15 research outputs found

    Dynamics in the Central Plant Cell Metabolism

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    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.status: publishe

    The Effect of Pasteurisation on the Physicochemical and Nutritional Quality of Soursop (Annona muricata L.) Juice

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    The effect of pasteurisation on the nutritional and physicochemical quality of soursop juice was investigated. Soursop juice was prepared from soursop fruit pulp and pasteurised at different temperatures (63, 71, 78, 83 and 95°C) for different durations. The effect of pasteurisation on ascorbic acid level, total phenolic content and total antioxidant capacity of the juice was analysed. Additionally, the changes in pH, total soluble solids, titratable acidity and colour (L*a*b*) were determined. The pH, total soluble solids and titratable acidity were not significantly affected by pasteurisation. Pasteurisation affected the total phenolic content and total antioxidant capacity of the juice. For the same pasteurisation temperature, an increased duration of pasteurisation resulted in a reduction of total antioxidant capacity. Ascorbic acid levels in the juice decreased with increased duration and temperature of pasteurisation. A first-order kinetic model was developed to explain the effect of pasteurisation on the degradation of ascorbic acid in soursop juice. A degradation rate constant of 0.035 min-1 and an activation energy of 83 kJ/mol were obtained

    Priming with Methylene Blue Enhances the Antioxidant Properties and Germination Power of Cowpea, Millet and Sorghum Seeds

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    Seed priming is the preparation of plants to overcome stress with the aim of improving the efficiency and rate of germination. The effect of priming cowpea, millet and sorghum seeds with methylene blue (MB) on germination power (GP) was investigated in this work. The seeds were primed by steeping for 6 h in different concentrations of MB solution, germinated over 48 h on wet cotton and the GP determined. Further, soluble protein and total phenolic contents, and the antioxidant properties [scavenging ability for hydrogen peroxide, nitric oxide, hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and iron chelation ability] of the seeds steeped in MB that gave the highest GP were determined. The highest GP was observed for cowpea and sorghum seeds steeped in 80 µM MB solution and 60 µM MB solution for millet seeds. At these MB concentrations, a faster rate of germination coupled with an increase in soluble protein and total phenolic content was observed. The increase in germination rate was also characterized by enhanced antioxidant properties of the seeds. The results of this study show that priming with MB can be used to improve the germination of these seeds

    Effect of Storage Temperature on the Microbial Quality of Fura

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    Fura, a semi-solid millet-based dumpling, is popularly consumed throughout West Africa. This work was aimed at evaluating the effect of storage temperature on the microbial and physicochemical properties of fura. Freshly prepared fura was stored at 30 and 4°C and sampled periodically to determine the changes in pH, titratable acidity, total soluble solids and total phenolic content. Additionally, the effect of storage temperature on the microbial (by enumerating aerobic mesophiles, lactic acid bacteria, Enterobacteriaceae, and yeast and moulds) quality was determined. Storage affected the acidity of fura with a decrease in pH and an increase in titratable acidity. The total soluble solids and total phenolic content were, however, not affected by storage temperature. Lactic acid bacteria were the predominant microbe present in fura. During storage at 30°C, faster growth of lactic acid bacteria and the other microbes was observed compared to storage at the lower temperature

    Characterization of the 3-D microstructure of mango (Mangifera indica L. cv. Carabao) during ripening using X-ray computed microtomography

