Tomonori Nakai

To study plant organs, it is necessary to investigate the three-dimensional (3D) structures of plants. In recent years, non-destructive measurements through computed tomography (CT) have been used to understand the 3D structures of plants. In this study, we use the Chrysanthemum seticuspe capitulum inflorescence as an example and focus on contact points between the receptacles and florets within the 3D capitulum inflorescence bud structure to investigate the 3D arrangement of the florets on the receptacle. To determine the 3D order of the contact points, we constructed slice images from the CT volume data and detected the receptacles and florets in the image. However, because each CT sample comprises hundreds of slice images to be processed and each C. seticuspe capitulum inflorescence comprises several florets, manually detecting the receptacles and florets is labor-intensive. Therefore, we propose an automatic contact point detection method based on CT slice images using image recognition techniques. The proposed method improves the accuracy of contact point detection using prior knowledge that contact points exist only around the receptacle. In addition, the integration of the detection results enables the estimation of the 3D position of the contact points. According to the experimental results, we confirmed that the proposed method can detect contacts on slice images with high accuracy and estimate their 3D positions through clustering. Additionally, the sample-independent experiments showed that the proposed method achieved the same detection accuracy as sample-dependent experiments.

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

In vitro reconstitution of whole cellular events is one of the important goals in synthetic biology. Using a cell-free protein synthesis (CFPS) system reconstituted with human translation factors and chaperones, we reproduced the biogenesis of β-actin, synthesis, folding, and polymerization in a test tube. This system enabled us to define which step of the β-actin biogenesis was defective in genetic mutations related to diseases. Hence, the CFPS system reconstituted with human factors may be a useful tool for analyzing proteostasis in eukaryotes.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.