Planta

Planta 135, 6 9 - 7 5 (1977)

9 by Springer-Verlag 1977

Coordination of Ionic Relations and Mannitol Concentrations in the Euryhaline Unicellular Alga, Platymonas subcordiformis(Hazen) after Osmotic Shocks G.O. Kirst Institut ftir Botanik, Fachbereich Biologic der Technischen Hochschule Darmstadt, Schnittspahnstrasse 10, D-6100 Darmstadt, Federal Republic of Germany

Abstract. Following small hypo-osmotic shocks, ion concentrations (Na +, K +, C1-) in Platymonas subcordiformis decreased; this was due mainly to an increase of cell volume. With larger hypo-osmotic stresses, the decrease of ion concentration continued and, additionally, extrusion of mannitol was observed. The ion and mannitol concentrations were not regained after 240 rain. In contrast, following hyperosmotic shocks, the ion concentrations increased transitorily during the first 20-40 min. The same was true for K + following small hyperosmotic stresses and for Na + and - partially - C1- with larger shocks. Large hyperosmotic stresses caused permanent accumulation of mannitol, which levelled off after 60-80 min. Thus the transient increase of ions bridged the concentration gap until mannitol was accumulated to a high enough concentration to account for the osmotic adaptation of Platymonas, together with a basal level of the ions K +, Na +, C1-. Key words: Ionic relations - Mannitol - Osmoregulation - Platymonas

concentrations of organic compounds; turgor regulation involves changes in the concentration of ions in coenocytic and thalloid algae, the cells of which contain large vacuoles (see reviews by Cram, 1976, and Hellebust, 1976a). It is not certain if control of organic compounds in unicells or of ion composition in vacuolate algae is the sole mechanism of osmotic adaptation in the two groups of algae. It is unlikely that the ionic mechanism alone accounts for osmotic adaptation in the cytoplasm of vacuolate cells. A combination of the mechanisms has been described up to now only for Ochromonas malhamensis, the isofloridoside and the K + concentrations of which are involved in osmoregulation (Kauss et al., 1975), and for PIatymonas subcordiformis (see above; Kirst, 1977b) This paper presents investigations on ion composition and mannitol concentrations in Platymonas subjected to various hypo- and hyperosmotic shocks by changing the external NaC1 concentration. The regulation of the disturbance of osmotic equilibrium was followed to see to what extent ions and mannitol concentrations contribute and how these mechanisms are coordinated.

Introduction Materials and Methods It has been shown that Platymonas subcordiformis (Prasinophyceae) a euryhaline microalga common in the phytoplankton of coastal waters, responds to media of varying osmotic pressures by changing the cell volume (water potential) and, in addition, controlling the total number of osmotically active solutes, i.e., the ions K +, Na +, and CI-, and the polyol mannitol, the main product of photosynthesis (Kirst, 1975b, 1977a, b). It is known that osmoregulation in unicellular algae is based mainly on changes in endogenous Abbreviations: PS=photosynthesis; Resp. =respiration

Details of the procedures for culturing and harvesting Ptatymonas subcordiformis Hazen (strain 161-1 a from culture centre G6ttingen, W. Germany) have been given recently (Kirst, 1977 b). After rinsing twice the algae were resuspended in fresh culture solution (buffered with 20 mM Bicine-NaOH, pH 7.2) and preincubated for 30 min under experimental conditions: illumination was 400 W/m 2 (ca. 18,000 lx; 2 Philips argofoto-BM, 500 W). The temperature of the waterbath was 25 ~C, and the algal suspensions (10~0 x 106 cell/ml) were bubbled with air. Experiments were started by combining equal volumes of algal suspension and culture solution containing sufficient NaC1 to attain the desired concentration in the final solution. Other ion concentrations in the media remained constant (hyperosmotic stress=up shocks).

