Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages
1 Animal Physiological Ecology, Zoological Institute, University of
Tübingen, Konrad-Adenauer-Str. 20, D-72072 Tübingen,
Germany
2 Molecular Biology, Institute for Cell Biology, Auf der Morgenstelle 28,
D-72076 Tübingen, Germany
* Author for correspondence (e-mail: ralph.schill{at}uni-tuebingen.de)
Accepted 12 February 2004
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Summary |
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Key words: anhydrobiosis, cryptobiosis, Eutardigrada, heat-shock protein, stress protein, hsp70
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Introduction |
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Cryptobiosis in tardigrades and other invertebrates is characterized by
several major events that still remain largely unidentified. On the one hand,
research has focused on cryptobiotic cells that accumulate large amounts of
either one or both of the disaccharides trehalose or sucrose
(Clegg and Jackson, 1992;
Crowe et al., 1998
;
Viner and Clegg, 2001
;
Crowe, 2002
;
Oliver et al., 2002
;
Watanabe et al., 2003
). In
this context, the `water-replacement hypothesis' has been developed to explain
how cellular components may be protected during extreme drying. Essentially,
the hypothesis says that polyhydroxyl compounds, such as trehalose, replace
the shell of water around macromolecules, circumventing any damaging effects
during drying. However, the tardigrade Adorybiotus coronifer showed
rather low trehalose accumulation (1.6% of the dry mass) compared with several
anhydrobiotic species from other taxa, such as the nematode Aphelenchus
avenae, with a trehalose level of 12-13%, or cysts of the brine shrimp
Artemia franciscana, which contain 15-18%
(Liang et al., 1997
).
Nevertheless, Westh and Ramløv
(1991
) estimated the ability
of A. coronifer to accumulate the mentioned concentration of
trehalose approximately 10 times faster than A. avenae. Furthermore,
several stress proteins (Clegg et al.,
1999
; Liang and MacRae,
1999
; Clegg et al.,
2000
; Ramløv and Westh,
2001
; Viner and Clegg,
2001
; Willsie and Clegg,
2001
) and `late-embryogenesis-abundant' (LEA) proteins that have
been found in the nematode A. avenae
(Browne et al., 2002
) and in
plant seeds exhibiting desiccation tolerance during maturation
(Vertucci and Farrant, 1995
;
Ingram and Bartels, 1996
;
Chandler and Bartels, 1999
)
seem to be further keys in understanding the cryptobiotic mechanisms.
Drying of cells generally leads to massive damage to cellular membranes and
proteins, which eventually results in cell death and, consequently, the death
of the entire organism. Upon drying, intracellular proteins and membranes may
compensate the loss of hydrogen bonds to water by hydrogen bonds to other
molecules and can further compensate by protein-protein interactions
(Carpenter and Crowe, 1989;
Prestrelski et al.,
1993a
,b
;
Dong et al., 1995
). These
protein-protein interactions, however, can lead to irreversible conformational
changes and, in enzymes, to a loss of enzymatic activity
(Carpenter et al., 1987
).
Heat-shock proteins and their molecular partners are known to play diverse
roles, even in unstressed cells, in successful folding, assembly,
intracellular localization, secretion, regulation and degradation of other
proteins (Gething and Sambrook,
1992; Gething,
1997
). Alamillo et al.
(1995
) studied the resurrection
plant Craterostigma plantagineum, a desert species that expresses
heat-shock proteins in vegetative tissues during water stress; this expression
is thought to contribute to desiccation tolerance. Similarly, rice seedlings
express two proteins of the Hsp90 family upon exposure to water stress and
elevated salinity (Pareek et al.,
1997
). Several case studies in encysted brine shrimp (A.
franciscana) embryos showed that they undergo development arrest, in
which they may survive for years without environmental water or oxygen. These
cryptobiotic embryos accumulate enormous concentrations of a small heat-shock
protein p26, belonging to the
-crystallin family
(de Jong et al., 1998
) and
being restricted to this stage of the life history
(Jackson and Clegg, 1996
;
Liang and MacRae, 1999
). Clegg
et al. (1994
) also showed that
p26 underwent extensive stress-induced translocation to nuclei and other
sites. Information on the role of a major stress protein in cryptobiosis,
Hsp70, however, is still scarce.
In the present study, a complementary focus is on tardigrades undergoing stress in nature and on the roles of stress genes of the hsp70 family in the stress physiology of whole organisms in different life history stages. Using real-time RT-PCR, the levels of expression of hsp70 isoforms in the active and cryptobiotic animals and in intermediate stages have been studied in the tardigrade species M. tardigradum.
