A globular stage Arabidopsis embryo was fixed, stained and observed by a confocal microscope. A 3D reconstruction of a confocal stack was done by MorphoGraphX (https://www.morphographx.org/). The 3D reconstructed image of the confocal stack (upper left), a median longitudinal section of the 3D reconstructed confocal stack (bottom left), a 3D segmented embryo (upper right) and the median longitudinal section of the 3D segmented embryo (bottom right) are shown.

In recent decades, various interdisciplinary technologies promote plant imaging research, such as MEMS (Micro-Electro-Mechanical-Systems), organic synthesis, genomics, and mathematical modeling. We have organized a symposium entitled ‘Plant imaging research promoted by interdisciplinary collaborations’ at the 84th Annual Meeting of the Botanical Society of Japan on September 21^st, 2020. This symposium was co-sponsored by The Birth of New Plant Species (Scientific Research on Innovative Areas, a MEXT Grant-in-Aid Project), Integrated Imaging Research Support (IIRS), and The Japanese Society of Plant Morphology. We had five young expert researchers’ presentations to introduce their innovative research with cutting-edge techniques for imaging. Current issues in the plant research, their solutions by the new imaging technologies, and plant imaging potential were interactively discussed involving speakers and audiences. This symposium was held using the online conference tool ‘Zoom’ under the first trial of the online Annual Meeting of the Botanical Society of Japan due to the influence of the COVID-19.

Chloroplasts contain their own genome (chloroplast DNA), which interact with a wide variety of proteins to form chloroplast nucleoids. Chloroplast nucleoids serve as “chromosomes” in chloroplasts, functioning as a center for DNA replication, repair, and gene expression. With fluorescence microscopy, chloroplast nucleoids are generally detected as spherical structures of submicron order. Our live imaging technology using a microfluidic device has revealed that chloroplast nucleoids undergo a dramatic morphological shift from a spherical structure to a network-like structure during the chloroplast division. This process is presumably required for the equal distribution of chloroplast nucleoids to all daughter chloroplasts during chloroplast division. Analysis of a mutant with aberrantly aggregated chloroplast nucleoids, monokaryotic chloroplast (moc) 1, revealed that activation of MOC1 (= Holliday junction resolvase), is essential for the initiation of this process. The activity of MOC1 could be directly visualized by employing high-speed atomic force microscopy (AFM) and DNA origami techniques. Furthermore, detailed monitoring of chloroplast nucoeoids in moc1 mutants suggested that the hot spots of chloroplast nucleoid replication would be at the peripheral region of chloroplast nucleoids. These findings suggest a model for the structure of the chloroplast nucleoids that consists of a core composed of supercoiled DNA and DNA-binding proteins and a periphery where relaxed DNA, which tends to be a template for replication.

We have developed a system that can quantitatively analyze the dynamics of mineral elements in living plants by visualizing radioactive tracers moving within the plant. This system can visualize various types of radioactive tracers and track the dynamics of a wide variety of elements. Two types of systems have been developed according to the field of view. The macro-imaging system has a field of view of 10 x 20 cm with a resolution in the order of millimeters, whereas the microscopic-imaging system has a field of view of 600 x 600 µm with 100 µm resolution. Since these two systems use living plants for analysis, it is possible to change the plant growth environment during imaging and analyze plant responses to environmental changes. The visualization by this system is expected to be applied not only in the field of plant nutrition but also to various fields in bioscience such as plant morphology.

Morphology of plants is controlled by coordinated patterns of cell division and growth. Understanding the molecular mechanisms that regulate plant architecture requires careful observation and detailed analysis at the level of cells and tissues. In this review, we introduce recently developed microscopy protocols and computational tools to obtain and analyze cellular level data for research on plant morphology and development. We mainly focus on protocols for imaging plant structures by confocal laser scanning microscopes, followed by quantitative analysis based on 3D segmentation of cells layers/tissues, and computational modelling to explore fundamental questions in plant developmental biology.

The timing of plant reproduction and inflorescence architecture have a large impact on yield of crops. Reproductive development in temperate cereals such as barley and wheat is regulated by two developmental transitions: switching of the identity in the shoot apical meristem (SAM) from vegetative to reproductive phase and conversion of the developmental fate of lateral shoot meristems generated from SAM. Although genetic studies have clarified genetic components and environmental factors that affect inflorescence architecture and its developmental process, little is known about the cellular behavior at the shoot apex including SAM and lateral primordia during development especially in Triticeae crops. We established the 3D-imaging system of barley shoot apex using a clearing method to analyze the cellular dynamics during their developmental process. In this review, we summarize the findings about the morphological characters of the shoot apex in barley and introduce our method of 3D-imaging using the shoot apex of barley.

