Thesis (Ph.D.)--Chulalongkorn University, 2003

Effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC), were investigated in a unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in the growth rate when the concentration of NaCl was raised up to 550 mM. Instead, the growth rate of the cells decreased at 300 mM sorbitol, and inhibition of growth occurred at or higher than 700 mM sorbitol. Salt and sorbitol stresses affected the soluble and insoluble polyamine titers in the Synechocystis cells at 10 days cultivation. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. The cellular polyamine contents rapidly increased at early stage of cultivation of up to 4 days and decreased afterwards during growth for 20 days. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a 6-fold increase of enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to that observed for ADC. An analysis by reverse transcription-PCR revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was partly attributed to its slower turnover rate under both stress conditions. The attempt to study the effect of salt and osmotic stresses on SAMDC mRNA level was unsuccessful since no putative gene for SAMDC could be identified in the genome of Synechocystis sp. PCC 6803 whencomparing to known SAMDC gene sequences from other organisms. Effects of ionic and osmotic stresses on photosynthetic systems were investigated using 10 days Synechocystis cells culture. High salt stress did not affect the photosystem II (PSII) activity whereas high sorbitol stress (700 mM sorbitol) completely inhibited both PSII activity and whole photosynthesis. Importantly, darkness was found to inactivate the photosynthetic oxygen evolution and this dark-inactivation was enhanced by a concomitant sorbitol stress. The abundance of psbA mRNA slightly decreased under salt stress and more under sorbitol stress, especially under 700 mM sorbitol condition. The abundance of psaA transcripts was also decreased under salt stress as well as under sorbitol stress. The stability of psbA mRNA was unaffected by salt stress whereas psaA mRNA stability was decreased. On the other hand, sorbitol stress decreased the stability of both psbA and psaA mRNAs. Salt stress had no effect on the amounts of D1, D2 and PSI proteinswhereas the ndhF3 protein amount was slightly decreased. The decrease of D1, D2 and ndhF3 proteins were induced by sorbitol stress while no change of PSI protein level was observed under the same condition. In conclusion, three groups of proteins could be distinguished according to their steady-state amounts under the stress conditions studied: 1) PSII proteins are down regulated only under osmotic stress 2) PSI protein is not affected under any stress studied 3) ndhF3 protein is down regulated under both ionic and osmotic stresses.