Planta (1986)167:536-543
P l a n t a 9 Springer-Verlag 1986
The role of p-dimethylsulphoniopropionate, glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis D.M.J. Dickson* and G.O. Kirst Universitfit Bremen, Fachbereich Biologie, NW 2, Leobenerstrasse, D-2800 Bremen 33, Federal Republic of Germany
Abstract. The tertiary sulphonium compound, fi-
dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organi c solutes from the cells. The ions K +, Na +, C1- and the above organic solutes can account for the osmotic balance of the cells. Key words: fl-Dimethylsulphoniopropionate - Gly-
cine betaine - Homarine - Osmoacclimation -- Plalymonas.
Introduction
Platymonas (= Tetraselmis) subcordiformis is a prasinophyte unicellular flagellate alga commonly found in estuarine, coastal and marine waters. The cell is surrounded by an "envelope", the theca, which is composed of organic and inorganic residues (Kirst 1977c). The theca allows for the development of small turgor potentials. Summation of the individual osmotica, K +, Na +, CI-, HPO zand the polyhydric alcohol mannitol in cells maintained at steady-state salinities accounts for only about 60% of the assumed intracellular osmolality, rci (Kirst 1977a, b, c; 1980). Similar discrepancies between rq and the extracellular osmolality, rCo,are School of Biological Sciences, University College of Swansea, Swansea SA2 8PP, UK. Abbreviations: DMSP=fi-dimethylsulphoniopropionate; ~ = intracellular osmolality; ~o = extracellular osmolality * Present address:
evident in Dunaliella parva (Ginzburg 1981 ; Ginzburg et al. 1983) and in Dunaliella tertiolecta (Ehrenfeld and Cousin 1982, 1984). The imino acid proline is not detected in cell extracts of P. subcordiformis (Kirst, unpublished data) and the levels of total amino acids are low (about 30 m o l . m -3 cell osmotic volume) and change little with salinity variation (Kirst 1975). The apparent inability to account for an osmotic equilibrium between the cells and the external media has prompted Ahmad and Hellebust (1984) to call for a reassessment of the role of inorganic solutes in the osmoregulation of marine unicellular algae. Deficits in an estimation of rci might also arise from an assumption that low-molecular-weight carbohydrates and or proline are the only major intracellular organic osmotica synthesized by unicellular eukaryotic algae. Certainly, the range of reported organic osmotica accumulated by any one alga does not appear to exceed two to three solutes in number (Munns et al. 1983). In view of the sensitivity of in-vitro eukaryotic enzymes and the protein-synthesizing system to high electrolyte concentrations, it would be beneficial to both higher- and lower-plant cells to accumulate a range of "compatible" solutes at the expense of limiting intracellular electrolytes (Munns et al. 1983; Gibson et al. 1984). Quaternary ammonium and tertiary sulphonium compounds (= " o n i u m " solutes) could be involved in the osmotic adaptation of eukaryotic unicellular algae to saline environments. Quaternary ammonium compounds are synthesized in a number of higher plants subjected to NaC1 stress (Wyn Jones and Storey 1981) and the solutes occur in marine macroalgae (Blunden et al. 1981, 1982). Although the quaternary ammonium solute glycine betaine is involved in the osmoacclimation of a number of halotolerant cyanobacteria (Reed et al. 1984), the distribution of the compound in eukar-
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
537
yotic unicellular algae has not to our knowledge been reported. The tertiary sulphonium compound fl-dimethylsulphoniopropionate ( D M S P = dimethyl-fl-propiothetin: see Wyn Jones and Storey 1981, for chemical terminology) occurs in a number of marine macroalgae (Challenger 1959) and marine microalgae (Ackman et al. 1966) and is involved in the osmoacclimation of some marine macroalgae (Dickson et al. 1980, Reed 1983a, b). Since glycine betaine is found in a number of halotolerant algae and DMSP has been reported in Platymonas spp. (Ackman et al. 1966), it was of interest to investigate whether these or other " o n i u m " solutes are involved in the osmoacclimation of P. subcordiformis and therefore contribute to a lowering in the discrepancy between rci and
Organic extraction. Cells were sedimented at 1200 g and the pellets resuspended in 10 cm 3 of the test medium and centrifuged in graduated conical tubes at 5000 g for 30 s. The packed cell columes were noted (approx. 0.5 cm3); excess medium was removed by suction, and the inner walls of the tubes rinsed with distilled water. Samples were frozen at - 20 ~ C or immediately extracted in methanol: chloroform: water (12:5 : i ; by vol. ; G o r h a m et al. 1981).
7{0 .
Estimation of extracellular-space volume. The extracellular space
Material and methods Algal material and growth conditions. Cultures of Platymonas subcordiformis (Hazen) strain 161 l a (G6ttingen culture center, F R G ) were maintained in modified basal ASP medium (Provasoli et al. 1957) containing 500 mol-m1-3 NaC1, vitamin 8 mix and molybdenum, 0.2 gmol-dm -3. Basal medium contained: MgSO4, 2 5 m o l ' m t - 3 ; MgC12, 2 2 m o l m - 3 ; KCI, 8mol-m-3; KNO3, 2 m o l . m 3; CaCI2, 2 . 5 m o l . m 3; KzHPO4, 0.3 m o l . m - 3 ; NaiSiO3, 0.025 m o l - m -3. Cells were grown in an algal biostat (10 dm 3) operating at 22o-24 ~ C in continuous light ( 1 0 W . m - 2 ) and harvested 4-5 d later at 110 g, rinsed in fresh medium, recentrifuged and used to inoculate, 350-cm 3 flasks containing 250 cm 3 ASP medium (500 molt o - 3 NaC1). Initial celt concentrations were made to approx. 3.106 cells.cm -3. The flasks were incubated at 22~ with a 14-h light (10 W - m - Z ) : 10-h dark regime. Cultures were bubbled with air (approx. 3 dm 3.rain- 1). After 48 h of equilibration the cells were stressed with hypersaline or hyposaline media described below.
Identification and estimation of "onium '" compounds. Quaternary ammonium and tertiary sulphonium compounds were preliminarily identified and semi-quantatively estimated by the thinlayer-chromatography method described by G o r h a m et al. (1981 ; see also, Blunden et al. 1981, 1982).
Carbohydrate analyses. Carbohydrates in the organic extracts, were quantified as their trimethylsilyl ethers (TMS ethers) by gas-liquid chromatography as described by Holligan and Drew (1971). Xylitol was used as internal standard. (volume between packed cells in the algal pellet), was measured with dextran blue and dilution analyses as described by Zmiri and Ginzburg (1983). Estimates were made for cells centrifuged at 1200 g for 2 rain (Kirst 1977a) and at 5000 g for 30 s (present study) in 15-cm 3 graduated conical tubes.
Estimation of cell volume and non-osmotic volume. The cell volume and non-osmotic volume was estimated as described by Kirst (1977a). The non-osmotic volume was determined from the Boyle-van't Hoff plot of cell volume versus l/Zo (external osmolality) using NaC1 as the solute (Nobel 1974; Dick 1979). Recently the method has come under criticism as the values for the non-osmotic volume of a cell depends largely on the permeability of the plasmalemma to the solute (Ginzburg 1981 ; Reed 1984). Abrupt NaC1 hypersaline stress leads to an influx of N a + and C1- to the cells of P. subcordiformis and other algae. Thus the number of intracellular osmotica does not remain constant and the cells could not possible behave as perfect osmometers, as the influx of ions would mitigate the loss of cell-water through osmosis. It is also possible that NaCl-induced hypersaline stress leads to losses of intracellular K + (Reed 1984).
Hypersaline and hyposaline stress. Two hours after the onset of the light period the required mass of NaC1 was added to cultures to give final concentrations of 1000 or 750 m o l . m -3. For hyposaline stress, cells were sedimented at 200 g for 2 min and the pellets distributed in the appropriate medium (300 or 150 m o l - m - 3 NaC1). All experiments were duplicated.
Inorganic-ion analyses. Sedimented cells (1200 g for 2 rain) were resuspended in 150 cm 3 ice-cold isoosmotic Ca(NO3)2 solution and bubbled with air for 2 rain and recentrifuged. The procedure was repeated with 50 cm 3 ice-cold io'osmotic Ca(NO3)2. Finally the pellets were mixed with 10 cm 3 of the Ca(NO3)z solution, transferred to graduated 15-cm a conical tubes and centrifuged at 5000 g for 30 s. The Ca(NOr)2 solution was removed by suction, the pellet (approx. 0.5 cm 3) suspended in 5 cm 3 of deionised distilled water and disrupted in a French pressure cell at 200 MPa. Extracts, free of cell wall debris, were made up to 10 cm 3. The ions N a +, K + and Mg 2+ were estimated by atomic absorption spectroscopy; C1- by chloride titration and SO42 by the method of Jackson and McCandless (1978). The Ca(NO3)2 washing procedures were performed within 10 rain. Preliminary trials indicated no apparent changes in the packed cell volume of the NaCl-grown algae after the isoosmotic Ca(NO3)2 rinse.