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    In this study, X-ray computed microtomography (X-ray μCT) was applied to investigate the changes in the 3-D microstructure of mango during ripening at 20 °C. X-ray μCT provided a unique insight of the undamaged tissue and pore network during ripening. Analysis of the pore and tissue network revealed differences in the microstructure along the radial axis of the fruit and microstructural changes during ripening. Multivariate statistical analysis unveiled that ripening was associated with a decrease in pore size, and increase in pore fragmentation and pore specific surface area. These structural parameters have the highest discriminating ability, correctly classifying unripe from ripe fruit samples. The study concludes that ripening can be successfully characterized on the basis of its 3-D microstructure using X-ray microtomography. Industrial relevance: This study identified important parameters to describe the ripening process on the basis of microstructure. As today's microtomography technology allows for scanning only a small tissue sample from the fruit, the pace at which tomography technology is advancing will allow for scanning whole fruit with sufficient resolution and without any need for sample preparation. Results from this study could be applied for non-destructive determination of fruit microstructure for assessing fruit quality in relation to the ripening process.publisher: Elsevier articletitle: Characterization of the 3-D microstructure of mango (Mangifera indica L. cv. Carabao) during ripening using X-ray computed microtomography journaltitle: Innovative Food Science & Emerging Technologies articlelink: http://dx.doi.org/10.1016/j.ifset.2013.12.008 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved.status: publishe

    Influence of Partially Substituting Wheat Flour with Tiger Nut Flour on the Physical Properties, Sensory Quality, and Consumer Acceptance of Tea, Sugar, and Butter Bread

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    Tiger nut is a valuable source of fiber, lipids, minerals, and carbohydrates. However, avenues for incorporating tiger nuts into food remain underexplored, especially in several tropical countries where the plant grows well. The current study investigated the effects of partially substituting wheat flour (WF) with tiger nut flour (TNF) on the physical and sensory properties of different bread types to evaluate the more amenable system for tiger nut incorporation. The substitution was done at WF:TNF ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread (Bb), tea bread (Tb), and sugar bread (Sb). The results show that WF substitution with TNF increased bread brownness and color saturation and decreased lightness, showing the highest impact on Sb, followed by Tb and Bb. Additionally, bread-specific volume decreased significantly after 20% (Bb), 25% (Tb), and 30% (Sb) TNF substitution. Furthermore, substituting WF with 30% TNF increased crumb hardness from approx. 1.87 N to 3.64 N (Bb), 3.46 N to 8.14 N (Tb), and 6.71 N to 11.39 N (Sb) and caused significant increases to 17.80 N (Tb) and 21.08 N (Sb) after 3 d storage. Only a marginal effect on storage hardness (4.32 N) was observed for Bb. Substituting WF with 10% TNF for Bb or 25% TNF for Tb led to significantly higher consumer (N=56) scores for all attributes and overall acceptability. However, no significant effect on the overall acceptability of Sb was observed. Flash profiling showed frequently used descriptors for Bb as firm, moist, buttery, smooth, and astringent. After 10% TNF substitution, descriptors were chewy, firm, sweet, porous, dry, and caramel, and that of 30% TNF were grainy, chocolate, brown, nutty, and flaky. Substituting WF with TNF increased the lipids, fiber, and minerals content but decreased the protein and carbohydrate contents of bread. TNF substitution led to different physical and sensory effects depending on bread type, showing that Bb with 10% or Tb with 25% TNF is more comparable with the overall acceptance quality of 100% WF. The study is relevant for utilizing tiger nuts as an ingredient in bread products

    The effect of temperature on the metabolic response of lamb’s lettuce (Valerianella locusta, (L), Laterr.) cells to sugar starvation

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    © 2016 Elsevier B.V. Fresh leafy vegetables are highly perishable and may suffer from sugar starvation during postharvest storage. To fully understand their metabolic response to sugar starvation, isolated lamb's lettuce (Valerianella locusta, (L) Laterr.) cells were used as a model system to study biochemical and metabolic stress response to sugar starvation at 1 °C, 18 °C and 25 °C. The effect of sugar starvation was minimal at 1 °C. While the higher temperature showed clear impact of sugar starvation on the overall metabolic profile no significant differences were observed between the starvation at either 18 °C or 25 °C for the main sugars (glucose, glucose-6-phosphate, fructose, fructose-6-phosphate and sucrose). Biochemical and metabolic changes of the isolated cells upon sugar starvation involved a decrease in the levels of sugars, except for trehalose and ribose, as well as an increase in the levels of sugar alcohols. Sugar starvation altered the central metabolism by decreasing the levels of the intermediates of the glycolytic pathway, except for 3-phosphoglycerate and pyruvate. Increased levels of the intermediates of the tricarboxylic acid cycle were also observed. 13C labelling data showed a decreased label accumulation in almost all metabolites, except for mannitol, myo-inositol, and trehalose. The increase in the levels of free soluble amino and fatty acids with a corresponding decrease in their 13C label suggested a breakdown of protein and triacylglycerides.status: publishe

