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Dynamics in the Central Plant Cell Metabolism

As part of their development, plants have acquired adaptive mechanisms to cope with different stress situations they encounter in their environment. These adaptive mechanisms include the ability to alter their metabolism when faced with extreme environmental conditions such as low oxygen. In higher plants, oxygen (O2) availability is important for energy production through respiratory metabolism. Under conditions where O2 becomes limiting, respiratory metabolism can be impeded leading to impaired growth. Low O2 conditions in plants can be created by environmental and man-made factors such as soil flooding and control atmosphere (CA) storage, respectively. In addition, anatomical arrangements can create uneven gas distribution leading to low O2 conditions in cells located in the inner tissues of plants. The effect of low O2 stress on plants include stunted growth of field crops and development of storage disorders in fruits stored under CA. Taking into account that plants serve as an importantsource of food, it is important to study and understand how plant metabolism cope with low O2 stress.
The main objective of this thesis is to study the effect of low O2 on plants through metabolome and fluxome analysis. Metabolome analysis involves the comprehensive quantitative analysis of all low molecular weight metabolites in an organism while fluxome analysis measures the rates at which metabolites are distributed through a reaction network. Together, these two techniques can be used to study and understand the response of plants to low O2 stress.
Analysis of flux, especially using isotope labelling techniques, require feeding an organism with labelled substrate and measuring the incorporation of label in different metabolites. In whole plants, performing isotopic feeding experiments is limited by the long incubation times needed for metabolites to incorporate quantifiable amounts of label in their storage polymers like proteins and carbohydrates. To overcome this difficulty heterotrophic cell suspensions were used as a model system as they can be easily manipulated to grow on a defined medium, allowing a much faster incorporation of labelled substrate into their metabolome.
In the first part of this thesis, techniques were developed to establish cellsuspension from tomato leaves. Subsequently, a gas chromatography-mass spectrometry (GC-MS) based protocol for separating, identifying and quantifying intracellular polar metabolites and their label accumulation during 13C-label feeding experiments was developed. Finally, 13C-label feeding experiments were carried out to determine the effect of low O2 stress on the polar metabolic profile of tomato cell suspension and to analyse the changes in fluxes of the central carbon metabolism under both metabolic dynamic and steady-state conditions.
A cell suspension was established from dark grown friable callus of tomato leaves (Lycopersicum esculentum L. var </>cerasiforme). The growth of the cell suspension involved a lag period, a linear phase of growth and a stationary phase. Polar metabolites present in the cells were separated and detected on the GC-MS after methanol extraction and derivatisation using N,O-bis-(trimethylsilyl)trifluoroacetamide. A total of 70 polar metabolites could be identified and quantified with a GC-MS temperature programme of approximately 40 min duration. The polar metabolitespresent in the cells belonged to the functional groups of amino acids, organic acids, sugars and sugar alcohols. After performing feeding experiments, 13C-label accumulation could be detected in 47 of the 70 metabolites measured.
The metabolic response of plant cells following the induction of low O2 stress was studied by analysing the changes in polar metabolite after incubating cell suspension at different O2 levels in a bioreactor. The cells were incubated at O2 levels of 21, 1 and 0 kPa and cell samples taken every hour for a period of 12 h with a final sampling after 24 h of incubation. 13C-glucose was added to the mediumof the cells four hours after the start of the incubation. The changes in metabolite levels as well as the incorporation of 13C-label was measured with GC-MS. Low O2 altered the polar metabolic profile of the cells.There was a general reduction in the levels of most amino acids, organic acids and sugars and an increase in the intermediates of glycolysis, lactate and some sugar alcohols. The 13C-label data showed reduced label accumulation in almost all metabolites except lactate and some sugar alcohols under low O2 stress. The results indicated that low O2 in plant cells activated fermentative metabolism and sugar alcohol synthesis while inhibiting the activity of the TCA cycle. Also, the levels of metabolites whose precursors are derived from the intermediates of the central carbon metabolism such as amino acids were reduced upon the induction of low O2 stress.
To obtain a quantitative understanding ofthe response of the fluxome following the induction of low O2 stress inplant cells, the changing metabolite levels and 13C-label accumulation were used to construct a dynamic model of the central carbon metabolism.A compartmentalised metabolic network model containing glycolysis in the cytosol and plastid, the TCA cycle in the mitochondria and the syntheses of alanine, aspartate, lactate, glutamate, serine, sucrose and valinewas developed. The model contained differential equations describing both Michaelis-Menten and first-order kinetics and the model parameters were estimated using a non-linear least square optimisation approach. The dynamic modelling showed that incubating cell suspension under low O2 lead to a significant reduction in glucose uptake rate. Low O2 stress alsocaused a reduction in the activity of several enzymes involved in the TCA cycle resulting in the accumulation of intermediates of the glycolysis. An increase flux of lactate and ethanol synthesis was observed showing the enhanced role of fermentative metabolism in ensuring energy production under the low O2 stress. Analysis of energy production and utilisation showed similar amounts of ATP production at the different O2 levels even though the ATP produced under the low O2 conditions came at a cost of high substrate usage. Metabolic control analysis of glycolysis, fermentation and the TCA cycle showed that the uptake of external glucose controls most of the fluxes in the central carbon metabolism while thetransport of pyruvate into the mitochondria from the cytosol controls the activity of the TCA cycle. Also, enzymes which compete for a common substrate exerted negative control on each other.
Steady-state metabolic flux analysis was carried out using the 13C-label incorporated into free intracellular metabolites instead of the conventional approach of utilising the label being incorporated in proteinogenic amino acids. This was done to avoid the long incubation times needed to achieve metabolic and isotopic steady-state in proteinogenic amino acids. For steady-state flux analyses, cell suspensions were incubated in a bioreactor at O2 levels of 21, 8, 5 and 0 kPa until metabolic and isotopic steady-state was reached (24 h after the start of the experiment). Free intracellular metabolites were extracted with methanol, derivatised with N-(tert-butyldimethylsilyl)-trifluoroacetamide and analysed using GC-MS. 13C-labelpresent in metabolites of the central carbon metabolism, amino acids and sugars were determined for steady-state fluxes analyses. Fluxes were estimated using the 13CFLUX2 software. The steady-state response to low O2 stress was similar to the observations made under dynamic conditions with a decrease in substrate uptake, an increase increased fermentative metabolism and a reduced TCA cycle activity and amino acid synthesis. Based on the similarity in fluxes through the central carbonmetabolism, the dynamic and steady-state modelling approaches were compared. Dynamic modelling offers several advantages including providing more detailed information on the structure and regulation of metabolic networks under different stress conditions and providing a time dependent response of an organism to stress. Steady-state flux analysis is, however, useful in obtaining a quick overview of the changes in metabolismupon stress induction especially in systems where metabolic and isotopic steady-state can be ascertained.
Date:1 Oct 2009 →  2 Jul 2014
Keywords:Central plant cell metabolism
Disciplines:Plant biology
Project type:PhD project