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Stress Response Of Tomato Cell Cultures To Toxic Metals and Heat Shock: Differences and Similarities
J Plant Physiol. Vol. 146. pp. 736-742 (1995)
Stress Response of Tomato Cell Cultures to Toxic Metals and Heat Shock: Differences and Similarities R.
WOLLGIEHN*
and D.
NEUMANN
Institut fur Pflanzenbiochemie, Weinberg 3, D-06120 Halle/Saale, Germany Received February 1, 1995 . Accepted April 28, 1995
Summary
Cultured tomato cells were exposed to different metal ions and heat shock and the induction and intracellular localization of the low molecular weight heat shock proteins were examined. Treatment with cadmium, mercury, zinc and arsenite resulted in synthesis of heat shock proteins without essentially affecting synthesis of house keeping proteins. Under heat shock a higher level of HSPs was synthesized than under metal stress, and «normal» protein synthesis was drastically reduced. Under all stress conditions investigated, HSPs accumulated in the cytoplasm and in nuclei. The patterns of the low molecular weight HSPs observed after 2-dimensional electrophoresis were different in the cytoplasm and in nuclei, and in nuclei the pattern of HSPs 17 under heat shock was different from that observed after metal stress. The induction of hsp17 and hsp70 gene transcription could be detected in the nuclear run-on assay already after one min heat shock or metal ion stress of the cells. Under all stress conditions the maximum rates were reached within 2-5min. The transcription of the rRNA genes was drastically reduced only under heat stress, but not influenced under metal stress.
Key words: Lycopersicon peruvianum, cell cultures, electrophoresis, heat stress, heat stress proteins, immunob/ots, metal ion stress, run-on-transcription. Abbreviations: HS = heat shock; HMW/LMW HSPs = high/low molecular weight heat shock proteins; PMSF = phenylmethylsulfonyl fluoride; SDS = sodium dodecyl sulfate_ Introduction
The exposition of plants to toxic metal ions as a consequence of industrial environment causes reduced plant growth, coincidental with disturbance of several metabolic processes, impaired uptake of nutrients and changes in cell ultrastructure_ Long-term growth of plants on metal contaminated soils may generate the development of metal tolerance. At present, little is known about the molecular and cellular basis of the response to metal stress. Several mechanisms contribute to the metal tolerance, as metal deposition in different cell compartments or secretion by specific glands, exudation of metal-chelating substances and alteration in membrane structure and function (Ernst et al., 1992; Neumann et al., 1994). One of the most prominent meta-
*
Corresponding author.
© 1995 by Gustav Fischer Verlag, Stuttgart
bolic alterations during metal stress is the elevated expression of heat shock proteins (HSPs) in a similar way as observed under heat stress. The HSPs have a key role in the protection of proteins and cell membranes against irreversible damage caused by the stress (Nover, 1994). The plant heat stress response is characterized by the synthesis of a few high molecular weight (HMW) and a complex group of low molecular weight heat shock proteins (LMW HSPs) with molecular sizes ranging from 15 to 28 KD (Nover et aI., 1990; Vierling, 1991). LMW HSPs were found to be localized mainly in the cytoplasm as larger aggregates, referred to as «heat shock granules» (Nover et aI., 1983; Neumann et al., 1984; Mansfield and Key, 1988), but also associated with the endomembrane system of the cell (for references s. Vierling, 1991; Nover, 1991). A broad spectrum of LMW HSPs was found in isolated nuclei of soybean seedlings (Lin et aI., 1984), maize roots (Cooper and Ho, 1987)
Heat shock proteins under metal ion stress and tomato fruits (Kato et al., 1993). Additionally, we have previously reported on the localization of HSP 17 in nuclei of tomato cell cultures under heat shock not only by biochemical means, but also by immunfluorescence and immune electron microscopy (Wollgiehn et al., 1994). In comparison to our extensive knowledge on heat stress only a few reports about stress proteins induced under metal stress of plants have been published. Czarnecka et al. (1984) and Edelman et al. (1988) investigated Cd and As effects on soybean seedlings. Using Northern blot hybridization and protein analysis Edelman et al. (1988) detected induction and accumulation of stress protein mRNA and high and low mol. weight HSPs. The response to heat or metal stress was qualitatively similar, but they observed distinct quantitative and temporal differences. The regulatory mechanisms controlling transcription and translation reacted differently to the various stress treatments. Nover et al. (1990) also found the induction of hsp17 and hsp70 mRNA by Cd, Hg and As in tomato cell cultures. Metal ion induced synthesis of low and high mol. weight HSPs was also described for sorghum seedlings (Howarth, 1990), Petunia leaves (Winter et al., 1988), cell cultures of Datura (Delhaize et al., 1989), tomato (Neumann et aI., 1994) and alfalfa (Gyorgyey et aI., 1991). The results, obtained so far, show that the molecular stress responses of the plants to heat and metal ion stress are similar, but exhibit also distinct differences depending on the stress-conditions. Little is known about the intracellular localization of HSPs under metal stress. In soybean seedlings accumulation of HSPs in nuclei and other cell organelles was observed only under heat shock, but not under As-stress (Lin et aI., 1984). Also in As-treated Drosophila Kc cells HSP70 was found to remain largely cytoplasmic, whereas under heat shock it was also enriched in nuclei (Vincent and Tanguay, 1982). Here, we compare the effects of metal ions and heat shock on synthesis and intracellular localization of LMW HSPs, particularly their accumulation in nuclei, and the regulation of hsp-mRNA and rRNA synthesis at the transcriptional level.
Material and Methods
Plant material and incubation conditions
The cultivation of the Lycopersicon peruvianum cell suspension culture has been described by Nover et aI. (1982). Exponentially growing cells with a density (As78) of about 0.9, usually 2.5 days after inoculation, were used for the experiments. Propagation and incubation of control cultures were done at 24°C. The appropriate heat stress and metal stress regimes are described in connection with the experiments. The following optimized metal salt concentrations were used: 0.5 mM CdCb (Cd); 0.07 mM HgCb (Hg); 30 mM ZnCh (Zn); 0.5 mM NaAs0 2(As).