70 For hypo-osmotic stresses (down shocks) the algal suspension was centrufuged, the supernatent discarded, and the cells "#ere resuspended in twice the volume of medium with the desired NaC1 concentration to obtain cell numbers similar to those in up shock experiments. Controls were subjected to the same procedure (centrifugation, dilution of algal suspension) as the stressed algae: In most cases up shock and down shock experiments were run in parallel. During the experiments algae were incubated in a shaking watcrbath (under the same conditions as described previously) without bubbling with air. Five ml samples were usually collected at the times indicated in the Figures. To avoid contamination of the theca of Platymonas with external ions, the algae were washed in ice-cold isotonic sucrose solution with 0.05 N HC1, followed by two additional washes with sucrose solution without HC1 (Kirst, 1977b). Methods of extraction of the cells and ion estimation (Na +, K+: flame photometer; Mg 2+, Ca2+: atomic absorption spectrophotometer; CI-: chloride titrator) as welI as mannitol determination were described in detail by Kirst (1975b, 1977b). Cell volume was calculated from the volume of packed cells in hematocrit centrifuge tubes. The measurements were corrected for intercellular space ("inulin-space"); nonosmotic volume was not considered (Kirst, 1977a). Cell counts were made in a hemocytometer.

G.O. Kirst: Ionic Relations after Osmotic Shocks

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Results In the first series of experiments Platymonas was exposed to increasing and decreasing osmotic stresses to investigate threshold values at which external osmotic pressure concentrations of ions and mannitol were affected. Cultures grown in low (0.2 M NaCI) and high (0.5 M NaC1) salinity media were used. After 20 rain incubation in media with the NaC1 concentrations indicated (Fig. 1), the N a § concentration in Platymonas rose with increasing external NaC1 concentration. There was no difference in behavior between algae cultivated in 0.2 or in 0.5 M NaC1. With decreasing external NaCl concentrations, the Na + concentration in Platymonas dropped. Calculating the results on a cellular basis to cancel out the effect of changes in cell volume did not alter the direction of changes in cellular N a +. The results of this calculation are not shown here and will not be discussed further in this section (however, see Discussion). In contrast to N a +, the concentration of K + increased only in cells subjected to small hyperosmotic stresses: up to about 0.4-0.45 M external NaC1 in algae from 0.2 M NaC1 media and to not more than 0.55-0.575 M external NaC1 in algae adapted to high salinity. With additional hyperosmotic treatments, the K + concentration in Platymonas was greatly reduced (Fig. 1). Application of hypo-osmotic conditions caused a decrease of internal K + concentration that was almost directly proportional to the external NaCI concentration.

02

&

i.N Cl

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Fig. 1. Concentrations of Na + and K + in Plalymonas subcordiformis 20 rain after changing of the external NaC1 concentration. Algae were cultivated in 0.2 ( o - - I ) and 0.5 M NaC1 (o I o) media. Concentration of K + in the media: 8.2_+0.24 mM

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Fig. 2. Concentrations of Ca z+ and Mg z+ in P. subcordiformis. For further details see Figure 1. Concentration of the ions in the media: Ca 2+ 7.2_+0.15 raM; Mg 2+ 1.52_+0.02 mM

mannitot

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Fig. 3. Concentration of mannitol in P. subcordiformis. For further details, see Figure 1

G.O. Kirst: Ionic Relations after Osmotic Shocks

71

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Fig. 5. Time course of concentrations o f K + in P. subcordiformis grown in 0.5 M NaC1 (control) after hyperosmotic shock (e NaCI: , - - e ; final concentration 0 . 8 M ) and hypo-osmotic shock (e NaCI: o - - o ; final concentration 0.1 M). K § concentration of the media: 8.2 m M

contr. 0.5MNaCl O

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min

120

Fig. 4A and B. A Concentration of C1- in P. subcordifbrmis. For further details, see Figure 1. B Time course of concentration of C1- in P. subcordiformis grown in 0.5 M NaC1 (control: 9 9 after hyperosmotic shock (~ NaCI: + - + ; final concentration 1.0 M) and hypo-osmotic shock (e NaCI: o - - o ; final concentration 0.1 M)

The concentrations of the bivalent cations, Ca 2+ and Mg 2 +, were not affected significantly in the range of the lower osmotic stresses (Fig. 2). Extreme hypoand hyperosmotic shocks resulted in a decrease of Ca 2 + and Mg 1+ concentrations; this was particularly noticeable in cultures from 0.5 M NaC1 media. The cell envelope of Platymonas contains Ca 2+ and Mg 2+ associated with galacturonic acid, so the real internal concentrations of Ca 2+ and Mg 2+ are lower than those shown in Figure 2. Therefore, concentrations of the bivalent cations will not be considered for the present; this is further justified by their relatively low concentrations compared with the monovalent cations (Ca 2+ concentration corrected for the content of the theca would be 2 5 - 3 0 m M in 0.5 M NaC1 cultures; see Kirst, 1977 b). The internal concentration of the main external anion, CI-, is given in Figure 4A. With low hyperosmotic pressures (ca. plus 0.1 to 0.2 M NaCI in addition to the control level), C1- concentration remained almost constant in both the high (0.5 M NaC1) and low (0.2 M NaCI) saline cultures. When submitted to large hyperosmotic stesses, the C1- concentration increased with the external concentration. In the hig-