Recent studies on plant cell walls have shown that plants are excellent structural systems that can flexibly change their morphology in response to various external perturbations. In order to properly understand the flexibility of plants, a coordinated research design that carefully combines measurement techniques for plant mechanical properties, imaging techniques for accurately depicting shape, and theoretical biological approaches is required. In order to promote this kind of interdisciplinary collaborative research, we organized an interdisciplinary symposium at the 84th Annual Meeting of the Botanical Society of Japan under the co-hosting of the Japanese Society of Plant Morphology and the project team ‘Elucidation of the strategies of mechanical optimization in plants toward the establishment of the bases for sustainable structure system’.

We developed a near-infrared laser-induced surface deformation (NIR-LISD) microscope and applied it to the dynamic relaxation measurements of a living plant surface at single-cell level. In the microscope, the surface deformation is induced by a NIR laser beam, then the changes in intensity of probe beam reflected from the surface are measured. The obtained power spectrum reflects the viscoelasticity of the surface. The application of the NIR laser beam in LISD measurements decreased the photo-damages of plant cells, in comparison with the irradiation of a visible laser beam. The NIR-LISD microscope enabled us to discriminate the differences in power spectra between the subapical and lateral regions of tip-growing single rhizoids in Marchantia polymorpha. It has an advantage for the dynamic viscoelastic measurements of cells and tissues that could be damaged caused by the strong absorption to ultraviolet or visible light.

Clarification of mechanical states of plants are essential for understanding structural inevitability and environmental adaptability of growing plants. Atomic force microscopy (AFM) has attracted attention as a promising measurement tool for evaluating mechanical states with resolution of single-cell level. This review describes principle of the AFM device and method to estimate elasticity of the cell from force curves obtained by the AFM measurement. When we apply the measurement to a growing plant, the sample has to be held on the measurement substrate without inhibiting the growth. We also introduce a solution for this problem, in which a glass micro device is applied to the substrate.

The external mechanical forces on a floral meristem play a key role in floral development. A novel experimental system was developed to apply mechanical forces on the floral meristem of Arabidopsis thaliana, causing various morphological changes in floral development. A micromanipulator with a silicon device (= micro device), which is shaped to fit the contour of the abaxial side of the young primordium on a floral meristem, is used to apply contact pressure on the floral primordia. Mechanical forces were successfully applied on the floral primordia using this experimental system, and morphological changes were induced in floral development. One of the important issues in this experimental system is measuring the mechanical forces imposed on the floral primordia, and I tried to apply “Pollination Simulation” for the measurement. “Pollination Simulation” is a device which equips a double-bending load cell as its microforce sensor, and it was originally developed to measure the forces needed to release the lever mechanism in the genus Salvia exerted by pollinators. In this novel experimental system, the mechanical forces on the floral meristem were measured in several nano-Newton order by the device. The measurements depend on the shapes of the micro devices and floral primordia. These results demonstrate that the device “Pollination Simulation” is applicable to measure the mechanical forces on the floral meristem, inducing the morphological changes in floral development in the novel experimental system.

Regularity in phyllotaxis, or the arrangement of leaves around a stem, has attracted attention of researchers over centuries. Physicists and mathematicians as well as botanists have contributed to the phyllotaxis studies, giving a successful example of the continuous collaboration between observations, experiments, and theoretical works, which is rather rare in biological fields. Representative phyllotactic patterns including spiral phyllotaxis with the golden angle are reproduced by multiple mathematical models without depending on model details, indicating the universality of such patterns. Diversity and flexibility of patterns have been explained by results that the patterns can be switched by a small number of parameters, which can be interpreted to measurable values such as the size ratio between organ primordia and shoot apical meristem. In this review, I briefly describe the contribution of mathematical modelling approach on phyllotactic pattern formation. A mathematical model of phyllotaxis has been applied to floral development, and numerical simulations of the model have suggested non-intuitive transitions between patterns. Suggestions raised by theoretical studies would stimulate further experimental studies and advance our understandings on plant pattern formation.

Phyllotaxis of plants shows regular and beautiful patterns with a limited variety, which mostly fall within several types. Phyllotactic patterns have long been studied and described by morphological analysis. Now phyllotactic patterns are considered to be spontaneously generated through positional relationships between leaf primordia resulting from inhibition of new primordium formation by existing primordia at their vicinity, which has been supported by successful production of major phyllotactic patterns in computer simulations with mathematical models assuming such inhibitory effect. Recent molecular studies have identified the inhibitory effect as the competition for auxin at the molecular level, leading to the establishment of the framework of the mechanism of phyllotactic pattern formation. This framework is expected to enable search for determinants of a particular phyllotactic pattern from its morphological features. The aim of this article is to propose such new approach. Here we review morphological features of phyllotactic patterns and their measurement, introduce mathematical models of phyllotactic pattern formation, and discuss how to estimate the parameter values of the mathematical model from the morphological features of phyllotactic patterns.