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nuclei were sedimented and purified by sequential steps of centrifugation and flotation in Percoll containing media. Preparation 0/proteins
Total cell proteins and cytosolic proteins: Washed cells (O.4g) were suspended in 1.5 mL of a medium containing 50 mM TrisHCI, pH 7.8, 25mM KCI, 10mM MgCh, 1mM PMSF, O.lmM EDTA, 0.5 mM mercaptoethanol, 1 % SDS, and homogenized by sonication. After centrifugation 10 min at 4000 x g the proteins from the supernatant were precipitated by adding 0.5 mL 20 % TCA. The proteins were sedimented by centrifugation and washed with ethanoV20 mM Tris base, ethanol and dissolved for gel electrophoresis in sample buffer (50 mM Tris, 1.5 % sodium dodecylsulfate,S % mercaptoethanol, 20 % glycerol), and the pH was adjusted by adding 10 ~L 0.4 M HCI per 100 ~L. Cytosolic proteins were precipitated with 80 % aceton and dissolved in sample buffer. Nuclear proteins: Seventy ~L of a nuclear suspension were mixed with 500 ~L of a medium containing 20 mM Tris-HCl, pH 7.8, 20 mM MgCb, 200 mM Mg-acetate, 20 mM KCl, 0.5 mM mercaptoethanol and 1 mM PMSF (Scharf and Nover, 1987). After 2 x15 sec ultrasonic treatment, 1 mL acetic acid was added, and the suspension was shaken for 45 min in ice. After centrifugation at 1000 x g the proteins of the supernatant were precipitated with 7.5 mL acetone, washed with ethanol, dried and dissolved for gel electrophoresis in sample buffer. Gel electrophoresis and immunodetection
0/heat stress proteins
Conditions for the one- and two-dimensional gel electrophoretic separation of proteins and autoradiography were described by Nover and Scharf {1984}. For identification of HSP17 a chemoluminiscent protein detection system (Tropic Western-Light System, Serva Heidelberg) was utilized, using antibodies against HSP 17 as described by Neumann et aI. (1987). In vitro transcription Nuclei (1.5 x 107 per assay) were incubated in 200 ~L containing 40mM Tris-HCl, pH 7.8, O.5mM of each GTP, CTP and UTP, 5MBq np_ATP (110TBq/mM), ImM DTT, 10mM MgC12' 50mM (NH 4}2S04, 2.5 mM phosphoenol pyruvate, 10 ~g pyruvate kinase, 7 % glycerol and 10 mM vanadylribonucleosid complex as RNAse inhibitor. After incubation for 30 min at 30°C, the reaction was stopped by addition of 8 ~L 0.5 M EDTA. The transcripts were purified on Quiagen columns (tips 20) according to the suppliers application protocol (Qiagen, Hilden). DNA·RNA hybridization
Hybridization was carried out using slot blots as described earlier (Wollgiehn, 1991) with recombinant plasmids containing sequences for hsp17 (PhspI7, obtained from F. Schoff!, Tiibingen), hsp70 (pMON q575 from J. Winter Palo Alto, California) and a mixture of 25 S rRNA and 18 S rRNA (pBD 25 and pBD 18 from B. Dobrowolski, Halle). Results
Isolation
0/nuclei
Highly purified nuclei were isolated as described by Wollgiehn (1991). Briefly, cells were incubated for two hours with cellulase and macerozyme for partial enzymatic digestion of the cell walls. After complete homogenization of the cells with an Ultra-Turrax,
We used two approaches to visualize the patterns of the LMW heat shock proteins synthesized under metal ion and heat stress after two-dimensional gel electrophoresis: Autoradiography after in vivo labeling and Western blotting, using a serum raised against HSP17 (Neumann et al., 1987).
738
R. WOLLGIEHN and D. NEUMANN
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Fig. 1: Heat stress proteins from cultured cells and isolated nuclei. Cells (2 gin 10 mL) were cultivated for 2 hours at 39 °C (HS) or in the presence of Hg or As and of 38 MBq 35S-methionine for protein labeling. Total cell proteins and nuclear proteins were analysed by 2-dimensional PAA gel electrophoresis. The figure shows the region of the autoradiographs with the labeled LMW stress proteins (HSP 17).
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The labeling patterns of the LMW HSPs are shown in Fig. 1. LMW HSPs of three different molecular weights each of them with several isoforms - could be identified. They are termed as HSP17/1, 2 and 3, since the exact mol. masses of these three protein groups were not determined. The patterns of the labeled LMW HSPs in the total cell protein fractions were similar under the different stress conditions (Fig. 1, left). However, the same time of stress resulted in stronger labeling of HSP 17 under heat shock than under metal ion influence. As already described for soybean (Edelman et al., 1988) and sorghum seedlings (Howarth, 1990) «normal» protein synthesis was drastically repressed during heat shock, but not under metal stress (not shown). The results of the labeling experiments were confirmed by Western blotting probed with HSP17-antiserum. Fig. 2 (left, total proteins) shows the similarities but also quantitative differences in the HSP17 patterns under heat shock and stress with the four metals Cd, Hg, Zn and As. These differences
Fig. 2: Western blots of the HSP17 heat stress proteins from cultured cells and isolated nuclei. Cells were cultivated for 2 hours at 39 °C (HS) or in the presence of Cd, Hg, Zn or As. Total cell proteins and nuclear proteins were analysed by 2-dimensional PAA gel electrophoresis. The WeStern blots were probed with HSP 17 antibodies. Note that different amounts of proteins were analysed to obtain optimal resolution and registration of all LMW stress proteins. However, if the same amounts of proteins isolated from cells after heat or metal stress were applied to the gels, the blots showed that under metal Stress HSP17 was accumulated at levels much lower than under heat stress.
appear in the number of isoforms, but also in the quantitative relations between HSP17/1, 2 and 3. Control samples from untreated cells exhibit no signal with the antibodies (data not shown). The serum was raised against proteins of group 1 and crossreacts with the whole series of HSP 17 (group 1-3). Therefore, we used HSP17 as a collective term for all proteins in this Mr-region of the electropherograms and Western blots (Neumann et al., 1987). As described earlier, HSP17 of cultured tomato cells was found to be localized under heat stress in the cytoplasmic «heat shock granules» and in the nuclei (Neumann et al., 1987; Wollgiehn et al., 1994). To examine the subcellular distribution of LMW HSPs under metal stress we analysed the labeling patterns and the Western blots of the LMW HSPs from highly purified nuclei, isolated from cells stressed with the different metal ions or by heat shock. The autoradiographs (Fig. 1, right) and the Western blots (Fig. 2, right) show that under heat stress the main nuclear HSPs 17 are the
Heat shock proteins under metal ion stress isoforms of protein 1, whereas under metal stress considerably more HSPs/2 and 3 accumulated. Additionally, there were apparent differences in the relative amounts of the three nuclear HSPs17 with their isoforms depending on the various metals, e.g. between Cd and As. After heat shock the patterns of the nuclear and total cell LMW HSPs were similar (Fig. 1 and 2) with HSP17/1 as the dominating protein group. However, after metal stress the HSP patterns of nuclear and total cell proteins were different since HSPs 2 and 3 specifically accumulated in the nuclei. Most of the LMW HSP of the cell is localized within the cytoplasm, therefore, the total cell HSP pattern should represent mainly cytoplasmic HSP. This is shown in Fig. 3 for Hg in comparison to heat stress. Additionally to total and nuclear proteins the cytosolic proteins were analyzed. Three findings result from this experiment: (1) As expected, the Western blots of total cell proteins and cytoplasmic proteins were similar. (2) In agreement with Fig. 1 and 2 HPS 17/3 was significantly enriched in the nuclear fraction under metal stress, but only scarcely after heat shock. (3) After heat stress the HSP17 patterns of cytosol and nuclei were identical, consisting of mainly proteins 1 and 2 and only low amounts of protein 3. After metal stress the patterns were different, since additionally HSP 1713 accumulated in nuclei. Under heat stress the relative amounts of the three nuclear HSPs17 were found to depend on the stress regime. For example, during recovery following a heat stress, HSP 1711 is depleted from the nuclei faster than the other HSPs 17 (W011giehn et al., 1994). Neumann et al. (1994) have already shown, that after Cd stress not only LMW HSPs, but also HSP70 is accumulated in tomato cells. We also have determined HSP70 after heat
Total protein
Cytopl. protein
Nuclear protein
Fig.3: Intracellular distribution of HSP 17 in cultured cells. Cells were cultivated for 2 hours at 39°C (HS) or in the presence of Hg. Total cell proteins, nuclear proteins and cytosolic proteins (from the supernatant after sedimentation of the nuclei) were analysed by 1-dimensional PAA gel electrophoresis. The figure shows the Western blots probed with HSP 17 antibodies.
