Within plant cells, many events occur simultaneously to sustain life, involving various genes and substances. This makes it difficult to identify which genes and substances are involved in a particular event and how. Professor Minako Ueda of the Graduate School of Life Sciences at Tohoku University has formed an interdisciplinary team with Assistant Professor Satoru Tsugawa of the Faculty of Systems Science and Technology at Akita Prefectural University and their colleagues. By combining dimensionality reduction focused on changes in plant cell "shape" with mechanical modeling and simulation techniques, they are working to elucidate complex life systems.
Focusing on the Simplest Fertilized Egg: Arabidopsis thaliana as the Research Subject
Research involving analysis of the genetic sequences of various organisms is progressing. However, even if all genes are identified and catalogued, much remains unknown about which genes work when and what substances they change in constructing complex life systems. Professor Minako Ueda of Tohoku University studies the mechanisms of plant development, investigating what roles their respective genes and substances play in the formation of the roots, stems, and leaves that constitute plants.
To reduce the complexity of events occurring simultaneously in life systems, Ueda focuses solely on changes in plant cell "shape". In other words, she has pursued "dimensionality reduction" to construct mechanical models. By comparing actual observational data with simulation results from mechanical models, she attempts to elucidate the mysteries of plant development based on a methodology of comprehensively understanding life systems. Ueda's current research subject is the fertilized egg of Arabidopsis thaliana. A. thaliana is a model plant whose genome has been completely sequenced and is used in research in various fields. "In states where cell division has progressed to some extent, the effects of genes are complexly intertwined, making it difficult to focus on specific phenomena. Therefore, I decided to focus on the fertilized egg at the simplest stage, which is the division from one cell to two cells."
Elucidating changes in "shape" through high-precision observation and mechanical models
When observing fertilized egg cells, a technique called "live imaging" is used to observe cells while they are alive. Ueda has established a method for highly accurate observation of changes occurring deep within tissues during fertilization and embryo development through live imaging using two-photon excitation microscopy, which has excellent transmissivity. Her laboratory is the only one in the world that utilizes this technique. The laboratory has two two-photon excitation microscopes, enabling long-term observations (Figure 1).
However, observing how cells change through live imaging alone cannot elucidate the workings of genes and substances. Therefore, she is collaborating with Assistant Professor Satoru Tsugawa of Akita Prefectural University, who specializes in "plant mechanics," representing plant mechanisms as mechanical models and simulating them, and is also knowledgeable in image analysis. They advance their research by analyzing high-precision image data obtained through live imaging and constructing simulations of mechanical models.
In their current JST CREST project, Ueda leads a team that includes Tsugawa, Professor Takumi Higaki of the Faculty of Advanced Science and Technology at Kumamoto University, who specializes in biological image analysis, and Professor Koichi Fujimoto of the Graduate School of Integrated Sciences for Life at Hiroshima University, who specializes in theoretical biology. "Since each person has a different background, we started by teaching and learning each other's specialized terminology. Even now, we continue to have discussions at monthly meetings," says Tsugawa.
Elucidating the mechanism creating "inside and outside" through trial and error with laser intensity and other parameters
Now, around three years after the start of the project, Ueda's team has already published many research findings. In 2023, they succeeded in precisely capturing changes in shape and velocity during the process of plant fertilized egg cell elongation leading to the first division. They discovered that unlike general plant cells, fertilized eggs elongate in a special manner called "tip growth." The following year, they derived a mechanical model that reproduces growth patterns and determined that fertilized egg elongation can be explained by two mechanical elements: pressure from inside the cell and surface flexibility (Figure 2).
They also discovered that when genes working in the outermost layer during plant embryo development are disrupted, embryos are created with mixed characteristics of inner and outer tissues (Figure 3-A). Through live imaging, they successfully observed embryo development from the first division of the fertilized egg until the establishment of the inner-outer axis (Figure 3-B). Furthermore, through mechanical model simulations, they identified a mathematical rule determining that the direction of cell division is decided based on cell shape and nuclear position after the first division (Figure 3-C). They elucidated the mechanism that first creates the "inside and outside" of plants.
B shows live imaging of wild-type embryo development (numbers in upper right indicate time in minutes).
C shows how division direction estimated through simulation based on embryo cell shape and nuclear position matches actual division planes.
Behind these diverse achievements lay various struggles and innovations. Since fertilized eggs are hidden deep within seeds, initially they could not observe fertilized eggs in detail. Through trial and error, they developed a method for clearly observing the inside of living fertilized eggs by combining plants whose fertilized eggs alone were made to glow brightly with two-photon excitation microscopy capable of viewing deep within tissues (Figure 4). There were other challenges: for example, if the near-infrared laser intensity used for the microscope light source is too strong, it damages cells, so Ueda recalls struggling to adjust the laser output for irradiation.
Additionally, in live imaging, there was a problem where fertilized eggs moved, causing slight blurring in images and making precise quantification of data difficult. Seeds containing fertilized eggs are placed in liquid medium and are in the process of growing, causing subtle movements. Therefore, Tsugawa succeeded in stabilizing cell contours by extracting cell outlines from images, establishing feature points, and performing parallel translation based on these points and rotation around the cell axis (Figure 5). By resolving positional displacement problems, even slight growth that was difficult to discern visually could be measured, enabling precise quantification of data.
Potential applications in crop improvement and environmental science: Further interdisciplinary integration under consideration
The researchers' recent achievements include research on microtubules, substances involved in cell shape changes. In A. thaliana, when fertilized eggs grow, a ring-shaped "microtubule band" is created near the cell tip and moves toward the tip as the cell grows. While this phenomenon itself had been confirmed through live imaging, the mechanism by which it moved toward the tip was unknown.
Therefore, Ueda and her colleagues quantified the width and velocity of microtubule bands from live imaging image analysis. By comparing with mechanical model simulation results, they determined the mechanism by which microtubule bands move. While microscopes are effective for observing phenomena inside cells, they are limited when it comes to the precise observation of microtubules at the nanoscale, which is even smaller than cells. This research demonstrated that simulations incorporating observational data are effective in bridging cellular-scale behavior with molecular-scale behavior.
Knowledge gained from this research will also be useful for crop improvement and environmental science. For example, even when crossing a "disease-resistant variety" with a "good-tasting variety," embryos may die or exhibit poor growth. If what happens inside fertilized eggs can be elucidated, the success rate of crossbreeding could be improved. Tsugawa's eyes light up as he explains that simulations of collective cell motion through mechanical models could potentially be applied to fiber-reinforced materials and composite materials manufactured through control of fine fibers.
On the topic of their current achievements, Ueda evaluates that they have finally begun to see the big picture. "In the future, I want to advance research not only in low dimensions but also in high dimensions. Additionally, I'd like to consider further interdisciplinary integration, such as teaming up with mechanical engineering experts to directly touch cells with precise manipulators to observe changes, or using different fluorescent dyes with chemistry researchers," she expresses with enthusiasm.
(Article: Noriko Higo, Photography: Haruka Watarai)
