Het onthullen van weefselspecifieke en zuurstofafhankelijke reacties van het centraal koolstofmetabolisme in tomatenvruchten

Tomato is a widely studied model plant with a complex fruit structure composed of multiple anatomically and physiologically distinct tissues, including the pericarp, septa, placenta, and columella. While the pericarp has been the focal point of most tomato fruit research, the spatial organization of internal tissues suggests potential for tissue-specific functions and inter-tissue metabolic interactions. Notably, tomato fruit experience internally low-O2 conditions, which can profoundly affect the central carbon metabolism. However, it remains unclear how individual tissues respond to low-O2 stress at the metabolic level and whether inter-tissue metabolite exchange contributes to the development spatial metabolic patterns. This PhD thesis investigates whether the central carbon metabolism and low-O2 stress responses are different across fruit tissues, and whether metabolic cooperation occurs between them. A tissue-disc-based experimental platform was developed to control O2 levels (21, 1, and 0 kPa) and uniformly supply 13C6 labeled glucose to columella, septa, and mesocarp tissues. Tissue specific metabolite concentrations and their 13C-label enrichment were quantified using GC-MS. In response to O2, columella tissue consistently showed the highest respiration rate and the strongest shifts in metabolite pools and 13C labeling, indicating higher metabolic activity and O2 sensitivity compared to septa and mesocarp. Low O2 triggered a common stress response in all tissues, characterized by the accumulation of glycolytic intermediates, fermentation products (lactate, ethanol, acetic acid), and amino acids (alanine, glycine, GABA), alongside reduced 13C6 glucose uptake from the medium. Nonetheless, labeling efficiency increased in key stress-responsive metabolites, especially in the columella, revealing low-O2 induced metabolic reprogramming. To assess the potential role of inter-tissue metabolite exchange, a complementary feed-and-cut approach was employed, feeding intact fruit slices after which the three tissues were separated and analyzed. Despite differences in metabolite pool sizes, likely due to reduced wounding, labeling dynamics were comparable to corresponding cut-and-feed tissue, where tissues were mechanically separated before being fed and analyzed. This suggests that spatial metabolic differences are primarily driven by intrinsic tissue properties and local O2 environments, rather than by the exchange of non-gaseous metabolites between the three tissues. To mechanistically interpret the metabolic adjustments in columella tissue, the most active tissue in the fruit, a compartmentalized kinetic model of the central metabolism was developed. The model was calibrated using time-series data of metabolite concentrations and 13C label enrichment for the columella tissue kept at 21, 1, and 0 kPa O2. Simulations confirmed that under low O2, glucose uptake is reduced, while glycolysis and fermentation pathways are enhanced to maintain ATP production and NAD⁺ regeneration. The model shows how vacuolar sugars are being mobilized to the cytosol to sustain the carbon supply. To mitigate cytosolic acidification and carbon loss associated with fermentation, alanine biosynthesis and the GABA shunt are being activated. At the same time, mitochondrial redox imbalance impairs TCA cycle flux and shifts carbon flow toward amino acid biosynthesis. In conclusion, this study demonstrates that the different tomato fruit tissues exhibit distinct central carbon metabolism and metabolic responses to low-O2 stress, with the columella being the most active and sensitive. The spatial variation in central carbon metabolism arises from intrinsic tissue regulation rather than inter-tissue metabolite exchange. The kinetic model provides a mechanistic framework for understanding how low-O2 stress alters fruit metabolism and lays the groundwork for future quantitative studies of metabolic adaptation in plant tissues.