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Materials and methods |
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Thermogravimetric analysis of the dried tardigrades to measure the residual
moisture content was not performed, although other authors observed a maximum
residual moisture content in the range of 6-10% in different anhydrobiotic
organisms when the above-mentioned drying method was used
(Potts, 1994;
Lapinski and Tunnacliffe,
2003
).
RNA/DNA extraction
RNA was extracted from individual tardigrades (N=5) in the
different stages (I-VI) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA).
Specimens were incubated in 200 µl TRIzol reagent for 5 min at room
temperature to achieve complete dissociation of nucleoprotein complexes. 40
µl of chloroform was added, the tubes were shaken vigorously by hand for 15
s and incubated for a further 5 min at room temperature. After centrifugation
(15 min, 12 000 g, 4°C), the aqueous phase containing RNA
was separated from the other phases, which were stored for DNA preparation
(see below). The colorless upper aqueous phase was transferred into fresh
vials to precipitate the RNA by addition of 100 µl isopropyl alcohol. The
samples were incubated for 10 min and centrifuged (20 min, 12 000
g, 4°C). The RNA precipitates were then washed twice with
75% ethanol (in DEPC-treated water), air-dried and resolved in DEPC-treated
water for the DNA digestion with RNase-free DNase I (Promega, Madison, WI,
USA).
DNA in the interphase and phenol phase of the initial homogenate was pooled and isolated by precipitation with ethanol and centrifugation (10 min, 2000 g, 4°C). The pellets were washed twice with 0.1 mol l-1 sodium citrate in 10% ethanol and centrifuged (5 min, 2000 g, 4°C). Following these two washes, the pellet was suspended in 75% ethanol, centrifuged (5 min, 2000 g, 4°C), air-dried and resolved in DEPC-treated water for RNA digestion with RNase H (Invitrogen).
Species-specific PCR primers
To obtain specific hsp70 gene family members, PCR was carried out
using degenerated oligonucleotide primers
(Table 1a), specified in
Köhler et al. (1998), for
a highly conserved region of the hsp70 gene. As an internal standard,
beta-actin was chosen as the housekeeping gene. Conserved
beta-actin primers (Table
1b) were designed by hand, based on the National Center for
Biotechnology Information (NCBI) GenBank (beta-actin accession no.
BC014861). After using DNA of M. tardigradum as a template, the PCR
products were cloned with the TOPO TA Cloning® Kit for Sequencing
(Invitrogen), and the inserts checked by digestion with EcoRI. Clones
of interest were sequenced twice and identified by BLAST® (Basic Local
Alignment Search Tool) in the NCBI GenBank. Partial sequences of three
hsp70 family genes and a beta-actin family gene were
described for the first time in tardigrades. Species-specific oligonucleotide
primers were designed with the Primer 3 software
(Rozen and Skaletsky, 2000
)
based on these partial gene sequences, which have been submitted to the NCBI
GenBank (hsp70 isoform 1, accession no. AJ579531; hsp70
isoform 2, accession no. AJ579532; hsp70 isoform 3, accession no.
AJ579533) and for beta-actin (accession no. AJ579530). The used
primers, purchased from MWG Biotech AG (Ebersberg, Germany), are summarized in
Table 1c-f.
|
Real-time RT-PCR
Reverse transcription (RT) of first-strand cDNA was performed with the
total RNA of each specimen. The RNA was incubated with 10 mmol l-1
dNTP mix and 50 ng oligo(dT)12-18 primer at 65°C for 5 min.
After cooling on ice, the reaction mixture [0.1 mol l-1
dithiothreitol, 5x 1st strand buffer and 40 units of RNaseOUTTM
(Invitrogen)] was added, mixed gently and incubated at 42°C for 2 min. 50
units of SuperScriptTM II (Invitrogen) were added and incubated
at 42°C for 50 min and inactivated by heating to 70°C for 15 min. The
cDNA was precipitated in 75% ethanol and washed twice with 75% ethanol,
air-dried and resolved in DEPC-treated water.
