Institute of Plant Physiology, Russian Academy of Science, Botanicheskaya Street 35, 127276 Moscow, Russian Federation
Correspondence
Dmitry A. Los
losnet{at}ippras.ru
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ABSTRACT |
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INTRODUCTION |
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It is well known that Ca2+ plays a role as a secondary messenger in stimulusresponse coupling in eukaryotic organisms, including plants, and it is involved in acquisition of their tolerance to a variety of environmental stress conditions (Knight et al., 1997; Sanders et al., 1999
; Trewavas & Malho, 1998
). In prokaryotic cells, the role of Ca2+ in regulation of stress responses has not been clearly demonstrated, but evidence of its involvement in regulation of temperature stress responses, pathogenesis, chemotaxis, differentiation and the cell cycle is now being accumulated (Norris et al., 1996
; Holland et al., 1999
; Jones et al., 1999
).
Some reports indicate that the resting level of cytosolic free Ca2+ in bacterial (Jones et al., 1999) and cyanobacterial cells (Torrecilla et al., 2000
) is in the submicromolar range, i.e. similar to that found in eukaryotic cells. In addition, direct evidence that Ca2+ signalling exists in cyanobacteria has become available recently (Torrecilla et al., 2000
). However, little is known about the Ca2+ transporters, including Ca2+ channels, that are responsible for the maintenance of Ca2+ homeostasis in cyanobacteria and other prokaryotes (Torrecilla et al., 2000
). Most likely, the Ca2+ status of cyanobacterial cells is primarily maintained by the activity of Ca2+ transport proteins in their plasma membrane (PM). Thus, the difference in the electrical potential across the PM might be essential in controlling the cellular level of Ca2+.
Some mechanosensitive ion channels were shown to be responsible for the permeability of cell membranes to Ca2+ (Ding & Pickard, 1993a, b
), and thus they might be involved in Ca2+ signalling under certain stress conditions, among which hypo-osmotic stress is well characterized. Cells of Synechocystis are equipped with the mechanosensitive ion channel MscL, which is located in their PM. However, the exact function of the channel in this freshwater cyanobacterium, which is normally not exposed to hypotonic stress, is unknown.
One of the approaches to reveal the activity of Ca2+ channels in the PM is to follow Ca2+ translocation across this membrane in response to perturbation of cellular Ca2+ homeostasis with agents that can depolarize the membrane. In the present study, we applied such an approach, with the use of the Ca2+-sensitive dye arsenazo III (Thomas, 1982), to test the functioning of Ca2+ transporters in the PM of wild-type Synechocystis and of a mutant deficient in the mechanosensitive ion channel MscL. We demonstrate that cells of Synechocystis are capable of releasing Ca2+ into the assay medium in response to depolarization of the PM caused by treatment of cells with valinomycin in the presence of K+ ions. Ca2+ release from the wild-type cells was very rapid, temperature-dependent, and inhibited by verapamil (Ca2+ channel blocker) and amiloride (mechanosensitive channel blocker). Ca2+ release strongly decreased in the
MscL mutant cells, and became practically temperature-independent. Our results suggest that MscL is involved in regulation of Ca2+ homeostasis in Synechocystis cells under temperature-stress conditions.
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METHODS |
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The MscL-deficient mutant of Synechocystis.
A single gene that encodes a homologue of the mechanosensitive channel MscL (Slr0875) in Synechocystis was identified by homology search in CyanoBase (http://www.kazusa.or.jp/cyano/Synechocystis/index.html) using the amino acid sequence of the MscL of Escherichia coli (Sukharev et al., 1994) as a query sequence. A DNA fragment that contained the mscL gene and its flanking regions was amplified from the genomic DNA of Synechocystis by PCR with the following primers: mecF (5'-GACACAAGCCCGGGTTAAAGTTGAAC-3') and mecR (5'-ACCAATCTAGAGAGTGTAATTGGTGC-3'). The MscL-null mutant (
MscL) of Synechocystis was produced by inserting a kanamycin-resistance gene, derived from plasmid pUC4KIXX (Pharmacia) by digestion with BamHI, into the unique BglII site of the slr0875 (mscL) gene of Synechocystis (Fig. 1
A). Transformation of Synechocystis cells was done as described by Williams (1988)
. Complete segregation of the recombinant chromosomes in the mutant strain was confirmed by PCR with the above-listed primers (Fig. 1B
).