Units. In our preliminary studies the water content:fresh weight ratios of the cells were found to be variable, whereas the estimated cell volumes and non-osmotic volumes gave consistent values. Therefore, and unless otherwise stated, the intracellular solute contents of the cells are expressed in terms of the osmotic volume; i.e. total cell volume corrected for the non-osmotic volume. Solute concentrations are given as gmol-cm 3 cell osmotic volume. We cannot exclude the possibility that the cell osmotic volume has been overestimated (see above) and, therefore, the calculated intracellular solute concentrations are likely to be in excess of the true values. The tables from Wyn Jones and G o t h a m (1983) were used to convert osmolalities from units of m o s m o l . k g -1 H 2 0 t o MPa. Chemicals. The hydrochloride salt of fl-dimethylsulphoniopropionate was prepared as described by Larher et al. (1977). All other chemicals and reagents were obtained commercially.
Results E x t r a c e l l u l a r - s p a c e volume. T h e e x t r a c e l l u l a r s p a c e for cells centrifuged
at 5 000 g for 30 s was found
538
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
to be 18-23% of the sediment volume. A value of 38-43% was found for cells centrifuged at 1200 g for 2 rain and the value is consistent with that observed by Kirst (1977a). Similar decreases of the extracellular-space volume of Dunaliella parva (var. 75) pellets with increasing centrifugal force were reported by Zmiri and Ginzburg (1983).
a
140-
total cell volume
130120
*c- 110o o 100.
u-o
90. 80-
Cell volume and non-osmotic volume. The non-os' motic volume of the cells, determined from the Boyle-van't Hoff plot, w a s 0 . 2 8 . 1 0 - 9 c m 3 (280 gin3). Criticisms regarding this method of estimating the non-osmotic volume of cells are given in the methods. The non-osmotic volume did not vary for cells exposed to or cultured in a range of external NaC1 concentrations (]50-1000 mol. m-3). The total volume of a cell grown in Provasoli medium containing 500 m o l . m -3 NaC1 was 0 . 4 3 2 - ] 0 - 9 c m 3 (=432 gm3; Table 1). The values given here for the volume and non-osmotic volume of cells are consistent with those reported by Kirst (1977a). Changes in the total cell volume of P. subcordiformis were followed as a function of time after exposing the algae to various external NaC1 concentrations (Fig. 1 a). The data are corrected for the non-osmotic volume and shown in Fig. ] b. The initial changes of cell volume are similar to those reported by Kirst (1977a). However, in this study, volume changes were recorded over 24 h. A comparison of Fig. 1 a and ] b shows that the changes of total cell volume are less than those for the cell osmotic volume. The data indicate that
70d
015
i
Ilk
3
4
5
6
24
hours
200-
~
180160.
b
/~b-~ --
cell osmotic volume
//'-% 120_
~ 1oo_ r - - -
-'"-
=~-~
~
~
80_
60_ 40. (~ 0]5 1' l/ :~ 3[ d g g 1/--24 hours Fig. 1 a, b. Time courses of the changes in the total cell volume (uncorrected for the non-osmotic volume - a) and cell osmotic volume (corrected for the non-osmotic volume - b) of P. subcordiformis exposed to hypersaline media 1000 (A), and 750 (z0 mol.m -3 NaC1; and hyposaline media 300 (o) and 150 (e) mol.m -3 NaC1. Control, 500mol-m -3 NaC1 medium (,,). Each point represents the mean of four duplicated experiments
TaMe 1. Steady-state intracellular ion levels of P. subcordiformis cells cultured in the test NaC1 media. Values are for cells washed in ice-cold isoosmotic Ca(NO3)2 solutions, as described in material and methods, and are the means of 12 separate determinations. The 95% confidence intervals are given for the ion concentrations corrected for the non-osmotic volume. Algae were sampled during the light period (1.107 pW-m-2). The data of Kirst (1977c) for cells washed in ice-cold isotonic sucrose solutions are shown in parentheses NaCI in medium (mol' m -3) 150
300
500
Total cell volume (gin 3) Non-osmotic volume (l~m3)
495 280
451 280
432 280
Ion levels K + g m o l ' c m -3 cell osmotic volume K + gmol.cm -3 packed cells K + fmol-cel1-1
255_+9 111 (116) 55 (65)
370 _+13 140 (125) 63 (60)
463 _+11 163 (150) 71 (60)
Na + gmol.cm -3 cell osmotic volume Na + gmol.cm -3 packed cells Na + fmol- cell- 1
23_+4 10 (8) 6 (4)
39+5 15 (25) 7 (15)
C1- I~mol' c m - 3 cell osmotic volume C1- pmol.cm -3 packed cells C1 fmol.cell- 1 M g 2 + fmol. cell- 1 SO4z- fmol.cell a
28 4- 3 12 (9) 6 (8) 7 (7) 3
52___3 20 (13) 9 (8) 7 3
(7)
750
1000
429 280
410 280
(200) (60)
558 _ 12 194 83
712_+ 14 226 92
42+5 15 (60) 7 (25)
(100) (36)
46_+6 16 7
51_+5 16 7
624-4 22 (33) 9 (13)
(41) (13)
83_+5 28 12
88+_5 31 12
7 (7) 3
7 (7) 3
7 3
600
(7)
(7)
D.M.J. Dicksonand G.O. Kirst: Quaternary ammoniumand tertiary sulphonium solutes of Platymonas
P. subcordiformis cells cultured in 500 m o l . m -3 NaC1 media partially regulate the cell volumes to within the range of control cells when exposed to abrupt changes of salinity in the range of 300-1000 t o o l . m - 3 NaC1. The subsequent increases of cell volumes after hypersaline stresses followed a biphasic curve towards the control values; i.e. a primary (coarse) adjustment within 2 h was followed by a secondary (fine) adjustment. A biphasic restoration of cell volume was also observed for algae exposed to the 300 tool. m -3 NaC1 hyposaline medium. In contrast to the above, cell volumes of algae exposed to the 150mo1.m -3 NaCI medium remained larger than those of the control cells and showed little tendency for regulation. The apparent incomplete cell-voume regulation of cells transferred to low salinities (namely, 1 0 0 - 1 5 0 m o l ' m -3 NaC1 media) is primarily caused by their forming large intracellular vacuoles; the cytoplasmic and chloroplastic volumes recover within minutes to those values of the control cells (Kirst and Kramer 1981). The observations indicate that the volume of the protoplasm rather than the vacuoles is regulated. The vacuoles have been proposed to act as sinks for excessive ions and organic solutes in algae exposed to hyposaline media, thus leading to increased water fluxes from the chloroplast-cytoplasm to the vacuole. Inorganic-ion levels. The steady-state intracellular ion levels of P. subcordiformis cells obtained by washing cells in ice-cold isoosmotic Ca(NO3)2 solutions, as described in the methods, are shown in Table 1. Data are given in g m o l - c m - 3 cell osmotic volume (corrected for the non-osmotic volume), gmol. c m - 3 packed cells (uncorrected for the non-osmotic volume) and ion content (fmol) per cell. Algal pellets suspended in ice-cold isoosmotic Ca(NOa)2 solutions for 2-10 rain yielded similar ion levels as those of Table 1 (data not shown). For comparative purposes the values for the ion composition of the cells washed in isotonic sucrose solutions (see Kirst 1977 b, c) are given in parentheses. Table 1 indicates that the Na + and C1- concentrations of cells washed in isoosmotic Ca(NO3)2 solutions increased as a function of external salinity, but that both ions expressed in terms of content (fmol) per cell remained more or less constant. In contrast to Na + and Cl-, the K + content of the cells increased and the intracellular K +/Na + ratios of the algae grown in the test salinities were in excess of a value of 10. In the earlier studies (Kirst 1977b, c) the K + content of P. subcordiformis did not change in response to higher salinities, and the Na + levels increased re-
539
sulting in low intracellular K + / N a + ratios (namely, 2 to 3 for the algae grown in 600 tool-m-3 NaC1 medium - see Table 1). The differences between the ion data shown in the current study and those given by Kirst (1977b, c) appear to be related to whether the algae were rinsed in ice-cold isoosmotic Ca(NO3)z or ice-cold isotonic sucrose solutions. The two methods are evaluated in the Discussion.
