Perezadams8907

Recent advancements in microscopy and biological technologies have allowed scientists to study dynamic plant developmental processes with high temporal and spatial resolution. Pavement cells, epidermal cells found on leaf tissue, form complex shapes with alternating regions of indentations and outgrowths that are postulated to be driven by the microtubule cytoskeleton. Given their complex shapes, pavement cells and the microtubule contribution towards morphogenesis have been of great interest in the field of developmental biology. Here, we focus on two live-cell imaging methods that allow for early and long-term imaging of the cotyledon (embryonic leaf-like tissue) and leaf epidermis with minimal invasiveness in order to study microtubules throughout pavement cell morphogenesis. The methods described in this chapter can be applied to studying other developmental processes associated with cotyledon and leaf tissue.Leaf epidermis pavement cells develop complex jigsaw puzzle-like shapes in many plant species, including the model plant Arabidopsis thaliana. Due to their complex morphology, pavement cells have become a popular model system to study shape formation and coordination of growth in the context of mechanically coupled cells at the tissue level. To facilitate robust assessment and analysis of pavement cell shape characteristics in a high-throughput fashion, we have developed PaCeQuant and a collection of supplemental tools. The ImageJ-based MiToBo plugin PaCeQuant supports fully automatic segmentation of cell contours from microscopy images and the extraction of 28 shape features for each detected cell. These features now also include the Largest Empty Circle criterion as a proxy for mechanical stress. In addition, PaCeQuant provides a set of eight features for individual lobes, including the categorization as type I and type II lobes at two- and three-cell junctions, respectively. The segmentation and feature extraction results of PaCeQuant depend on the quality of input images. To allow for corrections in case of local segmentation errors, the LabelImageEditor is provided for user-friendly manual postprocessing of segmentation results. For statistical analysis and visualization, PaCeQuant is supplemented with the R package PaCeQuantAna, which provides statistical analysis functions and supports the generation of publication-ready plots in ready-to-use R workflows. In addition, we recently released the FeatureColorMapper tool which overlays feature values over cell regions for user-friendly visual exploration of selected features in a set of analyzed cells.Tensile testing is widely used to evaluate the mechanical properties of biological materials including soft primary plant tissues. Commercially available platforms for tensile testing are often expensive and limited in customizability. In this chapter, we provide a guide for the assembly and use of a simple and low-cost micromechanical testing apparatus suitable for research and educational purposes. The build of the setup is presented with scalability and universality in mind and is based on a do-it-yourself mind frame towards mechanical tests on plant organs and tissues. We discuss hardware and software requirements with practical details on required components, device calibration and a script to run the device. Further, we provide an example in which the device was used for the uniaxial tensile test of onion epidermis.How complicated cell activities produce characteristic tissue and organ morphologies is an important question in plant morphogenesis. To address this question, 3D morphometry of plant organs on multiscales is indispensable. In recent years, advances in confocal microscopy with fluorescent probes that mark the cell wall or plasma membrane enable the visualization of organ morphology with submicron precision. In parallel, new quantitative and correlative imaging pipelines realize 3D image processing on 2D curved surface, facilitating the study of cell and tissue behaviors in plant organogenesis. Here, we describe methods for 3D morphometry of Arabidopsis sepals, focusing on live imaging coupled with MorphoGraphX-based 3D image processing for cellular growth analysis.The exocytosis process delivers proteins, lipids, and carbohydrates to the plasma membrane or the extracellular space to sustain plant cell growth, development, and response to environmental stimuli. Plant exocytosis is highly dynamic and requires the coordinated functions of multiple cellular components such as tethering complexes, GTPase signaling, and vesicle fusion machinery. Accurate spatio-temporal control of plant exocytosis is critical for the proper functions of plant cells. Live-cell imaging of fluorescence-tagged cargo proteins allows for quantitative analysis of exocytosis dynamics in plant cells. Small molecule inhibitors that target important components in the exocytosis machinery allow for transient manipulation of the exocytosis process. In this chapter, we describe procedures that use Endosidin2 (ES2) and Brefeldin A (BFA) as small molecule inhibitors to disrupt plant exocytic processes and use fluorescent protein-tagged PIN-formed 2 (PIN2) and Cellulose Synthase (CESA) as cargo proteins to quantify exocytosis dynamics in plant cells.Plant growth and morphogenesis are tightly controlled processes of division and expansion of individual cells. To fully describe the factors that influence cell expansion, it is necessary to quantify the counteracting forces of turgor pressure and cell wall stiffness, which together determine whether and how a cell expands. selleck chemicals Several methods have been developed to measure these parameters, but most of them provide only values for one or the other, and thus require complex models to derive the missing quantity. Furthermore, available methods for turgor measurement are either accurate but invasive, like the pressure probe; or they lack accuracy, such as incipient plasmolysis or indentation-based methods that rely on information about the mechanical properties of the cell wall. Here, we describe a system that overcomes many of the above-mentioned disadvantages using growing pollen tubes of Lilium longiflorum as a model. By combining non-invasive microindentation and cell compression experiments, we separately measure turgor pressure and cell wall elasticity on the same pollen tube in parallel.