The real-time PCR reaction mixture contained the following items in a final volume of 20 µl: 50 ng cDNA, 1 unit Taq DNA Polymerase D1806 (Sigma-Aldrich, Inc., St Louis, MO, USA) with 10x reaction buffer supplemented to a final concentration of 3.9 mmol l-1 MgCl2, 0.2 mmol l-1 dNTP, 2 µmol l-1 of each oligonucleotide primer and 2 µl of SYBR Green® I 1:1000 (Molecular Probes, Inc., Eugene, OR, USA). The PCR amplification profiles were as follows:
hsp70 isoform 1: initial denaturation for 8 min at 95°C, followed by 45 cycles of 30 s at 94°C, 30 s at 63°C and 60 s at 72°C;
hsp70 isoform 2: 30 s at 94°C, 30 s at 64°C, 30 s at 72°C;
hsp70 isoform 3: 30 s at 94°C, 30 s at 50°C, 60 s at 72°C, and final extension of 8 min at 72°C;
beta-actin: 30 s at 94°C, 30 s at 64°C, 60 s at 72°C, and final extension of 8 min at 72°C.
Negative control reactions containing water in place of cDNA were included in each batch of PCR reactions to ensure that contamination was not a problem. For the positive control and standard curve, a standard of the particular sequences was amplified in three different dilutions (102, 103 and 104 sequence copies).
Product analysis
In the iCycler iQTM Real-Time PCR Detection System (BioRad
Laboratories, Hercules, CA, USA), analysis of the real-time fluorescence
signal of SYBR Green® I (Molecular Probes, Inc.) bound to double-stranded
DNA was performed using the iCycler iQTM Real-Time PCR Detection System
software (BioRad Laboratories). A threshold position was user-defined for the
samples, using the exponential growth phase and baseline cycles of the
fluorescent amplification plots. The quantity of RNA was expressed in relation
to the internal reference of beta-actin and compensated for variation
in the quantity and quality of the cDNA samples. Standard curves were
generated by plotting the log of the cDNA copy number against respective
threshold cycles (CT) and covering the orders of magnitude
in variation of cDNA template concentrations. Amplicon size and reaction
specificity were confirmed by agarose gel electrophoresis on a 2% gel in
Tris-borate-EDTA buffer and stained with CYBR Gold® (Molecular Probes,
Inc.).
Statistics
The statistical significance of differences in the hsp70
transcript levels between the samples was tested using Mann-Whitney-Wilcoxon's
U-test. Significance levels were P>0.05 (not
significant), 0.01<P<0.05 (weakly significant, *),
0.001<P<0.01 (significant, **), and
P<0.001 (highly significant, ***).
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Results |
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The expression of hsp70 isoform 1 (Fig. 2A) decreased in a highly significant manner from stage I to stage II, stage III and stage IV. The copy number of this sequence was very low at the transitional stage (II), cryptobiotic stage (III) and consecutive transitional stage (IV) compared with at the active stage. Between the cryptobiotic stage (III) and the transitional stage (IV) there was no significant difference. By contrast, the copy number of isoform 1 in the active stage (V) 90 min after the transitional stage (IV) was increased more than twofold compared with stage I, with a significant difference to stage IV and a weakly significant difference to the active stage (I). In contrast to hsp70 isoforms 2 and 3, the expression of isoform 1 in M. tardigradum, compared with the beta-actin housekeeping gene copy numbers, was very high (Table 2).
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The hsp70 expression of isoform 2 (Fig. 2B) showed the lowest level in the active stage (I) and increased to the second highest observed level in the transitional stage (II). During the cryptobiotic stage (III), the level was slightly reduced again but increased continuously in the course of stages IV and V. The maximum level was achieved in active stage V, with a highly significant elevated expression that was about eight times as high as in active stage I.
The lowest mRNA expression of hsp70 isoform 3 (Fig. 2C) was detected in M. tardigradum during the cryptobiotic stage (III), followed by a significant increase of expression during stage IV. By contrast, mRNA expression was relatively high during the active stage and reached about the same level in stage V. During the transitional stages (II and IV), mRNA expression showed no significant changes compared with the active stages (I and V). Nevertheless, transcription levels showed a clear decrease from the active stage via dehydration to the cryptobiotic stage and an increasing trend from the cryptobiotic stage via rehydration to the active stage again.
Expression of all hsp70 family member genes was significantly (isoform 3) or even highly significantly (isoform 1 and 2) elevated during a heat shock at 37°C for 90 min (Fig. 3). Isoforms 1 and 3 showed heat-inducible expression levels that were 6-8-fold higher than the levels of the non-stressed active stages, respectively. The highest relative elevation in the studied hsp70 isoforms was found in isoform 2, which showed a >20-fold higher level after heat shock.
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In summary, three different patterns of gene expression in the studied hsp70 mRNA isoforms were observed.
