A Robot-Based Platform to Measure Multiple Enzyme Activities in Arabidopsis Using a Set of Cycling Assays: Comparison of Changes of Enzyme Activities and Transcript Levels during Diurnal Cycles and in Prolonged Darkness[W]
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A platform has been developed to measure the activity of 23 enzymes that are involved in central carbon and nitrogen metabolism in Arabidopsis thaliana. Activities are assayed in optimized stopped assays and the product then determined using a suite of enzyme cycling assays. The platform requires inexpensive equipment, is organized in a modular manner to optimize logistics, calculates results automatically, combines high sensitivity with throughput, can be robotized, and has a throughput of three to four activities in 100 samples per person/day. Several of the assays, including those for sucrose phosphate synthase, ADP glucose pyrophosphorylase (AGPase), ferredoxin-dependent glutamate synthase, glycerokinase, and shikimate dehydrogenase, provide large advantages over previous approaches. This platform was used to analyze the diurnal changes of enzyme activities in wild-type Columbia-0 (Col-0) and the starchless plastid phosphoglucomutase (pgm) mutant, and in Col-0 during a prolongation of the night. The changes of enzyme activities were compared with the changes of transcript levels determined with the Affymetrix ATH1 array. Changes of transcript levels typically led to strongly damped changes of enzyme activity. There was no relation between the amplitudes of the diurnal changes of transcript and enzyme activity. The largest diurnal changes in activity were found for AGPase and nitrate reductase. Examination of the data and comparison with the literature indicated that these are mainly because of posttranslational regulation. The changes of enzyme activity are also strongly delayed, with the delay varying from enzyme to enzyme. It is proposed that enzyme activities provide a quasi-stable integration of regulation at several levels and provide useful data for the characterization and diagnosis of different physiological states. As an illustration, a decision tree constructed using data from Col-0 during diurnal changes and a prolonged dark treatment was used to show that, irrespective of the time of harvest during the diurnal cycle, the pgm mutant resembles a wild-type plant that has been exposed to a 3 d prolongation of the night.
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Data for maximal catalytic activities of phosphorylating NAD-GAPDH (93 μmol m−2 s−1) and nonphosphorylating NADP-GAPDH (0.33 μmol m−2 s−1) in Arabidopsis leaves were used to constrain the upper limit of the chloroplast TP-3PGA shuttle.
“Similarly, data for the maximal catalytic activities of phosphorylating NAD-GAPDH (93 μmol m−2 s−1; Gibon et al., 2004) and nonphosphorylating NADP-GAPDH (0.33 μmol m−2 s−1; Rius et al., 2006) in Arabidopsis leaves were used to constrain the upper limit of the chloroplast TP-3PGA shuttle.”
From Leaf Energy Balance Requires Mitochondrial Respiration and Export of Chloroplast NADPH in the Light (2019) · cites this paper as Gibon et al., 2004
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Metabolic Networks: How to Identify Key Components in the Regulation of Metabolism and Growth
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Leaf Energy Balance Requires Mitochondrial Respiration and Export of Chloroplast NADPH in the Light
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Self-citation
- Authors
- Gibon, Y., Blaesing, O. E., Hannemann, J., Carillo, P., Höhne, M., Hendriks, J. H. M., Palacios, N., Cross, J., Selbig, J., & Stitt, M.
- Affiliations
- Max Planck Institute of Molecular Plant Physiology (DE)
- Max Planck Institute of Molecular Plant Physiology, Science Park Golm, 14476 Golm-Potsdam, Germany
- Venue
- The Plant Cell
- Publisher
- Oxford University Press (OUP)
- Published
- 2004-12-01
- Volume
- 16
- Issue
- 12
- Pages
- 3304-3325
- ISSN
- 1040-4651 (ISSN-L)
- 1532-298X
- PMC
- PMC535875
- OpenAlex
- W2013085592
- Cited by
- 552
- References
- 56
- Citations by year
- 2026: 5 · 2025: 15 · 2024: 14 · 2023: 18 · 2022: 29 · 2021: 28 · 2020: 33 · 2019: 31 · 2018: 28 · 2017: 28 · 2016: 24 · 2015: 35 · 2014: 24 · 2013: 32 · 2012: 29
- FWCI
- 24.33
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- The Plant Cell (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- Archivio Istituzionale della Ricerca - Università degli Studi della Campania "Luigi Vanvitelli" (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- Max-Planck-Gesellschaft
- Bundesministerium für Forschung und Technologie
- Topics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant nutrient uptake and metabolism
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Light effects on plants
- Keywords
- enzyme activity assay, Arabidopsis thaliana, diurnal cycle, prolonged darkness, post-translational regulation, mRNA levels, central carbon metabolism, nitrogen metabolism, sucrose phosphate synthase, ADP-glucose pyrophosphorylase, glutamate synthase, shikimate dehydrogenase, nitrate reductase, decision tree, plant physiological state
- MeSH terms
- Biological Assay, Circadian Rhythm, Darkness, Decision Trees, Energy Metabolism, Enzymes, Phosphorylases, Mutation, Nitrate Reductases, Protein Processing, Post-Translational, Robotics, Transcription, Genetic, Gene Expression Regulation, Enzymologic, Arabidopsis, Gene Expression Regulation, Plant, Arabidopsis Proteins, Nitrate Reductase
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