Deactivation of Photosynthetic Activities is Triggered ...

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Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation- tolerant cyanobacterium, Nostoc commune, ...
Plant Cell Physiol. 45(7): 872–878 (2004) JSPP © 2004

Deactivation of Photosynthetic Activities is Triggered by Loss of a Small Amount of Water in a Desiccation-Tolerant Cyanobacterium, Nostoc commune Manabu Hirai, Ruriko Yamakawa, Junko Nishio, Takaharu Yamaji, Yasuhiro Kashino, Hiroyuki Koike and Kazuhiko Satoh 1 Department of Life Science, Graduate School of Life Science, University of Hyogo (Formerly: Himeji Institute of Technology), Harima Science Garden City, Hyogo, 678-1297 Japan ;

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccationtolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm′–F)/Fm′ values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss.

Introduction

Keywords: Desiccation tolerance — Hypertonic treatment — Nostoc commune — Photosystem I — Photosystem II — Terrestrial cyanobacterium. Abbreviations: 2,6-DCBQ, 2,6-dichloro-p-benzoquinone; DLE, millisecond delayed light emission; Fo and Fm, minimum and maximum levels of Chl fluorescence in the dark-adapted samples, respectively; Fm′ and F, maximum and steady state levels of Chl fluorescence in samples illuminated with actinic light, respectively; Fv, a variable part of Chl fluorescence (Fv = Fm – Fo); LCM, a core-membrane linker peptide; P700, a reaction center Chl dimer of PSI; PBS, phycobilisome.

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Corresponding author: E-mail, [email protected]; Fax, +81-791-58-0549. 872

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Nostoc commune, a well-known, terrestrial, and highly desiccation-tolerant cyanobacterium (Potts 2000), occupies an important ecological position in some areas on earth. It can survive for almost 100 years in a dry state in the dark (Cameron 1962). Under wet conditions, it can perform both photosynthetic CO2 fixation and nitrogen fixation under sunlight and grows relatively rapidly (Scherer et al. 1984). Therefore, N. commune has been thought to be a good material for studying desiccation-tolerance and also UV-absorbing substances, which protect the cell from strong sunlight (Ehling-Schalz et al. 1997, Hill et al. 1994). Drought tolerance of higher plants has been studied by many workers, and the increase in compatible solute inside the cells has been found to be important for this tolerance (for reviews, see Hoekstra et al. 2001). However, little work has been done on desiccation tolerance, especially on that of algae and cyanobacteria, and functions of compatible solutes in desiccation-tolerant cyanobacteria are not known. Recently we measured recovery processes of various photosynthetic reactions including energy transfer and photochemical reactions during rehydration of N. commune colonies (Satoh et al. 2002). During rehydration, phycobiliproteins and PSI complexes recovered their functional forms very quickly (within 1 min). PSII activities recovered rather slowly, but the amount of water which was needed to recover and maintain full PSII activities was only as much as twice the dry weight of the N. commune colonies. Inactivation of photochemical reactions of both photosystems (PSI and PSII), and quite large quenching of fluorescence from PSI and PSII complexes and phycobilisome (PBS), which can be attributed to structural changes of these pigment– protein complexes, were reported for the first time in desiccation-tolerant species (Satoh et al. 2002). For further understanding of the structural changes and inactivation mechanisms of PSI and PSII under drying processes and the function of compatible solutes in N. commune, studies on photosynthetic activities under hypertonic conditions should be useful because the cyanobacterium can be fixed at a certain stage of drying or rewetting process. In this work, therefore, we determined the osmotic pressure of the N. com-

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mune cells by measuring their water potential, measured photosynthetic activities of the cells under hypertonic conditions, and compared the effects of hypertonic treatments in N. commune with those in a desiccation-sensitive cyanobacterium, Synechocystis sp. PCC6803. N. commune cells had a rather low solute concentration, and the dissipation of light energy absorbed by pigment–protein complexes and deactivation of photochemical activities are induced by relatively mild hypertonic treatments, which may be closely related to the desiccation tolerance in N. commune.