Organic compounds. Three " o n i u m " compounds were detected in the organic extracts of P. subcordiformis cells. These were identified as the tertiary sulphonium compound DMSP, and the quaternary ammonium compounds glycine betaine and homarine by two-dimensional thin-layer chromatography with the authentic compounds. The distribution of DMSP and glycine betaine in a number of higher and lower plants is referred to in the Introduction. Homarine (sometimes referred to in the literature as N-methyl picolinic acid) occurs in a number of marine invertebrates (Campbell 1970), but does not appear to be widespread among marine algae. The compound has been identified in the red macroalga Trichocarpus crinitus (see Blunden et al. 1981). Mannitol was the major soluble carbohydrate in the organic extracts. Trace amounts of glycerol and erythritol were detected, and these polyhydric alcohols formed less than 2% of the total cellular mannitol content. The intracellular steady-state concentrations of all known major organic solutes of P. subcordiformis exposed to a range of NaC1 media for 4 d are shown in Fig. 2. The solutes are accumulated in response to the NaC1 concentration of the media. Homarine and glycine betaine showed similar increases in the cells with respect to the media salinity. Sodium-chloride media in excess of 300 m o l - m -3 NaC1 appeared to stimulate a rapid and linear increase of intracellular mannitol. In contrast to the polyhydric alcohol, steady increases of intracellular " o n i u m " compounds were observed in the algae exposed to the test salinities. Mannitol formed the largest single intracellular organic solute in algae exposed to the 1 000 mol. m - 3 NaC1 medium. The concentrations of DMSP were always lower than those of homarine and glycine betaine, but of similar content to mannitol in cells equilibrated at the low salinities (namely, 150-300 tool - m - 3 NaC1 media). Organic compounds - hypersaline stress. Changes of the intracellular concentrations of homarine, glycinebetaine, DMSP and mannitol were followed as a function of time when P. subcordiformis was
540
D.M.J. Dickson and G.O. Kirst : Quaternary ammonium and tertiary sulphonium solutes of P l a t y m o n a s 600-
_z 4 0 0 -
a
homarine
~,
b
glycine betaine
._o 500-
300-
0 9~
4oo-
E
3so-
O
3OO-
g
200-
'~ 100-
0i
250= 200" o
0
"6
E
6
~
160"
~
' 1'2J~4
'
100-
a
'
~
300.
6 oh ' ola ' ols ' 03 ' NaCI
1~"~4 -600
C
50-
o
'
hours
hours
d
DMSP
mannitol
250.
-500
~ 200.
-400
150.
-300
'
tool d m -3 m e d i u m
Fig. 2. Steady-state levels of mannitol (A), glycine betaine (o), homarine (e) and DMSP (-) of P. s u b c o r d i f o r m i s in the light (10 W - m -z) as a function of external salinity. Each point represents the mean of three duplicated experiments. Intracellular concentrations are corrected for the non-osmotic volume
transferred from the control medium (500 tool ' m - 3 NaC1) to the hypersaline media (750 and 1 000 m o l - m - 3 NaC1) in the light (Fig. 3 a-d). Intracellular increases in the levels of the " o n i u m " solutes were observed after a lag period of about 3 h. The final amount of solute synthesized was dependent upon the NaCI concentration of the medium, and equilibrium levels were reached within 9 h after the hypersaline stress. As homarine, glycine betaine and D M S P were accumulated during a period that was concurrent with little change in the restoration of cell volume (i.e. hours 3 to 12; see Figs. I b and 3 a-c), their rates of synthesis can be expressed in terms of gmol " o n i u m " compound synthesized, c m - 3 cell osmotic volume, h - 1. The homarine, glycine betaine and D S M P synthesis rates for cells exposed to 1000 m o l ' m -3 NaC1 were 13.8, 11.1, 13,3 ~tmol. c m - 3 h - 1, respectively; the solute synthesis rates for cells exposed to the 7 5 0 m o l . m -3 NaCI medium were 6.3, 6.6, 8.8 g m o l ' c m - 3 h - l , respectively. In contrast to the data shown for t h e " onium" compounds, mannitol was immediately synthesized and final intracellular levels were reached within 30-60min (Fig. 3d). Organic compounds - hyposaline stress'. Exposure of the cells to hyposaline media (300 and 150 mol. m - 3 NaC1) led to significant decreases in the intracellular organic solute concentrations (Fig. 3 a-d). The initial decreases in the levels of the solutes in cells exposed to the 300 t o o l - m - 3 NaC1 medium were a little larger than that predicted from the increase of the cell osmotic volume (namely,
i
-200
100
u
5od
-100
O_
6
'
4
' hours
~
'
1~/~4 hours
Fig. 3a--d. Time courses of the changes in intracellular homarine (a), glycine betaine (b), DMSP (c) and mannitol (d) of P. s u b c o r d i f o r r n i s cells exposed to hypersatine 1000 (e), 750 (o) tool . m - 3 NaCI media; and hyposaline 300 (zx), 150 (A) tool .m 3 NaC1 media. Control, 500 mol.m -3 NaCI medium (,.). Each point represents the mean _+95% confidence intervals (n = 6). Cells were sampled during the light (10 W . m 2) period. Algae were stressed at time 0
+ 64% ; see Fig. 1 b). Similarly, the initial decreases in the solute contents of the cells exposed to the 150 m o l . m 3 NaC1 medium, were greater than that which can be calculated from the increase of the cell osmotic volume (namely, + 8 4 % ; see Fig. 1 b). The partial restoration of the cell volume with time was not accompanied by increase in the intracellular concentrations of the organic solutes, and further losses and-or degradation are proposed to have occurred. In the hyposaline media, D M S P was detected (to be detailed elsewhere), and mannitol has been reported to leak out of algal cells exposed to dilute seawater concentrations (Kirst 1977b). We did not attempt to analyse the possibility of homarine and glycine betaine occurring in the hyposaline media. Discussion
In common with the cytoplasmic ion concentrations of many marine macroalgae and slightly vacuolated unicellular algae (Raven 1976; Munns
D.M.J. Dicksonand G.O. Kirst: Quaternary ammoniumand tertiary sulphonium solutes of Platymonas et al. 1983), P. subcordiformis cells contain high levels of K + relative to Na + (Table 1). The cells appear to contain lower levels of NaC1 and higher amounts of K + than previously estimated. Low intracellular NaC1 levels are suggested to be of adaptive importance in cells with those metabolic activities shown to be sensitive to the ions (Munns et al. 1983; Gibson et al. 1984). There are no data on the intracellular distribution o f K + in P. subcordiformis, but considering that the volumes of the vacuoles, chloroplast and cytoplasm are 6%, 61% and 22%, respectively, of the total cell volume of algae grown at 500 m o l . m -3 NaC1 (Kirst and Kramer 1981), much of the cation K + must reside in the cytoplasm and chloroplast. Generally, estimates of the Na + and C1- content of algal cells washed in isotonic non-electrolyte solutions, with or without low concentrations of M g 2 + and Ca + +, are substantially smaller than those levels measured from correcting for the amounts of contaminating ions in the extracellular space volume of the algal pellet (Ehrenfeld and Cousin 1982; Ginzburg and Ginzburg 1985). Criticisms regarding the use of the two methods are given by Munns et al. (1983). Measurements of extracellular-space volumes are also open to error, owing to the possible uptake by the cells of the radiolabelled marker (Ginzburg 1981; Ehrenfeld and Cousin 1982; Reed 1984). Dickson etal. (1980) and more recently Ritchie and Larkum (1985) used ice-cold isoosmotic Ca(NO3)2 rinsing techniques to remove extracellular Na +, K + and C1- from the cell walls of marine macroalgae. The authors have concluded that isoosmotic rinses with Ca(NOa)2 solutions do not appear to lead to losses of intracellular ions from the algae. Our present data (Table 1) might, therefore, indicate that the isotonic sucrose rinse does not completely remove extracellular Na + and CI-, or replace NaC1 media trapped between the plasmalemma and inner theca wall, and could even lead to a loss of intracellular K + from P. subcordiformis cells (see also, Reed 1984). Our results indicate that the tertiary sulphonium compound DMSP and the quaternary ammonium compounds glycine betaine and homarine are involved in the acclimation of P. subcordiformis to increased saline environments. After a lag period of about 3 h the intracellular " o n i u m " solute concentrations increased significantly in algae transferred from 500 tool-m-3 NaC1 medium to the 750 and 1 000 t o o l . m - 3 NaC1 media (Fig. 3 a c). The 3-h time lag indicates d e - n o v o synthesis of enzymes and suitably reduced sulphur and nitrogen intermediates required for the " o n i u m " solute
541
accumulation. The duration of the time lag is greater than that observed for the synthesis of proline (namely, 30-60 min) in the NaCl-stressed freshwater alga Chlorella emersonii (Greenway and Setter 1979). In contrast to the time-lagged synthesis of " o n i u m " solutes in the alga during hypersaline stress, mannitol was rapidly accumulated and equilibrium concentrations were reached within 30-60 min (Fig. 3d; see also Kirst 1977b). Similar time courses for the increases of a number of lowmolecular-weight carbohydrates have been reported for other eukaryotic unicellular algae stressed in media hypersaline to that of the growth medium (Munns et al. 1983), and the relatively instantaneous accumulation of these carbohydrates indicates activation rather than de-novo synthesis of enzyme. Our current observations for the effects of salinity variation on the cellular levels of " o n i u m " compounds in P. subcordiformis necessitate an evaluation of the integrated activities of inorganic and organic solutes in cell-volume regulation. Abrupt hypersaline stresses lead to passive transitory increases of NaCI concentrations in P. subcordiformis and after 2 h the Na § and CI- content of the cells are similar to those of the control algae (Kirst 1977b; and observed in the current study data not shown: see also references in Reed 1984; Ehrenfeld and Cousin 1984). These passively accumulated ions are considered to act as short-term osmotica in contrast to the uptake and retention of K § and synthesis of organic solutes. Thus it appears that within 2 h of stressing P. subcordiformis cells with hypersaline media, K +, Na § CIand mannitol effect the primary (coarse) adjustments of cell-volume regulation: the subsequent synthesis of " o n i u m " solutes (3 h plus) appears to contribute to the secondary (fine) adjustments of cell-volume restoration towards that of the control cells (see Fig. 1 a-b). Considering the limited vacuolar volume of P. subcordiformis cells (see above) much of the organic solute content of the alga must be distributed between the cytoplasm and chloroplast to allow for an osmotic equilibrium between the two compartments. Compatibility of glycine betaine, at concentrations of up to 500 m o l - m - 3 and more, with various higher-plant metabolic functions has been reported (Wyn Jones and Gorham 1983; Gibson et al. 1984). Further studies are required to assess the compatibility of homarine and DMSP with algal/higher-plant metabolic processes and these compounds should be considered as speculative compatible solutes. Mannitol is non inhibitory to several soluble enzymes from P. subcordiformis.