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Detection of putative Ca2+ stores within Synechocystis cells.
The presence of putative intracellular Ca2+ stores in the cyanobacterial cells was assayed by recording the increase in chlortetracycline (CTC) fluorescence during the uptake of this Ca2+ indicator by the cells (Dixon et al., 1984). The assay was performed in the same medium (1 ml) as used for detection of Ca2+ release from the cells but lacking magnesium sulfate and supplemented with CTC at 25 µM. The fluorescence measurements were carried out at room temperature in an unstirred 1 cm light-path cuvette of a Hitachi 850 fluorescence spectrophotometer set at 380 nm (excitation) and 530 nm (emission).
Detection of membrane potential on the PM of Synechocystis cells and its dissipation in the presence of valinomycin/K+ or tetraphenylphosphonium (TPP+).
The presence of membrane potential on the PM of Synechocystis cells was assayed by recording fluorescence changes of the potential-sensitive cyanine dye diS-C3-(5) (Waggoner, 1974). The assay medium (1 ml) had the same composition as that used for detection of Ca2+ release from the cells, but it was supplemented with 1 µM diS-C3-(5). Fluorescence of diS-C3-(5) was measured at desired temperature in an unstirred 1 cm light-path cuvette of a Hitachi 850 fluorescence spectrophotometer set at 620 nm (excitation) and 670 nm (emission).
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RESULTS |
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Ca2+ release induced by valinomycin appeared only in the presence of K+ ions in the assay medium, and no release was observed in the absence of K+ (Fig. 2A). This finding suggests that valinomycin catalyses the influx of K+ ions from the assay medium into the cells, thus leading to depolarization of the PM, i.e. a collapse of the membrane potential which is maintained by the cells under optimal growth conditions. To validate this suggestion, the putative sensitivity of membrane potential on the PM of the Synechocystis cells to the combined action of valinomycin and K+ ions in the assay medium was directly tested by using the potential-sensitive probe diS-C3-(5). Fig. 3
(A) shows the kinetics of distribution of this probe between the assay medium and the cells. It can be seen that valinomycin added to the cells after achieving a steady state of diS-C3-(5) fluorescence quenching had practically no effect on this value. However, subsequent addition of K+ ions to the cells initiated a pronounced increase in fluorescence of the probe, clearly indicating depolarization of the PM. Moreover, a similar, and even more marked, effect was also observed in the presence of TPP+, which is known as a simulator of the action of valinomycin/K+, suggesting that depolarization of the PM indeed occurs (Fig. 3A
).
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Detection of putative Ca2+ stores inside the cyanobacterial cells
The above findings strongly suggest that valinomycin/K+ treatment of the cells induced mobilization of Ca2+ from intracellular Ca2+ stores. Although the resting level of intracellular free Ca2+ in Synechocystis cells is expected to be low enough and similar to the intracellular Ca2+ levels reported for a variety of eukaryotic cells (Knight et al., 1997; Trewavas & Malho, 1998
), the presence of Ca2+ stores cannot be excluded. In order to test this suggestion, we attempted to detect the presence of such stores in the cells by following fluorescence of the Ca2+ indicator CTC. which is capable of monitoring sequestered Ca2+ at concentrations of 0·130 mM (Dixon et al., 1984
). Fig. 4
shows a gradual increase in CTC fluorescence that results from its passive equilibration with the entrapped Ca2+ within both the wild-type and mutant cells. The kinetics of this process for both types of cells was about the same, and the observed fluorescence signal after achieving a steady-state level is rapidly reversed by the subsequent addition of the Ca2+ chelator EGTA to the assay medium. Thus, the observed fluorescence emanated primarily from CTCCa2+ complexes bound to some unknown structures within the cells.
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Effect of temperature on the Ca2+ release and membrane potential of the cells
Since the activity of Ca2+ channels in plants may be modulated by temperature (Ding & Pickard, 1993b; Plieth, 1999
), we addressed the question whether the release of Ca2+ caused by depolarization of the PM is temperature-dependent in Synechocystis cells. Fig. 6
shows the Ca2+ release from the cells at 20 and 30 °C. It can be seen that the intensity of the observed response of arsenazo III in wild-type cells appeared to be markedly lower at 20 °C than that at 30 °C.