Results and Discussion Water potential of N. commune colonies Cells of higher plants are known to increase their solute concentrations under drying conditions, which will protect the cells from water loss. However, we did not find the osmolarity of N. commune cells to be so high. In order to determine the osmotic pressure, we measured changes in water potential of N. commune colonies during dehydration (Fig. 1). The water content of a fully wetted N. commune colony is usually more than 30 times the dry weight of the colony. Since most of the water was lost by air-drying, most of it (about 75%) was free water in the matrix of the colony. When the water of the colony decreased from around 30 to 8 (relative to its dry weight), there was almost no change in the water potential; it was close to zero. Data on colonies with water contents higher than 20 were omitted in Fig. 1. The decrease in the water potential by the loss of water started when the relative water content became 8,

and the subsequent decrease in the water potential showed two phases when reciprocals of the values were plotted as a function of the water content (Fig. 1, insert). The initial decrease in the water potential from zero might correspond to the decrease in the turgor pressure of the cells, and the break point at around 6 in the water content, to the point where the turgor pressure becomes zero. When the water content was 6, the water potential was about –0.4 MPa, which corresponded to 0.25 M sorbitol solution. By extrapolation of the straight line in the range of the water content from 0 to 6 (Fig. 1, insert) to the point where the decrease in the water potential started (8 in the water content), the water potential with full turgor pressure can be calculated to be –0.33 Mpa. This value corresponds to 0.22 M sorbitol and suggests that the intracellular solute concentration of fully wetted N. commune is around 0.22 M, which will be supported by the following experiments. In this figure, we neglected the water remaining in the air-dried colonies because its amount is only 5% of the dry weight calculated after oven drying. Fluorescence emission spectra at 77K of N. commune and Synechocystis PCC6803 at various sorbitol concentrations Fluorescence emission spectra at 77K of N. commune colonies treated with various concentrations of sorbitol are shown in Fig. 2A. Up to 0.6 M sorbitol, fundamentally no change in the emission spectra was observed except that the fluorescence intensity at 695 nm (F695, fluorescence from the PSII core complex) increased at 0.4 M sorbitol. At 0.8 M sorbitol, F695 decreased markedly with little effect on F685 (fluorescence

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Fig. 1 Changes in water potential during dehydration of wet N. commune colonies. Wet colonies of N. commune were air-dried in the dark, and the water potential and weight of the colonies were measured during the drying process. After the measurement, the colonies were airdried for 1 d, and the minimum dry weight of the colonies was determined. Insert: reciprocals of the values of water potential were plotted against the relative amount of water remaining in the colonies. Three different symbols represent three independent measurements.

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from both PBS core-membrane linker (LCM) and PSII), suggesting that energy transfer from LCM to the PSII core complex was inhibited (Kura-Hotta et al. 1986, Satoh et al. 2002), and that the PSII core complex became non-fluorescent. The latter idea was supported by a decrease in F695 even with blue excitation light (with Toshiba V-42 and Corning 4–96 filters), which is absorbed mainly by Chl a (data not shown). With an increase in the sorbitol concentration, F685 and F735 (fluorescence from PSI) started to decrease, and at 1.5 M sorbitol, fluorescence from phycocyanin (F645) also became very small. The emission spectra in 1.5 M, 0.8 M, and 0.6 M sorbitol corresponded to those of dry colonies, 1, and 60 min after rehydration, respectively (Satoh et al. 2002). The colonies were incubated with various concentrations of sorbitol for more than 10 min because changes in the fluorescence intensity due to the hypertonic treatments had completed within 10 min (data not

Fig. 3 Light-induced redox changes of P700 in N. commune colonies (A) and in Synechocystis PCC6803 cells (B) incubated with various concentrations of sorbitol. All samples were added with 10 µM DCMU. Duration of the actinic light was 1 s.