542
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
Table 2. The contribution of all major intracellular osmotica to a calculated hi, (~'ni =calc.) of P. subcordiformis cells equilibrated in the test NaC1 media. Intracellular solute concentrations are given in gmol. cm -3 cell osmotic volume and osmolalities in MPa. gb = glycine betaine; hom = homarine; mann = mannitol; no = external medium osmolality; ni cale. ions = calculated contribution of intracellular ions to Xn~ calc. ; nl calc. org. = calculated contribution of intracellular organics to Zni calc. Turgor pressure: P=Zn~ calc.-no. Solute concentrations are taken from Table l and Fig. 3
External salinity : NaC1 tool-m- 3
Solute concentration (gmol- c m - a cell osmotic volume)
Osmolality (MPa)
K+
Na +
CI-
gb
horn
DMSP
mann
150 300 500 750 1000
255 370 463 558 712
23 39 42 46 51
28 49 62 83 88
101 153 223 285 348
89 141 224 278 347
49 66 136 204 248
37 102 174 315 513
The contribution of all known intracellular osmotica to the 7q of the cells is shown in Table 2. Osmotic coefficients of 1.8 for NaC1 and 1.0 for the organic solutes are used to derive the cell osmolality. Difficulties are encountered in estimating an osmotic coefficient for K § as the cation is largely balanced by orthophosphate and polyphosphate (Kirst 1977c). The ratio of intracellular orthophosphate, PO 3 , to polyphosphate [O-(PnOn) n O - ] is approx. 1 : 1. Potassium ions bound to proteins or to polyphosphate bodies, which could largely be sequestered within unit-membrane vesicles (Kugel, personal communication), are likely to be less osmotically active than those associated with orthophosphate. The intracellular pH of the cells, 7.0-7.5 (Kugel, personal communication), would indicate that orthophosphate is present as HPO4z . For the sake of simplicity, we have assumed firstly that 70% of the intracellular K + is associated with H P O 2- and that the osmotic coefficient of KzHPOg is about 2.1 (our own measurements), and secondly, that 30% of the intracellular K + is bound to polyphosphate and that the cation has an osmotic coefficient of 0.9. For reasons stated in the methods (see "Units") the intracellular concentrations of all osmotica may be in doubt owing to a likely overestimation of the cell nonosmotic volume, and this could lead to further errors in deriving values for n~. The data in Table 2 indicate that intracellular K +, mannitol and the " o n i u m " solutes can account for the ni of P. subcordiforrnis cells exposed to all the external NaC1 media. At salinities above 500 m o l . m 3 NaC1 in the media the organic compounds contribute significantly more to the osmolality of the cells than do the inorganic solutes. The calculated turgor potentials across the plasmalemma of between 0.4 M P a and 0.6 M P a would indicate an overestimation of ni and clearly our values are too high for a marine unicellular flagellate alga. Table 2
no
nl calc. ions
ni calc. org.
Xnl calc.
P ($7~i-7c0)
0.98 1.55 2.53 3.91 5.23
0.68 1.01 1.37 1.75 2.16
0.68 1.15 1.76 2.67 3.60
1.36 2.16 3.13 4.42 5.76
0.38 0.61 0.60 0.51 0.53
does, however, indicate that all the currently identified osmotica could account for an osmotic balance in P. subcordiformis cells. Our present paper indicates a need to assess the role of quaternary ammonium and tertiary sulphonium compounds in the adaptation of eukaryotic microalgae to the saline environment. I f " onium" solutes are present in other halotolerant eukaryotic unicellular algae the compounds might allow for a calculable osmotic balance between the cells and the external media. This work was supported by a Royal Society of London European Exchange Research Felowship to David Dickson and a generous equipment grant from the Deutsche Forschungsgemeinschaft. We also thank Dr. H. Kugel (University of Bremen, FRG) for access to his unpublished data and Dr. Ursula Winter (University of Bremen) for performing the inorganic-ion analyses.
References Ackman, R.G., Tocher, C.S., McLachlan, J. (1966) Occurrence of dimethyl-fl-propiothetin in marine phytoplankton. J. Fish. Res. Board Can. 23, 357-364 Ahmad, I., Hellebust, J.A. (1984) Osmoregulation in the extremely euryhaline marine microalga, Chlorella autotrophiea. Plant Physiol. 74, 1010-1015 Blunden, G., E1 Barouni, M.M., Gordon, S.M., McLean, W.F.H., Rogers, D.J. (1981) Extraction, purification and characterization of Dragendorff-positive compounds from some British marine algae. Bot. Mar. 24, 451-456 Blunden, G., Gordon, S.M., McLean, W.F.H., Guiry, M.D. (1982) The distribution and possible taxonomic significance of quaternary ammonium and other Dragendorff-positive compounds in some genera of marine algae. Bot. Mar. 25, 563-567 Campbell, J.W. (1970) Comparative biochemistry of nitrogen metabolism. I. The invertebrates. Academic Press, London New York Challenger, F. (1959) Aspects of the organic chemistry of sulphur. Butterworths, London Dick, D.A.T. (1979) Structure and properties of water in the cell. In: Mechanisms of osmoregulation in animals: maintenance of cell volume, pp. 3-45, Gilles, R., ed. John Wiley & Sons, Chichester, New York Brisbane Toronto
D.M.J. Dickson and G.O. Kirst : Quaternary ammonium and tertiary sulphonium solutes of Platymonas Dickson, D.M.J., Wyn Jones, R.G., Davenport, J. (1980) Steady state osmotic adaptation in Ulva laetuca. Planta 150, 158 165 Ehrenfeld, J., Cousin, J-L. (] 982) Ionic regulation of the unicellular green alga Dunaliella tertiolecta. J. Membr. Biol. 70, 47-57 Ehrenfeld, J., Cousin, J-L. (1984) Ionic regulation of the unicellular green alga Dunaliella tertiolecta: response to hypertonic shock. J. Membr. Biol. 77, 45-55 Gibson, T.S., Speirs, J., Brady, C.J. (1984) Salt tolerance in plants. II. In vitro translation of m-RNA from salt-tolerant and salt sensitive plants on wheat germ ribosomes. Response to ions and compatible organic solutes. Plant Cell Environ. 7, 579 587 Ginzburg, M. (1981) Measurement of ion concentration and fluxes in Dunaliella parva. J. Exp. Bot. 32, 321-332 Ginzburg, M.E., Brownlee, C., Jennings, D.H. (1983) The Influence of NaC1 concentrations on the amino acid and neutral soluble carbohydrate content of Dunaliella. Plant Cell Environ. 6, 381 384 Ginzburg, M., Ginzburg, B.Z. (1985) Influence of age of culture and light intensity on solute concentrations in two Dunaliella strains. J. Exp. Bot. 36, 701-712 Gorham, J., Coughlan, S.J., Storey, R., Wyn Jones, R.G. (1981) Estimation of quaternary ammonium and tertiary sulphonium compounds by thin-layer electrophoresis and scanning reflectance densitometry. J. Chromatogr. 210, 550-554 Greenway, H., Setter, T.L. (1979) Accumulation of proline and sucrose during the first hours after transfer of Chlorella emersonii to high NaC1. Aust. J. Plant Physiol. 6, 69 79 Holligan, P.M., Drew, E.A. (1971) Routine analysis by gasliquid chromatography of soluble carbohydrates in extracts of plant tissues. New Phytol 70, 27t-297 Jackson, S.G., McCandless, E.L. (1978) Simple, rapid, turbidometric determination of inorganic sulfate and/or protein. Anal. Biochem. 90, 802-808 Kirst, G.O. (1975) Effect of different concentrations of NaC1 and other osmotic substances on CO2-fixation of the unicellular alga, Platyrnonas subcordiformis. Oecologia (Berlin) 20, 237 354 Kirst, G.O. (1977a) The cell volume of the unicellular alga, Platymonas subcordiJbrmis Hazen: effect of the salinity of the culture media and of osmotic stresses. Z. Pflanzenphysiol. 81,386-394 Kirst, G.O. (1977b) Co-ordination of ionic relations and mannitol concentrations in the euryhaline alga Platymonas subcordiformis (Hazen) after osmotic shock. Planta 135, 6975 Kirst, G.O. (1977c) Ion composition of unicellular marine and freshwater algae, with special reference to Platymonas subcordiformis cultivated in media with different osmotic strengths. Oecologia (Berlin) 28, 177-189 Kirst, G.O. (1980) Phosphate transport in Platymonas subcordi-
543