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The observed suppression of the Ca2+ release from the wild-type cells at 20 °C could be due to enhanced depolarization of the cell membranes at lower temperatures. We tested this possibility by direct recording of the membrane potential of the cells at different temperatures with the use of diS-C3-(5). Fig. 3(B) shows that both the wild-type and mutant cells exhibited pronounced depolarization of the PM in response to a temperature decrease from 32 to 20 °C, as judged by a marked reduction of the TPP+-induced increase in diS-C3-(5) fluorescence with a decrease in temperature. This figure also demonstrates another notable fact, already mentioned above, that the resting membrane potential of the mutant cells also declines with a decrease in temperature, and their behaviour in this respect differs only slightly from that of the wild-type cells.
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DISCUSSION |
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The release of Ca2+ from Synechocystis cells suggests that the Ca2+ gradient, which is directed towards the extracellular medium, exists on the PM. This gradient might be formed due to the presence of intracellular Ca2+ store(s) in the cells. This proposal is supported by the results showing the increase in fluorescence intensity of CTC resulting from its passive equilibration with the entrapped Ca2+ within the cells (Fig. 4). Although the nature of the detected Ca2+ store(s) remains unclear, the putative Ca2+ pool in the cells is most likely associated with the PM, because their treatment with EGTA resulted in a rapid disappearance of the fluorescence signal from CTC (Fig. 4
). A recent report on the cyanobacterium Anabaena PCC 7120 also indicated the presence of intracellular Ca2+ stores in these cells (Torrecilla et al., 2000
).
Nature of the putative Ca2+ channels in the PM of the cyanobacterial cells
The results obtained by application of the ion channel blockers verapamil and amiloride (Fig. 5) allow us to suggest that the release of Ca2+ from the intracellular space occurs through a special type of Ca2+ channels, most likely voltage-gated, in the PM. The transient nature of the verapamil/amiloride-sensitive Ca2+ release is in accordance with this conclusion, because there is evidence that during sustained depolarization voltage-gated Ca2+ channels undergo very rapid transition to a nonconducting, inactive state (Cens et al., 1999
). This feature of the Ca2+ release and the conditions that are required for its activation suggest that opening of voltage-gated Ca2+ channels in the PM results from its depolarization.
In eukaryotic cells, such channels are responsible, as a rule, for Ca2+ influx into the intracellular space under environmental stress conditions (Knight et al., 1991; Hamilton et al., 2000
). In cyanobacteria, however, nothing is known about the functioning of Ca2+ channels in the PM. It is interesting to note that in the cyanobacterium Anabaena PCC 7120, verapamil did not inhibit a transient burst of intracellular free Ca2+ induced by an increase in concentration of external Ca2+, but was able to maintain the resting value of free Ca2+ at a rather high level (Torrecilla et al., 2000
). However, it remains unclear whether this effect was, in fact, due to interaction of verapamil with some outward Ca2+ channels in the PM.
MscL as a Ca2+ channel
The data obtained with the MscL mutant of Synechocystis suggest that the MscL mechanosensitive ion channel operates in the PM of these cells as a substantial contributor to the Ca2+ release induced by the membrane depolarization. Ca2+ release was significantly suppressed in the mutant cells as compared to that observed in the wild-type cells (Figs 2, 5 and 6
). The data presented in Figs 3 and 4
show that alternative explanations for this result based on a lower value of the transmembrane Ca2+ gradient on the PM of the mutant cells, or a greatly increased initial depolarization of these cells as compared to those of the wild-type cells, can be excluded.
The temperature dependence of the Ca2+ release dramatically changed in the mutant cells as compared to that in the wild-type cells (Fig. 6). A possible explanation for this phenomenon is temperature dependence of the activity of the MscL (Ding & Pickard, 1993b
; Kikuyama & Tazawa, 2001
), which might be the reason for the observed temperature dependence of the Ca2+ release in the wild-type cells of Synechocystis PCC 6803.