Non-destructive three-dimensional reconstruction is required to precisely determine plant morphology and its changes. We developed a space-efficient and low-cost three-dimensional reconstruction system for plant architecture of Arabidopsis thaliana, which can be easily obtained in laboratories specializing in plant science. In this system, images of a plant rotating at low speed on a rotator are captured by a camera from multiple directions and a three-dimensional image is reconstructed based on the visual volume intersection method. The accuracy of the three-dimensional reconstruction was verified using a three-dimensional model of known size and shape. It was observed that 360 images captured at intervals of approximately 1° were sufficient to obtain accurate three-dimensional images with submillimeter resolution and high shape reproducibility. Our system is expected to contribute to the promotion of multi-dimensional phenotyping of plant architectures.

Plant shoot gravitropism is determined by gravity sensing, curvature sensing (proprioception), and the ability to uphold self-weight. Recently, data- and model-analyses have revealed the detailed morphology of shoot bending, while mechanical mechanism to cause the bending is still unknown. By making a best correspondence between data and model of shoot bending in wild-type and lazy1-like 1(lzy1) mutant Arabidopsis thaliana, we found that both the bending force (derived from the gravi-proprioceptive response) and the stretching force (derived from shoot axial growth) differ significantly between the wild type and mutant. Finally, we interpreted the mechanical forces associated with differential cell growth and provide a plausible mechanical explanation of shoot gravitropism.

The acquisition of chloroplasts was an important event in the evolution of plant cells. It is widely accepted that plastids (chloroplasts) evolved from engulfed endosymbiotic cyanobacteria. The plastids of green plants are thought to have lost their ability to produce peptidoglycan during evolution. However, we found that the moss Physcomitrella (Physcomitrium patens) possesses 11 genes required to generate peptidoglycan in plastids, and that single-gene knockouts of these genes trigger defects in chloroplast division. Moreover, we used a click chemistry-based metabolic labelling method to target peptidoglycan, and found that peptidoglycan surrounded the chloroplasts of P. patens. Recent results suggest that the chloroplasts of several streptophytes have a peptidoglycan wall. The evolution of chloroplasts with peptidoglycan walls is discussed.

The cells that comprise the outermost leaf epidermal tissue layer differentiate from the protodermal cells and acquire characteristic functions and morphogenetic features. Pairs of guard cells form small holes called stomata, and the adjacent jigsaw puzzle piece-shaped pavement cells interlock. These two different cell types cooperate to produce stomatal movement, and the macroscopic functions necessary for plant growth, such as gas exchange and transpiration, are acquired. The cell spatial organization in tissues is important for intercellular coordination, and in Arabidopsis thaliana, the pavement cell distribution does not allow adjacent stomata formation. Spatial organization is also important inside cells. For the acquisition of macroscopic functions, such as cell differentiation and maturation, the proper distribution and coordination among different organelles are necessary. In pavement cells, the localization of cortical microtubules and membrane trafficking, which are related to cell morphogenetic mechanisms, were observed. Thus, leaf epidermal tissues are very suitable for studying how spatial organization of organelle or cells contribute to the acquisition of macroscopic functions in higher layers such as cells or tissues. Here, I introduce microscopic observations, and the accompanying image analyses, of cell morphogenesis and distributions in the leaf epidermal tissue of A. thaliana.

During gynoecium development in Arabidopsis thaliana, two carpel primordia are initiated with their margins fused together, forming a hollow tube. The fused carpel margins, or carpel boundaries, generate ovules, septa, and repla, which are essential structures for successful reproduction and seed dispersal. The boundary-specific CUP-SHAPED COTYLEDON (CUC) genes CUC1, CUC2, and CUC3 serve overlapping and distinct roles in the development of various shoot organ boundaries. The redundant functions of CUC1 and CUC2 are particularly important for carpel margin development. Here we report genetic interactions between the CUC genes and BELL-class homeobox gene BELLRINGER (BLR), which is also required for carpel boundary development. The blr single mutant displayed a mild defect in septum formation, while the CUC gene single mutants exhibited little effect on septum morphology. However, the blr phenotype was enhanced by any of the CUC gene single mutations, indicating overlapping roles for the CUC and BLR genes during carpel boundary development. Furthermore, we found that the blr mutation also caused a mild increase in the number of carpel boundaries, and this phenotype was significantly enhanced by the cuc3 mutation. Together, our analyses indicate overlapping and unique functions of CUC and BLR in regulating at least two aspects of carpel boundary development: septum formation and the patterning of carpel boundaries.