shown). All these data suggest that water can move in and out of the cells very quickly and that the loss of a small amount of water from the cells is enough to cause a change of photosynthetic systems. Judging from the data that the cellular solute concentration corresponded to 0.22 M sorbitol, about 40% of intracellular water can be calculated to be withdrawn from the cells in 0.4 M sorbitol solution. We also measured the fluorescence emission spectra at 77K in Synechocystis sp. PCC6803, which has no desiccationtolerance (Fig. 2B). In contrast to N. commune, almost no change in emission spectra was observed even in Synechocystis PCC6803 cells incubated with up to 2.0 M sorbitol.

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Fig. 2 Fluorescence emission spectra at 77K of fragmented N. commune colonies (A) and of Synechocystis PCC6803 cells (B) incubated with various concentrations of sorbitol. For details, see Materials and Methods.

Desiccation and osmotic stresses in cyanobacteria

At 3.0 M and 3.6 M sorbitol, fluorescence intensities from PSI and PSII complexes and PBS became small, but ratios of the fluorescence intensities from these three pigment–protein complexes were almost the same. These results suggest that transfer of light energy absorbed by phycobilins and Chl molecules and the rate of energy dissipation to heat were not much changed by the loss of water in Synechocystis PCC6803 cells. The decrease in the fluorescence intensities in sorbitol solution higher than 2.0 M might be due to flattening effects caused by shrinkage of cells and thylakoid membranes. PSI and cyclic electron transport activities at various sorbitol concentrations Fig. 3 shows the time courses of light-induced redox changes of P700, a reaction center Chl dimer of PSI, in N. commune colonies (Fig. 3A) and Synechocystis PCC6803 cells (Fig. 3B) suspended in various concentrations of sorbitol. The maximum levels of P700 oxidized with 1 s of illumination are plotted in Fig. 4. In this experiment, in order to obtain the maximum photooxidation level of P700, we added 10 µM DCMU, which inhibits electron transport from PSII, to the surrounding media. As the sorbitol concentration increased, the maximum photooxidation level of P700 was decreased and reached a stationary level at the sorbitol concentration of 2.0 M in the both species. However, the stationary level in N. commune (Fig. 4, open circles) was almost a half of that in Synechocystis PCC6803 (Fig. 4, filled circles). On turning off the actinic light, P700 was re-reduced quickly in the absence of sorbitol (within 500 ms), showing a high activity of cyclic electron flow around PSI in both cyanobacteria (Fig. 3, 5). In N. commune, re-reduction of P700 was totally inhibited by the presence of 3.0 M sorbitol. On the other

Fig. 5 Extents of re-reduced P700 as a function of the sorbitol concentration. Extents of P700 re-reduced at 500 ms after turning off the actinic light relative to the extent of photooxidized P700 were measured in N. commune (open circles) and in Synechocystis PCC6803 (filled circles) after incubation in various concentrations of sorbitol.

hand, a high rate of P700 re-reduction was observed in Synechocystis PCC6803 at any sorbitol concentration tested (compare A and B in Fig. 3). Movement of plastocyanin or cytochrome c6 in the lumen of thylakoids must be retarded by the sorbitol treatment due to the increase in the lumen viscosity, but it seems to be rapid enough not to become a rate-limiting step in the cyclic electron flow. It is also possible to imagine that the cyclic electron flow was inhibited, but P700 was rereduced by back electron flow from an iron-sulfur center, Fx or FA/FB, in Synechocystis PCC6803. However, we think this rereduction was due to cyclic electron flow because electron transport in Synechocystis PCC6803 under hypertonic conditions is quite active (for details, see below). In terms of the time course of P700 photooxidation, it became biphasic in N. commune incubated in 0.2–1.0 M sorbitol (Fig. 3A). The rate of P700 photooxidation is affected by three main factors under usual dark-adapted conditions: the actinic light intensity, the rate of electron donation to oxidized P700, and the photoactivation state of an electron carrier on the reducing side of PSI (Satoh 1982). Because we used the same actinic light, and the re-reduction rate of P700 seemed not much changed in this sorbitol range (Fig. 4), we suppose that photoactivation of the electron carrier on the reducing side of PSI was decelerated. In any case, the data show that even changes in turgor pressure of the cells, which reflect the early dehydration stage, modified the electron transfer activities around P700. These results also support the idea that, in addition to the PSII activity, the PSI activity was actively deactivated during