rRNA
control-
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I min
3 min 15 min
30 min
60 min
-
HSP17
739
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HSP70
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2h 4h
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4h+ 4h 24°C 4h+ 8 h 24°C 4h+ 20 h 24°C
-
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Fig. 4: Effect of heat stress of cultured cells on the in vitro run-on transcription of rRNA, hsp 17 and hsp70 genes. After different times of heat stress at 39°C of the cells or heat stress followed by recovery periods, the nuclei were isolated and after run-on transcription RNA was purified and used for slot-blot hybridization with rRNA-, hsp 17- and hsp70-specific DNA.
and metal stress using Western blots with HSP70 antiserum. Under stress with all four metal ions HSP70 was accumulated in cells and in nuclei significantly above the control. Under heat shock the accumulation was higher than under metal stress (data not whown). In a previous report Nover et al. (1990) have shown that arsenite, Cd and Hg induce increased levels of hsp70 and hsp17 mRNAs in tomato cell cultures in a similar way as heat stress. In addition to these results we have investigated the kinetics of the induction of hsp mRNA by in vitro runon transcription assays. The nuclei were isolated for transcription measurement from cells after different heat stress regimes or different time of exposure to the metal ions. Fig. 4 shows the run-on transcription of HSP 17 genes in comparison to hsp70 and rRNA genes after heat stress. In nuclei from control cells rRNA and a low level of hsp70 mRNA was transcribed according to the synthesis of rRNA and a low level of the constitutive HSP70 in the cells. Already after one minute heat stress of the cells at 38 - 40°C the synthesis of hsp mRNAs 17 and 70 was induced and after a few minutes the maximum of transcriptional activity was reached. The activity remained constant for several hours, then the activity was reduced and finally completely stopped after about 8 hours. On the other hand, the transcriptional activity of the rRNA genes was stopped already after 30-
740
R. WOLLGIEHN and D. NEUMANN
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-• ... --•.. -- -•.' •- --,.. -•- .. -...... -- -- .. --rRNA
control 1 min 5 min 15mio 30 min 60mio 120 min
B
rRNA
control Cd
Hg Zn As
hsp17
hsp17
hsp70
hsp70
Fig. 5: Effect of metal stress of cells on the in vitro run-on transcription of rRNA, hsp17 and hsp70 genes. After metal stress of the cells the nuclei were isolated and used for run-on transcription. RNA was extracted and utilized for slot-blot hybridization. A. Time kinetics of the Cd stress. B. Cells were treated for 5 min with Cd, Hg, Zn or As.
60 min of heat stress. During recovery at 24°C the metabolism normalized slowly. The transcription of the heat stress genes ceased, synthesis of the rRNA started again. Under Cd stress the induction of transcription of hsp genes followed the same kinetics as under heat stress (Fig. 5 A). Already after one minute metal stress, induction of hsp genes could be observed, after 5 min the full activity was reached. Hg, Zn and As exhibit the same induction kinetics as Cd (data not shown). The maximum transcription activities of the hsp genes were observed already after 5 min treatment of the cells with the different metal ions (Fig. 5 B). However, in contrast to heat stress, the transcription of the rRNA genes remained stable for several hours of metal stress, as shown for Cd in Fig. 5 A. After long metal stress the cells were damaged (Neumann et aI., 1994) and the whole metabolism, including transcription of rRNA, hsp, and control-protein genes were reduced and finally ceased (data not shown). Long-term and recovery experiments with metals are not possible in the same way as under temperature stress. In contrast to heat stress with the possibility of immediate switch on and off of the stress it is
impossible to remove the metal ions of the cells quickly and controlled. Discussion
The results obtained so far give a manifold picture about synthesis and intracellular localization of the HSPs under metal stress in comparison to heat shock. In cell cultures of Datura innoxia (Delhaize et al., 1989) and Lycopersicon peruvianum (Neumann et al., 1994) the patterns of HSPs were found to be quite similar under heat and Cd stress. The same results were obtained with soybean seedlings under heat and As treatment (Lin et aI., 1984). On the other hand, exposure of sorghum seedlings to arsenite resulted in the synthesis of only HMW HSPs, but not of LMW HSPs (Howarth, 1990). Also in Drosophila Kc cells, arsenite in contrast to heat shock did not significantly induce the synthesis of LMW HSPs (Vincent and Tanguay, 1982). Our results with tomato cell cultures clearly demonstrate the synthesis (labeling experiments) and accumulation (immunodetection) of LMW HSPs under Cd, Hg, Zn, and As stress, only quantitatively different to the protein pattern observed after heat shock (Figs. 1 and 2). In this paper we restricted most of the experiments to the strictly stress dependent induced LMW HSPs, but previous reports from our laboratory and unpublished data have shown, that under toxic metal stress also HSP70 synthesis is induced (Nover et al., 1990; Neumann et al., 1994, but also Fig. 5). The function of the stress proteins, particularly for the acquisition of stress tolerance appears to depend not only on synthesis and pattern of HSPs, but also on their intracellular localization. The selective cellular localization of HSPs under heat stress was investigated in many details (for references see Nover, 1991; Wollgiehn et al., 1994). In tomato cells LMW HSPs were found in the cytoplasm, in «heat shock granules» (Nover et aI., 1983; Neumann et aI., 1984) and in nuclei (Wollgiehn et al., 1994). Less was known about the intracellular distribution of HSPs under metal stress. In Drosophila Kc cells HSP70 induced under heat stress was found to be localized in the cytoplasm and in nuclei, while As-induced HSP70 was found largely cytoplasmic unless these cells were further incubated at elevated temperature (Vincent and Tanguay, 1982). In soybean seedlings HSPs including LMW HSPs (15-18KD) were found in ribosomes, mitochondria and nuclei under heat stress, but the arseniteinduced HSPs were not organelle-associated at normal temperature, they became organelle-associated only during a subsequent HS at 40°C (Lin et al., 1984). In tomato cell cultures HSP17 accumulated under metal and under heat stress in the cytoplasm and in nuclei. After long heat stress the cytoplasmic HSP17 is concentrated in the granules (Nover et aI., 1983; Neumann et al., 1984), but under Cd-stress it is uniformly distributed in the cytoplasm. Only when the Cd-stress followed a short (15 min) heat shock, HSP17 containing granules are formed (Neumann et al., 1994). On the other hand, HSP17 is associated with tomato nuclei under metal stress directly as observed under heat stress (Figs. 1 and 2). This is in contrast to observations with soybean seedlings, where subsequent heat stress was
