Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer
Introduction
The mechanisms by which the so-called anhydrobiotic organisms preserve their cells under extreme desiccation have remained a challenge to biology. In most organisms, a severe drying of cells leads to irreversible damage to cellular structures, and eventually to death of the cell and the entire organism. However, anhydrobiotic organisms are apparently able to prevent such damage, or to successfully repair damage that arises. In previous research on the biochemistry of anhydrobiotic organisms, a strong focus has been on the role of polyhydroxy compounds, mainly carbohydrates, as membrane stabilizers in the dry state (see reviews in, e.g., Hoekstra et al., 1997, Crowe, 2002). More recently, a more diversified approach has emerged. In particular, the role of several stress proteins (heat-shock proteins and late embryogenesis proteins) in the protection against desiccation damage has been emphasized (e.g., Goyal et al., 2005, Clegg, 2005). Stress proteins act as molecular chaperones and bind to other proteins, thereby preventing aggregation or unfolding of the protein or promoting protein folding (Ellis and van der Vies, 1991, Gething and Sambrock, 1992, Kiang and Tsokos, 1998, Feder and Hofmann, 1999). Stress proteins may also protect cells from oxidative damage both in vivo and in vitro (Jacquier-Sarlin et al., 1994, Plumier et al., 1995). They may also interact with carbohydrates in protecting against cell damage (Elliott et al., 1996, Singer and Lindquist, 1998, Viner and Clegg, 2001, Ma et al., 2005). Late embryogenesis abundant (LEA) proteins are a complex of proteins originally identified in plant embryos in connection with water stress. Recently, Browne et al. (2002) described the induction of a group 3 LEA protein in the anhydrobiotic nematode Aphelenchus avenae in connection with desiccation. In vitro analyses indicate that this protein may serve a chaperone function, by preventing desiccation-induced aggregation of other proteins, and thus play an important role in the tolerance to desiccation in these animals.
The Hsp70 family is the largest and most conserved group of heat-shock proteins, participating in the folding of newly synthesized and denatured proteins, and inhibiting protein aggregation (e.g., Kiang and Tsokos, 1998, Fink, 1999). There is also evidence that it plays a role in gene regulation by interacting with transcription factors (Wickner et al., 1991). Hsp70 has been shown to interact with both membrane proteins (Fink, 1999), and membrane lipids (Arispe et al., 2002). In extreme desiccation-tolerant (anhydrobiotic) organisms, Hsp70 has not been investigated very much, but its strong connection to general stress and documented association to cell membranes suggests that it may well play a role in the desiccation tolerance machinery.
Tardigrades represent one of the three main invertebrate taxa where anhydrobiotic populations are wide-spread, the other two groups being nematodes and rotifers (Wright et al., 1992). In these taxa, the anhydrobiotic state may be induced over the whole life cycle, from the egg to the adult stage (holo-anhydrobiosis, Jönsson, 2005). The biochemical mechanisms and components behind anhydrobiotic survival in tardigrades are largely unknown, but the eutardigrade Richtersius coronifer was shown to accumulate the disaccharide trehalose at about 2.3% dry weight at the entrance of the anhydrobiotic state (Westh and Ramløv, 1991). Only two previous studies have investigated the induction of stress proteins in tardigrades. Ramløv and Westh (2001) described the appearance of a protein with a molecular mass of 71 kDa from anhydrobiotic specimens of the tardigrade R. coronifer and the absence of the protein from active tardigrades. In a more extensive study, Schill et al. (2004) documented three heat-shock protein (Hsp70 family) genes in the tardigrade species Milnesium tardigradum and their different expressions which are involved in the cycle of dehydration, cryptobiosis and rehydration.
Here we report a study on the induction of Hsp70 in response to desiccation, heat and ionising radiation, in the limno-terrestrial eutardigrade R. coronifer, one of the most well-studied tardigrades with respect to cryptobiosis (e.g., Westh and Ramløv, 1991, Ramløv and Westh, 2001, Jönsson and Rebecchi, 2002, Jönsson et al., 2001). We directly quantified the levels of induced Hsp70 using the immuno-westernblot method.
Section snippets
Specimen collection
Tardigrade specimens of R. coronifer were extracted from mosses (Orthotrichum cupulatum) collected dry from limestone rock on the Swedish island Öland, situated in the Southern Baltic Sea. The moss was hydrated for 24 h and the animals were then extracted with sieves (mesh size 250 μ and 40 μ). Only medium and large specimens (ca. 0.5–1.0 mm) with normal movements were used in the experiment. Sex cannot be determined in living tardigrades of this species (unless developing eggs are observed),
Results
All treatment groups deviated significantly (at the p < 0.05 level) from the control group (Fig. 1, Table 1, Table 2). Elevated values were recorded in all treatment groups except one, tardigrades in the desiccated state, that had a mean value about 60% of that in controls (Fig. 1). As expected, heat stress induced a strong Hsp70 response, with induced levels of more than twice that of the untreated active tardigrades. However, Hsp70 levels did not significantly deviate from that of the two
Discussion
Our analysis shows a clear induction of Hsp70 1 h after rehydration of desiccated tardigrades, while Hsp70 levels in the desiccated tardigrades are lower than in the active control animals. This result is similar to that of Schill et al. (2004) for their isoforms 1 and 3 of hsp70 mRNA expression in the eutardigrade M. tardigradum, where desiccated tardigrades also had significantly lower levels of mRNA expression compared to animals in the pre-desiccation active state. Thus, in these cases it
Acknowledgements
We are very grateful to J. Torudd for help with the irradiated samples, and to H.-R. Köhler for providing the facility for the densitometric image analysis. R.O. Schill was supported by a scholarship from the German Research Council (DFG, SCHI 865/1-1).
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The authors contributed equally to the publication.