hest salinity medium tested (1.5 M NaC1), the internal C1- concentration was almost equal to the Na + concentration, although the Na + level in Platymonas was about 1.5 to 3 times higher under less extreme conditions. Similarly to K + and Na +, the reported C1- concentration decreased under hypo-osmotic concentrations of external NaC1. For comparison, the variation of mannitol concentration with external NaC1 is presented in Figure 3. In a concentration range of about +0.1 M NaC1 close to the concentration of the control culture medium, the mannitol concentration of Platymonas was not altered. Further increase of the NaC1 concentration caused a linear increase of the mannitol concentration; under the most extreme hyperosmotic conditions little further increase occurred (0.5 M NaCI culture, Fig. 3). When Platymonas (0.5 M NaCI cultures) was exposed to hypoosmotic shocks, the mannitol content declined the moment the external NaC1 concentration was less than 0.45 M. At 0.3 M concentration of external NaC1, mannitol could be detected in the medium. The concentration of external mannitol increased with decreasing NaC1 concentrations. Microscopic observation showed that no cell rupture appeared, which happened in solutions with less than 0.01 M NaC1. In a second series of experiments the behavior of ion concentrations in Platymonas was studied as a function of time after subjecting the algae to a hypoosmotic or hyperosmotic shock. Platymonas grown in 0.5 M NaC1 media were stressed hyperosmotically w i t h 0.8 M NaCI final concentration. The K + concentration (Fig. 5) increased slightly within 10 min, followed by a decrease to the control level. A final concentration of

72

G.O. Kirst: Ionic Relations after Osmotic Shocks

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60

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120

Fig. 6. Time course of concentrations of Na + and of mannitol (~z--n) in P. subcord~formis. Same experiment as in Figures 4B and 5. Final concentrations of NaC1 after hyperosmotic shock: 0.8 M (s--e) and 1.0 M ( + - +); after hypo-osmotic shock: 0.1 M (o-- o)

1 M NaC1 did not produce a significant alteration in the K § concentration. The results were very similar to those in control cells, where K § remained constant throughout the experiment. For clarity, the values were not drawn in the graph Fig. 5). Hypo-osmotic stress (0.1 M NaC1 final concentration) resulted in a rapid decrease of K § which remained unchanged during the experimental time of 240 min. As opposed to K § the internal Na + concentration changed considerably more (Fig. 6). Under both the hyperosmotic stresses (0.8 and 1.0 M NaC1 final concentration) the Na + concentration of PIatymonas increased to a maximum that was attained 20-30 min after starting the experiment. In the following 30-40 rain the N a + concentration declined, returning to a level similar to that in the control in the case of the lower hyperosmotic shock (0.8 M NaC1). Algae exposed to the larger shock (1 M NaC1) exhibited an increased Na § level even after 120 or 240 min (results not shown). The response to a hypo-osmotic shock (0.1 M NaCt) was similar to that for K +. The time course of the intracellular mannitol concentration was investigated in detail by Kirst (1975b); this is shown for a large hyperosmotic shock (1 M NaCl) in Figure 6. The mannitol concentration increased rapidly from about 60 m M (control) and levelled off after 60-80 min at about 220 raM; this level was maintained. A similar time course of mannitol concentration was found in algae exposed to the lower hyperosmotic stress (0.8 M NaC1), but the final concentration after 120 min was only 180 mM.

The Cl- concentration showed a rapid increase in the first 20 min after hyperosmotic shock (Fig. 4B), decreasing then to levels slightly higher than in the control. This is similar to the observed action for Na § Throughout the experiments the concentration of C1- was lower than the corresponding Na + concentration. However, the differences between control levels and maximum levels (after 20 min hyperosmotic shock, Figs. 4B, 6) were from ca. 50 mM for C1- and ca. 60 m M for Na +, indicating an almost stoichiometric 1:1 uptake of Na + and C1- in the case of large hyperosmotic shocks. The time course of concentration of CI- obtained from algae after the 0.8 M NaC1 shock is not shown, because there was no difference from the control. The loss of C1from Platymonas submitted to hypo-osmotic shock, like that of K + and Na § showed no return to the control level within the time investigated.

