Year

1987

Degree Name

Doctor of Philosophy

Department

Department of Biology

Abstract

The growth characteristics of the parent strain and salt-sensitive HL 25/8 mutant of Dunaliella tertiolecta have shown that the mutant is sensitive to salt concentrations in a range (0.17 to 1.0 M NaCl) in which the parent strain is able to grow. The mutant grew in 0.17 M NaCl at about half the rate of the parent strain. The optimum growth temperature of the parent strain increased with salinity but did not do so in the mutant, and gassing of the culture with 5 % mixture of C02 in air extended the duration of exponential growth rate of both strains.

The mutant had lower carbonic anhydrase activity than the parent strain. It is suggested that this reduced carbonic anhydrase activity affects carbon transport and assimilation in a way that largely, if not wholly, diminishes the salt tolerance of the mutant. In both light and dark conditions, salt stress caused alkalization of the intracellular pH . The mutant had about half the photosynthetic activity (14C-fixation and photosynthetic oxygen evolution) of the parent strain, which increased with salinity in the parent strain but gradually decreased in the mutant. The results also suggest that the mutant had a higher photorespiratory activity than the parent strain.

The glycerol pool in Dunaliella has a turnover rate that is low in comparison to the Benson-Calvin cycle, therefore an osmoregulatory response to salt stress would require an accelerated glycerol synthesis. It is suggested that the synthesis and dissimilation of glycerol does not in any way constitute a metabolic cycle comparable to the established cycles such as the Krebs and Benson-Calvin cycles.

Dunaliella tertiolecta has a limited capacity of salt-induced photosynthetic oxygen evolution. Photosynthetic C02-fixation is stimulated by mild to moderate salt stress (0.17 to 0.7 M NaCl) but inhibited at severe stresses. The results show that increasing magnitude of salt stress leads to a preferential incorporation of photosynthetically fixed carbon into glycerol. During a salt stress, both photosynthesis and starch breakdown contribute carbon to the glycerol synthesis. The relative contribution of these two processes depends on the magnitude of salt stress. During a dilution stress glycerol does not leak into the medium, but it is dissmilated biochemically. During a salt stress the glycerol synthetic pathway is stimulated and the glycerol dissimilation pathway is suppressed, conversely, during a dilution stress glycerol dissimilation pathway is stimulated and glycerol synthetic pathway is suppressed.

The regulation of salt-induced conversion of starch to glycerol was investigated by examining the properties of some key enzymes of phosphorolysis, amylolysis, the oxidative pentose phosphate pathway, and glycerol synthesis as well as by following the changes in the concentrations of some metabolites. Phosphate concentrations which normally occur in vivo in the dark or after a salt stress, stimulate phosphorylase activity. Phospofructokinase is sensitive to DTT , whereas amylase activity requires thiol reagents for its maximal activity. Enzymes of both hydrolytic and phosphorolytic starch degradation are resistant to NaCl, whereas phosphofructokinase or enzymes involved in the oxidative pentose phosphate pathway or the enzymes of glycerol synthetic pathway are not. Both in the light and dark, concentrations of Pi, PGA , and glycerol-3-P increased upon a salt stress. It is suggested that the oxidative pentose phosphate pathway is switched on during a salt stress in the light.

Based on the enzymic activities of Dunaliella, other results of this thesis and the results of previous studies by other authors, possible pathways for the synthesis and dissimilation of glycerol during salt and dilution stress are proposed.

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