Institut für Biochemie und Biologie
Refine
Document Type
- Doctoral Thesis (2)
Language
- English (2)
Is part of the Bibliography
- yes (2)
Keywords
- Pflanze (2) (remove)
Institute
The energy required to drive photochemical reactions is derived from charge separation across the thylakoid membrane. As the consequence of difference in proton concentration between chloroplasts stroma and thylakoid lumen, a proton motive force (pmf) is generated. The pmf is composed out of the proton gradient (ΔpH) and membrane potential (ΔΨ), and together they drive the ATP synthesis. In nature, the amount of energy fueling photosynthesis varies due to frequent changes in the light intensity. Thylakoid ion transport can adapt the energy flow through a photosynthetic apparatus to the light availability by adjusting the pmf composition. Dissipation of ΔΨ reduces the charge recombination at the photosystem II, allowing for an increase in ΔpH component to trigger a feedback downregulation of photosynthesis. K+ Exchange Antiporter 3 (KEA3) driven K+/H+ antiport reduces the ΔpH fraction of pmf, thereby dampening a non-photochemical quenching (NPQ). As a result, it increases the photosynthesis efficiency during the transition to lower light intensity. This thesis aimed to find the answers for questions concerning KEA3 activity regulation and its role in plant development. Presented data shows that in plants lacking chloroplast ATP synthase assembly factor CGL160 with decreased ATP synthase activity, KEA3 has a pivotal role in photosynthesis regulation and plant growth during steady-state conditions. Lack of KEA3 in cgl160 mutant results in a strong growth impairment, as photosynthesis is limited due to increased pH-dependent NPQ and decreased electron flow through cytochrome b6f complex. Overexpression of KEA3 in cgl160 mutant increases charge recombination at photosystem II, promoting photosynthesis. Thus, during periods of low ATP synthase activity, plants benefit from KEA3 activity. The KEA3 undergoes dimerization via its regulatory C-terminus (RCT). The RCT responds to changes in light intensity as the plants expressing KEA3 without this domain show reduced photo-protective mechanism in light intensity transients. However, those plants fix more carbon during the photosynthesis induction phase as a trade-off for a long-term photoprotection, showing KEA3 regulatory role in plant development. The KEA3 RCT is facing thylakoid stroma, thus its regulation depends on light-induced changes in the stromal environment. KEA3 activity regulation overlaps with the stromal pH changes occurring during light fluctuations. The ATP and ADP has shown to have an affinity towards heterologously expressed KEA3 RCT. Such interaction causes conformational changes in RCT structure. The fold change of RCT-ligand interaction depends on the environmental pH value. With a combination of bioinformatics and in vitro approach, the ATP binding site at RCT was located. Introduction of binding site point mutation in planta KEA3 RCT resulted in antiporter activity deregulation during transition to low light. Together, the data presented in this thesis allowed us to assess more broadly a KEA3 role in photosynthesis adjustment and propose the models of KEA3 activity regulation throughout transition in light intensity.
Subcellular compartmentation of primary carbon metabolism in mesophyll cells of Arabidopsis thaliana
(2011)
Metabolism in plant cells is highly compartmented, with many pathways involving reactions in more than one compartment. For example, during photosynthesis in leaf mesophyll cells, primary carbon fixation and starch synthesis take place in the chloroplast, whereas sucrose is synthesized in the cytosol and stored in the vacuole. These reactions are tightly regulated to keep a fine balance between the carbon pools of the different compartments and to fulfil the energy needs of the organelles. I applied a technique which fractionates the cells under non-aqueous conditions, whereby the metabolic state is frozen at the time of harvest and held in stasis throughout the fractionation procedure. With the combination of non-aqueous fractionation and mass spectrometry based metabolite measurements (LC-MS/MS, GC-MS) it was possible to investigate the intracellular distributions of the intermediates of photosynthetic carbon metabolism and its products in subsequent metabolic reactions. With the knowledge about the in vivo concentrations of these metabolites under steady state photosynthesis conditions it was possible to calculate the mass action ratio and change in Gibbs free energy in vivo for each reaction in the pathway, to determine which reactions are near equilibrium and which are far removed from equilibrium. The Km value and concentration of each enzyme were compared with the concentrations of its substrates in vivo to assess which reactions are substrate limited and so sensitive to changes in substrate concentration. Several intermediates of the Calvin-Benson cycle are substrates for other pathways, including dihydroxyacetone-phosphate (DHAP,sucrose synthesis), fructose 6-phosphate (Fru6P, starch synthesis), erythrose 4-phosphate (E4P,shikimate pathway) and ribose 5-phosphate (R5P, nucleotide synthesis). Several of the enzymes that metabolise these intermediates, and so lie at branch points in the pathway, are triose-phosphate isomerase (DHAP), transketolase (E4P, Fru6P), sedoheptulose-1,7-bisphosphate aldolase (E4P) and ribose-5-phosphate isomerase (R5P) are not saturated with their respective substrate as the metabolite concentration is lower than the respective Km value. In terms of metabolic control these are the steps that are most sensitive to changes in substrate availability, while the regulated irreversible reactions of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase are relatively insensitive to changes in the concentrations of their substrates. In the pathway of sucrose synthesis it was shown that the concentration of the catalytic binding site of the cytosolic aldolase is lower than the substrate concentration of DHAP, and that the concentration of Suc6P is lower than the Km of sucrose-phosphatase for this substrate. Both the sucrose-phosphate synthase and sucrose-phosphatase reactions are far removed from equilibrium in vivo. In wild type A. thaliana Columbia-0 leaves, all of the ADPGlc was found to be localised in the chloroplasts. ADPglucose pyrophosphorylase is localised to the chloroplast and synthesises ADPGlc from ATP and Glc1P. This distribution argues strongly against the hypothesis proposed by Pozueta-Romero and colleagues that ADPGlc for starch synthesis is produced in the cytosol via ADP-mediated cleavage of sucrose by sucrose synthase. Based on this observation and other published data it was concluded that the generally accepted pathway of starch synthesis from ADPGlc produced by ADPglucose pyrophosphorylase in the chloroplasts is correct, and that the alternative pathway is untenable. Within the pathway of starch synthesis the concentration of ADPGlc was found to be well below the Km value of starch synthase for ADPGlc, indicating that the enzyme is substrate limited. A general finding in the comparison of the Calvin-Benson cycle with the synthesis pathways of sucrose and starch is that many enzymes in the Calvin Benson cycle have active binding site concentrations that are close to the metabolite concentrations, while for nearly all enzymes in the synthesis pathways the active binding site concentrations are much lower than the metabolite concentrations.