The Influence of Water Stress on the Metabolism of Diatoms

Institut fiir Botanik und Mikrobiologie, Lehrstuhl fiir Botanik, Technische Universitat, Miinchen, Germany

The Influence of Water Stress on the Metabolism of Diatoms II. Proline Accumulation Under Different Conditions of Stress and Light B. SCHOBERT With 10 figures Received July 19, 1977 . Accepted August 15, 1977

Summary The diatom Phaeodactylum tricornutum accumulates proline with different rates, according to the intensity of the osmotic stress applied. The accumulation is accomplished within 60 hours and the enhanced proline level is maintained during further stress incubation. NaCI was added to the medium as osmotically active substance, but several other salts are likewise effective. In contrast, KCI in higher concentrations proved toxic. Carbohydrate supply is not limiting for proline synthesis. Exogeneously added glucose was ineffective in enhancing the proline level. Light is required for proline synthesis with NO a- and NH/ as nitrogen source. Addition of glutamine in the dark promotes proline accumulation, but the enhanced level is not maintained over the whole incubation period. Restitution of photosynthetic activity after initiation of osmotic upshock and proline accumulation are connected together. In a model system it is shown that proline protects membranes against a salt-induced alteration in their hydration sphere. Key words: diatoms, osmotic stress, proline accumulation.

Introduction In a previous paper proline accumulation in the diatom Cyclotella meneghiniana due to osmotic stress was reported (SCHOBERT, 1974). This phenomenon was described earlier for the diatom Phaeodactylum tricornutum (BESNIER et a!., 1969) and more recently also for Cyclotella cryptica (LIU and HELLEBUST, 1976 c). The relation between water stress and an increase in cellular proline content was also established earlier with several higher plants (BARNETT and NAYLOR, 1966; THOMPSON et a!., 1966; SINGH et a!., 1973 a; SINGH et aI., 1973 b; STEWART and LEE, 1974; TREICHEL, 1975). In spite of these numberous investigations, several aspects of proline synthesis and the function of proline itself have not been fully elucidated until now. Therefore, further experiments have been carried out to obtain Z. Pjlanzenphysiol. Bd. 85. S. 451-461.1977.

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information on the rate and extent of proline synthesis under different stress conditions and on its connection with photosynthesis. Eight diatom species of different ecological types have been investigated with regard to proline accumulation and they provided identical responses. Thus, it was concluded that the described phenomenon is a general one for all diatoms. As Phaeodactylum tricornutum proved the best species for continuous cultivation, it was chosen as an object in the following experiments. Materials and Methods 1. Cultivation of the algae Phaeodactylum tricornutum was obtained from the culture collection of algae, Gottingen, FRG, under the stock number 1090-1 a. The algae were cultured at 3000 lux light intensity and 20°C in a medium, modified from DARLEY and VOLCANI (1969), consisting of: NaCI, 18.0 g; MgS04 • 7 H 20, 4.9 g; MgCl 2· 6 HP, 4.1 g; CaCI 2 1.1 g; KCI 0.75 g; KNO a 0.5 g; Na 2Si 20 s 0.1 g; Tris-(hydroxymethyl)-aminomethane, 1.2 g; Thiamine-HCI, 0.5 mg; Na2-tartrate, 1.5 mg; Fe(III)-citrate 1.0 mg; Na 2Mo04 0.25 mg and 10 ml trace-element solution in 1000 ml of deionised water. The solution was adjusted to pH 8 with HCI and autoclaved for 30 minutes. K 2HP0 4 0.1 gil and NaHCO a 0.85 gil were added at inoculation. Composition of trace-element-solution: Na 2-EDTA, 3.0 g; H3BOa 0.6 g; FeS04' 7 H 20, 0.2 g; MnCI 2 0.14 g; ZnS04 0.13 g; CoCI 2 0.02 g; CUS04 0.02 g in 1000 ml of deionised water. The culture was placed in 5 I capacity glass bottles and aerated continuously. 2. Experimental conditions for proline estimation Algae from stock culture were centrifuged and resuspended in fresh nutrient solution. The density of the suspension was adjusted by measuring its turbidity at 510 nm, a 1 : 50 dilution of the suspension resulting in E = 0.03. 60 ml of the algae suspension were mixed with 60 ml nutrient solution, containing the osmotically active substance, to give the final concentrations indicated. Unless otherwise stated, NaCI was used to enhance the osmotic concentrations of the culture solution. The osmolarities of the resulting media were checked with an osmometer (Knauer, Berlin) by freezing point depression. The suspension was placed in 250 ml glass bottles and kept under culture conditions. At the times indicated, a 10 ml sample was withdrawn for measuring the volume of the packed cells and a 5 ml sample for proline estimation. After centrifugation (5 min, 8000 X g), the supernatant was decanted and the pellet was extracted twice with hot (80 DC) 50 Ofo ethanol. The combined extracts were evaporated to dryness and the proline content in the residue was estimated as described elsewhere (SINGH et aI., 1973 a). The 15 ml samples withdrawn were replaced by 15 ml of fresh medium of the respective composition, to reduce impoverishment of the nutrient solution and to keep the density of the suspension nearly constant during the experiment. When glutamine was added to the nitrogen free culture solution, the pellet was washed twice with nitrogen free culture solution of identical osmolarity, in order to prevent the disturbing effect of glutamine in proline estimation. When NH 4CI was added, the concentration of Tris-buffer was doubled to prevent a subsequent acidification of the medium. Z. Pflanzenphysiol. Bd. 85. S. 451-461.1977.

