Crucial Role of Extracellular Polysaccharides in Desiccation and Freezing Tolerance in the Terrestrial Cyanobacterium Nostoc commune

Abstract

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O₂ evolution was not detected in desiccated colonies, the ability of the cells to evolve O₂ rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O₂ evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O₂, but O₂ evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O₂ evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution; it ranges from the tropics to the polar regions of the Earth (17). In its native habitats, N. commune forms macroscopic colonies that consist of extracelluar polysaccharides (EPS) and filamentous cells embedded in the EPS. N. commune colonies are naturally subjected to regular cycles of desiccation and wetting. It has been found that N. commune stored in a desiccated state remains able to grow for more than 100 years (2, 11). Since N. commune does not differentiate into akinetes (spores), this organism must have unique mechanisms for adaptation to desiccation.

Physiological changes in N. commune during the rewetting and drying processes have been studied previously (9, 18, 19). Respiration recovers rapidly (within 30 min) but photosynthesis recovers slowly (6 to 8 h) after rehydration, and recovery of nitrogen fixation takes place 120 to 150 h after rehydration (19). Recently, it has been shown that the light-harvesting antennae and photosystem I quickly recover their functional forms as soon as 1 min after rehydration, but recovery of the activity of photosystem II takes 4 h (18). Photosynthesis ceases under hypertonic conditions, and the deactivation of photosynthesis apparently takes place in response to water loss (9). The cessation of photosynthetic electron transport can be considered an acclimative response to desiccation, but the mechanism has not been elucidated yet.

Production of EPS is widespread in cyanobacteria (1, 5). EPS in N. commune colonies account for more than 60% of the dry weight (8) and are composed of various sugars, including glucose, galactose, and xylose, and uronic acid (6, 10). In spite of the importance of EPS, the structure of EPS remains to be elucidated.

It is believed that EPS in cyanobacteria play a major role in protecting cells from various stresses in severe habitats, although the experimental evidence for the functions of EPS is limited. Philippis and Vincenzini (16) have described the role of EPS in the attachment of cells to sediment in benthic cyanobacteria, in facilitating the homogeneous dispersion of trichomes, and in the protection of nitrogenase from the harmful effects of oxygen. The production of EPS responds to the growth regimen in some Nostoc species, and EPS may serve as a sink for excess fixed carbon under unbalanced C/N metabolism conditions (14, 15). During the rehydration of desiccated colonies conspicuous changes in EPS of N. commune occur (20), and based on in vitro experiments EPS is thought to contribute to the stability of membrane vesicles (8).

The role of EPS in the protection of photosynthesis in cyanobacteria has not been directly demonstrated. In this study, EPS-depleted cells of N. commune were prepared to determine the role of EPS in the desiccation tolerance of photosynthesis. To our knowledge, this is the first report of a physiological function of EPS for adaptation to terrestrial environments in cyanobacteria.

MATERIALS AND METHODS

Organisms.

Colonies of N. commune that naturally grew in the field were collected from the Kakuma Campus of Kanazawa University in Japan. The field samples were washed with tap water to remove the soil, air dried, and stored at room temperature until they were used for experiments. Laboratory strain KU002 of N. commune, which was isolated from the Kakuma Campus of Kanazawa University, was cultured at 25°C with constant illumination of 20 microeinsteins m⁻² s⁻¹ on modified BG11₀ solid medium (without nitrate) containing 1.2% Bacto agar (Difco) buffered with 20 mM HEPES-NaOH (pH 7.5) and supplemented with a vitamin mixture to give final concentrations of biotin, thiamine, and cyanocobalamin of 1 μg liter⁻¹, 2 mg liter⁻¹, and 1 μg liter⁻¹, respectively (3).

Preparation of ESP-depleted cells.

Desiccated colonies were rehydrated with 25 mM HEPES-NaOH (pH 7.0) overnight and then homogenized mechanically with a blender at low speed three times for 10 s. The homogenized sample was filtered twice with double coarse filters (Kimwipe; Crecia) by vacuum filtration, and the filtrate was centrifuged at 1,500 × g for 10 min at 20°C to collect the cells. The cells were washed twice with 25 mM HEPES-NaOH (pH 7.0) and collected by centrifugation at 1,700 × g for 10 min at 20°C. The EPS-depleted cells were suspended in 25 mM HEPES-NaOH (pH 7.0) and stored at room temperature until they were used.

