title: Simple and robust determination of the activity signature of key carbohydrate metabolism enzymes for physiological phenotyping in model and crop plants.
abstract: The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance, crop yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric kinetic assays. The comparison of extraction buffers and requirement for dialysis of crude protein extracts resulted in a universal protein extraction protocol, suitable for the preparation of protein extracts from different organs of various species. Individual published kinetic activity assays were optimized and adapted for a semi-high-throughput 96-well assay format. These assays proved to be robust and are thus suitable for physiological phenotyping, enabling the characterization and diagnosis of the physiological state. The potential of the determination of distinct enzyme activity signatures as part of a physiological fingerprint was shown for various organs and tissues from three monocot and five dicot model and crop species, including two case studies with external stimuli. Differential and specific enzyme activity signatures are apparent during inflorescence development and upon in vitro cold treatment of young inflorescences in the monocot ryegrass, related to conditions for doubled haploid formation. Likewise, treatment of dicot spring oilseed rape with elevated CO2 concentration resulted in distinct patterns of enzyme activity responses in leaves.
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Jammer
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Anna
authors.initials: A
authors.lastname: Gasperl
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Nora
authors.initials: N
authors.lastname: Luschin-Ebengreuth
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Elmien
authors.initials: E
authors.lastname: Heyneke
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Hyosub
authors.initials: H
authors.lastname: Chu
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Elena
authors.initials: E
authors.lastname: Cantero-Navarro
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Dominik K
authors.initials: DK
authors.lastname: Großkinsky
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Alfonso A
authors.initials: AA
authors.lastname: Albacete
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Edith
authors.initials: E
authors.lastname: Stabentheiner
authors.affiliation: Institute of Landscape and Plant Ecology, University of Hohenheim, August-von-Hartmann-Strasse 3, D-70599 Stuttgart, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Franzaring
authors.affiliation: Institute of Landscape and Plant Ecology, University of Hohenheim, August-von-Hartmann-Strasse 3, D-70599 Stuttgart, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Fangmeier
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria.
authors.forename: Eric
authors.initials: E
authors.lastname: van der  Graaff
authors.affiliation: Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria roitsch@plen.ku.dk.
authors.forename: Thomas
authors.initials: T
authors.lastname: Roitsch
mesh_terms: D050260:Carbohydrate Metabolism; D018556:Crops, Agricultural / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D040901:Proteomics / Q000379:methods*
chemical_list: D010940:Plant Proteins
keywords: Carbohydrate metabolism; dialysis; enzyme activities; kinetic assay; physiological phenotyping; physiological state; protein extraction; signatures.