Organic Osmoregulatory Solutes in Blue-green Algae

Organic Osmoregulatory Solutes in Blue-green Algae N.ERDMANN

Sektion Biologie, Wilhelm-Pieck-Universitat, Doberaner Str. 143, DDR-2S00 Rostock, G.D.R. Received November 30,1982· Accepted January 28,1983 Summary

Three blue-green algae were studied for their ability to produce organic osmoregulatory solutes when they were exposed to NaCl stress. Microcystis firma shifted its 14C02 fixation in favour of glucosylglycerol and accumulated it up to 24 % of the dry mass (external NaCI concentration 770 mmol·I- 1). Anabaena vanabilis raised the content of sucrose after increase in the external NaCl concentration (14 % of dry mass at 171 mmol·I- 1). Synechocystis aquatilis did usually not alter the 14C fixation pattern. The marked NaCI resistance of M firma was explained by the formation of glucosylglycerol. Key words: Cyanophyceae, 14C02fixation, glucosylglycerol, osmotic regulation, sucrose.

Introduction Survival in a saline environment is only possible if the organisms have developed special strategies to overcome the reduced water potential. These are mainly the regulation of the ion content in the cell and the formation of hydrophilic lowmolecular organic solutes, which are osmotically active (osmotic solutes). Schobert (1980) concluded that the organic solutes contribute less to the restoration of the osmotic potential inside the cells, but serve as water substitute for the hydration sphere of biopolymers and thereby maintain the metabolic activities. The number of osmotic solutes known at present amounts to more than ten (polyols, proline and quarternary ammonium compounds); they are well under study in the case of higher plants (reviews by Hellebust, 1976; Flowers et aI., 1977) and eukaryotic algae (reviews by Hellebust, 1976; Kauss, 1979; Spektorov and Stroganov, 1979), but very little information is available for blue-green algae. There are some indications demonstrating that blue-green algae likewise need the accumulation of osmotic solutes to withstand hypertonic stresses. For instance, Schiewer et al. (1978) have pointed out that in the dark Microcystis firma was unable to adapt to enhanced NaCI concentrations. Provided the ionic mechanism of osmotic regulation functions in the dark, too (d. Hellebust, 1976), in M firma another mechanism of osmotic regulation seems to be effective: the light-dependent synthesis of organic solutes. Using three blue-green algae marked by a different salt resistance, the aim of the current investigation was to examine the products of photoassimilation and to look for such osmotic solutes. Z. Pjlanzenphysiol. Bd. 110. S. 147-155. 1983.

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Materials and Methods Culture conditions The unicellular blue-green alga Microcystis firma (Bn:b. et Lenorm.) Schmidle, strain Gromov/Leningrad 398 and Synechocystis aquatilis Sauv., strain Gromov/Len. 428 as well as the filamentous blue-green Anabaena variabilis Kiitz., strain Lefevre/Lf;n. 305 were grown in batch or turbidostate culture under sterile conditions. The main culture parameters were: 20W· m- z (continuous light), 29°C (temperature) and COz-enriched air (5% v/ v). Further details were published elsewhere (d. Schiewer et ai., 1978). For a sudden NaCI stress (NaCl shock) exponentially growing algae from batch culture were harvested and resuspended in the basal medium (containing 2 mmol·I- 1 NaCI) and in NaCI-enriched basal media (86-770 mmol·I- 1 NaCI), respectively, and further cultured as described. Two to three days later, the 14COZ fixation experiments were performed. In the case of the turbidostate culture, the basal medium was gradually substituted by the NaCl-enriched medium according to the growth rate. During the transient stage (raising NaCl content) as well as after attaining a new steady state (7th day), the 14COz incorporation was analyzed.