Measurement of water content in air-dried N. commune colonies.

The colonies of N. commune were routinely air dried under ambient conditions in our laboratory (approximately 25°C and a relative humidity of 50 to 80%). The air-dried colonies were equilibrated with the atmosphere in the laboratory, and no changes in weight were observed. Approximately 0.5 g of these air-dried colonies was desiccated further with a drying oven (DX-300; Yamato) at 80°C or with a lyophilizer (FD-80 freeze dryer; Eyela) under reduced pressure (approximately 50 Pa). Air-dried N. commune colonies were also dehydrated by replacement of water with alcohol. Approximately 0.25 g of air-dried colonies was placed sequentially in 50% ethanol, 99.5% ethanol, and 99% t-butanol for 2 h to replace the water with alcohol. The t-butanol was removed by evaporation in a chemical hood, and the loss of weight of the colonies due to alcohol replacement was determined.

Desiccation tolerance of O₂ evolution in N. commune.

An air-dried colony (approximately 10 mg) was rehydrated with distilled water, and photosynthetic O₂ evolution was measured with a gas-phase Clark-type oxygen electrode (LD1; Hansatech Instruments Ltd.). A saturating level of CO₂ was supplied by 1 M NaHCO₃ in the chamber according to the manufacturer's protocol. Illumination was provided by an array of high-intensity light-emitting diodes with a peak wavelength of 660 nm.

An air-dried colony (approximately 10 mg) was rehydrated in an oxygen electrode chamber with 25 mM HEPES-NaOH (pH 7.0) supplemented with 10 mM NaHCO₃, and the recovery of photosynthetic O₂ evolution was monitored using an aqueous-phase Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, United Kingdom) with saturating actinic light (1,600 microeinsteins m⁻² s⁻¹) at 30°C.

The desiccation and rehydration treatments were repeated four times with the same colonies to determine the desiccation tolerance and recovery by rehydration of photosynthesis. An air-dried colony (approximately 10 mg) was rehydrated with 25 mM HEPES-NaOH (pH 7.0) for 30 min, and O₂ evolution was measured using an aqueous-phase Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, United Kingdom) with 10 mM NaHCO₃ as the final electron acceptor with saturating actinic light (1,600 microeinsteins m⁻² s⁻¹) at 30°C. After measurement of the initial level of O₂ evolution, the colony was removed from the oxygen electrode chamber, washed with distilled water, and air dried under ambient conditions for 2 days. The dehydrated colony was rehydrated with 25 mM HEPES-NaOH (pH 7.0), and O₂ evolution was measured again.

Desiccation tolerance of O₂ evolution in EPS-depleted cells of N. commune.

The photosynthetic O₂ evolution activity of EPS-depleted cells was measured in a suspension state using an aqueous-phase Clark-type oxygen electrode (Rank Brothers Ltd., Cambridge, United Kingdom) immediately after preparation to obtain the initial level of O₂ evolution in the untreated cells. EPS-depleted cells were blotted on cellophane film to form an artificial colony and placed in the atmosphere overnight for desiccation. After desiccation, the artificial colonies of EPS-depleted cells were rehydrated in 25 mM HEPES-NaOH (pH 7.0) to measure the remaining O₂ evolution activity. The air-dried artificial colonies were desiccated further with a drying oven at 80°C or using a lyophilizer for 1 h to examine the desiccation tolerance of the EPS-depleted cells. After these additional desiccation treatments, the remaining O₂ evolution activity was measured after rehydration in 25 mM HEPES-NaOH (pH 7.0).

EPS-depleted cells and EPS prepared from N. commune colonies were mixed to examine the mechanism of protection by EPS during desiccation. The residue obtained after vacuum filtration was treated at 80°C for 30 min to inactivate the O₂ evolution activity and was added to the EPS-depleted cells. The mixture was blotted on cellophane film and placed in the atmosphere for desiccation. After desiccation, the EPS-depleted cells mixed with EPS were rehydrated in 25 mM HEPES-NaOH (pH 7.0) to measure the remaining O₂ evolution activity.

