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Analysis of Cell Division and Cell Elongation in the Hypocotyls of Arabidopsis Heterotrimeric G Protein Mutants

The heterotrimeric G-proteins form classical signal transduction complexes conserved in all eukaryotes. The repertoire of G-protein signaling complex is much simpler in plants than in metazoans. One of the best understood functions for the plant G-protein complex is its modulation ...

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Biochemical Analysis of the Interaction Between Phospholipase D1 and GTP-Binding Protein -Subunit from Arabidopsis thaliana

Phospholipase Ds (PLDs) play diverse roles in plant lipid metabolism and cellular signaling processes. The sole canonical G-protein α-subunit (Gα) in Arabidopsis also plays multiple roles in plant growth and cellular signaling processes. Interestingly, overlapping functions ...

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Aequorin Luminescence-Based Functional Calcium Assay for Heterotrimeric G-Proteins in Arabidopsis

Heterotrimeric GTP-binding proteins (G-proteins) and G-protein-coupled receptors are important signaling components in eukaryotes. In plants, the G-proteins are involved in diverse physiological processes, some of which are exerted via changes in the level of cytosolic free c ...

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Analysis of Unfolded Protein Response in Arabidopsis

The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. M ...

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Fusarium oxysporum Infection Assays in Arabidopsis

Increased susceptibility to Fusarium oxysporum is one of the most conspicuous characteristics of the Arabidopsis mutants lacking the heterotrimeric G protein β and γ1 subunits. The molecular mechanisms placing these G proteins in the plant innate immunity network are yet to be discov ...

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Functional Analysis of Heterotrimeric G Proteins in Chloroplast Development in Arabidopsis

Functional analysis of G-proteins has been extensively carried out using their over-expressing lines and knockout mutants in plants. Since α subunit exists in an active or inactive form, overexpressing α subunit does not mean that G-protein signaling pathways are activated in the trans ...

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Essential Methods of Plant Sample Preparation for Light Microscopy

There are various preparatory techniques for light microscopy permitting access to the inner structure of plant body and its development. Minute objects might be processed as whole-mount preparations, while voluminous ones should be separated into smaller pieces. Hereby we summar ...

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Quantifying Cell Shape and Gene Expression in the Shoot Apical Meristem Using MorphoGraphX

Confocal microscopy is a technique widely used to live-image plant tissue. Cells can be visualized by using fluorescent probes that mark the cell wall or plasma membrane. This enables the confocal microscope to be used as a 3D scanner with submicron precision. Here we present a protocol using the 3D i ...

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High-Pressure Freezing and Low-Temperature Processing of Plant Tissue Samples for Electron Microscopy

Use of electron tomography methods improves image resolution of transmission electron microscopy especially in the z-direction, enabling determination of complicated 3D structures of organelles and cytoskeleton arrays. The increase in resolution necessitates preser ...

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Mechanical Measurements on Living Plant Cells by Micro-indentation with Cellular Force Microscopy

Indentation methods on the micro- and nanoscale are increasingly used to assess mechanical properties of living plant tissues. These techniques rely on recording the force resulting from indenting the cell surface with a small probe. Depending on the scale of indentation and the indenter ...

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Reconstructing Plant Cells in 3D by Serial Section Electron Tomography

In micrographs acquired with a transmission electron microscope, 3-dimensional (3D) objects are superimposed onto a 2D screen. This reduction in dimension necessarily leads to a degradation of image resolution. To overcome this problem, 3D microscopy techniques, such as tomograp ...

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Imaging Plant Nuclei and Membrane-Associated Cytoskeleton by Field Emission Scanning Electron Microscopy

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructur ...

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Immunogold Labeling of Resin-Embedded Electron Microscopical Sections

Knowledge about the spatio-temporal distribution patterns of proteins and other molecules of the cell is essential for understanding their function. A widely used technique is immunolabeling which uses specific antibodies to reveal the distribution of molecular components at v ...

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Morphological Analysis of Cell Growth Mutants in Physcomitrella

This protocol describes a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. The protocol can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Information is presen ...

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Live Cell Imaging of Arabidopsis Root Hairs

Root hairs are tubular extensions from the root surface that expand by tip growth. This highly focused type of cell expansion, combined with position of root hairs on the surface of the root, makes them ideal cells for microscopic observation. This chapter describes the method that is routinely us ...

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Plant Cell Lines in Cell Morphogenesis Research

Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studyi ...

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Antisense Oligodeoxynucleotide-Mediated Gene Knockdown in Pollen Tubes

Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy recently emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen tubes. Here, we describe the protocol for the successful employment of AODN ...

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Laser Microdissection of Plant Cells

Different plant cell types express unique transcriptomes, proteomes, and metabolomes. Therefore, the isolation of specific cell types prior to molecular analyses is important to understand the specification, differentiation, and function of these cells. Isolation of specif ...

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Lab-on-a-Chip for Studying Growing Pollen Tubes

A major limitation in the study of pollen tube growth has been the difficulty in providing an in vitro testing microenvironment that physically resembles the in vivo conditions. Here we describe the development of a lab-on-a-chip (LOC) for the manipulation and experimental testing of indiv ...

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Optical Trapping in Plant Cells

Optical tweezers allow noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi-)quantitative force measurements within a living cell can be performed. In this chapter, we prov ...

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