    Metabolic profiling reveals a coordinated response of isolated lamb's (Valerianella locusta, L.) lettuce cells to sugar starvation and low oxygen stress

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    © 2016 Elsevier B.V. Sugar starvation is a common phenomenon occurring in most leafy vegetables after harvest and storage. Additionally, leafy vegetables are subjected to low O2 stress when stored in modified atmosphere conditions. In this study, the metabolism of isolated lamb's lettuce cells was studied upon sugar starvation under O2 stress conditions, using 13C labelled glucose. Fast depletions of the soluble sugars were observed, being more pronounced under aerobic conditions than under low O2 stress conditions. Sugar starvation under aerobic conditions resulted in increased levels and decreased 13C label incorporation of TCA cycle intermediates and amino and fatty acids originating from glycolytic and TCA cycle pathways, compared to starving cells incubated under low O2 stress. On incubation under low O2 stress a switch in metabolism from aerobic to fermentation metabolism was observed. Under low O2 stress conditions, increased levels and 13C label incorporated in hexose phosphates, pyruvate, lactate, GABA, alanine, together with increased levels of acetaldehyde, ethanol and ethyl acetate was observed indicating fermentative metabolism was triggered.status: publishe

    Quality Changes during Storage of Burkina (a Millet and Milk-based) Drink

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    Burkina, a drink prepared from millet and milk, is gaining economic attention in Ghana due to its perceived nutritious nature and high energy content. The drink which is produced on a small-scale is usually vended without proper control of storage conditions leading to rapid loss of quality. The objective of this study was to investigate the effect of storing burkina at different temperatures (4 and 30°C) on the microbial and physicochemical (pH, titratable acidity, brix and phenolic content) quality of the drink. The pH, titratable acidity, brix and phenolic content of freshly prepared burkina were 3.65, 0.49%, 2.05 and 0.26 mg GAE/100 g, respectively. Although, changes were observed, storage temperature did not have a significant effect on the physicochemical quality of burkina. The initial load of aerobic mesophiles, lactic acid bacteria, Enterobacteriaceae, and yeast and moulds in the freshly prepared Burkina were 6.45, 5.49, 2.58 and 4.45 log cfu/mL, respectively. Storage at the higher temperature resulted in an increased microbial load within 48 h, leading to faster spoilage, with only marginal increases observed at the lower storage temperature

    Tissue breakdown of mango (Mangifera indica L. cv. Carabao) due to chilling injury

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    Chilling injury (CI) presents a major problem in postharvest preservation and marketing potential of mango. In this study, tissue breakdown caused by chilling injury was investigated using X-ray computed microtomography (X-ray μCT). This provided a unique insight in 3-D changes in tissue structure and pore networks during chilling injury development. Evidence of cell collapse and cavity formation in severely chill injured fruit was apparent. In addition, microstructural evidence supporting the occurrence of intracellular water leakage was found. Quantitative analysis revealed that chilling injury was associated with, first, a decrease in porosity, pore size and pore connectivity due to cell leakage and tissue breakdown, which was followed by an increase in pore size due to cavity formation. Multivariate statistics identified pore connectivity and Euler number as the most important parameter in relation to chilling injury. The results support the hypothesis that changes in tissue and pore structure in mango fruit contribute significantly to the development of postharvest tissue disorders by drastic changes in tissue aeration and water movement, identical to what has been observed in other fruit species.publisher: Elsevier articletitle: Tissue breakdown of mango (Mangifera indica L. cv. Carabao) due to chilling injury journaltitle: Postharvest Biology and Technology articlelink: http://dx.doi.org/10.1016/j.postharvbio.2016.11.009 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.status: publishe
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