Heat shock proteins under metal ion stress necessary for the nuclear accumulation of HSP17 (Lin et al., 1984). The patterns of the nuclear HSP17 after heat shock and under metal stress exhibit distinct differences. It remains open whether this reflects also different functions of the individual LMW HSPs within the cell. The accumulation of HSP17 in nuclei is not a heat or metal stress-dependent property. HSP17 is also expressed stress-independent in developing seeds and this HSP17 was mainly found in nuclei (zur Nieden et aI., 1995). There is no evidence for a nuclear localization signal in the proteins. A transport as a proteincomplex together with other nuclear proteins seems possible. The role of the LMW heat shock proteins in the cellular response to stress and during defined stages of development of different organisms was discussed in detail by Arrigo and Landry (1994). The HSP level of the cell detected after metal ion treatment is generally lower than after theat stress (see also Edelman et al., 1988; Delhaize et aI., 1989; Bournias-Vardiabasis et aI., 1990). The induction of the hsp gene transcription is extremely rapid in both cases and can be observed already after one min heat shock or metal stress. The maximum rates were achieved within 2 - 5 min (Figs. 4 and 5). Heat shock transcription factors were found to be activated in tomato cells also within 1.5 min of heat shock (Scharf et al., 1990). Therefore, different HSP levels of the cells under metal and heat stress require additional regulatory mechanisms, either on transcriptional or translational level. It seems also possible, that the stability of the hsp mRNAs is different under the various stress conditions (Edelman et aI., 1988; Kimpel et al.,1990). During heat shock drastic repression of normal protein synthesis is a general phenomenon. However, under comparable conditions the metal ion treated cells display only a slight inhibition of the normal translation pattern. This was also found under Cd and As stress in soybean seedlings (Edelmann et al., 1988), and under arsenite stress in sorghum seedlings (Howarth, 1990), and Tetrahymena periformis (Amaral et al., 1988). These differences were interpreted as translational control, which operates under heat shock (Storti et al., 1980) and failure of translational control under metal stress (Amaral et aI., 1988). Lindquist and McGarry (1986) suggested that specific leader sequences with a high affinity to ribosomes in the hsp mRNAs, which enable them to compete with «normal» messengers, could be responsible for specific translation of heat shock mRNAs. But this specificity should also function with hsp mRNAs induced by metal ions. As described earlier (Schoffl et aI., 1987; Vazquez et aI., 1993) transcription of rRNA genes is drastically reduced and finally completely stopped during heat stress and reactivated during recovery at normal temperature (Fig. 4). On the other hand, under metal stress transcription of rDNA is not influenced (Fig. 5). It seems possible, that in contrast to heat shock, under metal stress ribosomes and, therefore, the protein synthesizing system of the cell remains largely intact which results in synthesis of HSPs additionally to normal synthesis of the house keeping proteins. The mechanisms by which heat shock but not metal stress affects transcription of rRNA and other non-heat shock genes is not clear. A general inhibitory effect on the RNA polymerase molecules at high temperature, but also new initiation of transcription, altera-
741
tion of specific transcription factors or other mechanisms are possible (Vazquez et aI., 1993). Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 326). We are grateful to Prof. Lutz Nover (Frankfurt/ Main) for stimulating discussions. We also thank Dr. Norbert NaB for critical reading of the manuscript, Brigitte Werner and Karin Ohme for skillful technical assistance and Ruth Laue for kindly typing the manuscript.
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NEUMANN, D., O. LICHTENBERGER, D. GONTHER, K. TSCHIERSCH, and L. NOVER: Heat shock proteins induce heavy-metal tolerance in higher plants. Planta 194,360-367 (1994). NEUMANN, D., K.-D. SCHARF, and L. NOVER: Heat shock induced changes of plant cell ultrastructure and autoradiographic localization of heat shock proteins. Europ. J. Cell BioI. 34, 254-264 (1984). NEUMANN, D., U. ZUR NIEDEN, R. MANTEUFFEL, G. WALTER, K.-D. SCHARF, and L. NOVER: Intracellular localization of heat-shock proteins in tomato cell cultures. Europ. J. Cell BioI. 43, 71- 81 (1987). NIEDEN ZUR, U., D. NEUMANN, A. BUCKA, and L. NOVER: Tissue-specific localization of heat-stress proteins during embryo development. Plant a 196, 530-538 (1995). NOVER, L.: The heat stress response as part of the plant stress network: An overview with six tables. In: CHERRY, J. H. (ed.): Biochemical and Cellular Mechanisms of Stress Tolerance in Plants. pp. 3-45, Springer-Verlag, Berlin (1994). NOVER, L.: Heat Shock Response. CRC Press Boca Raton (1991). NOVER, L., E. KRANZ, and K.-D. SCHARF: Growth cycle of suspension cultures of Lycopersicon esculentum and L. peruvianum. Biochern. Physiol. Pflanzen 177, 483-499 (1982). NOVER, L., D. NEUMANN, and K.-D. SCHARF (eds.): Heat Shock and Other Stress Response Systems of Plants. Springer-Verlag, Berlin (1990). - - - Intracellular localization and related functions of heat shock proteins. In: NOVER, L. (ed.): Heat Shock Response. pp. 374-407, CRC Press, Boca Raton (1991). NOVER, L. and K.-D. SCHARF: Synthesis, modification and structural binding of heat shock proteins in tomato cell cultures. Eur. J. Biochem. 139, 303-313 (1984). NOVER, L., K.-D. SCHARF, and D. NEUMANN: Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves. Mol. Cell. BioI. 3,1648-1655 (1983).