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3. Estimation of the soluble chrysolaminarine content

10 ml were withdrawn from the algal suspension, prepared as described above, centrifuged and the pellet extracted once with 50 0 10 ethanol and twice with MCF-mixture (methanol : chloroform: formic acid 7 n = 12: 5 : 3). The combined extracts were evaporated to dryness. The residue was redissolved in 1 ml 0.1 M acetaie buffer pH 5.0 and incubated for 5 hours with 50 ,ul fJ-D-glucosidoglucohydrolase (Boehringer, Mannheim. 0.1 ml from this solution was used for glucose estimation with the GOD-Perid-method (Boehringer, Mannheim). No glucose was present when the enzyme was omitted. As chrysolaminarine is commercially not available, laminarine (Roth, Karlsruhe) was used for a standard curve.

4. Measurement of photosynthesis and respiration The algal suspensions were prepared as described above. At the times indicated, 1 ml samples were withdrawn and the rate of oxygen production was recorded with an oxygraph (Gilson, Dusseldorf) at 25°C and 6000 lux light intensity. Mitochondria from etiolated oat laminae were isolated as described elsewhere (HAMPP and WELLBURN, 1976). An aliquot of the suspension was mixed with isolation solution containing NaCI respectively proline, to give the final concentrations indicated. Succinate (8 ,uM/ml) was added as substrate for respiration and the relative rates of oxygen consumption were recorded with a Gilson oxygraph at 25°C.

Results and Discussion 1. Proline accumulation under different stress conditions Proline synthesis starts immediately after stress initiation with rates corresponding to the intensities of the osmotic stress applied (fig. 1). After 2 hours of incubation time, proline accumulation was not yet finished, but indicated a further increase. As proline plays an important role in water stress resistance (SINGH et aI., 1972; HUBAC and GUERRIER, 1972), it was expected that proline accumulation reaches a

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Fig. 1: Initial velocities of proline synthesis in Phaeodactylum tricornutum, according to different stress intensities. Osmotic strain was initiated by adding NaCl to the culture solution at the time zero. The different milli-osmolarity of the media is given in parentheses.

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constant level after some time, which is maintained during stress influence. Fig. 2 shows that proline accumulation is accomplished about 60 hours after stress initiation. Then, the different levels, induced by increasing salt concentrations, are nearly constant. An optimum level is effected with a medium concentration of approximately 2 osmolar. Higher concentrations result again in lower proline levels, probably because the stress is too severe and acts partially damaging to the algal cell.

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Fig. 2: Time course of proline accumulation in P. tricornutum due stresses. Conditions as described at fig. 1.