Determination of uronic acid content.

The amount of uronic acid was determined using the carbazole assay (4). Air-dried N. commune colonies were ground to a powder and suspended with 25 mM HEPES-NaOH (pH 7.0). Three milliliters of concentrated sulfuric acid was added to 0.5 ml of a sample containing approximately 1 mg of the Nostoc powder. After boiling in a water bath for 20 min to hydrolyze EPS, 100 μl of carbazole reagent containing 0.1% (wt/wt) carbazole in 95% ethanol was added to the acid extract and incubated for 2 h at room temperature, followed by measurement of the A₅₃₀. The uronic acid concentration was determined from a standard curve constructed with known concentrations (10 to 100 μg ml⁻¹) of d->(+)-glucuronolactone. The detection limit was 5 μg uronic acid in 1 mg of the Nostoc powder with this assay.

Alcian blue stain.

Cells were washed with distilled water, placed in 3% acetic acid for 30 min, and then placed in Alcian blue reagent (pH 2.5) containing 0.33% (wt/wt) Alcianblau 8GS (Chroma) in 3% (wt/wt) acetic acid to stain acid mucopolysaccharides. The samples were washed with distilled water to remove excess dye, and the stained samples were observed by light microscopy.

Vital staining of cells with FDA.

Fluorescein diacetate (FDA) was used to stain live cells (13). An equal volume of an FDA solution containing 0.01% FDA in 25 mM HEPES-NaOH (pH 7.0) was added to a cell suspension (approximately 1 mg cells ml⁻¹). After incubation for 5 min at room temperature, the cells were collected by centrifugation at 21,000 × g for 5 min and washed with 25 mM HEPES-NaOH (pH 7.0) to remove the excess dye. The fluorescein that accumulated in live cells was excited by blue light at 470 to 490 nm, and green fluorescence passing through a 515- to 550-nm band-pass filter was observed by fluorescence microscopy. The survival rate was calculated from the number of cells emitting green fluorescence divided by the total cell number.

Determination of chlorophyll a content.

Chlorophyll a (Chl a) was extracted from the cells with 100% methanol. The concentration of Chl a was calculated from the A₆₆₅ with an extinction coefficient of 78.741 liters g⁻¹ cm⁻¹ (12).

RESULTS

Water absorption and the recovery of photosynthesis after rehydration.

Water uptake by desiccated colonies of N. commune was measured (Fig. 1A). When air-dried colonies were placed in distilled water, the colonies started to swell, and the weight of the colonies increased rapidly due to water absorption. After 5 min of rehydration, the N. commune colonies had absorbed water equivalent to approximately five times the initial dry weight. The water absorption continued for 24 h, and the amount of water absorbed was equivalent to more than 20 times the initial dry weight (Fig. 1A). When wet Nostoc colonies naturally swelled after rain in the field were desiccated with a lyophilizer, their weight decreased to approximately 2% of the initial wet weight (data not shown). These results indicate that desiccated colonies of N. commune rapidly absorb water after rehydration and that the water content of wet colonies is approximately 98%.

FIG. 1.

Water absorption and recovery of photosynthesis upon rehydration in field-isolated, natural colonies of N. commune. (A) Air-dried N. commune colonies were rehydrated with distilled water, and the increase in weight due to water absorption was measured. The increased weight was normalized based on the initial dry weights of the colonies. The experiment was replicated at least three times, and the averages and standard deviations are shown. (B) Recovery of photosynthetic O₂ evolution after rehydration was examined using a gas-phase O₂ electrode with a saturating supply of CO₂. The experiment was repeated three times, and nearly identical results were obtained. The data from a single experiment are shown. (C) Recovery of photosynthetic O₂ evolution with illumination was examined using an aqueous-phase O₂ electrode with 10 mM NaHCO₃ as the final electron acceptor. At time zero, an air-dried Nostoc colony was placed in the chamber to initiate rehydrarion and recovery of photosynthesis. Saturated actinic light (1,600 microeinsteins m⁻² s⁻¹) was supplied by a halogen lamp during measurement. The experiment was repeated four times, and nearly identical results were obtained. The data from a single experiment are shown.