14COZ incorporation and separation ofthe labeled compounds The culture suspension was centrifuged (2200 xg) and resuspended in the fresh medium (optical density at 750nm about 0.6). A sample of Iml was preincubated at 20W ' m- z for 10 min and then 3.7 MBq NaH 14C03 (specific activity 1670 MBq' mmol- I) were added. After 5 min illumination the assimilation was terminated by the addition of 5 ml boiling ethanol. The ethanol water mixture was evaporated by a rotary evaporator at 40°C. The dry residue (remaining water would disturb the thin-layer chromatography of NaCl-containing samples) was taken up by 400 pl ethanol (96 %) and incubated for 2 hat 60 0C. An aliquot of 10/11 was subjected to thin-layer chromatography according to Feige et al. (1969). The labeled photosynthetic products were detected by autoradiography (Orwo-Rontgenfilm TF 14, VEB Filmfabrik Wolfen) after spraying the plates with 1 % PPO and exposing for 2-4 weeks. The localized compounds were eluted and were either rechromatographed in different solvent systems for identification or the radioactivity was measured by liquid scintillation counting (XyloliTriton-X 100,2: 1; 6g .1- 1 butyl-PBD). Another 10/11 aliquot was acidified with 40/11 HCI (2 mol· I-I) and sparged with air for 20 min followed by the determination of the total acid-stable radioactivity by scintillation counting. Determination ofglucose, glucosylglycerol and sucrose 2 ml algae suspension were centrifuged. The cells were broken by the use of a cell mill (Vibrogen-Zellmiihle, E. Biihler, Tiibingen, FRG) in the presence of glass balls (diameter < 0.1 mm). After 2 washings and centrifugation aliquots of the supernatant were used for the enzymatic assay of the three compounds. By the test used (blood sugar test combination Fermognost~, VEB Arzneimittelwerk Dresden) glucose could only be determined. Therefore, it was only practicable if glucosylglycerol and sucrose, respectively, were the single glucose source per sample. At first glucose was estimated as ordered by the producer's instruction. Then for the determination of glucosylglycerol and sucrose, respectively, fresh aliquots were acidified with HCI to a final concentration of 0.5 mol· I-I and hydrolyzed at 100 °C for 120 and 30 min, respectively. After neutralization the glucose content in each sample was determined and the respective contents of glucosylglycerol and sucrose calculated from the difference. Cell number and diameter Both parameters were only ascertained for the unicellular algae M. firma and S. aquatilis. They were obtained in one run by the use of a coulter counter (Laborscale, Medicor, Budapest, Hungary). The diameter values corresponded to those of volume-equivalent spheres. Z. Pjlanzenphysiol. Ed. 110. S. 147-155. 1983.

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Chlorophyll a and dry mass The determination of chlorophyll a was performed spectrophotometrically at 663 nm after the cells had been broken by glass balls and extracted three times with acetone (90%). The extinction coefficient of Jeffrey and Humphrey (1975) was used. Dry mass was determined as given previously (Schiewer et al., 1978).

Results and Discussion In the Figures 1 and 2 the pattern of 14C-Iabeled ethanol-soluble CO 2 fixation products is shown for the three blue-green algae. When M firma and S. aquatilis were grown in the basal medium the pattern was the same for both algae after 5 minutes of illumination (Fig. 1 a). A. variabilis produced a similar pattern of 14C fixation products, except for sucrose which belonged to the most heavily labeled compounds in the basal medium (about 22 % of the total 14C activity incorporated in the ethanolsoluble fraction - Fig. 2 a, Table 1). This high incorporation rate was not reflected in the sucrose content per dry mass which was far lower (1.46% - Fig. 3). Such a difference is not uncommon as 14C labelling is rather an indication of the turnover rate than of the absolute amount of a metabolite. Sucrose is an unusual photosynthate in the case of most blue-green algae and as yet, if at all, detected only in small amounts. A nabaena belongs to the exceptions (Tsusue and Fujita, 1964). After transferring the three blue-green algae into a NaCl-containing medium with NaCI concentrations near the resistance limit (M firma about 1 mol. 1-1; S. aquatilis