Discussion

The results presented here clearly show that control of the ion composition and the mannitol concentration, superimposed by osmotic changes in volume, are involved in osmoregulatory processes. The contributions of the various changes are coordinated and depend on the kind (hypo- or hyperosmotic) and the extent of the osmotic stress. Concerning the extent of the stress some comments are necessary: small osmotic stresses (hypo- and hyperosmotic: - and + ) are thought to be not greater than _+0.1 M NaC1 of the control level (0.5 M NaCI cultures=32.6%o salinity; standard sea water salinity 34.3%~ This range may be found in the environment of the phytoplankton. High stresses, i.e., _+0.1-0.4 M NaC1, may occur in the tide pools and in estuaries. Extreme stresses are primarily of academic interest to study the ability to survive. A simplified survey of the coordination and the tim e lapse of the osmoregulatory mechanisms is given in Table 1. Swelling or shrinkage of the cell volume, i.e., increasing or decreasing the water potential, is the first response to osmotic shocks observed in Platymonas (Kirst, 1977a), in Ochromonas (fresh water alga: Kauss et al., 1975), and in Dunaliella (Ben-Amotz, 1974). The plasmalemma is affected immediately and hence the sensor, which transduces the pressure or the tension into the physiologic events, is widely held to be located there (Hellebust, 1976a; Cram, 1976). Particularly for very low changes in external concentrations (less than _+0.075 M NaC1), the behavior of Platymonas as an osmometer with a resulting increase or decrease of concentrations of internal osmotic active solutes can largely account for regaining osmotic

73

G.O. Kirst: Ionic Relations after Osmotic Shocks Table 1. Coordination of osmoregulatory mechanisms in Platymonasafter osmotic stresses Summary of results reported in: Kirst, 1975a, b; 1977a, b; Kirst and Keller, 1976. Hyperosmotic stress (up shock)

Further shrinkage of cell volume limited (high percentage of non osmotic volume)

Cell volume not recovered, Na + and Cl- concentrations increase; mannitol concentration increases

Cell volmne not recovered, Na + and Cl- concentrations regained (or slightly increased)

Large

PS inhibited

K + concentration not affected (extreme stress: decreases)

Mannitol accumulated

Resp. stimulated (extreme stress: inhibited)

PS inhibited later Resp. stimulated on: start of recovery

PS recovered Resp. partially recovered

Shrinkage of cell volume (decrease of water potential)

Cell volume recovers slowly

Cell volume not fully recovered

K + concentration increases

K + concentration regained

Mannitol concentration not or only slightly affected

Mannitol concentration regained (mannitol concentration slightly enhanced)

Small

Small

Large

PS slightly inhibited Resp. stimulated

Resp.

Seconds

Minutes

Hours = equilibrium

Swelling of cell volume (increase of water potential)

Cell volume recovers slowly; ion (Na +, K +, C1 ) concentrations decreased

Cell volume not fully recovered Ion concentrations remained lowered

}

start of recovery

PS slightly inhibited Resp. stimulated

Ps } Resp.

Swelling of cells limited (restricted elasticity of the theca ; development of turgor)

Cell volume remained swollen Ion concentrations decreased

start of recovery

Mannitol concentration decreased (efflux of mannitol)

PS inhibited Resp. less stimulated Hypo-osmotic stress (down shock)

Ps } Resp.

slowly starting to recover

Resp.

