Abstract
The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor. Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected. This indicated that superoxide anions were formed during freezing and thawing. This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite. Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme. Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap. Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain.
Highlights
The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase, Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, ␥-glutamylcysteine synthetase and Yap1 transcription factor
Freeze-Thaw Injury Is Most Extensive in Mutants with Defective Cytoplasmic Cu,Zn-SOD—We have previously shown that H2O2 pretreatment could induce freeze-thaw tolerance of yeast cells [7]
Mn-SOD in the freeze-thaw stress survival of yeast cells. They confirm in a more direct way that oxidative stress is a cause of lethal damage to cells, as has been suggested in other systems [5, 6], and further show that the main damage in aerobic systems occurs in the cytoplasmic compartment and results from generation of O2
Summary
(Received for publication, December 23, 1997, and in revised form, May 13, 1998). From the ‡School of Biochemistry & Molecular Genetics and Cooperative Research Center for Food Industry Innovation, University of New South Wales, Sydney, New South Wales 2052 and ¶Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, New South Wales 2050, Australia. The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain. Oxidative damage has been considered to be a factor since an oxidative burst has been predicted to occur during thawing [5], and this would lead to the generation of reactive oxygen species (ROS) and oxidative damage to cellular components This is supported by the observation that antioxidant defense systems of reptiles are activated by freezing stress [5] and that overexpression of superoxide dismutase enhances the freezing tolerance of transgenic Alfalfa [6].
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