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This volume presents current methods to detect and measure the activity of proteolytic enzymes in organisms ranging from unicellular algae to flowering plants. Chapters detail in vitro production, characterization of plant proteases, tools for in vivo modifications, proteomic approaches for identification of substrates, inhibitors and interacting partners, and the function of plant proteases in plant programmed cell death. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Plant Proteases: Methods and Protocols aims to be a useful practical guide to researches to help further their study in this field.
List of contents
Expression and purification of the type I metacaspase from a cryptophyte Guillardia theta, GtMCA-I.- Expression and purification of the type II metacaspase from a unicellular green alga Chlamydomonas reinhardtii.- Expression in Escherichia coli, refolding, and purification of plant aspartic proteases.- Production of functional plant legumain proteases using the Leishmania tarentolae expression system.- Purification of His-tagged proteases from the apoplast of agroinfiltrated N. benthamiana.- Identification of cognate protease/substrate pairs by use of class-specific inhibitors.- Peptide backbone modifications for the assessment of cleavage site relevance in precursors of signaling peptides.- Detection of apoplastic protease inhibitors using convolution activity based proteome profiling.- Cysteine reactivity profiling to unveil redox regulation in phytopathogens.- Determination of caspase-like activities in roots by the use of fluorogenic substrates.- Detection of damage-activated metacaspase activity by Western blot in plants.- Sensitive plant N-terminome profiling with HUNTER.- Profiling sequence specificity of proteolytic activities using proteome-derived peptide libraries.- Monitoring Cell Death via Ion leakage and PAM Fluorometry.- Measuring and perturbing ferroptosis in plants.- Detection and quantification of the hypersensitive response cell death in Arabidopsis thaliana.- Monitoring of ATG4 protease activity during autophagy in the model microalga Chlamydomonas reinhardtii.- Oxidative stress in roots - detection of lipid peroxidation and total antioxidative capacity.- Imaging of potassium and calcium distribution in plant tissues and cells to monitor stress response and programmed cell death.- Monitoring Pseudomonas syringae growth in agroinfiltrated leaves: the 'agromonas' assay.- Analysis of virus spread around the cell death zone at spatiotemporal resolutionusing confocal microscopy.- A user guide to validation, annotation and evaluation of N-terminome datasets with MANTI.- Exploring posttranslational modifications with the Plant PTM Viewer.