formia after osmotic stresses. Z. Pflanzenphysiol. 97, 289-297 Kirst, G.O., Kramer, D. (1981) Cytological evidence for cytoplasmic volume control in Platymonas subcordiforrnis after osmotic stress. Plant Cell Environ. 4, 455-462 Larher, F., Hamelin, J., Stewart, G.R. (1977) L~ dimethylsulfonium-3-propanoique de Spartina anglica. Phytochemistry 16, 2019-2020 Munns, R., Greenway, H., Kirst, G.O. (1983) Halotolerant eukaryotes. In: Encyclopedia of plant physiology, N.S., vol. 12C: Physiological plant ecology III, pp. 5%135, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer, Berlin Heidelberg New York Nobel, P.S. (1974) Introduction to biophysical plant physiology. Freeman, San Francisco Provasoli, L., McLaughlin, J.J.A_, Droop, M.R. (1957) The development of artificial media for marine algae. Arch. Microbiol. 25, 39~428 Raven, J.A. (1976) Transport in algal cells. In: Encyclopedia of plant physiology, N.S., vol. 2A: Cells, pp. 129-182, Liittge, U., Pitman, M.G., eds. Springer, Berlin Heidelberg New York Reed, R.H. (1983a) Measurement and osmotic significance of fl-dimethylsulphoniopropionatein marine macroalgae. Mar. Sci. Lett. 4, 173-181 Reed, R.H. (1983 b) The osmotic responses of Polysiphonia Ianosa (L) Tandy from marine and estuarine sites: evidence for incomplete recovery of turgor. J. Exp. Mar. Biol. Ecol. 68, 169-193 Reed, R.H. (1984) Transient breakdown in the selective permeability of the plasma membrane of Chlorella emersonii in response to hyperosmotic shock: implications for cell water relations and osmotic adjustment. J. Membr. Biol. 82, 83-88 Reed, R.H., Chudek, J.A., Foster, R., Stewart, W.D.P. (1984) Osmotic adjustment in cyanobacteria from hypersaline environments. Arch. Microbiol. 138, 333-337 Ritchie, R.J., Larkum, A.W.D. (1985) Potassium transport in Enteromorpha intestinalis (L). Link. J. Exp. Bot. 36, 63 78 Wyn Jones, R.G., Gorham, J. (1983) Osmoregulation. In: Encyclopedia of plant physiology, N.S., vol. I2C: Physiological plant ecology III, pp. 35-58, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer, Berlin Heidelberg New York Wyn Jones, R.G., Storey, R. (1981) Betaines. In: Physiology and biochemistry of drought resistance in plant, pp. 171204, Pateg, L.G., Aspinal, D., eds. Academic Press, London New York Zmiri, A., Ginzburg, B.Z. (1983) Extracellular space and cellular sodium content in pellets of Dunaliella parva (Dead Sea, 75). Plant Sci. Lett. 30, 211-218 Received 16 July; accepted 21 November 1985
P l a n t a 9 Springer-Verlag 1986
The role of p-dimethylsulphoniopropionate, glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis D.M.J. Dickson* and G.O. Kirst Universitfit Bremen, Fachbereich Biologie, NW 2, Leobenerstrasse, D-2800 Bremen 33, Federal Republic of Germany
Abstract. The tertiary sulphonium compound, fi-
dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organi c solutes from the cells. The ions K +, Na +, C1- and the above organic solutes can account for the osmotic balance of the cells. Key words: fl-Dimethylsulphoniopropionate - Gly-
cine betaine - Homarine - Osmoacclimation -- Plalymonas.
Introduction
Platymonas (= Tetraselmis) subcordiformis is a prasinophyte unicellular flagellate alga commonly found in estuarine, coastal and marine waters. The cell is surrounded by an "envelope", the theca, which is composed of organic and inorganic residues (Kirst 1977c). The theca allows for the development of small turgor potentials. Summation of the individual osmotica, K +, Na +, CI-, HPO zand the polyhydric alcohol mannitol in cells maintained at steady-state salinities accounts for only about 60% of the assumed intracellular osmolality, rci (Kirst 1977a, b, c; 1980). Similar discrepancies between rq and the extracellular osmolality, rCo,are School of Biological Sciences, University College of Swansea, Swansea SA2 8PP, UK. Abbreviations: DMSP=fi-dimethylsulphoniopropionate; ~ = intracellular osmolality; ~o = extracellular osmolality * Present address:
evident in Dunaliella parva (Ginzburg 1981 ; Ginzburg et al. 1983) and in Dunaliella tertiolecta (Ehrenfeld and Cousin 1982, 1984). The imino acid proline is not detected in cell extracts of P. subcordiformis (Kirst, unpublished data) and the levels of total amino acids are low (about 30 m o l . m -3 cell osmotic volume) and change little with salinity variation (Kirst 1975). The apparent inability to account for an osmotic equilibrium between the cells and the external media has prompted Ahmad and Hellebust (1984) to call for a reassessment of the role of inorganic solutes in the osmoregulation of marine unicellular algae. Deficits in an estimation of rci might also arise from an assumption that low-molecular-weight carbohydrates and or proline are the only major intracellular organic osmotica synthesized by unicellular eukaryotic algae. Certainly, the range of reported organic osmotica accumulated by any one alga does not appear to exceed two to three solutes in number (Munns et al. 1983). In view of the sensitivity of in-vitro eukaryotic enzymes and the protein-synthesizing system to high electrolyte concentrations, it would be beneficial to both higher- and lower-plant cells to accumulate a range of "compatible" solutes at the expense of limiting intracellular electrolytes (Munns et al. 1983; Gibson et al. 1984). Quaternary ammonium and tertiary sulphonium compounds (= " o n i u m " solutes) could be involved in the osmotic adaptation of eukaryotic unicellular algae to saline environments. Quaternary ammonium compounds are synthesized in a number of higher plants subjected to NaC1 stress (Wyn Jones and Storey 1981) and the solutes occur in marine macroalgae (Blunden et al. 1981, 1982). Although the quaternary ammonium solute glycine betaine is involved in the osmoacclimation of a number of halotolerant cyanobacteria (Reed et al. 1984), the distribution of the compound in eukar-
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
537
yotic unicellular algae has not to our knowledge been reported. The tertiary sulphonium compound fl-dimethylsulphoniopropionate ( D M S P = dimethyl-fl-propiothetin: see Wyn Jones and Storey 1981, for chemical terminology) occurs in a number of marine macroalgae (Challenger 1959) and marine microalgae (Ackman et al. 1966) and is involved in the osmoacclimation of some marine macroalgae (Dickson et al. 1980, Reed 1983a, b). Since glycine betaine is found in a number of halotolerant algae and DMSP has been reported in Platymonas spp. (Ackman et al. 1966), it was of interest to investigate whether these or other " o n i u m " solutes are involved in the osmoacclimation of P. subcordiformis and therefore contribute to a lowering in the discrepancy between rci and
Organic extraction. Cells were sedimented at 1200 g and the pellets resuspended in 10 cm 3 of the test medium and centrifuged in graduated conical tubes at 5000 g for 30 s. The packed cell columes were noted (approx. 0.5 cm3); excess medium was removed by suction, and the inner walls of the tubes rinsed with distilled water. Samples were frozen at - 20 ~ C or immediately extracted in methanol: chloroform: water (12:5 : i ; by vol. ; G o r h a m et al. 1981).
7{0 .
Estimation of extracellular-space volume. The extracellular space
Material and methods Algal material and growth conditions. Cultures of Platymonas subcordiformis (Hazen) strain 161 l a (G6ttingen culture center, F R G ) were maintained in modified basal ASP medium (Provasoli et al. 1957) containing 500 mol-m1-3 NaC1, vitamin 8 mix and molybdenum, 0.2 gmol-dm -3. Basal medium contained: MgSO4, 2 5 m o l ' m t - 3 ; MgC12, 2 2 m o l m - 3 ; KCI, 8mol-m-3; KNO3, 2 m o l . m 3; CaCI2, 2 . 5 m o l . m 3; KzHPO4, 0.3 m o l . m - 3 ; NaiSiO3, 0.025 m o l - m -3. Cells were grown in an algal biostat (10 dm 3) operating at 22o-24 ~ C in continuous light ( 1 0 W . m - 2 ) and harvested 4-5 d later at 110 g, rinsed in fresh medium, recentrifuged and used to inoculate, 350-cm 3 flasks containing 250 cm 3 ASP medium (500 molt o - 3 NaC1). Initial celt concentrations were made to approx. 3.106 cells.cm -3. The flasks were incubated at 22~ with a 14-h light (10 W - m - Z ) : 10-h dark regime. Cultures were bubbled with air (approx. 3 dm 3.rain- 1). After 48 h of equilibration the cells were stressed with hypersaline or hyposaline media described below.
