Unravelling the metabolic effects of sugar starvation on leafy vegetable cells

status: publishe

Unravelling the metabolic effect of sugar starvation on leafy vegetable cells

After harvest, fresh leafy vegetables are stored under conditions of low temperature in combination with controlled atmosphere (CA) and/or modified atmosphere (MAP) conditions to preserve their quality, while prolonging their postharvest storage life. In reality, these conditions are hardly met since these fresh produce are stored to situations were photosynthetically active radiation is limited or completely absent and the low oxygen conditions in the MAP or CA could result in fermentation. These will lead to sugar starvation and other unwanted physiological disorders, leading to premature economic losses. The main objective of this PhD is to study and understand the effect(s) of sugar starvation on metabolic and biochemical changes on the respiratory metabolism of leafy vegetables cells through metabolomics and(13C-)fluxomics analysis. To meet the main objective and the scientific research questions of this thesis, the following sub-objectives were defined: To optimise a protocol for the isolation and characterization of lamb’s lettuce cells. To optimise measurement and experimental setups in the bioreactor. To characterise the changes in the metabolome and fluxome of the isolated cells in response to sugar starvation at different temperatures and low oxygen stress conditions, respectively, using GC-MS metabolic profiling approach. To construct an integrated compartmentalised dynamic model of the central carbon metabolism to estimate fluxes and to investigate the control of these fluxes in the different pathways as affected by low O2 stress and temperature, respectively.status: publishe

Kinetic modelling of the metabolic response of isolated lamb’s lettuce (Valerianella locusta L.) cells to sugar starvation

Leafy vegetables are highly perishable, as they are exposed to carbohydrate starvation during their postharvest storage life, leading to premature quality loss. An in-depth knowledge of the fundamental metabolic regulation and control mechanisms of these leafy vegetables to sugar starvation is beneficial in understanding how they modify their metabolism to survive in the absence of sugar. To this end, isolated lamb's lettuce cells were incubated in the dark to trigger sugar starvation at different temperatures (25, 18 and 1°C) to monitor the dynamic changes in the metabolome over time. To understand which pathways were up- or downregulated, the isolated cells were preloaded with uniformly labelled 13C-glucose and the distribution of the 13C label throughout the different metabolic pathways was monitored. The metabolite and the corresponding 13C label data were used to construct an integrated compartmentalised central metabolic model, containing both first-order and Michaelis-Menten kinetics, to quantify the effect of sugar starvation on the kinetic parameters and, thus, to calculate fluxes of the respiratory metabolism and to analyse the metabolic control mechanisms that plants have to adopt upon sugar starvation as a function of storage temperature. From this study, a decrease in flux was observed in the respiratory metabolism of cells when they were incubated at 1°C compared with 25 and 18°C. When sugar starvation was induced at 18 and 25°C, a decrease in the fluxes was observed in the glycolytic pathway metabolites as compared with that of the tricarboxylic acid cycle metabolites, which increased during sugar starvation. Moreover, an increased flux coming from protein and phospholipid breakdown was observed, resulting in increased concentrations of free amino and fatty acids, suggesting proteolytic and β-oxidation reactions.status: publishe

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

© 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

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

© 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

3D printing of plant tissue for innovative food manufacturing: encapsulation of alive plant cells into pectin based bio-ink

3D food printing allows creation of foods by depositing food material according to computer aided designs. However, the number of printable materials for food is still low which limits the possibilities of creating specific structures and textures. A novel approach is tested of using food printing materials incorporating plant cells in order to print foods that resemble plant tissues in various ways. A 3D printing method was developed based on the extrusion of bio-inks composed of a low-methoxylated pectin gel and embedded lettuce leaf cells. Bovine serum albumin was added in order to increase the air fraction in the printed gel matrix. Objects containing up to 5 × 106 cells/mL were successfully 3D printed. The mechanical strength increased by the pectin concentration and decreased with the increase of air fraction and concentration of encapsulated cells. The viability of the encapsulated plant cells depended on the pectin concentration and varied from 50 to 60 %.status: accepte

The metabolic response of cultured tomato cells to low oxygen stress

The storage of fruits and vegetables under a controlled atmosphere can induce low oxygen stress, which can lead to post-harvest losses through the induction of disorders such as core breakdown and browning. To gain better understanding of the metabolic response of plant organs to low oxygen, cultured tomato cells (Lycopersicum esculentum) were used as a model system to study the metabolic stress response to low oxygen (0 and 1 kPa O2). By adding 13C labelled glucose, changes in the levels of polar metabolites and their 13C label accumulation were quantified. Low oxygen stress altered the metabolite profile of tomato cells, with the accumulation of the intermediates of glycolysis in addition to increases in lactate and sugar alcohols. 13C label data showed reduced label accumulation in almost all metabolites except lactate and some sugar alcohols. The results showed that low oxygen stress in tomato cell culture activated fermentative metabolism and sugar alcohol synthesis while inhibiting the activity of the TCA cycle and the biosynthesis of metabolites whose precursors are derived from central metabolism, including fluxes to most organic acids, amino acids and sugars.status: publishe