}

PSsp.}

recovered

recovered

Cell volume decreased slightly Ion and mannitol concentrations remained lowered

Ps} Resp.

partially recovered

Extreme stress: cell rupture

PS= photosynthesis; Resp.=respiration

equilibrium. This is s u p p o r t e d by the findings that, u n d e r h y p o - o s m o t i c stresses, i o n c o n c e n t r a t i o n s were l o w e r e d i m m e d i a t e l y while the c o n t e n t p e r cell rem a i n e d fairly c o n s t a n t ( e s t i m a t i o n o f the t h r e s h o l d values, Figs. 1, 4 A ) . M a n n i t o l , u n l i k e the ions, is m e t a b o l i z e d ; this m a y e x p l a i n the surprising cons t a n c y o f its c o n c e n t r a t i o n (Fig. 3). I n fact the energy s u p p l y i n g processes, p h o t o s y n t h e s i s a n d respiration, are slightly a n d t r a n s i e n t l y influenced (Kirst, 1975a, K i r s t a n d Keller, 1976), i n d i c a t i n g a d d i t i o n a l m e t a b o lical regulation, a l t h o u g h the v o l u m e c h a n g e is passive a n d seemed to be sufficient in the r a n g e o f very low o s m o t i c stresses. In a d d i t i o n to the swelling o f the volume, which is limited by the theca (Kirst, 1977a), the general

r e s p o n s e to increasing h y p o - o s m o t i c stress is the r a p i d decrease o f c o n c e n t r a t i o n s o f ions a n d m a n n i t o l ( o t h e r o r g a n i c c o m p o u n d s were n o t tested, b u t they m a y also be involved). T h e loss o f m a n n i t o l in diluted sea w a t e r has been described for a n o t h e r species: Platymonas suecica (Hellebust, 1976b). T h e r a p i d i t y of this loss suggests t h a t the p l a s m a l e m m a b e c a m e leaky. A l t h o u g h o n e h o u r a n d m o r e after the start o f the e x p e r i m e n t p h o t o s y n t h e s i s a n d r e s p i r a t i o n h a d at least p a r t i a l l y recovered, i n d i c a t i n g a recovery o f osm o t i c e q u i l i b r i u m , the i o n c o n c e n t r a t i o n s r e m a i n e d low. C o m p a r i n g these with the i o n c o m p o s i t i o n s of Platymonas g r o w n in 0.1 M NaC1 o r o f fresh water algae (Kirst, 1977b), N a § a n d C1- c o n t e n t s were similar. I n t e r n a l K + , however, was left i m u s u a l l y low

74 (external concentration was unchanged), indicating severe damage to the mechanism regulating the internal K + level (K + influx pump?). Possibly this is one of the reasons why large hypo-osmotic shocks are more harmful to algae than hyperosmotic stresses of the same magnitude. Regulation after hyperosmotic stress is much more complex because of the involvement of several processes with opposing effects; coordinate regulation within the experimental periods, however, is clear (Table 1). With small hyperosmotic stresses the enhancement of K § content and, hence, of the concentration was the main response observed in addition to volume decrease (Figs. 1 and 5). The contribution of K § to osmoregulatory mechanisms is known from Ochromonas (Kauss et al., 1975) and particularly from coenocytic and thalloid marine and fresh water algae, e.g., Chaetomorpha (Kessler, 1954; Zimmermann and Steudle, 1971), Valonia (Gutknecht, 1968), and Nitella (Nakagawa et al., 1974). However, in contrast to these cases, increase of K § concentration in Platymonas was found to be transient. The range of external NaC1 concentrations over which changes of K § are effective in osmotic equilibration is smaller in highly saline cultures (0.5 M NaCl) than in algae adapted to low salinity (0.2 M NaC1) (Fig. 1). This is consistent with former findings that internal K § concentrations are sufficient for osmotic balance in Platymonas grown in low saline media (Kirst, 1977b). Overlapping of increasing ion and mannitol concentrations, as mentioned previously, was much clearer under large hyperosmotic stresses. Instead of K § the concentrations of Na + increased remarkably in parallel with the mannitol concentrations (Figs. 4B and 6). C1- was affected at large osmotic stresses only (Fig. 4A). With external concentrations of more than 1 M NaC1, the influxes of Na § and CI- were almost equal. The additional fluxes occurring during osmotic stress (Figs 4B and 6; 1 M NaC1 final concentration) were calculated and found to be of a reasonable magnitude. For appraising the surface, a Platymonas cell was assumed to be a sphere whose radius was either the largest or the least half axis of the cell, thus yielding, respectively, an over- and an underestimated value. The range of the fluxes for both Na + and C1- was from 0.16 to 11 lamol m -z s- 1. C1 influxes from medium to cytoplasm in other marine algae were calculated to be 2-8 gmol m -2 s -1 (Acetabularia: Saddler, 1970) and 10 gmol m -2 s -~ (Chaetomorpha: Findlay et al., 1971). The corresponding values for Na + fluxes were from 0.11-0.5 gmol m - 2 s- 1 (Acetabularia : Saddler, 1970) and 1.1 gmol m - 2 s - 1 (Chaetomorpha: Dodd et al.,