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The increase in cellular proline content is nearly lO-fold with the highest concentration applied (fig. 2). Earlier, several authors reported a 10 to 100 fold rise in proline with water deficient leaves, compared to the irrigated control (PALFI and JUHASZ, 1970; BARNETT and NAYLOR, 1966). Such a high increase was not confirmed in recent experiments. Very different results have been reported for the proline response after stress initiation. Investigations with excised turnip leaves exhibited a 3-fold increase in proline level within 24 hours, followed by a decline until 96 hours. It was suggested by the authors that this phenomenon is due to insufficient supply of carbohydrates in the leaves (STEWART et a!., 1966). In contrast, excised barley leaves showed a linear rise in proline over an incubation period of 48 hours (SINGH et a!., 1973 b). Experiments with intact plants resulted in a proline increase which is not finished within several days (TREICHEL, 1975); a decrease of the enhanced proline level during upshock conditions was reported for Cyclotella cryptica (HELLEBUST, 1976). All these results indicate that the proline responses differ widely with the investigated objects and the applied conditions. Z. PJlanzenphysiol. Bd. 85. S. 451-461.1977.

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Fig. 3: Time course of proline accumulation in P. tricorrzutum, induced by the addition of different salts at the time zero. The milli-osmolarity of the media is given in parentheses.

To demonstrate that proline synthesis is induced by a reduction in cellular water potential, the osmotic concentration of the medium was raised with salts other than NaCl, which is ecologically most important. MgCl 2 and Na 2S04 exhibited results which are consistent with NaCl, whereas KCl was toxic and effected gradually a damage of P. tricornutum (fig. 3). The mode of action of K+ is not restricted to osmotic stress. Displacement in the relation of Na+ and K+ to higher K+ values in the culture solution (0.77 osmolar) resulted likewise in an irreversible damage of the algal cells within several hours. Consequently, depending on its relative concentration, K+ penetrates the cells either passively or actively without a feedback control. It seems highly probable that the rapid recovery of cell volume in KCl - and glycerol solutions, described for Cyclotella cryptica (LIU and HELLEBUST, 1976 b), is not due to a rapid osmotic balance, but rather to a fast destruction of the cell. The toxicity of glycerol, as a consequence of its low reflexion coefficient, was indicated earlier (SCHOBERT, 1974). Since drought and saline conditions likewise cause proline accumulation in several higher plants, the effect of air drying on proline response in diatoms has been investigated. Fig. 4 shows that only little proline production occurred, decreasing continuously after 2 hours of stress initiation. Presumably this response is only the «attempt of a proline synthesis», a water stress in air with 60 % relative humidity is too severe to be endured for several hours. It is interesting to note that this result is similar to those described by STEWART et al. (1966) with excised wilted leaves. Obviously, the applied conditions were far from optimum for proline synthesis. 2. The influence of carbohydrate supply on proline accumulation

It was established by several authors (BARNETT and NAYLOR, 1966; THOMPSON et aI., 1966; PALFI and JUHASZ, 1970; SCHOBERT, 1974; LIU and HELLEBUST, 1976 a) Z. P/lanzenphysiol. Bd. 85. S. 451-461. 1977.

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algal suspension were filtered off on filter paper with a Buchner funnel, then placed on watchglasses and dried in light (3000 lux) at 20°C and 60 % relative humidity. At the times indicated, a sample of the series was extracted with ethanol to estimate the proline content (see methods).

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that proline accumulation is not due to hydrolysis of a proline-rich protein, but to a new synthesis from reserve substances respectively glucose as the carbon source. An accelerated loss of starch due to wilting or even an exhaustion in carbohydrate reserves in leaves was reported (PALFI and JUHASZ, 1970; STEWART, 1971). After an exogeneous supply of carbohydrates, the proline level in these leaves was enhanced (STEWART et aI., 1966; SINGH et ai., 1973 b). Therefore, the content of soluble reserve substances in P. tricornutum under different osmotic stresses has been examined next. Under the applied conditions, no depletion of chrysolaminarine was shown, although its content is lowered with increasing stress intensities (fig. 5). Exogeneously added glucose is consumed by the cells of P. tricornutum (checked with He-glucose; see also LIU and HELLEBUST, 1976 a), but up to a 0.1 M final concentration it is not effective in enhancing the accumulated proline level. These results indicate that during the stress incubation period the diatoms are sufficiently supplied with carbohydrates, glucose not being a limiting component in proline synthesis. From this it is obvious that proline does not function as a storage compound for reduced carbon, as was assumed earlier (BARNETT and NAYLOR, 1966).

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Fig. 5: Content of soluble reserve substance in P. tricornutum under conditions of different stress intensities (the milli-osmolarity of the solution is indicated in parentheses).

