Defining key proteins of the primary carbohydrate metabolism in Arabidopsis thaliana
Carrera, Dániel Árpád
- Doctoral Thesis
Rights / licenseIn Copyright - Non-Commercial Use Permitted
Photosynthetic assimilation of atmospheric carbon dioxide (CO2) is the major carbon fixation mechanism. Thereby the captured light energy is used to produce carbohydrates within the Calvin-Benson cycle. A portion of the synthesized carbohydrates is exported out of the chloroplast into the cytosol, where sucrose can be produced. Sucrose is a non-reducing, neutral disaccharide, which can be transported throughout the plant, providing the necessary carbon and energy for development. The Calvin-Benson cycle and cytosolic sucrose metabolism build a complex network consisting of many highly regulated biochemical reactions. One characteristic of primary carbohydrate metabolism in plants is the apparent high level of genetic redundancy, especially among the cytosolic sucrose-metabolic enzymes. While the biochemical reactions are relatively well described, the role of the single isoforms is still unclear, and the apparent redundant isoforms might have a metabolic and physiological role. The aim of this work was to unravel and clarify the function of these (iso)enzymes and, if possible, attribute specific functions to them. First, homozygous single knock-out mutants were screened for phenotypic differences under normal growth conditions. This rarely revealed significant alterations compared to the wild type, suggesting that the isoenzymes may actually be redundant. However, as plants are naturally constantly exposed to environmental fluctuations, the roles of the isoforms may become apparent during acclimation. We therefore established a hydroponic platform, where plants could be grown in well-defined nutrient-deficient media (either without nitrate or phosphate) and under mild long-term abiotic stresses (cold, warm, salt or osmotic stress). This allowed me to perform a comparative proteomic analysis in wild type plants, where the plants’ stress responses to different treatments were examined in both the shoot and the root (Chapter 1). My results suggested that the plants response to the different stresses in a very specific manners. Furthermore, these responses were distinct in the root and the shoot. To link the changes in the proteome with changes at the metabolic level, we also measured carbohydrates in the hydroponically grown plants (Chapter 2). Moreover, I selected promising candidates based on the proteomic analyses, i.e. sucrose-metabolic proteins that were either up- or downregulated in one of the stress responses. The corresponding single mutants were then grown under the stress treatments and analyzed for their growth and early development (Chapter 2). One isoform among the four sucrose phosphate phosphatases (SPP2) and one fructokinase (FRK) turned out to be essential in seed development, the complete knock-out of them resulted in aborted seed development and non-viable plants. The knock-out mutant of another fructokinase (FRK7), which had been downregulated during the salt-stress treatment, grew slower under normal conditions, but was not further impaired during this stress. Further, a cytosolic fructose 1,6-bisphosphate aldolase (FBA8) appeared as the major isoform of its gene family in the cytosol. In the final part of this work (Chapter 3), I characterized additional fructose 1,6-bisphosphate aldolases. The FBA family is known to have diverse functions in plant physiology (i.e. glycolysis, sucrose biosynthesis, Calvin-Benson cycle), but I focused on their role in the Calvin-Benson cycle and investigated the three plastidial isoforms (FBA1, FBA2 and FBA3). While the fba1 mutant was indistinguishable from the wild type, the fba2 and fba3 single mutants grew considerably slower and displayed additional distinct phenotypes: The fba2 mutant showed a marked decrease in total FBA activity and a low level of starch coupled with a high sugar content. The fba3 mutant had an extremely high carbohydrate content, displaying five times elevated sugar and starch levels at the end of the day, but the total FBA activity was altered only little. GUS-reporter lines suggested that FBA3 was predominantly expressed in vascular tissues. This was in contrast to FBA1 and FBA2, which were expressed predominantly in the photosynthesizing cells of the leaf lamina. These data indicate that FBA1 and FBA2 catalyze the canonical reactions in the Calvin-Benson cycle, with FBA2 being the major isoform. By contrast, FBA3 is probably functional in non-photosynthesizing, heterotrophic tissues, especially in the vasculature. The knock-out of FBA3 created a barrier between the photosynthesizing source tissues and the heterotrophic sink tissues. Show more
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ContributorsExaminer: Zeeman, Samuel C.
Examiner: Streb, Sebastian
Examiner: Gibon, Yves
Organisational unit03707 - Zeeman, Samuel C. / Zeeman, Samuel C.
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