Recovery of photosynthesis was measured using a gas-phase O₂ electrode with CO₂ as the final electron acceptor (Fig. 1B). Photosynthetic O₂ evolution activity and respiratory O₂ consumption were not detected in the air-dried colonies of N. commune, indicating that neither process occurred in the desiccated cells. When the desiccated colonies were placed in distilled water for 5 min, photosynthetic O₂ evolution was detected (Fig. 1B). The recovery of photosynthetic O₂ evolution was monitored using an aqueous-phase oxygen electrode; a maximum level of O₂ evolution was observed after 10 min (Fig. 1C). It is worth noting that the recovery of O₂ evolution took place with strong illumination and that photoinhibition was not evident. The level of O₂ evolution recovered after rehydration was similar to the level of O₂ evolution of wet colonies collected from the field after rain (data not shown), although the activities observed after 30 min of rehydration varied (90 to 1,800 μmol O₂ g [dry weight]⁻¹ h⁻¹) when approximately 140 field-isolated, natural colonies were examined. These results indicate that the photosynthetic activity rapidly recovers in dehydrated N. commune cells upon rehydration.

Water content of air-dried colonies.

Natural colonies of N. commune were routinely collected in the field and stored for the experiments after air drying. To determine the water content of the air-dried colonies, N. commune colonies were desiccated further in a drying oven at 80°C. Approximately 10% (wt/wt) of the initial weight was lost after 1 h of treatment with a drying oven at 80°C, and the removal of water reached an equilibrium in 1 h. When the colonies were placed under ambient conditions after the desiccation treatment at 80°C for 1 h, the weight of the colonies immediately began to increase, showing that the colonies quickly absorbed water from the atmosphere.

The results observed after oven drying were consistent with the results obtained when the air-dried colonies were desiccated with a lyophilizer under reduced pressure. Approximately 10% (wt/wt) of the weight was lost after overnight treatment (data not shown). The weight also decreased after alcohol dehydration. These results suggest that the decreases in weight observed with these treatments are due to removal of water from the Nostoc colonies and that the water content accounts for approximately 10% of the weight of air-dried colonies.

Tolerance of N. commune colonies to the air drying-rehydration cycle.

As shown in Fig. 1, photosynthetic O₂ evolution recovered rapidly when air-dried colonies were rehydrated in the laboratory, and the level of O₂ evolution was nearly identical to that of the wet colonies which were collected in the field after a rainfall. Repeated cycles consisting of air drying and rehydration treatments were used to assess the desiccation stress tolerance of photosynthesis in colonies of N. commune isolated from the field. After measurement of the initial level of O₂ evolution, the colonies were air dried. When the air-dried colonies were rehydrated for the second time, nearly identical levels of O₂ evolution were detected. This cycle of air-drying and rehydration was repeated up to four times with the same colonies, and nearly identical levels of O₂ evolution were observed throughout the treatments. These results strongly suggest that the photosynthetic activity in N. commune is highly desiccation tolerant and that O₂ evolution capacity is maintained during desiccation and is rapidly recovered when the colonies are rehydrated.

Removal of EPS from cells.

In this study, a method to remove the EPS from cells in Nostoc colonies was developed, and EPS-depleted cells were prepared by filtering the homogenized Nostoc colonies with coarse paper filter as described in Materials and Methods. To assess the removal of EPS by this method, prepared cells were examined microscopically (Fig. 2). Filaments of cells embedded in an extracellular matrix were observed in field-isolated, natural colonies. For the EPS-depleted cells, no extracellular matrix was visible around the filaments of cells (data not shown). The Alcian blue reagent stains acid mucopolysaccharides, which are a known component of EPS (6, 10, 20). When stained by Alcian blue, all of the extracellular matrix in a colony of N. commune appeared to be blue (Fig. 2A). In the EPS-depleted cells, blue staining of the extracellular matrix was not visible, although small particles stained by Alcian blue were occasionally seen in the field of view (Fig. 2B). These results indicate that EPS is successfully removed by mechanical shearing.