SCHARF, K.-D. and L. NOVER: Control of ribosome biosynthesis in plant cell cultures under heat shock conditions II. Ribosomal proteins. Biochim. Biophys. Acta 909, 44-57 (1987). SCHARF, K.-D., S. ROSE, W. ZOTT, F. SCH6FFI., and L. NOVER: Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF. The EMBO Journal 9, 4495-4501 (1990). SCH6FFL, F., I. RossoL, and S. ANGERMOlLER: Regulation of the transcription of heat shock genes in nuclei from soybean (Glycine max) seedlings. Plant Cell and Environment 10, 113 -119 (1987). STORTI, R. V., M. P. SCOTT, A. RICH, and M. L. PARDUE: Translational control of protein synthesis in response to heat shock in D. melanogaster. Cell 22, 825-834 (1980). VASQUEZ, J., D. PAULI, and A. TISSIERES: Transcriptional regulation in Drosophila during heat shock: A molecular run-on analysis. Chromosoma 102, 233-248 (1993). VIERLING, E.: The roles of heat shock proteins in plants. Annu. Rev. Plant Physiol. Plant Mol. BioI. 42, 579-620 (1991). VINCENT, M . and R. M. TANGUAY: Different intracellular distributions of heat-shock and arsenite-induced proteins in Drosophila Kc cells. J. Mol. BioI. 162, 365-378 (1982). WINTER, J., R. WRIGHT, N. DUCK, C. GASSER, R . FRALEY, and D . SHAH: The inhibition of Petunia hsp70 mRNA processing during CdCh stress. Mol. Gen. Genet. 211, 315-319 (1988). WOLLGIEHN, R.: Conditions for gene-specific transcription in isolated nuclei from tomato cell cultures. Biochem. Physiol. Pflanzen 187, 305-315 (1991). WOLLGIEHN, R., D. NEUMANN, U. ZUR NIEDEN, A. MOSCH, K.-D. SCHARF, and L. NOVER: Intracellular distribution of small heat shock proteins in cultured cells of Lycopersicon peruvianum. J. Plant Physiol. 144,491-499 (1994).
Stress Response of Tomato Cell Cultures to Toxic Metals and Heat Shock: Differences and Similarities R.
WOLLGIEHN*
and D.
NEUMANN
Institut fur Pflanzenbiochemie, Weinberg 3, D-06120 Halle/Saale, Germany Received February 1, 1995 . Accepted April 28, 1995
Summary
Cultured tomato cells were exposed to different metal ions and heat shock and the induction and intracellular localization of the low molecular weight heat shock proteins were examined. Treatment with cadmium, mercury, zinc and arsenite resulted in synthesis of heat shock proteins without essentially affecting synthesis of house keeping proteins. Under heat shock a higher level of HSPs was synthesized than under metal stress, and «normal» protein synthesis was drastically reduced. Under all stress conditions investigated, HSPs accumulated in the cytoplasm and in nuclei. The patterns of the low molecular weight HSPs observed after 2-dimensional electrophoresis were different in the cytoplasm and in nuclei, and in nuclei the pattern of HSPs 17 under heat shock was different from that observed after metal stress. The induction of hsp17 and hsp70 gene transcription could be detected in the nuclear run-on assay already after one min heat shock or metal ion stress of the cells. Under all stress conditions the maximum rates were reached within 2-5min. The transcription of the rRNA genes was drastically reduced only under heat stress, but not influenced under metal stress.
Key words: Lycopersicon peruvianum, cell cultures, electrophoresis, heat stress, heat stress proteins, immunob/ots, metal ion stress, run-on-transcription. Abbreviations: HS = heat shock; HMW/LMW HSPs = high/low molecular weight heat shock proteins; PMSF = phenylmethylsulfonyl fluoride; SDS = sodium dodecyl sulfate_ Introduction
The exposition of plants to toxic metal ions as a consequence of industrial environment causes reduced plant growth, coincidental with disturbance of several metabolic processes, impaired uptake of nutrients and changes in cell ultrastructure_ Long-term growth of plants on metal contaminated soils may generate the development of metal tolerance. At present, little is known about the molecular and cellular basis of the response to metal stress. Several mechanisms contribute to the metal tolerance, as metal deposition in different cell compartments or secretion by specific glands, exudation of metal-chelating substances and alteration in membrane structure and function (Ernst et al., 1992; Neumann et al., 1994). One of the most prominent meta-
*
Corresponding author.
© 1995 by Gustav Fischer Verlag, Stuttgart
bolic alterations during metal stress is the elevated expression of heat shock proteins (HSPs) in a similar way as observed under heat stress. The HSPs have a key role in the protection of proteins and cell membranes against irreversible damage caused by the stress (Nover, 1994). The plant heat stress response is characterized by the synthesis of a few high molecular weight (HMW) and a complex group of low molecular weight heat shock proteins (LMW HSPs) with molecular sizes ranging from 15 to 28 KD (Nover et aI., 1990; Vierling, 1991). LMW HSPs were found to be localized mainly in the cytoplasm as larger aggregates, referred to as «heat shock granules» (Nover et aI., 1983; Neumann et al., 1984; Mansfield and Key, 1988), but also associated with the endomembrane system of the cell (for references s. Vierling, 1991; Nover, 1991). A broad spectrum of LMW HSPs was found in isolated nuclei of soybean seedlings (Lin et aI., 1984), maize roots (Cooper and Ho, 1987)
Heat shock proteins under metal ion stress and tomato fruits (Kato et al., 1993). Additionally, we have previously reported on the localization of HSP 17 in nuclei of tomato cell cultures under heat shock not only by biochemical means, but also by immunfluorescence and immune electron microscopy (Wollgiehn et al., 1994). In comparison to our extensive knowledge on heat stress only a few reports about stress proteins induced under metal stress of plants have been published. Czarnecka et al. (1984) and Edelman et al. (1988) investigated Cd and As effects on soybean seedlings. Using Northern blot hybridization and protein analysis Edelman et al. (1988) detected induction and accumulation of stress protein mRNA and high and low mol. weight HSPs. The response to heat or metal stress was qualitatively similar, but they observed distinct quantitative and temporal differences. The regulatory mechanisms controlling transcription and translation reacted differently to the various stress treatments. Nover et al. (1990) also found the induction of hsp17 and hsp70 mRNA by Cd, Hg and As in tomato cell cultures. Metal ion induced synthesis of low and high mol. weight HSPs was also described for sorghum seedlings (Howarth, 1990), Petunia leaves (Winter et al., 1988), cell cultures of Datura (Delhaize et al., 1989), tomato (Neumann et aI., 1994) and alfalfa (Gyorgyey et aI., 1991). The results, obtained so far, show that the molecular stress responses of the plants to heat and metal ion stress are similar, but exhibit also distinct differences depending on the stress-conditions. Little is known about the intracellular localization of HSPs under metal stress. In soybean seedlings accumulation of HSPs in nuclei and other cell organelles was observed only under heat shock, but not under As-stress (Lin et aI., 1984). Also in As-treated Drosophila Kc cells HSP70 was found to remain largely cytoplasmic, whereas under heat shock it was also enriched in nuclei (Vincent and Tanguay, 1982). Here, we compare the effects of metal ions and heat shock on synthesis and intracellular localization of LMW HSPs, particularly their accumulation in nuclei, and the regulation of hsp-mRNA and rRNA synthesis at the transcriptional level.