Identification and estimation of "onium '" compounds. Quaternary ammonium and tertiary sulphonium compounds were preliminarily identified and semi-quantatively estimated by the thinlayer-chromatography method described by G o r h a m et al. (1981 ; see also, Blunden et al. 1981, 1982).
Carbohydrate analyses. Carbohydrates in the organic extracts, were quantified as their trimethylsilyl ethers (TMS ethers) by gas-liquid chromatography as described by Holligan and Drew (1971). Xylitol was used as internal standard. (volume between packed cells in the algal pellet), was measured with dextran blue and dilution analyses as described by Zmiri and Ginzburg (1983). Estimates were made for cells centrifuged at 1200 g for 2 rain (Kirst 1977a) and at 5000 g for 30 s (present study) in 15-cm 3 graduated conical tubes.
Estimation of cell volume and non-osmotic volume. The cell volume and non-osmotic volume was estimated as described by Kirst (1977a). The non-osmotic volume was determined from the Boyle-van't Hoff plot of cell volume versus l/Zo (external osmolality) using NaC1 as the solute (Nobel 1974; Dick 1979). Recently the method has come under criticism as the values for the non-osmotic volume of a cell depends largely on the permeability of the plasmalemma to the solute (Ginzburg 1981 ; Reed 1984). Abrupt NaC1 hypersaline stress leads to an influx of N a + and C1- to the cells of P. subcordiformis and other algae. Thus the number of intracellular osmotica does not remain constant and the cells could not possible behave as perfect osmometers, as the influx of ions would mitigate the loss of cell-water through osmosis. It is also possible that NaCl-induced hypersaline stress leads to losses of intracellular K + (Reed 1984).
Hypersaline and hyposaline stress. Two hours after the onset of the light period the required mass of NaC1 was added to cultures to give final concentrations of 1000 or 750 m o l . m -3. For hyposaline stress, cells were sedimented at 200 g for 2 min and the pellets distributed in the appropriate medium (300 or 150 m o l - m - 3 NaC1). All experiments were duplicated.
Inorganic-ion analyses. Sedimented cells (1200 g for 2 rain) were resuspended in 150 cm 3 ice-cold isoosmotic Ca(NO3)2 solution and bubbled with air for 2 rain and recentrifuged. The procedure was repeated with 50 cm 3 ice-cold io'osmotic Ca(NO3)2. Finally the pellets were mixed with 10 cm 3 of the Ca(NO3)z solution, transferred to graduated 15-cm a conical tubes and centrifuged at 5000 g for 30 s. The Ca(NOr)2 solution was removed by suction, the pellet (approx. 0.5 cm 3) suspended in 5 cm 3 of deionised distilled water and disrupted in a French pressure cell at 200 MPa. Extracts, free of cell wall debris, were made up to 10 cm 3. The ions N a +, K + and Mg 2+ were estimated by atomic absorption spectroscopy; C1- by chloride titration and SO42 by the method of Jackson and McCandless (1978). The Ca(NO3)2 washing procedures were performed within 10 rain. Preliminary trials indicated no apparent changes in the packed cell volume of the NaCl-grown algae after the isoosmotic Ca(NO3)2 rinse.
Units. In our preliminary studies the water content:fresh weight ratios of the cells were found to be variable, whereas the estimated cell volumes and non-osmotic volumes gave consistent values. Therefore, and unless otherwise stated, the intracellular solute contents of the cells are expressed in terms of the osmotic volume; i.e. total cell volume corrected for the non-osmotic volume. Solute concentrations are given as gmol-cm 3 cell osmotic volume. We cannot exclude the possibility that the cell osmotic volume has been overestimated (see above) and, therefore, the calculated intracellular solute concentrations are likely to be in excess of the true values. The tables from Wyn Jones and G o t h a m (1983) were used to convert osmolalities from units of m o s m o l . k g -1 H 2 0 t o MPa. Chemicals. The hydrochloride salt of fl-dimethylsulphoniopropionate was prepared as described by Larher et al. (1977). All other chemicals and reagents were obtained commercially.
Results E x t r a c e l l u l a r - s p a c e volume. T h e e x t r a c e l l u l a r s p a c e for cells centrifuged
at 5 000 g for 30 s was found
538
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
to be 18-23% of the sediment volume. A value of 38-43% was found for cells centrifuged at 1200 g for 2 rain and the value is consistent with that observed by Kirst (1977a). Similar decreases of the extracellular-space volume of Dunaliella parva (var. 75) pellets with increasing centrifugal force were reported by Zmiri and Ginzburg (1983).
a
140-
total cell volume
130120
*c- 110o o 100.
u-o
90. 80-
Cell volume and non-osmotic volume. The non-os' motic volume of the cells, determined from the Boyle-van't Hoff plot, w a s 0 . 2 8 . 1 0 - 9 c m 3 (280 gin3). Criticisms regarding this method of estimating the non-osmotic volume of cells are given in the methods. The non-osmotic volume did not vary for cells exposed to or cultured in a range of external NaC1 concentrations (]50-1000 mol. m-3). The total volume of a cell grown in Provasoli medium containing 500 m o l . m -3 NaC1 was 0 . 4 3 2 - ] 0 - 9 c m 3 (=432 gm3; Table 1). The values given here for the volume and non-osmotic volume of cells are consistent with those reported by Kirst (1977a). Changes in the total cell volume of P. subcordiformis were followed as a function of time after exposing the algae to various external NaC1 concentrations (Fig. 1 a). The data are corrected for the non-osmotic volume and shown in Fig. ] b. The initial changes of cell volume are similar to those reported by Kirst (1977a). However, in this study, volume changes were recorded over 24 h. A comparison of Fig. 1 a and ] b shows that the changes of total cell volume are less than those for the cell osmotic volume. The data indicate that
70d
015
i
Ilk
3
4
5
6
24
hours
200-
~
180160.