FIG. 2.

Field-isolated, naturally grown cells with EPS and EPS-depleted cells of N. commune. (A) Alcian blue-stained N. commune colony with EPS. (B) Alcian blue-stained EPS-depleted cells. (C) EPS-depleted cells emitting green fluorescence from fluorescein after vital staining. (D) EPS-depleted cells stained with FDA visualized by red fluorescence from chlorophyll. It is notable that almost all EPS-depleted cells stained with FDA emitted both green and red fluorescence (compare panels C and D).

In order to examine the effects of the treatment to remove EPS on the viability of the cells, vital staining with FDA was carried out with EPS-depleted cells (Fig. 2C). FDA stained nearly all of the cells; a total of more than 200 cells were examined, and approximately 96% of the cells emitted both green fluorescence from fluorescein and red fluorescence from chlorophyll. This result indicates that the treatment to remove EPS did not severely damage the cells and that the function of the plasma membrane was still maintained.

To assess the remaining polysaccharides in the EPS-depleted cells quantitatively, the Chl a and uronic acid contents were determined (Table 1). In the EPS-depleted cells, the level of Chl a was significantly higher (approximately fivefold higher) than the Chl a level in N. commune colonies. Consistent with the increased level of Chl a, the amount of uronic acid decreased remarkably; only 30% of the uronic acid found in colonies was detected in the EPS-depleted cells. Laboratory strain KU002 contained a higher level of Chl a and had a lower concentration of uronic acid, indicating that little EPS was produced under the laboratory culture conditions used for N. commune. It has been reported that EPS may account for up to approximately 70% of the dry weight of cyanobacteria (1, 5); thus, the removal of EPS should result in a decrease in the dry weight. These results suggest that EPS removal can be assessed quantitatively by measurement of the Chl a content in a preparation.

TABLE 1.

Evaluation of EPS removal and effects of EPS removal on photosynthesis^a

Parameter	Colonies with EPS	EPS-depleted cells	KU002
Chl a content (μg mg [dry wt]−1)b	1.4 ± 0.3	7.0 ± 0.2	8.3 ± 2.4
Uronic acid content (μg mg [dry wt]−1)c	291 ± 48	95 ± 1.0	77 ± 2.0
O2 evolution (μmol O2 mg Chl a−1 h−1)d	111 ± 36	81 ± 17	152 ± 40

^aColonies with EPS naturally grown in the field, EPS-depleted cells, and laboratory culture strain KU002 of N. commune were examined. The experiment was done in triplicate, and the averages ± standard deviations are shown.

^bChl a was extracted with 100% methanol, and the Chl a content was determined spectrophotometrically.

^cThe uronic acid content was determined by the carbazole assay.

^dPhotosynthetic oxygen evolution was measured using a Clark-type oxygen electrode with 10 mM NaHCO₃ as the final electron acceptor.

To examine the effects of the removal of EPS on photosynthesis, O₂ evolution was measured with EPS-depleted cells. A moderate level of O₂ evolution (approximately 81 μmol O₂ mg Chl a⁻¹ h⁻¹) was detected for the cells containing a high level of Chl a (approximately 7.0 μg mg [dry weight]⁻¹) and a very small amount of EPS, as prepared by our method (Table 1). The laboratory culture of N. commune strain KU002, which also contained a smaller amount of EPS, exhibited a high level of photosynthetic O₂ evolution (Table 1). These results indicate that the treatment to remove EPS does not impair photosynthetic O₂ evolution and that EPS does not have an apparent role in photosynthesis.

Restored O₂ evolution activity after desiccation treatment.