Material and Methods
Plant material and incubation conditions
The cultivation of the Lycopersicon peruvianum cell suspension culture has been described by Nover et aI. (1982). Exponentially growing cells with a density (As78) of about 0.9, usually 2.5 days after inoculation, were used for the experiments. Propagation and incubation of control cultures were done at 24°C. The appropriate heat stress and metal stress regimes are described in connection with the experiments. The following optimized metal salt concentrations were used: 0.5 mM CdCb (Cd); 0.07 mM HgCb (Hg); 30 mM ZnCh (Zn); 0.5 mM NaAs0 2(As).
737
nuclei were sedimented and purified by sequential steps of centrifugation and flotation in Percoll containing media. Preparation 0/proteins
Total cell proteins and cytosolic proteins: Washed cells (O.4g) were suspended in 1.5 mL of a medium containing 50 mM TrisHCI, pH 7.8, 25mM KCI, 10mM MgCh, 1mM PMSF, O.lmM EDTA, 0.5 mM mercaptoethanol, 1 % SDS, and homogenized by sonication. After centrifugation 10 min at 4000 x g the proteins from the supernatant were precipitated by adding 0.5 mL 20 % TCA. The proteins were sedimented by centrifugation and washed with ethanoV20 mM Tris base, ethanol and dissolved for gel electrophoresis in sample buffer (50 mM Tris, 1.5 % sodium dodecylsulfate,S % mercaptoethanol, 20 % glycerol), and the pH was adjusted by adding 10 ~L 0.4 M HCI per 100 ~L. Cytosolic proteins were precipitated with 80 % aceton and dissolved in sample buffer. Nuclear proteins: Seventy ~L of a nuclear suspension were mixed with 500 ~L of a medium containing 20 mM Tris-HCl, pH 7.8, 20 mM MgCb, 200 mM Mg-acetate, 20 mM KCl, 0.5 mM mercaptoethanol and 1 mM PMSF (Scharf and Nover, 1987). After 2 x15 sec ultrasonic treatment, 1 mL acetic acid was added, and the suspension was shaken for 45 min in ice. After centrifugation at 1000 x g the proteins of the supernatant were precipitated with 7.5 mL acetone, washed with ethanol, dried and dissolved for gel electrophoresis in sample buffer. Gel electrophoresis and immunodetection
0/heat stress proteins
Conditions for the one- and two-dimensional gel electrophoretic separation of proteins and autoradiography were described by Nover and Scharf {1984}. For identification of HSP17 a chemoluminiscent protein detection system (Tropic Western-Light System, Serva Heidelberg) was utilized, using antibodies against HSP 17 as described by Neumann et aI. (1987). In vitro transcription Nuclei (1.5 x 107 per assay) were incubated in 200 ~L containing 40mM Tris-HCl, pH 7.8, O.5mM of each GTP, CTP and UTP, 5MBq np_ATP (110TBq/mM), ImM DTT, 10mM MgC12' 50mM (NH 4}2S04, 2.5 mM phosphoenol pyruvate, 10 ~g pyruvate kinase, 7 % glycerol and 10 mM vanadylribonucleosid complex as RNAse inhibitor. After incubation for 30 min at 30°C, the reaction was stopped by addition of 8 ~L 0.5 M EDTA. The transcripts were purified on Quiagen columns (tips 20) according to the suppliers application protocol (Qiagen, Hilden). DNA·RNA hybridization
Hybridization was carried out using slot blots as described earlier (Wollgiehn, 1991) with recombinant plasmids containing sequences for hsp17 (PhspI7, obtained from F. Schoff!, Tiibingen), hsp70 (pMON q575 from J. Winter Palo Alto, California) and a mixture of 25 S rRNA and 18 S rRNA (pBD 25 and pBD 18 from B. Dobrowolski, Halle). Results
Isolation
0/nuclei
Highly purified nuclei were isolated as described by Wollgiehn (1991). Briefly, cells were incubated for two hours with cellulase and macerozyme for partial enzymatic digestion of the cell walls. After complete homogenization of the cells with an Ultra-Turrax,
We used two approaches to visualize the patterns of the LMW heat shock proteins synthesized under metal ion and heat stress after two-dimensional gel electrophoresis: Autoradiography after in vivo labeling and Western blotting, using a serum raised against HSP17 (Neumann et al., 1987).
738
R. WOLLGIEHN and D. NEUMANN
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Fig. 1: Heat stress proteins from cultured cells and isolated nuclei. Cells (2 gin 10 mL) were cultivated for 2 hours at 39 °C (HS) or in the presence of Hg or As and of 38 MBq 35S-methionine for protein labeling. Total cell proteins and nuclear proteins were analysed by 2-dimensional PAA gel electrophoresis. The figure shows the region of the autoradiographs with the labeled LMW stress proteins (HSP 17).
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The labeling patterns of the LMW HSPs are shown in Fig. 1. LMW HSPs of three different molecular weights each of them with several isoforms - could be identified. They are termed as HSP17/1, 2 and 3, since the exact mol. masses of these three protein groups were not determined. The patterns of the labeled LMW HSPs in the total cell protein fractions were similar under the different stress conditions (Fig. 1, left). However, the same time of stress resulted in stronger labeling of HSP 17 under heat shock than under metal ion influence. As already described for soybean (Edelman et al., 1988) and sorghum seedlings (Howarth, 1990) «normal» protein synthesis was drastically repressed during heat shock, but not under metal stress (not shown). The results of the labeling experiments were confirmed by Western blotting probed with HSP17-antiserum. Fig. 2 (left, total proteins) shows the similarities but also quantitative differences in the HSP17 patterns under heat shock and stress with the four metals Cd, Hg, Zn and As. These differences
Fig. 2: Western blots of the HSP17 heat stress proteins from cultured cells and isolated nuclei. Cells were cultivated for 2 hours at 39 °C (HS) or in the presence of Cd, Hg, Zn or As. Total cell proteins and nuclear proteins were analysed by 2-dimensional PAA gel electrophoresis. The WeStern blots were probed with HSP 17 antibodies. Note that different amounts of proteins were analysed to obtain optimal resolution and registration of all LMW stress proteins. However, if the same amounts of proteins isolated from cells after heat or metal stress were applied to the gels, the blots showed that under metal Stress HSP17 was accumulated at levels much lower than under heat stress.