b
/~b-~ --
cell osmotic volume
//'-% 120_
~ 1oo_ r - - -
-'"-
=~-~
~
~
80_
60_ 40. (~ 0]5 1' l/ :~ 3[ d g g 1/--24 hours Fig. 1 a, b. Time courses of the changes in the total cell volume (uncorrected for the non-osmotic volume - a) and cell osmotic volume (corrected for the non-osmotic volume - b) of P. subcordiformis exposed to hypersaline media 1000 (A), and 750 (z0 mol.m -3 NaC1; and hyposaline media 300 (o) and 150 (e) mol.m -3 NaC1. Control, 500mol-m -3 NaC1 medium (,,). Each point represents the mean of four duplicated experiments
TaMe 1. Steady-state intracellular ion levels of P. subcordiformis cells cultured in the test NaC1 media. Values are for cells washed in ice-cold isoosmotic Ca(NO3)2 solutions, as described in material and methods, and are the means of 12 separate determinations. The 95% confidence intervals are given for the ion concentrations corrected for the non-osmotic volume. Algae were sampled during the light period (1.107 pW-m-2). The data of Kirst (1977c) for cells washed in ice-cold isotonic sucrose solutions are shown in parentheses NaCI in medium (mol' m -3) 150
300
500
Total cell volume (gin 3) Non-osmotic volume (l~m3)
495 280
451 280
432 280
Ion levels K + g m o l ' c m -3 cell osmotic volume K + gmol.cm -3 packed cells K + fmol-cel1-1
255_+9 111 (116) 55 (65)
370 _+13 140 (125) 63 (60)
463 _+11 163 (150) 71 (60)
Na + gmol.cm -3 cell osmotic volume Na + gmol.cm -3 packed cells Na + fmol- cell- 1
23_+4 10 (8) 6 (4)
39+5 15 (25) 7 (15)
C1- I~mol' c m - 3 cell osmotic volume C1- pmol.cm -3 packed cells C1 fmol.cell- 1 M g 2 + fmol. cell- 1 SO4z- fmol.cell a
28 4- 3 12 (9) 6 (8) 7 (7) 3
52___3 20 (13) 9 (8) 7 3
(7)
750
1000
429 280
410 280
(200) (60)
558 _ 12 194 83
712_+ 14 226 92
42+5 15 (60) 7 (25)
(100) (36)
46_+6 16 7
51_+5 16 7
624-4 22 (33) 9 (13)
(41) (13)
83_+5 28 12
88+_5 31 12
7 (7) 3
7 (7) 3
7 3
600
(7)
(7)
D.M.J. Dicksonand G.O. Kirst: Quaternary ammoniumand tertiary sulphonium solutes of Platymonas
P. subcordiformis cells cultured in 500 m o l . m -3 NaC1 media partially regulate the cell volumes to within the range of control cells when exposed to abrupt changes of salinity in the range of 300-1000 t o o l . m - 3 NaC1. The subsequent increases of cell volumes after hypersaline stresses followed a biphasic curve towards the control values; i.e. a primary (coarse) adjustment within 2 h was followed by a secondary (fine) adjustment. A biphasic restoration of cell volume was also observed for algae exposed to the 300 tool. m -3 NaC1 hyposaline medium. In contrast to the above, cell volumes of algae exposed to the 150mo1.m -3 NaCI medium remained larger than those of the control cells and showed little tendency for regulation. The apparent incomplete cell-voume regulation of cells transferred to low salinities (namely, 1 0 0 - 1 5 0 m o l ' m -3 NaC1 media) is primarily caused by their forming large intracellular vacuoles; the cytoplasmic and chloroplastic volumes recover within minutes to those values of the control cells (Kirst and Kramer 1981). The observations indicate that the volume of the protoplasm rather than the vacuoles is regulated. The vacuoles have been proposed to act as sinks for excessive ions and organic solutes in algae exposed to hyposaline media, thus leading to increased water fluxes from the chloroplast-cytoplasm to the vacuole. Inorganic-ion levels. The steady-state intracellular ion levels of P. subcordiformis cells obtained by washing cells in ice-cold isoosmotic Ca(NO3)2 solutions, as described in the methods, are shown in Table 1. Data are given in g m o l - c m - 3 cell osmotic volume (corrected for the non-osmotic volume), gmol. c m - 3 packed cells (uncorrected for the non-osmotic volume) and ion content (fmol) per cell. Algal pellets suspended in ice-cold isoosmotic Ca(NOa)2 solutions for 2-10 rain yielded similar ion levels as those of Table 1 (data not shown). For comparative purposes the values for the ion composition of the cells washed in isotonic sucrose solutions (see Kirst 1977 b, c) are given in parentheses. Table 1 indicates that the Na + and C1- concentrations of cells washed in isoosmotic Ca(NO3)2 solutions increased as a function of external salinity, but that both ions expressed in terms of content (fmol) per cell remained more or less constant. In contrast to Na + and Cl-, the K + content of the cells increased and the intracellular K +/Na + ratios of the algae grown in the test salinities were in excess of a value of 10. In the earlier studies (Kirst 1977b, c) the K + content of P. subcordiformis did not change in response to higher salinities, and the Na + levels increased re-
539
sulting in low intracellular K + / N a + ratios (namely, 2 to 3 for the algae grown in 600 tool-m-3 NaC1 medium - see Table 1). The differences between the ion data shown in the current study and those given by Kirst (1977b, c) appear to be related to whether the algae were rinsed in ice-cold isoosmotic Ca(NO3)z or ice-cold isotonic sucrose solutions. The two methods are evaluated in the Discussion.
Organic compounds. Three " o n i u m " compounds were detected in the organic extracts of P. subcordiformis cells. These were identified as the tertiary sulphonium compound DMSP, and the quaternary ammonium compounds glycine betaine and homarine by two-dimensional thin-layer chromatography with the authentic compounds. The distribution of DMSP and glycine betaine in a number of higher and lower plants is referred to in the Introduction. Homarine (sometimes referred to in the literature as N-methyl picolinic acid) occurs in a number of marine invertebrates (Campbell 1970), but does not appear to be widespread among marine algae. The compound has been identified in the red macroalga Trichocarpus crinitus (see Blunden et al. 1981). Mannitol was the major soluble carbohydrate in the organic extracts. Trace amounts of glycerol and erythritol were detected, and these polyhydric alcohols formed less than 2% of the total cellular mannitol content. The intracellular steady-state concentrations of all known major organic solutes of P. subcordiformis exposed to a range of NaC1 media for 4 d are shown in Fig. 2. The solutes are accumulated in response to the NaC1 concentration of the media. Homarine and glycine betaine showed similar increases in the cells with respect to the media salinity. Sodium-chloride media in excess of 300 m o l - m -3 NaC1 appeared to stimulate a rapid and linear increase of intracellular mannitol. In contrast to the polyhydric alcohol, steady increases of intracellular " o n i u m " compounds were observed in the algae exposed to the test salinities. Mannitol formed the largest single intracellular organic solute in algae exposed to the 1 000 mol. m - 3 NaC1 medium. The concentrations of DMSP were always lower than those of homarine and glycine betaine, but of similar content to mannitol in cells equilibrated at the low salinities (namely, 150-300 tool - m - 3 NaC1 media). Organic compounds - hypersaline stress. Changes of the intracellular concentrations of homarine, glycinebetaine, DMSP and mannitol were followed as a function of time when P. subcordiformis was
540
D.M.J. Dickson and G.O. Kirst : Quaternary ammonium and tertiary sulphonium solutes of P l a t y m o n a s 600-
_z 4 0 0 -
a
homarine
~,
b
glycine betaine
._o 500-
300-
0 9~
4oo-
E
3so-
O
3OO-
g
200-
'~ 100-
0i
250= 200" o
0
"6
E
6
~
160"
~
' 1'2J~4
'
100-
a
'
~
300.
6 oh ' ola ' ols ' 03 ' NaCI
1~"~4 -600
C
50-
o
'
hours
hours
d
DMSP
mannitol
250.
-500
~ 200.
-400
150.
-300
'
tool d m -3 m e d i u m
Fig. 2. Steady-state levels of mannitol (A), glycine betaine (o), homarine (e) and DMSP (-) of P. s u b c o r d i f o r m i s in the light (10 W - m -z) as a function of external salinity. Each point represents the mean of three duplicated experiments. Intracellular concentrations are corrected for the non-osmotic volume
transferred from the control medium (500 tool ' m - 3 NaC1) to the hypersaline media (750 and 1 000 m o l - m - 3 NaC1) in the light (Fig. 3 a-d). Intracellular increases in the levels of the " o n i u m " solutes were observed after a lag period of about 3 h. The final amount of solute synthesized was dependent upon the NaCI concentration of the medium, and equilibrium levels were reached within 9 h after the hypersaline stress. As homarine, glycine betaine and D M S P were accumulated during a period that was concurrent with little change in the restoration of cell volume (i.e. hours 3 to 12; see Figs. I b and 3 a-c), their rates of synthesis can be expressed in terms of gmol " o n i u m " compound synthesized, c m - 3 cell osmotic volume, h - 1. The homarine, glycine betaine and D S M P synthesis rates for cells exposed to 1000 m o l ' m -3 NaC1 were 13.8, 11.1, 13,3 ~tmol. c m - 3 h - 1, respectively; the solute synthesis rates for cells exposed to the 7 5 0 m o l . m -3 NaCI medium were 6.3, 6.6, 8.8 g m o l ' c m - 3 h - l , respectively. In contrast to the data shown for t h e " onium" compounds, mannitol was immediately synthesized and final intracellular levels were reached within 30-60min (Fig. 3d). Organic compounds - hyposaline stress'. Exposure of the cells to hyposaline media (300 and 150 mol. m - 3 NaC1) led to significant decreases in the intracellular organic solute concentrations (Fig. 3 a-d). The initial decreases in the levels of the solutes in cells exposed to the 300 t o o l - m - 3 NaC1 medium were a little larger than that predicted from the increase of the cell osmotic volume (namely,
i
-200
100
u
5od
-100
O_
6
'
4
' hours
~
'
1~/~4 hours
Fig. 3a--d. Time courses of the changes in intracellular homarine (a), glycine betaine (b), DMSP (c) and mannitol (d) of P. s u b c o r d i f o r r n i s cells exposed to hypersatine 1000 (e), 750 (o) tool . m - 3 NaCI media; and hyposaline 300 (zx), 150 (A) tool .m 3 NaC1 media. Control, 500 mol.m -3 NaCI medium (,.). Each point represents the mean _+95% confidence intervals (n = 6). Cells were sampled during the light (10 W . m 2) period. Algae were stressed at time 0
