Photorespiration Methods and Protocols

This volume provides methods on researching photorespiration to provide a better understanding of this vital pathway in plants. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

 

Authoritative and cutting-edge, Photorespiration: Methods and Protocols aims to ensure successful results in the further study of this vital field.

High throughput phosphoglycolate phosphatase activity assay using crude leaf extract and recombinant enzyme to determine kinetic parameters Km and Vmax using a microplate reader-  Measuring phosphoglycolate phosphatase activity using NMR detection of glycolate.- Determination of phosphoglycolate phosphatase activity via a coupled reaction using recombinant glycolate oxidase.- Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.- Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) – expression, purification, activity and redox regulation.-Spectrophotometric Assays for Measuring Hydroxypyruvate Reductase Activity.- High throughput glycerate kinase activity assay using crude leaf extract and recombinant enzyme to determine kinetic parameters K_m and V_max using a microplate reader.- Purification of recombinant N-terminal histidine-tagged Arabidopsis thalianaphosphoglycolate phosphatase 1, glycolate oxidase 1 and 2, and hydroxypyruvate reductase 1.- Measuring and Quantifying Characteristics of the Post-Illumination Burst.- Low-cost system for maintaining low atmospheric CO₂ levels during Arabidopsis cultivation.- Measuring the release of ammonia from leaves.- Using gas exchange to study CO₂ release during photosynthesis with steady- and non-steady-state approaches.- Measurement of photorespiratory oxygen exchange by membrane inlet mass spectrometry.- Using Dynamic Gas Exchange Measurements During Oxygen Transients to Study Non-steady-state Photorespiration.- Analysis of photorespiratory intermediates under transient conditions by mass spectrometry.- Combining gas exchange and rapid quenching of leaf tissue for mass spectrometry and NMR analysis using an external chamber.- Combining gas exchange and rapid quenching of leaf tissue for mass spectrometry analysis directly in gas exchange cuvette.- Reaction-kinetic modeling of photorespiration using modelbase.- Using publicly available RNA-seq data for expression analysis of genes of interest.- Identification and functional characterization of cis-regulatory elements of key photorespiratory genes in response to short-term abiotic stress conditions.- Identification of auxiliary organellar targeting signals for plant peroxisomes using bioinformatic analysis of large protein sequence datasets followed by experimental validation.