The intensive Ca2+ release from the wild-type cells at 32 °C caused by the valinomycin/K+-induced depolarization of the PM and relatively low intensity of such release at 20 °C might be due to the fact that cold stress itself causes depolarization of the PM and thus simulates the valinomycin/K+ effect (Fig. 3B). Such an action of cold stress was most likely responsible for the observed attenuation of the valinomycin/K+-induced Ca2+ release from the cells. Since the mutation in MscL only strongly inhibited (by about 50 %) but did not abolish completely the depolarization-induced Ca2+ release, it is reasonable to suggest that additional Ca2+ channels other than MscL are present in the PM of the cells of Synechocystis. Taking into account the fact that in the
MscL mutant cells the Ca2+ release was also observed and it was sensitive to verapamil and amiloride, it is possible that other putative Ca2+ channels also have a mechanosensitive nature.
In the MscL mutant cells of Synechocystis, the Ca2+ release was only slightly affected by temperature, suggesting that the MscL might be the main channel that controls the Ca2+ release under temperature stress conditions.
At present, the mechanisms that activate the MscL as a result of depolarization of the PM of Synechocystis remain unclear. At the same time, despite the generally accepted model of stretch-induced opening of the MscL (Blount & Moe, 1999; Sukharev, 1999
; Martinac, 2001
; Sukharev et al., 2001
), some recent reports demonstrate both the voltage-induced and stretch-independent activation of mechanosensitive ion channels, as well as voltage-induced changes in membrane tension (Gil et al., 1999
; Reifarth et al., 1999
; Zimmerman & Sentenac, 1999
). The latter effect, which is probably caused by changes in electromechanical compression of the lipid bilayer (Needham & Hochmuth, 1989
; Menconi et al., 2001
), provides a possible explanation for our results as well. Such an interpretation is in accordance with the known cross-sensitivity of cell membrane ion channels to factors of different physical nature, such as temperature and osmotic stresses (Ding & Pickard, 1993a
, b
; Martinac et al., 1990
; Jones et al., 2000
; Marchenko & Sage, 2000
; Kikuyama & Tozawa, 2001
). Based on the data cited above, it could be expected that the MscL of the freshwater Synechocystis may be activated, albeit to different extents, by different stress factors.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cens, T., Restituito, S., Galas, S. & Charnet, P. (1999). Voltage and calcium use the same molecular determinants to inactivate calcium channels. J Biol Chem 274, 54835490.
Ding, J. P. & Pickard, B. G. (1993a). Mechanosensory calcium-selective cation channels in epidermal cells. Plant J 3, 83110.[CrossRef][Medline]
Ding, J. P. & Pickard, B. G. (1993b). Modulation of mechanosensitive calcium-selective cation channels by temperature. Plant J 3, 713720.[CrossRef][Medline]
Dixon, D., Brandt, N. & Haynes, D. H. (1984). Chlorotetracycline fluorescence is a quantitative measure of the free internal Ca2+ concentration achieved by active transport. In situ calibration and application to bovine cardiac sarcolemmal vesicles. J Biol Chem 259, 1373713741.
Gil, Z., Silberberg, S. D. & Magleby, K. L. (1999). Voltage-induced activation of mechanosensitive cation channel in oocytes of Xenopus laevis. Proc Natl Acad Sci U S A 96, 1459414599.
Glatz, A., Vass, I., Los, D. A. & Vígh, L. (1999). The Synechocystis model of stress: from molecular chaperones to membranes. Plant Physiol Biochem 37, 1112.
Hamill, O. P., Lane, J. W. & McBride, D. W., Jr (1992). Amiloride: a molecular probe for mechanosensitive channels. Trends Pharmacol Sci 13, 373376.[CrossRef][Medline]
Hamilton, D. W. A., Hills, A., Kohler, B. & Blatt, M. R. (2000). Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid. Proc Natl Acad Sci U S A 97, 49674972.