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Fig. 1: NaCI effect on the 14C0 2 incorporation pattern after 5-min photosynthesis. (A: S. aquatilis, basal medium; B: S. aquatilis, 231 mmol·I- 1 NaCl; C: M. firma, 770 mmol·I- 1 NaCl; the pattern of M. firma grown in basal medium is exactly the same as for S. aquatilis and is not presented. Autoradiograms after two-dimensional thin-layer chromatographic separation according to Feige et al. [1969]. 1: sucrose; 2: aspartate; 3: serine + glycine; 4: glutamine; 5: glutamate; 6: glucosylglycerol; 7: glycoleate; 8: citrulline; 9: threonine; 10: alanine; 11: tyrosine + proline.)

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Fig. 2: NaCI effect on the 14C02 incorporation pattern of A. variabilis after 5-min photosynthesis. (A: basal medium, B: 171 mmol·l- I NaCl; for further details see Fig. 1.) Table 1: 14C incorporation into osmoregulatory solutes after 5 min of photoassimilation (in % of the total 14C labelling on the plate after thin-layer chromatography according to Feige et al., 1969). Confidence intervals are calculated according to Student's t-test for 95 % probability. Osmoregulatory NaCI concentration of the medium (mmol·l- I ) solute 171 257 2 S6

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I) Turbidostate culture (transient stage). 2) Including glutamic acid which is not much affected by NaCI stress, as is also true for S. aquatilis (Figs. 1 a and b). 3) Batch culture.

260 mmol·l- 1; A. variabilis 200 mmol·l- 1}, remarkable changes in the 14C distribution took place (Figs. 1 and 2). Three distinct photosynthates suffered most conspicuous alterations (see below). The mutual relations of the other compounds remained rather constant, but they were partly masked by the decrease of the 14C incorporation after NaCl stress. Cells of M firma adapted to 770 mmol·l- 1 NaCl fixed, on a dry mass basis, only about 60 % 14C02 as compared with a NaCl-poor culture; but when the 14C incorporation was related to cell number and to chlorophyll a content, no significant difference was observed (Table 2). This distinction can be explained by the fact that after transfer to the NaCl medium, cell dry mass and, therefore, cell diameter were increased while, on the other hand, chlorophyll a content per cell remained fairly constant (data not shown). Further details on the NaCl dependZ. Pjlanzenphysiol. Bd. 110. S. 147-155. 1983.

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Table 2: Effect of NaCI on 14C02 incorporation per cell number, dry mass and chlorophyll a, respectively. Turbidostate-adapted cells. NaCI concentrations: basal medium 2, NaCl-enriched 231 (S. aquatilis) and 770 mmol·I- 1(M.firma). Alga

Reference basis

S. aquatilis

cell number (dpm· h- 1) 0.245± 0.016 dry mass (dpm· mg- 1. h- 1) 12.73 ± 1.01 1 1 chlorophyll a (dpm· mg- . h- ) 774.7 ± 71.0 cell number (dpm· h- 1) 0.146± 0.070 dry mass (dpm· mg- 1. h- 1) 29.22 ± 5.99 chlorophyll a (dpm· mg- 1. h- 1) 2255 ±381