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Fig. 4 Extents of photooxidized P700 as a function of the sorbitol concentration. Maximum levels of photooxidized P700 shown in Fig. 3 were plotted for N. commune (open circles) and Synechocystis PCC6803 (closed circles).

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dehydration in a desiccation-tolerant cyanobacterium, N. commune (Satoh et al. 2002). Although we cannot determine the exact deactivated site from the present data, the function of an electron transport component(s) in the cyclic electron flow might be controlled by the water content inside the N. commune cells, a phenomenon not observed in desiccation-sensitive Synechocystis PCC6803 cells. PSII activities at various concentrations of sorbitol Although the fluorescence intensities from phycobiliproteins are high in cyanobacteria, they usually do not change during illumination. Therefore, the Fv/Fm and (Fm′–F)/Fm′ values, in cyanobacteria, can be regarded to reflect (not equal) the maximal efficiency of the PSII photochemical reaction and the quantum yield of PSII in the light-adapted state, respectively (Inoue et al. 2001, Satoh et al. 2002). Fig. 6 shows changes in these fluorescence parameters in N. commune colonies and in Synechocystis PCC6803 cells incubated with various concentrations of sorbitol. In the N. commune colonies, the Fv/Fm value started to decrease at 0.6 M sorbitol and reached zero at 1.0 M sorbitol. The (Fm′–F)/Fm′ value started to decrease at lower concentrations and reached zero at 0.8 M sorbitol. The decrease in these fluorescence parameters corresponded to that of the fluorescence intensity at 695 nm, which is emitted by PSII core complexes (Fig. 2A). The cause of the increase in the Fv/Fm value at around 0.6 M sorbitol was not clear (Fig. 6). However, the increase in F695 at 0.4 M sorbitol (Fig. 2A) suggests that state transition is related to this phenomenon. Stamatakis and Papageorgiou (2001) showed that the osmotic pressure of the suspending medium affects energy transfer from PBS to PSI and PSII. By contrast, Synechocystis

Fig. 7 Effects of the sorbitol concentration on oxygen evolution activities supported by NaHCO3 (squares) or 2,6-DCBQ (circles) in fragmented N. commune colonies (open symbols) or Synechocystis PCC6803 cells (filled symbols). After incubation of the samples with various concentrations of sorbitol, oxygen evolution was measured in the same sorbitol solution. The rate of oxygen evolution in the presence of NaHCO3 or 2,6-DCBQ at 0 M sorbitol was 165 or 75.6 µmol O2 (mg Chl)–1 for Synechocystis PCC6803 and 51.9 or 23.2 µmol O2 (mg dry weight)–1 for N. commune, respectively.