appear in the number of isoforms, but also in the quantitative relations between HSP17/1, 2 and 3. Control samples from untreated cells exhibit no signal with the antibodies (data not shown). The serum was raised against proteins of group 1 and crossreacts with the whole series of HSP 17 (group 1-3). Therefore, we used HSP17 as a collective term for all proteins in this Mr-region of the electropherograms and Western blots (Neumann et al., 1987). As described earlier, HSP17 of cultured tomato cells was found to be localized under heat stress in the cytoplasmic «heat shock granules» and in the nuclei (Neumann et al., 1987; Wollgiehn et al., 1994). To examine the subcellular distribution of LMW HSPs under metal stress we analysed the labeling patterns and the Western blots of the LMW HSPs from highly purified nuclei, isolated from cells stressed with the different metal ions or by heat shock. The autoradiographs (Fig. 1, right) and the Western blots (Fig. 2, right) show that under heat stress the main nuclear HSPs 17 are the
Heat shock proteins under metal ion stress isoforms of protein 1, whereas under metal stress considerably more HSPs/2 and 3 accumulated. Additionally, there were apparent differences in the relative amounts of the three nuclear HSPs17 with their isoforms depending on the various metals, e.g. between Cd and As. After heat shock the patterns of the nuclear and total cell LMW HSPs were similar (Fig. 1 and 2) with HSP17/1 as the dominating protein group. However, after metal stress the HSP patterns of nuclear and total cell proteins were different since HSPs 2 and 3 specifically accumulated in the nuclei. Most of the LMW HSP of the cell is localized within the cytoplasm, therefore, the total cell HSP pattern should represent mainly cytoplasmic HSP. This is shown in Fig. 3 for Hg in comparison to heat stress. Additionally to total and nuclear proteins the cytosolic proteins were analyzed. Three findings result from this experiment: (1) As expected, the Western blots of total cell proteins and cytoplasmic proteins were similar. (2) In agreement with Fig. 1 and 2 HPS 17/3 was significantly enriched in the nuclear fraction under metal stress, but only scarcely after heat shock. (3) After heat stress the HSP17 patterns of cytosol and nuclei were identical, consisting of mainly proteins 1 and 2 and only low amounts of protein 3. After metal stress the patterns were different, since additionally HSP 1713 accumulated in nuclei. Under heat stress the relative amounts of the three nuclear HSPs17 were found to depend on the stress regime. For example, during recovery following a heat stress, HSP 1711 is depleted from the nuclei faster than the other HSPs 17 (W011giehn et al., 1994). Neumann et al. (1994) have already shown, that after Cd stress not only LMW HSPs, but also HSP70 is accumulated in tomato cells. We also have determined HSP70 after heat
Total protein
Cytopl. protein
Nuclear protein
Fig.3: Intracellular distribution of HSP 17 in cultured cells. Cells were cultivated for 2 hours at 39°C (HS) or in the presence of Hg. Total cell proteins, nuclear proteins and cytosolic proteins (from the supernatant after sedimentation of the nuclei) were analysed by 1-dimensional PAA gel electrophoresis. The figure shows the Western blots probed with HSP 17 antibodies.
rRNA
control-
~
I min
3 min 15 min
30 min
60 min
-
HSP17
739
...
HSP70
'.
2h 4h
8h
4h+ 4h 24°C 4h+ 8 h 24°C 4h+ 20 h 24°C
-
.. -
Fig. 4: Effect of heat stress of cultured cells on the in vitro run-on transcription of rRNA, hsp 17 and hsp70 genes. After different times of heat stress at 39°C of the cells or heat stress followed by recovery periods, the nuclei were isolated and after run-on transcription RNA was purified and used for slot-blot hybridization with rRNA-, hsp 17- and hsp70-specific DNA.
and metal stress using Western blots with HSP70 antiserum. Under stress with all four metal ions HSP70 was accumulated in cells and in nuclei significantly above the control. Under heat shock the accumulation was higher than under metal stress (data not whown). In a previous report Nover et al. (1990) have shown that arsenite, Cd and Hg induce increased levels of hsp70 and hsp17 mRNAs in tomato cell cultures in a similar way as heat stress. In addition to these results we have investigated the kinetics of the induction of hsp mRNA by in vitro runon transcription assays. The nuclei were isolated for transcription measurement from cells after different heat stress regimes or different time of exposure to the metal ions. Fig. 4 shows the run-on transcription of HSP 17 genes in comparison to hsp70 and rRNA genes after heat stress. In nuclei from control cells rRNA and a low level of hsp70 mRNA was transcribed according to the synthesis of rRNA and a low level of the constitutive HSP70 in the cells. Already after one minute heat stress of the cells at 38 - 40°C the synthesis of hsp mRNAs 17 and 70 was induced and after a few minutes the maximum of transcriptional activity was reached. The activity remained constant for several hours, then the activity was reduced and finally completely stopped after about 8 hours. On the other hand, the transcriptional activity of the rRNA genes was stopped already after 30-
740
R. WOLLGIEHN and D. NEUMANN
A
-• ... --•.. -- -•.' •- --,.. -•- .. -...... -- -- .. --rRNA
control 1 min 5 min 15mio 30 min 60mio 120 min
B
rRNA
control Cd
Hg Zn As
hsp17
hsp17
hsp70
hsp70
Fig. 5: Effect of metal stress of cells on the in vitro run-on transcription of rRNA, hsp17 and hsp70 genes. After metal stress of the cells the nuclei were isolated and used for run-on transcription. RNA was extracted and utilized for slot-blot hybridization. A. Time kinetics of the Cd stress. B. Cells were treated for 5 min with Cd, Hg, Zn or As.
