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Monitoring primary carbon metabolism in plants using heavy isotope labelling and mass spectrometry

¹³CO₂ labelling, detection and estimation in intact plants

Lindén, Pernilla (2015). Monitoring primary carbon metabolism in plants using heavy isotope labelling and mass spectrometry. Diss. (sammanfattning/summary) Umeå : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae, 1652-6880 ; 2015:58
ISBN 978-91-576-8314-4
eISBN 978-91-576-8315-1
[Doctoral thesis]

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Abstract

This thesis covers the possibilities and limitations of studying primary metabolism in intact plants, with special focus on heavy isotope labelling and mass spectrometry methodology. In paper I, a series of Arabidopsis thaliana mutants lacking one or both genes of mitochondrial malate dehydrogenase (mMDH characterised) were characterised. We found that mMDH has a complex respiration-controlling role. In paper II, we continued to study one of the single mutants, mmdh1. We developed a method using ¹³CO₂ to label whole plants and analyse their metabolic profiles by gas and liquid chromatography coupled to mass spectrometry (GC- and LC-MS). The results show that mmdh1 had a metabolic phenotype that revealed an altered flux through photorespiration, especially under low CO₂ conditions. Combining incorporation data with metabolite pool size deepened our understanding regarding the role of mMDH in photorespiration, respiration, and cellular redox balance. The practical and theoretical aspects of ¹³C-labelling of plants learned from this study were used for designing the experimental setup of paper III, a labelling study of developing wood in hybrid aspen. The labelling strategy, time resolution, and sampling had to be adapted to suit woody plants.

Two months old trees were labelled with ¹³CO₂ in a 4 h burst and then sampling of source leaves, stem phloem, and developing wood continued over 24 hours. Since sucrose is the main carbon transporter the analysis was concentrated around this metabolite. We found previously unrecognised temporal patterns in wood biosynthesis and an indication that the diurnal cycle serves as a cue in the regulation of carbon allocation in developing wood.

To study systems with complex structures such as plants, it is informative to study specific cell types rather than whole plants or whole organs. In paper IV, a method was developed for analysis of metabolite profiles in cell type specific cells. Isolated protoplast from Arabidopsis roots were sorted by fluorescence-activated cell sorting (FACS) and their metabolite profiles were analysed by GC-MS. The method is fast, robust and reliable for analysis of cell type specific metabolic profiling and a beginning to meet the call for new metabolite analysis techniques with a high spatial resolution.

Authors/Creators:Lindén, Pernilla
Title:Monitoring primary carbon metabolism in plants using heavy isotope labelling and mass spectrometry
Subtitle:¹³CO₂ labelling, detection and estimation in intact plants
Series/Journal:Acta Universitatis agriculturae Sueciae (1652-6880)
Year of publishing :2015
Depositing date:12 May 2015
Volume:2015:58
Number of Pages:75
Papers/manuscripts:
NumberReferences
ITomaz T*, Bagard M*, Pracharoenwattana I, Lindén P, Lee CP, Carroll AJ, Stroher E, Smith SM, Gardestrom P, Millar AH (2010). Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis. Plant Physiology 154: 1143-1157
IILindén P, Keech O, Stenlund H, Gardeström P, Moritz T. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by ¹³C-labelling (manuscript).
IIIMahboubi A*, Lindén P*, Hedenström M, Moritz T, Niittylä T (2015). Carbon-13 tracking after ¹³CO2 supply revealed diurnal patterns of wood formation in aspen. Plant Physiology in press.
IVPetersson S*, Lindén P*, Moritz T, Ljung K. Cell-type specific metabolic profiling of Arabidopsis thaliana protoplasts as a tool for plant systems biology (submitted).
Place of Publication:Umeå
Publisher:Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences
ISBN for printed version:978-91-576-8314-4
ISBN for electronic version:978-91-576-8315-1
ISSN:1652-6880
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:F Plant production > F60 Plant physiology and biochemistry
Subjects:(A) Swedish standard research categories 2011 > 4 Agricultural Sciences > 404 Agricultural Biotechnology > Plant Biotechnology
Agrovoc terms:carbon, metabolism, plants, photorespiratory pathway, carbon cycle, carbon dioxide, isotope labelling, climatic change, mass spectrometry
Keywords:Plant Primary Carbon Metabolism, Mass Spectrometry, Heavy Isotope Labelling, ¹³CO₂, Photorespiration, Metabolomics, Carbon Allocation, Cell Sorting, Multivariate Statistical Analysis
URN:NBN:urn:nbn:se:slu:epsilon-e-2645
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-2645
ID Code:12212
Faculty:S - Faculty of Forest Sciences
Department:(S) > Dept. of Forest Genetics and Plant Physiology
External funders:FORMAS
Deposited By: Pernilla Lindén
Deposited On:12 May 2015 10:05
Metadata Last Modified:12 May 2015 10:05

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