Doctor of Philosophy
Department of Biological Sciences
Shariati, Mansoor, Manipulation of the glycerol content of dunaliella electroporation: response of photosynthesis, cell volume and glycerol content, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 1994. http://ro.uow.edu.au/theses/1050
The aim of the first part of this project, was to determine the effects of salt stress photosynthesis and respiration in the green algae D. tertiolecta and D. salina. A previously reported burst in oxygen evolution after salt addition was investigated. Following salt stress, no burst in photosynthesis was found under any conditions for both species in contradiction to the earlier reports. This might be due to strain or species differences. High salt stress (increasing salinity by a factor of 5.88) inhibited photosynthesis in D. tertiolecta. Low salt stress (increasing salinity by a factor of 1.66) caused in D. tertiolecta a small increase in photosynthesis but had no substantial effects in D. salina. Salt stress decreased respiration in D. tertiolecta, more markedly at high salt stress. In D. salina, salt stress by a factor of 1.66 caused respiration to increase significantly. In general, the magnitude of salt stress was more important than the final salt concentration. No significant differences were found between phosphate and Hepes buffers.
In the second part of this project, the aim was to use electroporation to lower the intracellular glycerol concentration by a small proportion (10 - 20%) while the external osmotic pressure was kept constant. The effects of electroporation on photosynthetic O2 evolution, mitochondrial O2 uptake, release of soluble protein and release of intracellular glycerol to the medium were measured. Electroporation conditions were optimised to release about 10% of intracellular glycerol to the external medium with minimal apparent effects on metabolism. The results showed that glycerol release did not originate from the total rupture of a small proportion of cells. The uptake of mannitol, the major solute in the electroporation medium, was determined and found to be less than 20% of glycerol release. Following the release of glycerol by electroporation, D. tertiolecta cells were incubated in isotonic Hepes buffer under illumination. The intracellular glycerol content then increased to reach pre-electroporation values after about 30 minutes. The cell volume, measured on motile cells by video microscopy, reduced by 23% immediately after electroporation but returned to pre-electroporation values after about 30 minutes. Because the cells were maintained at constant external osmotic pressure throughout the procedure, it is concluded that the regulatory mechanism responsible for setting the intracellular glycerol content does not sense external osmotic pressure per se. These findings suggest that the intracellular glycerol content is set by a mechanism that senses cell volume or some parameter linked directly to cell volume.