To examine the desiccation tolerance in N. commune cells, the restored O₂ evolution activities after the extreme desiccation treatments were measured (Table 2). Photosynthetic O₂ evolution in field-isolated, natural colonies of N. commune was highly heat tolerant when the colonies were in a dry state; approximately 60% of the initial activity remained after extreme desiccation treatment with a drying oven at 80°C (Table 2). It is important to note that no activity was detected when wet colonies were directly desiccated with a drying oven at 80°C (data not shown). When the wet colonies were treated with a water bath at 80°C for 1 min, the color of the colonies visibly changed from green to brown due to the denaturation of phycobiliproteins by heat, and no O₂ evolution activity remained. To test the heat tolerance of photosynthesis in wet colonies, N. commune colonies were treated in a water bath, and the remaining activity was measured at 30°C. When treated at 50°C for 10 min, cells retained 50% of the initial activity, and the activity was only 10% of the initial activity after 30 min of treatment at 50°C. These results indicate that N. commune cells embedded in EPS are heat tolerant in a desiccated state but not in a wet state and that N. commune can be classified as a mesophilic cyanobacterium.

TABLE 2.

Desiccation tolerance of photosynthesis in field-isolated, natural colonies with EPS and EPS-depleted cells of N. commune^a

Desiccation treatment	% of control
	Colonies with EPS	EPS-depleted cells	Mixture of EPS and EPS-depleted cells
Air dried	100 ± 30b	33 ± 12c	34 ± 6d
Baked at 80°C for 1 he	67 ± 14b	NDg	NTh
Desiccated with lyophilizerf	86 ± 26b	9 ± 1c	NT

^aColonies with naturally occurring EPS isolated from the field, EPS-depleted cells, and a mixture of EPS and EPS-depleted cells were air dried at room temperature overnight. The air-dried samples were desiccated further with a drying oven or a lyophilizer. After these desiccation treatments, the remaining activities recovered after rehydration were determined. O₂ evolution was measured using an aqueous-phase oxygen electrode with 10 mM NaHCO₃ as the final electron acceptor. The experiment was done in triplicate, and the averages ± standard deviations are shown.

^bThe initial level of O₂ evolution was determined for rehydrated colonies, and the normalized (100%) level in the untreated colonies with EPS ranged from 95 to 144 μmol O₂ mg Chl a⁻¹ h⁻¹.

^cThe normalized (100%) level was determined for the suspension of EPS-depleted cells immediately after preparation and ranged from 105 to 136 μmol O₂ mg Chl a⁻¹ h⁻¹.

^dThe normalized (100%), level was determined for the suspension of EPS-depleted cells immediately after preparation and ranged from 530 to 980 μmol O₂ g (dry weight)⁻¹ h⁻¹. After measurement of the initial level of O₂ evolution, the EPS-depleted cells were mixed with the EPS fraction and air dried.

^eAfter air drying, the sample was desiccated with a drying oven at 80°C for 1 h.

^fAfter air drying, the sample was desiccated with a lyophilizer for 1 h.

^gND, not detected.

^hNT, not tested.

In EPS-depleted cells, little activity remained after air drying, and photosynthetic activity was completely abolished after the additional desiccation treatment at 80°C for 1 h (Table 2). In the laboratory culture KU002, O₂ evolution was significantly reduced after air drying; only 20% of the activity remained after desiccation (data not shown). These results indicate that EPS-depleted cells and culture strain KU002 are sensitive to desiccation.

The efficiency of EPS removal varied because of the degree of homogenization and because of the condition of colonies collected from the environment. Thus, cells with various amounts of EPS and Chl a concentrations ranging from 2 to 8 μg mg (dry weight)⁻¹ were prepared, and the restored O₂ evolution was measured using these cells. In spite of the difference in the amounts of remaining EPS, O2 evolution of approximately 100 μmol O₂ mg Chl a⁻¹ h⁻¹ was routinely detected in a suspension immediately after preparation of the EPS-depleted cells. These results indicate that the amount of EPS remaining has little or no effect on photosynthesis.

The effects of remaining EPS on the desiccation tolerance of photosynthesis were examined, and the restored O₂ evolution activities were plotted as a function of Chl a content (Fig. 3). When the level of Chl a increased, the restored activity decreased significantly. When the Chl a content was approximately 2.1 μg mg (dry weight)⁻¹, which is equivalent to the level in the field colonies of N. commune, the restored level of O₂ evolution was as high as the initial level, suggesting that there was little or no damage to photosynthesis. Only 10% of the activity remained when the Chl a content was approximately 8.6 μg mg (dry weight)⁻¹, which was approximately sixfold higher than the Chl a content of N. commune colonies. These results strongly suggest that the amount of EPS is associated with the desiccation tolerance of photosynthesis in N. commune.