Holland, I. B., Jones, H. E., Campbell, A. K. & Jacq, A. (1999). An assessment of the role of intracellular free Ca2+ in E. coli. Biochimie 81, 901907.[CrossRef][Medline]
Hosey, M. M. & Lazdunski, M. (1988). Calcium channels: molecular pharmacology, structure and regulation. J Membr Biol 104, 81105.[Medline]
Jones, H. E., Holland, I. B., Baker, H. L. & Campbell, A. K. (1999). Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium. Cell Calcium 25, 265274.[CrossRef][Medline]
Jones, S. E., Naik, R. R. & Stone, M. O. (2000). Use of small fluorescent molecules to monitor channel activity. Biochem Biophys Res Commun 279, 208212.[CrossRef][Medline]
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3, 109136.[Medline]
Kikuyama, M. & Tazawa, M. (2001). Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis. Plant Cell Physiol 42, 358365.
Knight, M. R., Campbell, A. K., Smith, S. M. & Trewavas, A. J. (1991). Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352, 524526.[CrossRef][Medline]
Knight, H., Trewavas, A. J. & Knight, M. R. (1997). Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J 12, 10671078.[CrossRef][Medline]
Los, D. A. & Murata, N. (1999). Responses to cold-shock in cyanobacteria. J Mol Microbiol Biotechnol 2, 221230.
Los, D. A. & Murata, N. (2000). Regulation of enzyme activity and gene expression by membrane fluidity. Science's Signal Transduction Knowledge Environment http://www.stke.org/cgi/content/full/OC_sigtrans; 2000/62/pe1.
Marchenko, S. M. & Sage, S. O. (2000). Hyperosmotic but not hypoosmotic stress evokes a rise in cytosolic Ca2+ concentration in endothelium of intact rat aorta. Exp Physiol 85, 151157.[Abstract]
Martinac, B. (2001). Mechanosensitive channels in prokaryotes. Cell Physiol Biochem 11, 6176.[CrossRef][Medline]
Martinac, B., Adler, J. & Kung, C. (1990). Mechanosensitive ion channels of E. coli activated by amphipaths. Nature 348, 261263.[CrossRef][Medline]
Menconi, M. C., Pellegrini, M. & Pellegrino, M. (2001). Voltage-induced activation of mechanosensitive cation channels in neurons. J Membr Biol 180, 6572.[CrossRef][Medline]
Needham, D. & Hochmuth, R. M. (1989). Electro-mechanical permeabilization of lipid vesicles. Role of membrane tension and compressibility. Biophys J 55, 10011009.[Abstract]
Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F. N., Toth, I., Trinei, M., Modha, K. & Norman, R. I. (1996). Calcium signaling in bacteria. J Bacteriol 178, 36773682.
Plieth, C. (1999). Temperature sensing by plants: calcium-permeable channels as primary sensors a model. J Membr Biol 172, 121127.[CrossRef][Medline]
Reifarth, F. W., Clauss, W. & Weber, W. M. (1999). Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis. Biochim Biophys Acta 1417, 6376.[Medline]
Sanders, D., Brownlee, C. & Harper, J. F. (1999). Communicating with calcium. Plant Cell 11, 691706.
Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35, 171205.[Medline]
Sukharev, S. (1999). Mechanosensitive channels in bacteria as membrane tension reporters. FASEB J 13 Suppl, S55S61.
Sukharev, S. I., Blount, P., Martinac, B., Blattner, F. R. & Kung, C. (1994). A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265268.[CrossRef][Medline]
Sukharev, S., Betanzos, M., Chiang, C. S. & Guy, H. R. (2001). The gating mechanism of the large mechanosensitive channel MscL. Nature 409, 720724.[CrossRef][Medline]
Thomas, M. V. (1982). Metallochromic indicators. In Techniques in Calcium Research, pp. 90138. London: Academic Press.
Torrecilla, I., Leganes, F., Bonilla, I. & Fernandez-Pinas, F. (2000). Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant Physiol 123, 161176.
Trewavas, A. J. & Malho, R. (1998). Ca2+ signalling in plant cells: the big network! Curr Opin Plant Biol 1, 428433.[CrossRef][Medline]
Waggoner, A. S. (1974). Dye indicators of membrane potential. Annu Rev Biophys Bioeng 8, 4768.
Williams, J. G. K. (1988). Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol 167, 766778.
Zimmerman, S. & Sentenac, H. (1999). Plant ion channels: from molecular structures to physiological functions. Curr Opin Plant Biol 2, 477482.[CrossRef][Medline]
Received 25 October 2002;
revised 23 December 2002;
accepted 6 January 2003.