Abstract
Primary carbohydrate metabolism connects all parts of plant metabolism. When carbon fixed via photosynthesis is used for synthesis of sucrose, or when starch is broken down during the night and is used for e.g. sucrose synthesis, respiration or production of defence compounds or other secondary metabolites, it goes through core reactions in central carbohydrate metabolism. Consequently, the core reactions of carbohydrate metabolism are crucial for plant development and fitness upon environmental changes. One type of enzymes that holds a key position, is the cytosolic ATP-dependent phosphofructokinases (PFKs), of which plant typically have several isoforms. These enzymes phosphorylate the C-1 position of Fru-6-P, leading the flux of carbon in the glycolytic direction. Despite a central position in plant carbohydrate metabolism, there are no reports of active plant PFK expressed in a non-plant system. Furthermore, there are no published studies of stable transgenic plants with altered plant PFK activity. Therefore, knowledge on the impact of PFKs on overall carbon flux and especially on individual isoforms including their in planta significance, is so far very limited. In this PhD study, cytosolic A. thaliana PFKs were cloned and expressed in E. coli and initial experiments regarding purification and enzymatic activity were carried out. This revealed that at least one of the A. thaliana isoforms, can be purified as an enzyme with the anticipated PFK activity. In order to study the in planta significance of the individual cytosolic PFK isoforms, homozygous A. thaliana single and double knock-out lines were obtained. One of the double mutants was found to have a strong growth and development phenotype. Quantification of selected primary and secondary metabolites, as well as transcript analysis furthermore showed that this mutant display altered sugar homeostasis and possibly altered source-sink relations. This being despite the finding of only minor changes when looking at the short time flux of carbon.
Another enzyme central to plant carbohydrate metabolism is the bifunctional enzyme F2KP (fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase). This enzyme is responsible for making and degrading the signalling metabolite Fru-2,6-BP, which during photosynthesis controls the flux of carbon into sucrose and starch synthesis. This enzyme carries a long N-terminal extension that had so far received very little attention. In this PhD study, a putative N-terminal myristoylation site of A. thaliana F2KP was investigated. Heterologous expression and subsequent in vitro assays showed the protein to be a substrate for the N-myristoyl transferase 1 (NMT1), thereby confirming the presence of this putative site. The biological function of myristoylation of F2KP was investigated by studying transgenic plants overexpressing different versions of fluorophore tagged F2KP. This revealed that disruption of the NMT1 recognition site resulted in a change in cellular localisation. Thereby indicating that myristoylation of F2KP does take place in planta, and that it functions in localisation of the enzyme.
Another enzyme central to plant carbohydrate metabolism is the bifunctional enzyme F2KP (fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase). This enzyme is responsible for making and degrading the signalling metabolite Fru-2,6-BP, which during photosynthesis controls the flux of carbon into sucrose and starch synthesis. This enzyme carries a long N-terminal extension that had so far received very little attention. In this PhD study, a putative N-terminal myristoylation site of A. thaliana F2KP was investigated. Heterologous expression and subsequent in vitro assays showed the protein to be a substrate for the N-myristoyl transferase 1 (NMT1), thereby confirming the presence of this putative site. The biological function of myristoylation of F2KP was investigated by studying transgenic plants overexpressing different versions of fluorophore tagged F2KP. This revealed that disruption of the NMT1 recognition site resulted in a change in cellular localisation. Thereby indicating that myristoylation of F2KP does take place in planta, and that it functions in localisation of the enzyme.
| Originalsprog | Engelsk |
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| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Status | Udgivet - 2018 |