyledon epidermal cell morphogenesis, but these methods require expensive microscopes and can be technically demanding. Here, we propose a silver-nano-ink coating method for time-lapse imaging and quantification of morphological changes in the epidermal cells of growing Arabidopsis thaliana cotyledons. To obtain high-resolution and wide-area cotyledon surface images, we placed the seedlings on a biaxial goniometer and adjusted the cotyledons, which were coated by dropping silver ink onto them, to be as horizontal to the focal plane as possible. The omnifocal images that had the most epidermal cell shapes in the observation area were taken at multiple points to cover the whole surface area of the cotyledon. The multi-point omnifocal images were automatically stitched, and the epidermal cells were automatically and accurately segmented by machine learning. Quantification of cell morphological features based on the segmented images demonstrated that the proposed method could quantitatively evaluate jigsaw puzzle-shaped cell growth and morphogenesis. The method was successfully applied to phenotyping of the bpp125 triple mutant, which has defective pavement cell morphogenesis. The proposed method will be useful for time-lapse non-destructive phenotyping of plant surface structures and is easier to use than the conversional methods that require fluorescent dye labeling or transformation with marker gene constructs and expensive microscopes such as the confocal laser microscope.

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Smooth Elongation of Pavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions of the Cotyledon in Arabidopsis thaliana

Kikukawa,

Takigawa-Imamura,

Soga

et al. 2023

Plant and Cell Physiology

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The interdigitated pavement cell shape is suggested to be mechanically rational at both the cellular and tissue levels, but the biological significance of the cell shape is not fully understood. In this study, we explored the potential importance of the jigsaw puzzle-like cell shape for cotyledon morphogenesis in Arabidopsis. We used a transgenic line overexpressing a Rho-like GTPase-interacting protein, ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1), which causes simple elongation of pavement cells. Computer-assisted microscopic analyses, including virtual reality observation, revealed that RIC1 overexpression resulted in abnormal cotyledon shapes with marginal protrusions, suggesting that the abnormal organ shape might be explained by changes in the pavement cell shape. Microscopic, biochemical and mechanical observations indicated that the pavement cell deformation might be due to reduction in the cell wall cellulose content with alteration of cortical microtubule organization. To examine our hypothesis that simple elongation of pavement cells leads to an abnormal shape with marginal protrusion of the cotyledon, we developed a mathematical model that examines the impact of planar cell growth geometry on the morphogenesis of the organ that is an assemblage of the cells. Computer simulations supported experimental observations that elongated pavement cells resulted in an irregular cotyledon shape, suggesting that marginal protrusions were due to local growth variation possibly caused by stochastic bias in the direction of cell elongation cannot be explained only by polarity-based cell elongation, but that an organ-level regulatory mechanism is required.

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Four-dimensional imaging with virtual reality to quantitatively explore jigsaw puzzle-like morphogenesis of <i>Arabidopsis</i> cotyledon pavement cells

Cited by 5 publications

References 33 publications

Wide-Range Segmentation of Cotyledon Epidermal Cells for Morphometrical Analysis and Mechanical Simulation

Wide-Range Segmentation of Cotyledon Epidermal Cells for Morphometrical Analysis and Mechanical Simulation

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis

Smooth Elongation of Pavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions of the Cotyledon in Arabidopsis thaliana

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