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Fig. 6: Time course of proline accumulation in starved and non-starved cells of P. tricornutum under identical stress intensities (1800 milli-osmoles). Starved algae have been kept in dark for 48 hours before stress initiation. starved; starved, +glucose 0,01 M; 6.-6. starved, +glutamine 0.1 M in nitrogen free culture solution; X--X non-starved.

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Starved leaves are reported to accumulate no proline (STEWART et aI., 1966). To reduce the internal carbohydrate pool of P. tricornutum, one portion of the algal suspension was kept in the dark for 48 hours. As is evident from fig. 6, in the light period applied throughout the experiment, starved diatoms accumulate less proline than nonstarved, the level of which is not raised by the addition of glucose and only little with glutamine. However, the ability of proline synthesis is not reduced, but only delayed. 120 hours after stress initiation the proline level in the starved algae was identical with the non-starved sample. In the beginning, the chlorophyll content in the diatoms, kept in dark before, was lower than in non starved ones. At the end of the experiment their chlorophyll content was approximately equal. As a connection between chlorophyll concentration and proline accumulation was already suggested earlier (SINGH et aI., 1973 b), it seems very probable that the result, exhibited in fig. 6, is primarily due to an insufficient amount of reducing power, provided by photoreduction, rather than to carbohydrate exhaustion. 3. The influence of light on proline synthesis

The results described before led to an investigation on the role of light in proline synthesis. The biosynthetic pathway via glutamate to proline is well established in microorganisms and several higher plants (STRECKER, 1957; SPLITTSTOESSER and SPLITTSTOESSER, 1973; BENDER, 1975). 2 moles of NAD(P)H 2 per mole of proline and also ATP are involved in the conversion of glutamate to proline. Presumably, reducing power and also A TP are supplied by photoreduction and photophosphorylation. Earlier reports on the effect of light indicated that proline synthesis is inhibited in the dark (MIZUSAKI et aI., 1964; NAGUCHI et aI., 1968; SINGH Z. Pjlanzenphysiol. Bd. 85. S. 451-461.1977.

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et aI., 1973 bj Lru and HELLEBUST, 1976 c). These results were confirmed in experiments with diatoms (fig. 7). Furthermore it is shown that the enhanced proline level is not maintained in a subsequent dark period, whereas light induces proline synthesis also after 48 hours of darkness. The proline decline in the dark demonstrates that the accumulated amount is maintained by an equilibrium between synthesis and decomposition. ~

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As N0 3- is the sole nitrogen source in the algal medium, it was possible that darkness impaired only the reduction of N0 3 - to NH/ and not the further proline pathway. Therefore NH 4Cl, equimolar to KNO a, was added as the sole nitrogen source, but no difference to the result shown in fig. 7 was exhibited. As the glycolytic pathway also provides reducing power and ATP, the effect of exogeneously added glucose on proline synthesis in the dark was examined. Glucose does not enhance proline accumulation, neither with NO a- nor with NH/ as nitrogen source, and is likewise ineffective in the light, as was outlined earlier (fig. 8). However, glutamine as nitrogen source in the dark is much more effective than the light sample supplied with N0 3-, but the proline level is not maintained and decreases after 60 hours of accumulation (fig. 8). A low synthesis of proline from 14C-glutamate in the dark was observed by Lru and HELLEBUST (1976 c). These results indicate that proline synthesis from NO a- or NH/ in the dark is too costly for the cell, whereas the pathway starting from glutamate is possible, at least for some time. The connection of proline enhancement with different nitrogen sources will be reported in a subsequent publication. As was established earlier (SCHOBERT, 1974), 02-evolution in photosynthesis is greatly impaired at the beginning of osmotic upshock, followed by a recovery phase. Z. PJlanzenphysiol. Bd. 85. S. 451-461.1977.

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To demonstrate a possible interaction between proline accumulation and the restitution of the photosynthetic activity, both reactions have been investigated together. Fig. 9 shows that photosynthesis is completely inhibited with the beginning of osmotic stress, followed by a rapid increase which is obviously not due to proline accumulation. However, after 20 minutes of stress incubation, different responses of the light and dark sample are evident. In the dark, where proline accumulation is low, the restitution of the photosynthetic activity is likewise much lower than in the light sample. Therefore it was concluded that proline accumulation and a reversible altering of membrane structure in chloroplasts are connected in some way. To obtain further information on this interaction, a model system was studied. Unfortunately, the isolation of a 02-evolving chloroplast preparation from spinach 3 ~

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leaves was not successful. As the proline effect was supposed to be connected with membranes, mitochondria preparations have been examined instead and provided good respiratory activity. Addition of 0.1 M NaCl to the mitochondria suspension reduced the respiratory activity, but the simultaneous presence of 1 M proline completely compensated the negative salt effect (fig. 10).