FIG. 3.

Desiccation tolerance of photosynthesis in EPS-depleted cells of N. commune. The photosynthetic activities remaining after desiccation treatment are plotted as a function of the Chl a content in EPS-depleted cells. Immediately after preparation of the EPS-depleted cells, the initial level of O₂ evolution (defined as 100%) was measured in a cell suspension; this level was approximately 100 μmol O₂ mg Chl a⁻¹ h⁻¹. The EPS-depleted cells were air dried overnight and then rehydrated as described in Materials and Methods. The Chl a content in the EPS-depleted cells was measured for each preparation. The regression equation was y = 2.8635x² − 42.529x + 175.41 (r² = 0.54, P < 0.01).

A reconstruction experiment was performed in order to examine the protection of photosynthesis by EPS during desiccation (Table 2). The photosynthetic O₂ evolution in a mixture of EPS-depleted cells and EPS was still sensitive to desiccation, suggesting that EPS must be associated with the cells appropriately to enhance desiccation tolerance, although we could not rule out the possibility that the protective compounds were inactivated by heat treatment under our experimental conditions.

Freeze-thaw stress tolerance in EPS-depleted cells.

In cells of N. commune colonies grown in the natural environment, O₂ evolution was strongly tolerant to freeze-thaw treatment. A nearly identical level of O₂ evolution was detected when frozen colonies were thawed at 30°C to measure the activity remaining after freeze-thaw treatment (data not shown). The effects of the remaining EPS on the freeze-thaw tolerance of photosynthesis were examined. Figure 4 shows that O₂ evolution remained after the freeze-thaw treatment in EPS-depleted cells with various levels of Chl a. The level of restored activity decreased as the Chl a content increased significantly. In the cells with approximately 2.0 μg Chl a mg (dry weight)⁻¹, which is equivalent to the level in the field colonies, almost all activity remained. The cells with approximately 8.6 μg Chl a mg (dry weight)⁻¹ exhibited only 20% of the initial activity. These results suggest that EPS also has a role in the freeze-thaw tolerance of photosynthesis in N. commune.

FIG. 4.

Freeze-thaw stress tolerance of photosynthesis in EPS-depleted cells of N. commune. The O₂ evolution activities remaining after freeze-thaw treatment are plotted as a function of the Chl a content in EPS-depleted cells. After preparation of the EPS-depleted cells, the initial level of O₂ evolution (defined as 100%) was measured in a cell suspension; this level was approximately 100 μmol O₂ mg Chl a⁻¹ h⁻¹. The EPS-depleted cells were frozen and kept at −80°C overnight, and the O₂ evolution after thawing at 30°C was measured. The regression equation was y = −10.572x + 131.4 (r² = 0.33, P < 0.02).

DISCUSSION

The terrestrial cyanobacterium N. commune is subjected to desiccation regularly in natural environments because it inhabits open areas and is completely air dried over periods of time (17). Multiple roles for EPS have been suggested, including stress tolerance and as a sink for excess energy (14). In this study, EPS was mechanically removed without damaging the cells (Table 1 and Fig. 2), and the role of EPS in the stress tolerance of photosynthesis was examined using EPS-depleted cells of N. commune (Table 2 and Fig. 3 and 4). We concluded that the desiccation tolerance of photosynthesis in N. commune is strongly associated with EPS, and this is the first report to demonstrate the physiological role of EPS in stress tolerance in N. commune directly. We also characterized the physiological characteristics of N. commune. The water content in naturally air-dried colonies of N. commune was approximately 10%, and the remaining water was insufficient for activation of photosynthesis and respiration; no photosynthetic O₂ evolution activity or respiratory O₂ consumption was detected in the desiccated colonies of N. commune without water (Fig. 1). After rehydration of the desiccated colonies, the photosynthetic activity rapidly recovered, and the level was similar to the level detected in wet colonies grown in the field (Fig. 1B and C). With extreme desiccation in a drying oven at 80°C, the remaining water in air-dried colonies was removed, but this water was not directly associated with maintenance of the photosynthetic machinery in the desiccated state since a high level of O₂ evolution was observed after rehydration of the excessively desiccated colonies (Table 2). It is possible that a small amount of water molecules is tightly bound in the photosynthetic machinery to sustain the function under these extreme desiccation conditions.

