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Bimolecular-Fluorescence Complementation Assay to Monitor KinaseSubstrate Interactions In Vivo

Enzyme–substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, ...

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Fluorescence Correlation Spectroscopy and Fluorescence Recovery After Photobleaching to Study Receptor Kinase Mobility In Planta

Plasma-membrane-localized receptor kinases are essential for cell–cell communication and as sensors for the extracellular environment. Receptor function is dependent on their distribution in the membrane and interaction with other proteins that are either membrane-loc ...

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Modulating and Monitoring MAPK Activity During Programmed Cell Death in Pollen

Signal transduction through mitogen-activated protein kinase (MAPK) cascades regulates many cellular responses. One example of a stimulus-mediated MAPK signaling network in plants is the self-incompatibility (SI) response in Papaver rhoeas, which represents an important ...

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Quantifying Degradation Rates of Transmembrane Receptor Kinases

Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor levels at the cellular sur ...

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Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-Competitive Molecules

The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically ...

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Fluorescence Fluctuation Analysis of Receptor Kinase Dimerization

Receptor kinases are essential for the cellular perception of signals. The classical model for activation of the receptor kinase involves dimerization, induced by the binding of the ligand. The mechanisms by which plant receptors transduce signals across the cell surface are largely u ...

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Substrate Analysis of Arabidopsis PP2C-Type Protein Phosphatases

Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abundant among them. PP2Cs are ...

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Bacterial Assay to Study Plant Sensor Histidine Kinases

Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stream located elements, for ...

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Use of Phospho-Site Substitutions to Analyze the Biological Relevance of Phosphorylation Events in Regulatory Networks

Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephos ...

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Mitogen-Activated Protein Kinase Activity and Reporter Gene Assays in Plants

Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs are known to phosphorylate speci ...

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Expression and Purification of Active Protein Kinases from Wheat Germ Extracts

In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and �enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins rem ...

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Measurement of Plant Cyclin-Dependent Kinase Activity Using Immunoprecipitation-Coupled and Affinity Purification-Based Kinase Assays and the Baculovi

Orderly progression of the eukaryotic cell cycle is governed by a coordinated response to intrinsic and extracellular cues through activation of cyclin-dependent kinases (CDKs). It is therefore important to verify the kinase activity of distinct types of CDKs during the cell cycle. The i ...

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Dehydration Tolerance in Plants

Dehydration tolerance in plants is an important but understudied component of the complex phenotype of drought tolerance. Most plants have little capacity to tolerate dehydration; most die at leaf water potentials between −5 and −10MPa. Some of the non-vascular plants and a small percent ...

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Gene Regulation During Cold Stress Acclimation in Plants

Cold stress adversely affects plant growth and development and thus limits crop productivity. Diverse plant species tolerate cold stress to a varying degree, which depends on reprogramming gene expression to modify their physiology, metabolism, and growth. Cold signal in plants is tr ...

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Guide to the Book Plant Kinases

The growing number of fully annotated genomes of model and nonmodel plant species, such as Arabidopsis, Brachypodium, �grapevine, maize, rice, rape seed, soybean, tomato, and others, has led to a tremendous increase in sequence information. The novel genome information has been translat ...

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The Age of Protein Kinases

Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a ...

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Profiling of Hormones and Related Metabolites in Seed Dormancy and Germination Studies

Seed dormancy and germination are regulated by several plant hormones, such as abscisic acid, gibberellin, auxin (indole-3-acetic acid), ethylene, and brassinosteroid. Endogenous concentrations of a hormone are determined by the balance between biosynthesis and deactivati ...

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Approaches to the Identification of ABAR as an Abscisic Acid Receptor

Abscisic acid (ABA) is a vital phytohormone that regulates seed maturation and germination, seedling growth, and adaptation to environmental stresses. ABA functions through a complex network of signaling pathways, where the cell response is initiated by an ABA receptor which trigge ...

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Production of Seed Samples for the Effective Molecular Analysis of Dormancy Cycling in Arabidopsis

Most often, the samples used for molecular analysis of dormancy are populations of seeds. An essential survival characteristic of seed populations inhabiting the variable surface layers of the soil is that individuals in the population do not behave uniformly. In addition, seed dormancy ...

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Eyeing Emergence: Modified Treatments for Terminating Dormancy of Conifer Seeds

Many seeds of coniferous species display a deep primary dormancy at maturity and require several weeks of pretreatment to produce seed populations that germinate in a vigorous and timely manner. Facilitating an efficient transition from dormancy to germination by devising improved ...

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