+ 64% ; see Fig. 1 b). Similarly, the initial decreases in the solute contents of the cells exposed to the 150 m o l . m 3 NaC1 medium, were greater than that which can be calculated from the increase of the cell osmotic volume (namely, + 8 4 % ; see Fig. 1 b). The partial restoration of the cell volume with time was not accompanied by increase in the intracellular concentrations of the organic solutes, and further losses and-or degradation are proposed to have occurred. In the hyposaline media, D M S P was detected (to be detailed elsewhere), and mannitol has been reported to leak out of algal cells exposed to dilute seawater concentrations (Kirst 1977b). We did not attempt to analyse the possibility of homarine and glycine betaine occurring in the hyposaline media. Discussion
In common with the cytoplasmic ion concentrations of many marine macroalgae and slightly vacuolated unicellular algae (Raven 1976; Munns
D.M.J. Dicksonand G.O. Kirst: Quaternary ammoniumand tertiary sulphonium solutes of Platymonas et al. 1983), P. subcordiformis cells contain high levels of K + relative to Na + (Table 1). The cells appear to contain lower levels of NaC1 and higher amounts of K + than previously estimated. Low intracellular NaC1 levels are suggested to be of adaptive importance in cells with those metabolic activities shown to be sensitive to the ions (Munns et al. 1983; Gibson et al. 1984). There are no data on the intracellular distribution o f K + in P. subcordiformis, but considering that the volumes of the vacuoles, chloroplast and cytoplasm are 6%, 61% and 22%, respectively, of the total cell volume of algae grown at 500 m o l . m -3 NaC1 (Kirst and Kramer 1981), much of the cation K + must reside in the cytoplasm and chloroplast. Generally, estimates of the Na + and C1- content of algal cells washed in isotonic non-electrolyte solutions, with or without low concentrations of M g 2 + and Ca + +, are substantially smaller than those levels measured from correcting for the amounts of contaminating ions in the extracellular space volume of the algal pellet (Ehrenfeld and Cousin 1982; Ginzburg and Ginzburg 1985). Criticisms regarding the use of the two methods are given by Munns et al. (1983). Measurements of extracellular-space volumes are also open to error, owing to the possible uptake by the cells of the radiolabelled marker (Ginzburg 1981; Ehrenfeld and Cousin 1982; Reed 1984). Dickson etal. (1980) and more recently Ritchie and Larkum (1985) used ice-cold isoosmotic Ca(NO3)2 rinsing techniques to remove extracellular Na +, K + and C1- from the cell walls of marine macroalgae. The authors have concluded that isoosmotic rinses with Ca(NOa)2 solutions do not appear to lead to losses of intracellular ions from the algae. Our present data (Table 1) might, therefore, indicate that the isotonic sucrose rinse does not completely remove extracellular Na + and CI-, or replace NaC1 media trapped between the plasmalemma and inner theca wall, and could even lead to a loss of intracellular K + from P. subcordiformis cells (see also, Reed 1984). Our results indicate that the tertiary sulphonium compound DMSP and the quaternary ammonium compounds glycine betaine and homarine are involved in the acclimation of P. subcordiformis to increased saline environments. After a lag period of about 3 h the intracellular " o n i u m " solute concentrations increased significantly in algae transferred from 500 tool-m-3 NaC1 medium to the 750 and 1 000 t o o l . m - 3 NaC1 media (Fig. 3 a c). The 3-h time lag indicates d e - n o v o synthesis of enzymes and suitably reduced sulphur and nitrogen intermediates required for the " o n i u m " solute
541
accumulation. The duration of the time lag is greater than that observed for the synthesis of proline (namely, 30-60 min) in the NaCl-stressed freshwater alga Chlorella emersonii (Greenway and Setter 1979). In contrast to the time-lagged synthesis of " o n i u m " solutes in the alga during hypersaline stress, mannitol was rapidly accumulated and equilibrium concentrations were reached within 30-60 min (Fig. 3d; see also Kirst 1977b). Similar time courses for the increases of a number of lowmolecular-weight carbohydrates have been reported for other eukaryotic unicellular algae stressed in media hypersaline to that of the growth medium (Munns et al. 1983), and the relatively instantaneous accumulation of these carbohydrates indicates activation rather than de-novo synthesis of enzyme. Our current observations for the effects of salinity variation on the cellular levels of " o n i u m " compounds in P. subcordiformis necessitate an evaluation of the integrated activities of inorganic and organic solutes in cell-volume regulation. Abrupt hypersaline stresses lead to passive transitory increases of NaCI concentrations in P. subcordiformis and after 2 h the Na § and CI- content of the cells are similar to those of the control algae (Kirst 1977b; and observed in the current study data not shown: see also references in Reed 1984; Ehrenfeld and Cousin 1984). These passively accumulated ions are considered to act as short-term osmotica in contrast to the uptake and retention of K § and synthesis of organic solutes. Thus it appears that within 2 h of stressing P. subcordiformis cells with hypersaline media, K +, Na § CIand mannitol effect the primary (coarse) adjustments of cell-volume regulation: the subsequent synthesis of " o n i u m " solutes (3 h plus) appears to contribute to the secondary (fine) adjustments of cell-volume restoration towards that of the control cells (see Fig. 1 a-b). Considering the limited vacuolar volume of P. subcordiformis cells (see above) much of the organic solute content of the alga must be distributed between the cytoplasm and chloroplast to allow for an osmotic equilibrium between the two compartments. Compatibility of glycine betaine, at concentrations of up to 500 m o l - m - 3 and more, with various higher-plant metabolic functions has been reported (Wyn Jones and Gorham 1983; Gibson et al. 1984). Further studies are required to assess the compatibility of homarine and DMSP with algal/higher-plant metabolic processes and these compounds should be considered as speculative compatible solutes. Mannitol is non inhibitory to several soluble enzymes from P. subcordiformis.
542
D.M.J. Dickson and G.O. Kirst: Quaternary ammonium and tertiary sulphonium solutes of Platymonas
Table 2. The contribution of all major intracellular osmotica to a calculated hi, (~'ni =calc.) of P. subcordiformis cells equilibrated in the test NaC1 media. Intracellular solute concentrations are given in gmol. cm -3 cell osmotic volume and osmolalities in MPa. gb = glycine betaine; hom = homarine; mann = mannitol; no = external medium osmolality; ni cale. ions = calculated contribution of intracellular ions to Xn~ calc. ; nl calc. org. = calculated contribution of intracellular organics to Zni calc. Turgor pressure: P=Zn~ calc.-no. Solute concentrations are taken from Table l and Fig. 3
External salinity : NaC1 tool-m- 3
Solute concentration (gmol- c m - a cell osmotic volume)
Osmolality (MPa)
K+
Na +
CI-
gb
horn
DMSP
mann
150 300 500 750 1000
255 370 463 558 712
23 39 42 46 51
28 49 62 83 88
101 153 223 285 348
89 141 224 278 347
49 66 136 204 248
37 102 174 315 513
The contribution of all known intracellular osmotica to the 7q of the cells is shown in Table 2. Osmotic coefficients of 1.8 for NaC1 and 1.0 for the organic solutes are used to derive the cell osmolality. Difficulties are encountered in estimating an osmotic coefficient for K § as the cation is largely balanced by orthophosphate and polyphosphate (Kirst 1977c). The ratio of intracellular orthophosphate, PO 3 , to polyphosphate [O-(PnOn) n O - ] is approx. 1 : 1. Potassium ions bound to proteins or to polyphosphate bodies, which could largely be sequestered within unit-membrane vesicles (Kugel, personal communication), are likely to be less osmotically active than those associated with orthophosphate. The intracellular pH of the cells, 7.0-7.5 (Kugel, personal communication), would indicate that orthophosphate is present as HPO4z . For the sake of simplicity, we have assumed firstly that 70% of the intracellular K + is associated with H P O 2- and that the osmotic coefficient of KzHPOg is about 2.1 (our own measurements), and secondly, that 30% of the intracellular K + is bound to polyphosphate and that the cation has an osmotic coefficient of 0.9. For reasons stated in the methods (see "Units") the intracellular concentrations of all osmotica may be in doubt owing to a likely overestimation of the cell nonosmotic volume, and this could lead to further errors in deriving values for n~. The data in Table 2 indicate that intracellular K +, mannitol and the " o n i u m " solutes can account for the ni of P. subcordiforrnis cells exposed to all the external NaC1 media. At salinities above 500 m o l . m 3 NaC1 in the media the organic compounds contribute significantly more to the osmolality of the cells than do the inorganic solutes. The calculated turgor potentials across the plasmalemma of between 0.4 M P a and 0.6 M P a would indicate an overestimation of ni and clearly our values are too high for a marine unicellular flagellate alga. Table 2
no
nl calc. ions
ni calc. org.
Xnl calc.
P ($7~i-7c0)
0.98 1.55 2.53 3.91 5.23
0.68 1.01 1.37 1.75 2.16
0.68 1.15 1.76 2.67 3.60
1.36 2.16 3.13 4.42 5.76
0.38 0.61 0.60 0.51 0.53
does, however, indicate that all the currently identified osmotica could account for an osmotic balance in P. subcordiformis cells. Our present paper indicates a need to assess the role of quaternary ammonium and tertiary sulphonium compounds in the adaptation of eukaryotic microalgae to the saline environment. I f " onium" solutes are present in other halotolerant eukaryotic unicellular algae the compounds might allow for a calculable osmotic balance between the cells and the external media. This work was supported by a Royal Society of London European Exchange Research Felowship to David Dickson and a generous equipment grant from the Deutsche Forschungsgemeinschaft. We also thank Dr. H. Kugel (University of Bremen, FRG) for access to his unpublished data and Dr. Ursula Winter (University of Bremen) for performing the inorganic-ion analyses.
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