Abstract
BackgroundThe ubiquitous occurrence of inducible Heat Shock Proteins (Hsps) up-regulation in response to cold-acclimation and/or to cold shock, including massive increase of Hsp70 mRNA levels, often led to hasty interpretations of its role in the repair of cold injury expressed as protein denaturation or misfolding. So far, direct functional analyses in Drosophila melanogaster and other insects brought either limited or no support for such interpretations. In this paper, we analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp70 gene.Principal FindingsWe found that larvae of both strains show similar patterns of Hsps complex gene expression in response to long-term cold-acclimation and during recovery from chronic cold exposures or acute cold shocks. No transcriptional compensation for missing Hsp70 gene was seen in Hsp70- strain. The cold-induced Hsps gene expression is most probably regulated by alternative splice variants C and D of the Heat Shock Factor. The cold tolerance in Hsp70- null mutants was clearly impaired only when the larvae were exposed to severe acute cold shock. No differences in mortality were found between two strains when the larvae were exposed to relatively mild doses of cold, either chronic exposures to 0°C or acute cold shocks at temperatures down to -4°C.ConclusionsThe up-regulated expression of a complex of inducible Hsps genes, and Hsp70 mRNA in particular, is tightly associated with cold-acclimation and cold exposure in D. melanogaster. Genetic elimination of Hsp70 up-regulation response has no effect on survival of chronic exposures to 0°C or mild acute cold shocks, while it negatively affects survival after severe acute cold shocks at temperaures below -8°C.
Highlights
Insects, as very small ectotherms with little possibility to maintain body heat, have evolved different and complex physiological mechanisms to cope with low body temperatures [1]
We focused on cold tolerance in fully grown 3rd instar larvae that were acclimated (LTA) at low temperatures (15°C followed by 6°C) in order to express their maximum cold tolerance [14]
The expression levels of Hsp70 mRNA transcripts and another 19 genes belonging to Heat Shock Proteins (Hsps) complex plus 4 other genes (Frost, Menin, Cold shock protein, and Starvin) potentially linked to cold acclimation/cold injury repair, were quantified by quantitative Real Time PCR (qRT-PCR) but we found no compensation response for missing Hsp70 gene in Hsp70- strain
Summary
As very small ectotherms with little possibility to maintain body heat, have evolved different and complex physiological mechanisms to cope with low body temperatures [1] These mechanisms help to prevent occurrence of various types of cold injury: (a) indirect chilling injury: accumulates over long, chronic exposures (days to months) to mild cold (hypothermic temperatures around or above zero) and is mechanistically linked most likely with disturbed coordination of various metabolic pathways, accumulation of toxic intermediates including reactive molecules (oxidative stress), depletion of free chemical energy, and consequent disturbance of ion homeostasis [2,3,4,5,6]; (b) direct chilling injury: results from brief, acute exposures (minutes to hours) to severe cold (cryothermic temperatures close to supercooling point but without freezing), which causes dissociation of multimeric macromolecular complexes, loss of enzymatic activity, protein denaturation [7,8,9], phase transitions in membrane lipids, massive ion leakage, and rapid cell death [10,11]; and (c) freezing injury: is linked with growing ice crystals causing mechanical damage to subcellular structures, freeze-concentration of solutes that may exceed toxic limits, and severe freeze-dehydration that may cause fusions of incompatible membraneous compartments [12]. We analyze the cold tolerance and the expression levels of 24 different mRNA transcripts of the Hsps complex and related genes in response to cold in two strains of D. melanogaster: the wild-type and the Hsp70- null mutant lacking all six copies of Hsp gene
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