Constitutive expression of a stress-inducible heat shock protein gene, hsp70, in phylogenetically distant Antarctic fish

Previous research on Antarctic notothenioid fishes demonstrated the loss of the heat-shock response characterized by the rapid synthesis of molecular chaperones in response to increasing pools of damaged proteins. We determined that this loss was the result of constitutive expression of the inducible hsp70 gene. In this study, we examined the extent of this unique expression pattern in Antarctic fish by comparing the expression of two genes, the constitutive hsc71 gene and the inducible hsp70 gene, in tissues from Trematomus bernacchii to expression in tissues of Pagothenia borchgrevinki, a second Antarctic notothenioid, and Lycodichthys dearborni, a phylogenetically distant Antarctic species. Our study indicated that the expression of hsc71 is similar in all species; however, the constitutive expression of the inducible hsp70 gene was also manifested in these species. These data further suggest that cold denaturation of proteins at ecologically relevant temperatures may be contributing to this change in expression of the hsp70 gene.     

Inducible heat tolerance in Antarctic notothenioid fishes

Significant increases in heat tolerance (time of survival at 14°C) were observed for some, but not all, species of notothenioid fishes collected from McMurdo Sound, Antarctica (77°51′S) following acclimation to 4°C. The increase in thermal tolerance was rapid in Trematomus bernacchii, developing within 1–2 days of acclimation to 4°C. Long-term (6–8 weeks) acclimation to 4°C led to greater heat tolerance in Trematomus pennellii than in T. bernacchii. Unlike its demersal congeners, the cryopelagic notothenioid Pagothenia borchgrevinki did not increase heat tolerance during warm acclimation. A deep-living zoarcid fish, Lycodichthys dearborni, also failed to increase heat tolerance, but survived significantly (> threefold) longer at 14°C than the notothenioids.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

Differential expression of heat shock protein 70 in relation to stress type in the flatworm <Emphasis Type="Italic">Schmidtea polychroa</Emphasis>

Most heat shock proteins help to cope with stress in organisms ranging from bacteria to vertebrates. Many stress types acting on the intensity of intracellular protein can induce expression of heat shock proteins. Here, we studied changes in expression level of heat shock protein 70 (Hsp70), one of the best investigated stress proteins, in response to five potential stress factors in the planarian flatworm Schmidtea polychroa: (1) homogenized planarian tissue, which releases an alarm substance that signals predation injury, (2) physical damage by puncturing, (3) a simulation of ecological competition by adding a mixture of naturally co-occurring species: one Dendrocoelum and two Polycelis flatworms, one Asellus water louse and one leech, and (4) magnesium chloride, which inhibits regeneration ability. We found that alarm substance (1), physical harm (2), and magnesium chloride (4) led to increased expression of Hsp70, while interspecific competition (3) did not result in elevated Hsp70 expression. There was no difference between the experimental negative control and two temporal controls immediately after collection and just before the experiment. Results show that Schmidtea polychroa is not sensitive to sampling and lab maintenance. However, planarian homogenate, magnesium chloride and physical harm all caused Hsp70-inducing stress. We conclude that Hsp70 quantification is appropriate to study the current stress level in planaria in response to specific conditions.     

Gene expression profiles during short‐term heat stress in the red sea coral Stylophora pistillata

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide‐spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic ‘r’ strategist. It can thrive in relatively unstable environments and is considered a stress‐tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short‐term (13‐day) heat stress. The results allowed us to identify a two‐step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential ‘early warning genes’ and ‘severe heat‐related genes’. Our findings suggest that during short‐term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER‐unfolded protein response (UPR) and ER‐associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.     

Real‐time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real‐Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL‐TcA‐CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV‐A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up‐regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real‐time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.     

Heat stress-induced H2O2 is required for effective expression of heat shock genes in Arabidopsis

The mechanisms of sensing and signalling of heat and oxidative stresses are not well understood. The central question of this paper is whether in plant cells oxidative stress, in particular H₂O₂, is required for heat stress- and heat shock factor (HSF)-dependent expression of genes. Heat stress increases intracellular accumulation of H₂O₂ in Arabidopsis cell culture. The accumulation was greatly diminished using ascorbate as a scavenger or respectively diphenyleneiodonium chloride (DPI) as an inhibitor of reactive oxygen species production. The mRNA of heat shock protein (HSP) genes, exemplified by Hsp17.6, Hsp18.2, and the two cytosolic ascorbate peroxidase genes Apx1, Apx2, reached similar levels by moderate heat stress (37°C) or by treatment with H₂O₂, butylperoxide and diamide at room temperature. The heat-induced expression levels were significantly reduced in the presence of ascorbate or DPI indicating that H₂O₂ is an essential component in the heat stress signalling pathway. Rapid (15 min) formation of heat shock promoter element (HSE) protein-binding complex of high molecular weight in extracts of heat-stressed or H₂O₂-treated cells and the inability to form this complex after ascorbate treatment suggests that oxidative stress affects gene expression via HSF activation and conversely, that H₂O₂ is involved in HSF activation during the early phase of heat stress. The heat stress induction of a high mobility HSE-binding complex, characteristic for later phase of heat shock response, was blocked by ascorbate and DPI. H₂O₂ was unable to induce this complex suggesting that H₂O₂ is involved only in the early stages of HSF activation. Significant induction of the genes tested after diamid treatment and moderate expression of the sHSP genes in the presence of 50 mM ascorbate at 37°C occurred without activation of HSF, indicating that other mechanisms may be involved in stress signalling. Electronic Supplementary Material Supplementary material is available for this article at http//dx.doi.org/10.1007/s11103-006-0045-4 Roman A. Volkov and Irina I. Panchuk contributed equally