Oxygen deficiency is a severe stress for the plant and results in an energy crisis. Hypoxia can occur naturally in plant tissues with high meristematic activity but may also be a result of flooding. Plants developed numerous genetic, metabolic and anatomical adaptations to escape or endure low oxygen. Adventitious root growth or aerenchyma formation are some examples of anatomical modifications to help the plant survive submergence. On the cellular level, the mitochondrial respiration is inhibited due to the oxygen limitation and ATP content decreases dramatically. To cope with the energetic crisis, the expression of genes coding for proteins involved in fermentation, like the alcohol dehydrogenase and pyruvate decarboxylase (PDC), is induced. During hypoxic stress, Pyrophosphate (PPi) is considered as an alternative energy source to ATP. While it is a byproduct of many biochemical processes, PPi-dependent enzymes use PPi instead of ATP for the phosphorylation of their target molecules. Several of these enzymes are induced during hypoxia in rice including the pyrophosphate-fructose 6-phosphate-1-phosphotransferase (PFP). The aim of this study was to investigate the role of the PFP in the model organisms Arabidopsis thaliana and Oryza sativa under oxygen deficiency. First of all, rice transformation was established and PFP RNAi mutants were created in rice. Comparison of the metabolism of PFP mutants in Arabidopsis thaliana and Oryza sativa should reveal the physiological relevance of the enzyme in these organisms. Therefore, carbohydrate contents and the survival under submergence were examined and PFP/PFK activities were determined. The data suggest that in contrast to Arabidopsis thaliana, the PFP of rice might have an essential role for the viability of the plant. Furthermore, functional analysis of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K), an enzyme producing fructose-2,6-bisphosphate (Fru2,6BP) was performed. Fru2,6BP is a potent activator of the PFP. The metabolite content, PFP activities and survival rate were assessed in two independent A. thaliana T-DNA deletion mutants under submergence to investigate the role of PFP for the survival of the plants in hypoxic conditions. Additionally, phosphofructokinase (PFK), an enzyme of the glycolysis is phosphorylating fructose-6-phosphate using ATP. Several Arabidopsis thaliana PFKs are induced under oxygen deficiency, showing a similar expression pattern to the PDC, a marker gene of hypoxic stress response. T-DNA insertion lines for Arabidopsis thaliana PFKs were analyzed. It was shown that all Arabidopsis thaliana PFKs and the PF2K probably do not contribute to the fitness of the plant under hypoxia. The chloroplast contains a complex redox regulation system harnessing the reducing power of the photosynthesis in order to modify the activity of many plastidic proteins by reducing their disulfide bridges. Several previous published studies showed that PFK activity in Pisum sativum and Spinacia oleracea chloroplasts was regulated by light. During this PhD project the PFK5 amino acid sequence was analysed. Using site directed mutagenesis and changing cysteines to serines, two cysteines involved in the light-dependent redox regulation of the enzyme were identified. Protein modelling revealed the localization of the cysteines on a flexible loop emerging out of the surface of the PFK5 protein, being easily accessible to redox-regulating proteins such as thioredoxins. Further analysis showed that PFK5 activity was sensitive to the reducing agent Dithiothreitol (DTT) and modulated by light.
|School:||Universitaet Bayreuth (Germany)|
|Source:||DAI-C 81/4(E), Dissertation Abstracts International|
|Keywords:||Hypoxic stress, Oxygen deficiency, Plants, Hypoxia|
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