The air-dried colonies of N. commune had high water-absorptive properties (Fig. 1A). Consistent with the previously reported water absorption kinetics of desiccated colonies (18, 20), water absorption took place in two phases (Fig. 1A), and photosynthetic O₂ evolution recovered in the initial phase of water absorption in 10 min upon rehydration (Fig. 1B and C) (18). Water absorption kinetics in desiccated colonies are dependent on the physical and chemical structure of EPS (20). It has been shown that uronic acid is present in the EPS of N. commune (6, 10), and large amounts of uronic acid were detected in the colonies in our study (Table 1). Uronic acid is a highly hydrophilic substance and thus contributes to the highly water-absorptive character of EPS from N. commune colonies. This may help maintain water that is indispensable for survival during desiccation. It is interesting that N. commune was heat resistant in a dry state (Table 2), although N. commune was determined to be a mesophilic cyanobacterium.

In EPS-depleted cells, photosynthetic O₂ evolution was significantly damaged by desiccation (Table 2), and the viability of the EPS-depleted cells decreased; approximately 60% of the cells were positively stained after air drying when they were examined by vital staining with FDA (data not shown). Since it has been reported that the EPS of N. commune can prevent fusion of membrane vesicles in vitro at low relative humidities in the presence of a reducing sugar such as trehalose or sucrose (8), EPS might be important for maintaining the structure and functions of biological membranes during desiccation. In support of the idea that EPS protect biological membranes from irreversible and lethal changes during desiccation, the damage in the EPS-depleted cells was indicated by visible leakage of phycobiliproteins after the air-drying treatment (data not shown), and the surface of the EPS-depleted cells appeared to be damaged after the desiccation treatment with a drying oven at 80°C, as visualized by scanning electron microscopy (data not shown). EPS also have a function in freeze tolerance; the tolerance to freeze-thaw treatment was significantly decreased by removal of EPS (Fig. 4). It has been reported that the cellular membrane systems are the primary site of freeze-induced injury in higher plant cells and that cellular dehydration occurs upon ice formation (21); thus, EPS may also protect membranes during freezing in N. commune. Two important questions remain to be answered in future studies: (i) what are the extracellular components directly involved in the desiccation tolerance of the cells, and (ii) how does EPS protect the photosynthetic activity.

Nostoc cells produce EPS in natural environments in response to stress conditions. It has been reported that the production of EPS in Nostoc sp. appears to respond to unbalanced C/N metabolism and can be controlled by adjusting the C/N balance in a culture (14, 15). Culture strain KU002 contained a smaller amount of uronic acid (Table 1), suggesting that little EPS was produced when there was a sufficient supply of inorganic nutrients and light under laboratory culture conditions. Since strain KU002 was genetically identical to the Nostoc cells grown in natural environments according to a 16S rRNA gene analysis (N. Horiguchi and T. Sakamoto, unpublished data), production of EPS is thought to be controlled by the growth regimen, although the laboratory conditions that induce EPS production remain to be characterized for strain KU002. Characterization of the stress responses using culture strain KU002 of N. commune may help elucidate complex processes, including production of the EPS, stress-induced gene expression, and accumulation of compatible solutes. We plan to study the physiological roles of trehalose and sucrose in the stress tolerance of photosynthesis because N. commune tolerates a low salt concentration (Takani and Sakamoto, unpublished data) and trehalose and sucrose are thought to be compatible solutes in N. commune (7). The mechanisms for protection of living cells embedded in EPS against stress conditions may be relevant for development of a new technology for long-term storage of cells in a desiccated state.