PCC6803 maintained high Fv/Fm values even at quite high concentrations of sorbitol (Fig. 6, filled circles). Although the (Fm′–F)/Fm′ value in Synechocystis PCC6803 decreased in the same concentration range of sorbitol as in N. commune, it did not reach zero at 1.0 M sorbitol. In the sorbitol concentration range from 0.8 M to 3.0 M, the (Fm′–F)/Fm′ value was almost constant; about one-fourth of the initial value. We also measured the Fv/Fm and (Fm′–F)/Fm′ values in Fischerella muscicola, which is aquatic and has no drought-tolerance. F. muscicola gave results similar to those provided by Synechocystis PCC6803 (data not shown). Photosynthetic activities were also measured in fragmented N. commune colonies and in Synechocystis PCC6803 cells using a Clark-type oxygen electrode in the presence of NaHCO3 or 2,6-dichloro-p-benzoquinone (2,6-DCBQ) (Fig. 7), which is known as an efficient artificial electron acceptor of PSII (Satoh et al. 1992). In N. commune colonies, O2 evolution in the presence of NaHCO3 and 2,6-DCBQ decreased to zero at 0.8 M sorbitol. On the other hand, in Synechocystis PCC6803, O2 evolution in the presence of NaHCO3 could be detected at up to 2.5 M sorbitol, and a relatively high rate of O2 evolution in the presence of 2,6-DCBQ was observed even at 3.5 M sorbitol. With NaHCO3, unexpected increases and decreases in oxygen evolution at around 0.2 M sorbitol were observed for Synechocystis PCC6803 and N. commune, respectively, the reasons of which are unknown. However, these results are funda-

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Fig. 6 Fv/Fm (circles) and (Fm′–F)/Fm′ (squares) values in N. commune colonies (open symbols) or Synechocystis PCC6803 cells (filled symbols) incubated with various concentrations of sorbitol. These values are not equal but reflect the maximal efficiency of the PSII photochemical reaction and the quantum yield of PSII in the light-adapted state, respectively.

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mentally consistent with the data on the Fv/Fm and (Fm′–F)/ Fm′ values shown in Fig. 6. Intensity of delayed light emission at various concentrations of sorbitol The millisecond delayed light emission (DLE) is produced by the charge recombination between an oxidized electron donor and a reduced acceptor of PSII located on the opposite side of the thylakoid membranes (Itoh et al. 1971). Therefore, it is affected by the electrochemical potential difference that consists of the difference in H+ concentrations (∆pH) and the electric field (∆ψ) across the membranes; thus, it provides information about the high energy state of the membranes (Lavorel 1975, Malkin 1977). Fig. 8 shows time courses of DLE in N. commune colonies and Synechocystis PCC6803 cells incubated with various concentrations of sorbitol. In the N. commune colonies, the DLE intensity started to decrease at 1.0 M sorbitol and became almost null at 4.0 M sorbitol. By contrast, Synechocystis PCC6803 maintained a high DLE intensity at quite high concentrations of sorbitol. These results suggest that the semipermeability of the thylakoid membranes was not affected by the water loss due to hypertonic treatment with up to 4.0 M sorbitol. In N. commune, Fv decreased to almost zero at 1.0 M sorbitol (Fig. 6), but the cyclic electron flow around PSI can be clearly observed at this sorbitol concentration (Fig. 3A, 5). This could be the reason why DLE is more resistant to water loss than the Fv/Fm value in N. commune. The high DLE intensity

in Synechocystis PCC6803 even at 4.0 M sorbitol is consistent with its high PSII and cyclic electron flow activities at high sorbitol concentrations. In conclusion, we believe that a certain stage of a hydration or dehydration process in N. commune can be constantly and stably obtained by changing the sorbitol concentration of the solution in which the N. commune colonies are incubated, and that the solute concentration in fully wetted N. commune cells is not high (corresponds to around 0.22 M sorbitol). This idea is consistent with the result that (Fm′–F)/Fm′ decreased largely at 0.4 M sorbitol (Fig. 6), which suggests that the solute concentration is less than 0.4 M. It has also become clear that desiccation tolerance of N. commune is not brought about by preservation of water, instead, it obtained an ability to deactivate photosynthetic processes on sensing the decrease in the water content. It sounds peculiar that desiccation-tolerant N. commune is more sensitive to water loss than sensitive species with regard to the photochemical reaction center activities. However, when photosynthetic CO2 fixation is inhibited, active reaction centers will produce active oxygen species and destroy the cells (Anderson and Barber 1996). Excited Chl molecules can also produce singlet oxygen, which causes oxidation of lipids, etc. Under natural conditions, N. commune grows in open areas and is frequently irradiated by strong sun light after slight rain or dewfall. If N. commune has a capacity of photochemical reactions in excess of its CO2 fixation activity as in the case of desiccation-sensitive species under low water contents (Fig. 6, 7), it might be unable to grow or even survive under these

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Fig. 8 Millisecond DLE in fragmented N. commune colonies (traces a–e) and in Synechocystis PCC6803 cells (traces f–j) at various concentrations of sorbitol. After incubation of the samples with various concentrations of sorbitol, DLE was measured in the same sorbitol solution. The concentrations of sorbitol used are shown below the time courses of DLE.

