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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MAAP (Multidimensional Analysis of Axis Patterning in Plant Embryo)

Teaser

Summary

Understanding the mechanisms of morphogenesis is a fundamental challenge in biology. Embryogenesis encompasses the earliest stages of tissue morphogenesis in plants. Therefore it presents good opportunities to address this challenge. During embryogenesis, the shape of the embryo changes from radial/spherical to bilateral/heart-shaped and at the culmination of embryogenesis body axes and the root and shoot meristems are established. Unlike animal cells, plant cells are surrounded by stiff cell walls and as such are immobile. Thus positional information has an important role in regulating development of form. How does positional information guide axes formation? In previous studies the host group, showed that tight regulation of the homeobox proteins; PHABULOSA (PHB) and PHAVOLUTA (PHV) is important for apical basal and central peripheral axes formation during embryogenesis. However how this happens is unknown.
The embryo is simple and it consists of a limited numbers of cells providing a good platform to study this problem and understand how differential cell growth mediates PHB/PHV-dependent axes delimitation. However, because the embryo is contained inside the ovule, and it is of small size therefore cannot easily manipulate and study how gene action influences growth. The objective of this project was to establish a 4D time-lapse system of embryogenesis, and to perform cellular-level quantitative analysis of growth, cell division, and gene expression to understand the morphogenesis of embryos. We have optimized the image acquisition and image processing methods and succeeded in preforming 4D time-lapse imaging of embryogenesis for the first time. We have also established other new protocols to observe morphology of embryo in detail and we are now generating a 4D growth map of embryo. Thus, the project has contributed significantly to the field of plant embryology and we have generated a wealth of tools that will be broadly relevant for the field of plant development.

Work performed

To establish a 4D time-lapse system of Arabidopsis embryogenesis, we have optimized image acquisition for observation of live embryos under the microscope, we tested various microscopes, (1-photon confocal, 2-photon microscopes, light sheet), imaging settings, medium for ovule culture, tool to fix ovules during culture and marker lines that give an appropriate plasma membrane signal enabling 3D segmentation of obtained images. To be able to 3D-segment the images acquired in the time-lapse experiments, we tested various procedures for 3D segmentation in MorphographX, as well as the machine learning-based image processing software ilastik. To further improve the quality of obtained images, we tested deconvolution software. To develop a protocol to observe cellulose, we tested the use of a super resolution microscope, and worked with computational biologists to create custom solutions for quantification of cellulose fibers. To observe PHB:GFP expression, several marker lines were observed both in Arabidopsis and in Cardamine, and the lines that showed strong expression were selected. To perform laser ablation in embryo, the settings for ablation were tested. To perform 4D time-lapse in Cardamine, we screened for optimal plasma membrane marker lines and settings for imaging.

To develop computational models of embryo development we collaborated with Dr. Smithâ€™s group, resulting in a detailed 2D growing model of early embryogenesis as well as 3D pressurized templates of early embryonic developmental stages. These models are used to better understand the interaction between growth, gene expression and cell mechanics.
Taken together, the protocols we developed will provide tools to better understand how combination of orientated cell division, cell growth and cell fate determination drive embryonic patterning and establish axes in different Brassicaceae spices.
The results of this project were presented/discussed by the researcher (1 conference, 2 workshops) and collaborators (1 conference). The researcher published two publications related to the subject/techniques required in this project. Publication from this project itself is currently under preparation.

Final results

The protocol for time-lapse imaging, 3D segmentation and cellular quantitative analysis will be useful for the researchers working on embryology and in general for researchers in the field of plant developmental biology. The protocol for observation of cellulose fibers, which uses dyes to stain and visualize the fibers can be used for various plant tissues, organs and species. The modeling frameworks created have wider application in plant embryology. The project has set new standards in visualization and quantification of embryogenesis at the cellular and sub-cellular level. These contributions will help in furthering our understanding of how plant architecture is generated, ultimately bringing us closer to engineering plant shape and size.