Biotic and abiotic stress in microorganisms and plants: Some Mechanisms of Stress and Programmed Cell Death. Genetic Engineering Approaches Towards Improved Stress Tolerance

Sammanfattning: Various forms of stress, such as biotic and abiotic stress, were studied in microorganisms and plants to learn more about the underlying mechanisms of such stress and potential genetic engineering strategies towards stress tolerance. An antifreeze protein from winter flounder, expressed in E. coli, was subjected to directed evolution with NaCl as selective pressure. The resulting clones were tested for their ability to confer osmotolerance and antifreeze activity, and the amino acid substitutions are discussed. Proline is a compatible osmolyte and in the light of this a bifunctional enzyme was constructed from g-glutamyl kinase and g-glutamyl phosphate reductase. These are two enzymes in the biosynthetic pathway from glutamate to proline. The importance of chaneling is discussed as the intermediate g-glutamyl phosphate is labile. The free amino acid profile in response to frost, especially the concentrations of proline, glutamine and asparagine, of a frost tolerant potato variety in comparison with a frost sensitive variety was analyzed. The effect of proline in freezing tolerance is discussed. Programmed cell death is suggested to be an integral part of plant pathogen defense. Programmed cell death triggered by a pathogen-induced oxidative burst showed not only to be a local phenomena at the site of infection, but also to be part of a systemic response. The implications of this phenomena in the establishment of plant immunity is discussed. Programmed cell death in plants are also shown to have similarities with animal apoptosis and was studied morphologically and molecularly by artificial triggering and modulation of signaling pathways. Differentially expressed cDNA fragments were identified by differential display using an antibody to separate cells in different states. Separating cells from an embryogenic carrot cell suspension culture using the antibody resulted in two pools, one of which is destined to undergo programmed cell death. The potential of cloning genes involved in programmed cell death in this system is discussed.