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Discussion |
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To the best of our knowledge, this is the first report on different heat-shock (hsp70) gene transcripts stored in cryptobiotic stages in tardigrades. It is questionable whether the transcripts represent remnant mRNAs produced during transition from the active stage to the cryptobiotic stage, which will be destroyed without use or otherwise will be translated during the following rehydration, once the translation activity is restored.
It is apparent that all three isoforms seem to be true hsps since they could be clearly induced by temperature elevation. In addition, all three isoforms show a constitutive basic level, even without stress, and therefore fulfil this requirement for heat-shock cognate (hsc) genes.
Santomenna and Colberg-Poley
(1990) showed the effect of
heat-shock treatment upon human cytomegalovirus (HMCV) induction of
hsp70 RNA and Hsp70 protein expression. They found that there was a
several-hour delay between the time of hsp70 RNA induction and the
time of increased inducible Hsp70 protein expression. A correlation between
hsp70 RNA and Hsp70 protein in cerebral tissue of birds after heat
stress has also been shown by Dionello et al.
(2001
). We conclude that the
hsp70 RNA of tardigrades will be translated into Hsp70 proteins to a
comparable extent. Thus, the difference between hsp and hsc obviously has not
been established in tardigrades, a rather basic group of `prearthropods'
(Garey et al., 1996
). A
similar situation was found in Diplopoda
(Knigge, 2003
), another
phylogenetically `old' group.
All three isoforms are endogenously regulated, following the steps active-cryptobiotic-active. Based on absolute copy numbers and on the expression pattern, isoform 1 seems to be the dominant hsp70 isoform in active tardigrades. Expression of isoform 1 seems to cease prior to the transitional stage, and a presumably short half-life of this isoform leads to a rapid decrease in isoform 1 level. Eventually, transcription starts again at the end of stage II, right before formation of the cryptobiotic stage, but it is questionable whether the observed significant difference between stages II and III is biologically relevant. Transcription of isoform 1 starts again when tardigrades have reached the active state again. However, we regard isoform 1 as being the most important isoform in `normal' (i.e. non-cryptobiotic) metabolism (i.e. a `heat-inducible hsc'). The pattern of isoform 3 is similar to that of isoform 1 but at a much lower level. In contrast to the expression pattern of isoform 1, the pattern of isoform 3 is strongly induced by high temperature. Thus, isoform 3 can be regarded as a true hsp, with a rather low constitutive level and a more than eightfold increase by heat shock. As with isoform 1, isoform 3 does not seem to have a specific function for cryptobiosis, even though cessation of transcripts seems to take place later than in isoform 1: the transitional stages showed slightly and non-significantly lower levels than the active stages. Concomitantly, transcription of isoform 3 definitely starts upon `awakening' from the cryptobiotic stage in the transitional stage (IV).
We regard isoform 2 as the classical hsp since it showed a very large
constitutively transcribed copy number and a 20-fold inducibility by heat
shock. Furthermore, this isoform is inducible by the stress posed to the
individual when undergoing cryptobiosis. Transcription of isoform 2 is
significantly induced in transitional stage II, resulting in a comparatively
high mRNA copy number. The copy number remains constant throughout the
cryptobiotic stage and the transitional stage of `awakening' tardigrades, thus
implying that isoform 2 is the most relevant hsp70 gene for
cryptobiosis. Based on the induction cascade of true hsps by malformed and
nascent polypeptide strains, transcription of isoform 2 is elevated further
when the tardigrades turn from cryptobiosis into a new active stage, and,
consequently, the formation of overall new proteins starts. In the present
study, stage V represents the first 90 min of the new active stage only;
therefore, it is supposed that expression of isoform 2 will decrease after a
while and remain constantly low, as shown for active stage I. Presumably,
isoform 1 will take over some time after the return to the active stage and
will cover the tasks of isoform 2, which remains at a very low level further
on. Ramløv and Westh
(2001) described the
appearance of protein bands with a molecular mass of 71 kDa from cryptobiotic
tardigrades [Adorybiotus (Richtersius) coronifer]
and the absence of bands from active animals. However, they are not certain
that the observed de novo protein synthesis was a heat-shock protein
belonging to the Hsp70 family, but one can speculate the highly inducible
protein form, deriving from hsp70 isoform 2.
Like most nematodes, tardigrades regularly show cell constancy
(Greven, 1980), and
increasingly the cellular structure has to be secured by protecting
mechanisms. We have shown that different expression of hsp70 genes is
involved in the cycle of dehydration, cryptobiosis and rehydration, but the
role of other stress genes in this process still remains to be clarified.
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Acknowledgments |
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