G.O. Kirst: Ionic Relations after Osmotic Shocks 1966; further examples are listed by Raven, 1975, 1976). Considering the concentration gradient of Na § a passive component of influx is likely. In several algal species tested, K + efflux and Na + influx are downhill, while there are active components of Na § efflux and (usually) K + influx (Raven, 1976). Although measurements of electrical potentials (PD) in Platymonas were not yet available, the potentials could be calculated by use of the Nernst equation: for Na § the PD was found to be in the range of +49 to + 5 4 mV for both the 0.2 and 0.5 M NaC1 cultures. Comparing this with measured potentials of some - 4 0 mV of Chlorella species (Barber, 1968; Langmfiller and Springer-Lederer, 1974), the assumption of passive influx and active efflux of Na + mentioned previously is supported. If Platymonas has the Na + efflux pump generally found in plant cells (Gutknecht and Dainty, 1968; Raven, 1975, 1976), the results reported for high hyperosmotic stresses can be explained as follows: the 1:1 stoichiometry of Na + and CI- influxes observed during large hyperosmotic shocks and the large increase in Na + and C1- concentrations (Figs. 1 and 4B) suggest a transient breakdown of the low permeability of the plasmalemma due to the extreme stress. Thus, the influx of Na § ions together with the counter CI- ions exceeds the capacity of the Na + efflux pump. The ion concentration increase and the concomitant accumulation of mannitol increase the internal osmotic pressure (~'0, mitigating the external shock (no). Subsequently the poor permeability of the plasmalemma to ions is restored, and the rapid ion influx is prevented. By the activity of the Na § efflux pump, which is probably stimulated by high internal Na § concentrations (Saddler, 1970; Barber and Shieh, 1973; Tromballa, 1974), the Na + content is lowered while the mannitol concentration is further increased, thus maintaining high 7ci. C1 is thought to be linked to the Na + fluxes as counterion. C1influx is generally associated with net cation influx (Raven, 1976). Unlike the vacuolate marine algae, which have an active uptake of C1- (e.g., Bisson and Gutknecht, 1975) and hence high internal concentrations, the marine unicells have low internal C1concentrations and appear to exclude C1 (Kirst, 1977b). The consensus from investigations of a wide range of algae (Gutknecht and Dainty, 1968; Raven, 1975, 1976) is that chloride is pumped in a n d - p r e s u m a b l y - l e a k s out ( " p u m p and l e a k " - R a v e n , 1976). Considering the driving forces on C1- distribution in Platymonas, the ratio of ClJC!o shown in Figure 4A can be explained in the absence of active exclusion if the electrical potential of the cytoplasm is about - 8 0 mV, which does not seem unreasonable

G.O. Kirst: Ionic Relations after Osmotic Shocks

for a marine alga. However, the time course of the concentration of internal C1- (Fig. 4B) causes problems for a passive distribution, although the actual changes in PD that are needed to account for the net C1- fluxes are about 10-20 mV for the hyperosmotic shock. In conclusion, Platymonasbalances hyperosmotic shocks by a transient increase of ion content: K § under low zo and Na + - partially balanced by C1- under high ~o. In addition, mannitol is accumulated at high rco values and is the main osmotically active solute (together with K § the main cation), while ion concentration is regained thus serving as an osmoregulatory mechanism to bridge the concentration gap immediately after the osmotic shock until sufficient mannitol is synthesized. Calculating the balance of ion (K +, Na § C1-) and mannitol concentrations 20 rain after hyperosmotic shock (1 M NaCI final concentration), it was found that mannitol contributed ca. 20% to zi. After 120 min the percentage contribution of mannitol to osmotic equilibrium increased to about 45%. This is in agreement with the finding that the importance of mannitol as an osmotically active particle increases with increasing external salt concentration (Kirst, 1977b). The author thanks E. Curdts for skillful and keen technical assistance and Dr. J.A. Raven (University of Dundee) for reading the manuscript and for valuable suggestions. This research was supported by the Deutsche Forschungsgemeinschaft.

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Received 7 January," accepted 4 February 1977