M·firma

Basal medium

N aCI-enriched absolute % 0.161± 0.020 9.50 ± 2.56 616.7 ± 77.7

65.7 74.6 79.6

0.167± 0.055 17.00 ± 2.95 2122 ±336

114.4 58.2 94.1

ence of 14C incorporation and photosynthesis were reported earlier (Schiewer et al., 1978). Irrespective of the reference basis, in S. aquatilis the 14C incorporation was always diminished after a NaCI stress (Table 2); the basis dry mass was no exception since the increase of dry mass was quite low; thus, the diameter of the cells did not rise significantly (Table 4). Regarding the 14C fixation products with marked intensity changes, glutamine should be mentioned first. M firma as well as S. aquatilis strongly labeled this compound when grown under NaCl-poor conditions (Fig. 1 a), but considerably less after NaCI stress (Figs. 1 band 1 c). For S. aquatilis, this was the only pronounced alteration in the 14C02 assimilation pattern (Fig. 1 b). In contrast, NaCl-stressed (770 mmol·I- 1) M firma accumulated more than 80 % of the 14C radioactivity (Table 1) in a compound localized at the position of glutamic acid and some carbohydrates (fructose, pentoses, hexitols - Fig. 1 c). The compound could be characterized as a glycoside that splits into two fragments after hydrolyzation (2 hours at 100°C, 0.5 mol. I-I Hel). By means of thin-layer chromatography and thin-layer electrophoTable 3: RG values of glucosylglycerol and related polyols (glucose as reference compound). Thin-layer electrophoresis of the borate complexes (cellulose MN 300 - Macherey, Nagel & Co, Duren, FRG - 3 h at 300 V; electrolyte: 0.2 mol· I-I borate buffer according to Steiner and Maas, 1957) fructose glucose glucosylglycerol glycerol sucrose

0.90±0.11 1.00 0.36±0.04 0.57±0.06 0.26±0.09

Thin-layer chromatography on silica gel (Silufol®, Kavalier, Prague, CSSR - solvent: acetonitrillwater 85: 15 according to Gauch et aI., 1979) 1.08 1.00 0.800

1.32 0.69

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resis the fragments were identified to be glucose and glycerol. Additionally the identity of the glucose moiety was proved by an enzymatic test with glucose oxidase. Therefore, with high probability the compound is a glucosylglycerol. Table 3 summarizes the position of this compound relative to other polyols in two separation systems with the highest resolution power. Recently a glucosylglycerol (2-0-a-D-glucopyranosylglycerol) was first discovered in the blue-green alga Agmenellum quadruplicatum (Kollman et aI., 1979) whereas galactosylglycerols have been known in eukaryotic algae for a long time (d. Kauss, 1979). Thereafter, Borowitzka et al. (1980) found the same glycoside in a strain of Synechococcus (Cyanophyceae) and were able to demonstrate that its content rose proportionally to an increasing external NaCI concentration; therefore, they postulated that it was the major organic osmoregulatory solute in Synechococcus. This was also true for the glucosylglycerol found in M. firma. As seen from Fig. 3, its content was positively correlated to the strength of the NaCI stress and attained about 24 % of the dry mass at the highest concentration used (770 mmol·I- 1). The increase in cell diameter observed (Fig. 3) is probably a result of this accumulation. Small amounts of glucosylglycerol were present in S. aquatilis, too (Table 4). But when NaCI stressed, S. aquatilis did not synthesize glucosylglycerol to a greater

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A. variabilis and on cell diameter of M. firma (batch culture). Z. Pjlanzenphysiol. Ed. 110. S. 147-155. 1983.

Osmotic solutes in blue-green algae

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extent and thereby did not significantly enhance its diameter (Table 4). Sometimes, however, even in S. aquatilis NaCI stress brought about a modulation of the 14C incorporation in favour of glucosylglycerol. In turbidostate cultures adapted to 231 mmol·I- 1 NaCI for a long period, up to 5.6% of the dry mass consisted of glucosylglycerol. The reason for this variable response of S. aquatilis is unknown. The formation of glucosylglycerol was a photosynthetic reaction since in darkness no glycoside could be obtained: it was neither observed in the 14C fixation pattern after NaCI application to a turbidostate culture in the dark (not shown here), nor did it constitute essentially to the cell dry mass when batch cultures of M firma were shocked with NaCI in the dark for 24 hours; accordingly the cell volume did not enlarge (Table 4). In contrast, when NaCI shock was performed under light conditions, the content of glucosylglycerol increased up to 10 % of the dry mass and the cell diameter rose by 10% (Table 4). Table 4: NaCl effect on the content of glucosylglycerol and glucose and on the cell diameter of s. aquatilis and M. firma (batch culture). Alga Osmoregulatory NaCl concentration of the medium (mmol·I- 1) solute 2 86 171 257 S. aquatilis