60 min of heat stress. During recovery at 24°C the metabolism normalized slowly. The transcription of the heat stress genes ceased, synthesis of the rRNA started again. Under Cd stress the induction of transcription of hsp genes followed the same kinetics as under heat stress (Fig. 5 A). Already after one minute metal stress, induction of hsp genes could be observed, after 5 min the full activity was reached. Hg, Zn and As exhibit the same induction kinetics as Cd (data not shown). The maximum transcription activities of the hsp genes were observed already after 5 min treatment of the cells with the different metal ions (Fig. 5 B). However, in contrast to heat stress, the transcription of the rRNA genes remained stable for several hours of metal stress, as shown for Cd in Fig. 5 A. After long metal stress the cells were damaged (Neumann et aI., 1994) and the whole metabolism, including transcription of rRNA, hsp, and control-protein genes were reduced and finally ceased (data not shown). Long-term and recovery experiments with metals are not possible in the same way as under temperature stress. In contrast to heat stress with the possibility of immediate switch on and off of the stress it is
impossible to remove the metal ions of the cells quickly and controlled. Discussion
The results obtained so far give a manifold picture about synthesis and intracellular localization of the HSPs under metal stress in comparison to heat shock. In cell cultures of Datura innoxia (Delhaize et al., 1989) and Lycopersicon peruvianum (Neumann et al., 1994) the patterns of HSPs were found to be quite similar under heat and Cd stress. The same results were obtained with soybean seedlings under heat and As treatment (Lin et aI., 1984). On the other hand, exposure of sorghum seedlings to arsenite resulted in the synthesis of only HMW HSPs, but not of LMW HSPs (Howarth, 1990). Also in Drosophila Kc cells, arsenite in contrast to heat shock did not significantly induce the synthesis of LMW HSPs (Vincent and Tanguay, 1982). Our results with tomato cell cultures clearly demonstrate the synthesis (labeling experiments) and accumulation (immunodetection) of LMW HSPs under Cd, Hg, Zn, and As stress, only quantitatively different to the protein pattern observed after heat shock (Figs. 1 and 2). In this paper we restricted most of the experiments to the strictly stress dependent induced LMW HSPs, but previous reports from our laboratory and unpublished data have shown, that under toxic metal stress also HSP70 synthesis is induced (Nover et al., 1990; Neumann et al., 1994, but also Fig. 5). The function of the stress proteins, particularly for the acquisition of stress tolerance appears to depend not only on synthesis and pattern of HSPs, but also on their intracellular localization. The selective cellular localization of HSPs under heat stress was investigated in many details (for references see Nover, 1991; Wollgiehn et al., 1994). In tomato cells LMW HSPs were found in the cytoplasm, in «heat shock granules» (Nover et aI., 1983; Neumann et aI., 1984) and in nuclei (Wollgiehn et al., 1994). Less was known about the intracellular distribution of HSPs under metal stress. In Drosophila Kc cells HSP70 induced under heat stress was found to be localized in the cytoplasm and in nuclei, while As-induced HSP70 was found largely cytoplasmic unless these cells were further incubated at elevated temperature (Vincent and Tanguay, 1982). In soybean seedlings HSPs including LMW HSPs (15-18KD) were found in ribosomes, mitochondria and nuclei under heat stress, but the arseniteinduced HSPs were not organelle-associated at normal temperature, they became organelle-associated only during a subsequent HS at 40°C (Lin et al., 1984). In tomato cell cultures HSP17 accumulated under metal and under heat stress in the cytoplasm and in nuclei. After long heat stress the cytoplasmic HSP17 is concentrated in the granules (Nover et aI., 1983; Neumann et al., 1984), but under Cd-stress it is uniformly distributed in the cytoplasm. Only when the Cd-stress followed a short (15 min) heat shock, HSP17 containing granules are formed (Neumann et al., 1994). On the other hand, HSP17 is associated with tomato nuclei under metal stress directly as observed under heat stress (Figs. 1 and 2). This is in contrast to observations with soybean seedlings, where subsequent heat stress was
Heat shock proteins under metal ion stress necessary for the nuclear accumulation of HSP17 (Lin et al., 1984). The patterns of the nuclear HSP17 after heat shock and under metal stress exhibit distinct differences. It remains open whether this reflects also different functions of the individual LMW HSPs within the cell. The accumulation of HSP17 in nuclei is not a heat or metal stress-dependent property. HSP17 is also expressed stress-independent in developing seeds and this HSP17 was mainly found in nuclei (zur Nieden et aI., 1995). There is no evidence for a nuclear localization signal in the proteins. A transport as a proteincomplex together with other nuclear proteins seems possible. The role of the LMW heat shock proteins in the cellular response to stress and during defined stages of development of different organisms was discussed in detail by Arrigo and Landry (1994). The HSP level of the cell detected after metal ion treatment is generally lower than after theat stress (see also Edelman et al., 1988; Delhaize et aI., 1989; Bournias-Vardiabasis et aI., 1990). The induction of the hsp gene transcription is extremely rapid in both cases and can be observed already after one min heat shock or metal stress. The maximum rates were achieved within 2 - 5 min (Figs. 4 and 5). Heat shock transcription factors were found to be activated in tomato cells also within 1.5 min of heat shock (Scharf et al., 1990). Therefore, different HSP levels of the cells under metal and heat stress require additional regulatory mechanisms, either on transcriptional or translational level. It seems also possible, that the stability of the hsp mRNAs is different under the various stress conditions (Edelman et aI., 1988; Kimpel et al.,1990). During heat shock drastic repression of normal protein synthesis is a general phenomenon. However, under comparable conditions the metal ion treated cells display only a slight inhibition of the normal translation pattern. This was also found under Cd and As stress in soybean seedlings (Edelmann et al., 1988), and under arsenite stress in sorghum seedlings (Howarth, 1990), and Tetrahymena periformis (Amaral et al., 1988). These differences were interpreted as translational control, which operates under heat shock (Storti et al., 1980) and failure of translational control under metal stress (Amaral et aI., 1988). Lindquist and McGarry (1986) suggested that specific leader sequences with a high affinity to ribosomes in the hsp mRNAs, which enable them to compete with «normal» messengers, could be responsible for specific translation of heat shock mRNAs. But this specificity should also function with hsp mRNAs induced by metal ions. As described earlier (Schoffl et aI., 1987; Vazquez et aI., 1993) transcription of rRNA genes is drastically reduced and finally completely stopped during heat stress and reactivated during recovery at normal temperature (Fig. 4). On the other hand, under metal stress transcription of rDNA is not influenced (Fig. 5). It seems possible, that in contrast to heat shock, under metal stress ribosomes and, therefore, the protein synthesizing system of the cell remains largely intact which results in synthesis of HSPs additionally to normal synthesis of the house keeping proteins. The mechanisms by which heat shock but not metal stress affects transcription of rRNA and other non-heat shock genes is not clear. A general inhibitory effect on the RNA polymerase molecules at high temperature, but also new initiation of transcription, altera-
741
tion of specific transcription factors or other mechanisms are possible (Vazquez et aI., 1993). Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 326). We are grateful to Prof. Lutz Nover (Frankfurt/ Main) for stimulating discussions. We also thank Dr. Norbert NaB for critical reading of the manuscript, Brigitte Werner and Karin Ohme for skillful technical assistance and Ruth Laue for kindly typing the manuscript.
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