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natural conditions. Therefore, deactivation of the PSII reaction center activity and cessation of energy transfer from PBS to PSII, which occurred in an early stage of desiccation, and quenching of light energy absorbed by pigment–protein complexes must be very important for desiccation tolerance in N. commune. Further work to confirm this view is in progress.

Materials and Methods

Measurements of water potential The water potential of various concentrations of sorbitol and N. commune colonies was measured at 25°C using a WP4-T Potentia Meter (Decagon Devices, WA, U.S.A.). Values of water potential 30– 60 min after setting the samples into the potentiometer were transferred to a personal computer. The amount of water remaining in the dried colonies was estimated by the decrease in the weight after incubation of the air-dried colonies in a dry sterilizing oven at 120°C for 30 min. Measurements of photosynthetic activities Chl fluorescence was measured using a PAM 101/3 Chl fluorometer (Walz, Germany) as stated by Yamane et al. (1997). The intensity of the measuring light was 0.17 µmol photons m–2 s–1. Light-induced redox changes of P700 were also measured with a PAM 101/3 fluorometer, equipped with a dual-wavelength emitter-detector unit, ED-P700DW. The intensity of the actinic light was 350 µmol photons m–2 s–1. Photosynthetic CO2 fixation or the PSII oxygen-evolving activity was calculated from oxygen evolution measured with a Clark-type oxygen electrode under saturating white light. The reaction mixture contained BG11 medium supplemented with 5 mM NaHCO3 or 1.0 mM 2,6-DCBQ. For N. commune, fragmented colonies corresponding to 7.5 mg dry weight ml–1 were used. Millisecond DLE was measured with the passage of time at 25°C as reported by Satoh and Katoh (1983). Measurements of fluorescence emission spectra Fluorescence emission spectra at 77K were obtained using a laboratory-constructed spectrophotometer as reported by Yamane et al. (1997). The excitation light from a 12 V, 100 W halogen lamp was passed through a Corning 4–96 filter. Where indicated, a Toshiba V-42 filter was also used together with a Corning 4–96 filter. N. commune colonies on quartz glass fiber were quickly frozen at 77K by dipping the glass fiber into liquid nitrogen. Cell suspensions of Synechocystis PCC6803 and F. muscicola were put into a brass cuvette and were frozen at 77K as reported by Yamane et al. (1997).

The present work was partly supported by a grant (21st Century Center of Excellence Program) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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(Received December 9, 2003; Accepted April 12, 2004)

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Materials and preparation of fragmented colonies Colonies of N. commune were collected in and around the Harima Science Garden City Campus of Himeji Institute of Technology, Hyogo Prefecture, Japan (134.5°E, 35′N). Unless otherwise stated, the colonies were dried for more than 1 week at 25°C under room light (humidity, 61%). In some measurements, these dried colonies were cut into small pieces using a blender with little damage to the individual cells. Synechocystis sp. PCC6803 was grown at 30°C in BG11 medium as reported previously (Inoue et al. 2000). Cells corresponding to 15 µg Chl ml–1 were used in all the measurements. F. muscicola Gomont was isolated from a hot spring of Gunai, Hokkaido, Japan and was grown as in Synechocystis PCC6803 except that the growth temperature was increased to 40°C. The cells were suspended in BG11 medium containing various concentrations of sorbitol and were incubated for more than 10 min before measurements of activities.

Acknowledgments