Mfirma

glucosylglycerol 1) (% dry mass) 0.11 ±0.02 0.18 ±0.10 glucose 1) (% dry mass) 0.020±0.002 0.054±0.058 diameter 1) (I'm) 3.73 ±0.17 3.68 ±0.06

glucosylglycerol (% dry mass) diameter (I'm)

0.76 ±0.14 0.198±0.088 3.70 ±0.13

hypertonic shock for 24 h in the light

in the dark

10.65 ± 1.04 2.71

0.63 ±0.29 2.47

0.68 ±0.22 0.301 ± 0.029 3.94 ±0.22

2 )

1) Mean of 3 experiments (at the 2th, 3th and 4th day after NaCl stress with 3 determinations each). 2) Mean of 5 experiments. Earlier Schiewer et al. (1978) have deduced from other results that M. firma cells are unable to adapt to hypertonic conditions in the dark. It seems quite reasonable to postulate a causal relationship between salt resistance of M. firma and the lightdependent formation of glucosylglycerol. Two other light-dependent osmotic solutes are known: 1,2,4,5-cyclohexanetetrol (d. Hellebust, 1976) and proline (Greenway and Setter, 1979), whereas the majority of osmotic solutes are also formed in the dark though often in a reduced quantity. In A. variabilis no glucosylglycerol could be detected. A NaCI stress brought about a shift of the 14C incorporation pattern in favour of sucrose (Figs. 2 a and 2 b); at the highest NaCI concentration tested roughly 80 % of the total label was localized in sucrose (Table 1). This change was accompanied by an increase of the sucrose content Z. Pjlanzenphysiol. Bd. 110. S. 147-155. 1983.

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in the dry mass according to the NaCI concentration in the medium (Fig. 3). Consequently sucrose has to be regarded as an osmoregulatory solute in A. variabilis. A similar role of sucrose is documented in higher plants (d. Gorham et aI., 1981) and green algae (Wetherell, 1963; Hiller and Greenway, 1968; Muller and Wegmann, 1978; Greenway and Setter, 1979; Kirst, 1980), but, as yet, not for blue-green algae. In the three algae examined the glucose content was quite low and amounted to less than 0.5 % of the dry mass. After NaCI stress the amount exceeded scarcely 1 %; thus, glucose hardly contributes to the osmotic regulation. Apart from glucosylglycerol and sucrose a third organic compound is thought to have an osmoregulatory function in blue-green algae: mannosidomannitol occurring in the lichen Lichina pygmaea (Feige, 1975). But it seems likely that in respect to salt resistance of blue-green algae glucosylglycerol is most important: it is now established in four species; two of them (Agmenellum quadruplicatum and 5ynechococcus, strain RRIMP N 100) are of marine origin. The third one (M firma) is also salt resistant; obviously, the high salt resistance of this species is caused by the ability to synthesize glucosylglyceroi. The fourth one (5. aquatilis) produces this solute to a considerably lower extent and is therefore only slightly salt resistant. A. variabilis exhibits a similarly low salt resistance; possibly sucrose is less able to adapt the cell to a reduced water potential than glucosylglycerol does. Acknowledgements The author is gratefully indepted to Prof. Dr. E. Libbert for critical reading of the manuscript, to Doz. Dr. U. Schiewer for performing the algae cultures as well as to Chem.-Ing. K. Sommerey and 1. Dorr for the valuable technical assistance.