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Earlier, it was reported that proline protects isolated thylakoids against inactivation by freezing (HEBER et al., 1971). Salt- and freezing injury of membranes both involve an alteration of the hydration sphere, therefore it seems very possible that proline acts by restitution of that water structure which is necessary for the functional state of the membranes. It was first suggested by several authors (PROTSENKO et al., 1968; VLASYUK et al., 1968) that proline increases the hydration of protoplasma in case of water deficiency. This question is discussed in detail elsewhere (SCHOBERT, 1977) and is under further investigation. Acknowledgement This work was supported by a grant from the Deutsche Forschungsgemeinschaft. The author wishes to thank Dr. R. HAMPP for supplying the mitochondria preparatiom and Prof. Dr. H. ZIEGLER and Dr. P. TANSWELL for their critical reading of this paper. References BARNETT, N. M. and A. W. NAYLOR: Plant Physiol. 41, 1222 (1966). BENDER, D. A.: Amino Acid Metabolism, John Wiley & Sons, New York, 1975. BESNIER, V., M. BAZIN, .T. MARCHELIDON, and M. GENEVET: Bull. Soc. Chim. BioI. 51, 1255 (1967). DARLEY, W. M. and B. E. VOLCANI: Exptl. Cell. Res. 58, 334 (1969). HAMPP, R. and A. R. WELLBURN: Planta 131, 21 (1976). Z. PJlanzenphysiol. Bd. 85. S. 451-461.1977.

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HEBER, U., L. TYANKOVA, and K. A. SANTARIUS: Biochim. Biophys. Acta 241, 578 (1971). HELLEBUST, J. A.: Ann. Rev. Plant Physioi. 27, 485 (1976). HUBAC, C. and D. GUERRIER: Ecoi. Plant. 7, 147 (1972). LlU, M. S. and J. A. HELLEBUST: Can. J. Bot. 54, 930 (1976 a). - - Can. J. Bot. 54, 938 (1976 b). - - Can. J. Bot. 54,949 (1976 c). MlZUSAKI, S., M. NOGUCHI, and E. TAMAKI: Arch. Biochem. Biophys. 105, 599 (1964). NOGUCHI, M., A. KOIWAI, M. YOKOYAMA, and E. TAMAKI: Plant Cell Physioi. 9,35 (1968). PALFI, G. and J. JUHASZ: Acta Agron. Acad. Scient. Hung. 19, 79 (1970). PROTSENKO, D. F.,!. G. SMATKO, and E. A. RUBANYUK: Fisioi. Rast. 15,680 (1968). SCHOBERT, B.: Z. Pflanzenphysioi. 74, 106 (1974). - J. theoret. BioI., 68, 17 (1977). SINGH, T. N., D. ASPINALL, and L. G. PALEG: Nature New BioI. 236, 188 (1972). - - - Aust. J. bioI. Sci. 26, 45 (1973 a). SINGH, T. N., D. ASPINALL, L. G. PALEG, and S. F. BOGGESS, Aust. J. biol. Sci. 26, 57 (1973 b). SPLITTSTOESSER, S. A. and W. E. SPLITTSTOESSER: Phytochem. 12, 1565 (1973). STEWART, C. R., C. J. MORRIS, and J. F. THOMPSON: Plant Physiol. 41,1585 (1966). STEWART, C. R.: Plant Physiol. 48, 792 (1971). STEWART, G. R. and J. A. LEE: Plant a 120, 279 (1974). STRECKER, H. J.: J. BioI. Chern. 225, 825 (1957). THOMPSON, J. F., C. R. STEWART, and C. J. MORRIS: Plant Physioi. 41,1578 (1966). TREICHEL, S.: Z. Pflanzenphysiol. 76, 56 (1975). VLASYUK, P. A., L. G. SMATKO, and E. A. RUBANYUK: Fisiol. Rast. 15, 281 (1968).

Dr. B. SCHOBERT, Botanisches Institut der Technischen Universitat Miinchen, ArcisstraBe 16, D-8000 Miinchen.

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