References BOROWITZKA, L. J., S. DEMMERLE, M. A. MACKAY, and R. S. NORTON: Carbon-13 nuclear magnetic resonance study of osmoregulation in a blue-green alga. Science 210, 650-651 (1980). FEIGE, B., H. GIMMLER, W. D. JESCHKE und W. SIMONIS: Eine Methode zur diinnschichtchromatographischen Auftrennung von 14C_ und 32P-markierten Stoffwechselprodukten. J. Chromatog. 41, 80-90 (1969). - - - - Untersuchungen zur Okologie und Physiologie der marinen Blaualgenflechte Lichina pygmaea AG. III. Einige Aspekte der photosynthetischen C-Fixierung unter osmoregulatorischen Bedingungen. Z. Pflanzenphysiol. 77, 1-15 (1975). FLOWERS, T. J., P. F. TROKE, A. R. YEO: The mechanism of salt tolerance in halophytes. Ann. Rev. Plant Physiol. 28, 89-121 (1977). GAUCH, R., U. LEUENBERGER, and E. BAUMGARTNER: Quantitative determination of mono-, diand trisaccharides by thin-layer chromatography. J. Chromatog. 174, 195-200 (1979). GREENWAY, H. and T. L. SETTER: Accumulation of proline and sucrose during the first hours after tranfer of Chiarella emersanii to high NaC!. Aust. J. Plant Physiol. 6, 69-79 (1979). GORHAM, J., U. HUGHES, and R. G. WYN JONES: Low-molecular-weight carbohydrates in some salt-stressed plants. Physiol. Plant. 53, 27-33 (1981). HELLEBUST, J. A.: Osmoregulation. Ann. Rev. Plant Physiol. 27, 485-505 (1976). HILLER, R. G. and H. GREENWAY: Effects of low water potentials on some aspects of carbohydrate metabolism in Chiarella pyrenaidasa. Planta (Berl.) 78, 49-59 (1968). JEFFREY, S. W. and G. F. HUMPHREY: New spectrophotometric equations for determining chlo-

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rophylls a, b, Cl and C2 in higher plants, algae and natural phytoplankton. Biochem. Physio!. Pflanzen 167, 191-194 (1975). KAuss, H.: Osmotic regulation in algae. In: L. REINHOLD (Ed.): Progress in phytochemistry Vo!' 5, pp. 1-27. Pergamon Press Oxford, 1979. KIRST, G. 0.: 14COr Fixation in Valonia utricularis subjected to osmotic stress. Plant Science Letters 18, 155-160 (1980). KOLLMAN, V. H., J. L. HANNERS, R. E. LONDON, E. G. ADAME, and T. E. WALKER: Photosynthetic preparation and characterization of l3C-labeled carbohydrates in Agmenellum quadruplicatum. Carbohydr. Res. 73, 193-202 (1979). MULLER, W. and K. WEGMANN: Sucrose biosynthesis in Chlorella. 1. Thermic and osmotic regulation. Planta (Berl.) 141, 155-158 (1978). SCHIEWER, U., N. ERDMANN, and K.-H. KUHNKE: Influence of different NaCl concentrations on the photosynthetic intensity of the blue-green algae Microcystis firma and Synechocystis aqua· tilis. Biochem. Physio!. Pflanzen 172, 351-368 (1978). SCHOBERT, B.: The importance of water activity and water structure during hyperosmotic stress in algae and higher plants. Biochem. Physio!. Pflanzen 175, 91-103 (1980). SPEKTOROV, C. S. and B. P. STROGANOV: Mechanisms of marine and freshwater alga tolerance to changes in osmotic pressure of the external medium. Fizio!' Rast. 26, 967-977 (1979). STEINER, M. und E. MAAs: Nachweis und Trennung von Zuckeralkoholen in PflanzenpreBsaften. Naturwiss. 44, 90-91 (1957). TsusUE, Y. and Y. FUJITA: Mono- and oligo-saccharides in the blue-green alga, Tolypothrix tenuis. J. Gen. App!. Microbio!' 10, 283-294 (1964). WETHERELL, D. F.: Osmotic equilibration and growth of Scenedesmus obliquus in saline media. Physio!. Plant. 16, 82-91 (1963).

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