Loss of Intracellular Glycerol from Dunaliella tertiolecta after Decreasing the External Salinity

Loss of Intracellular Glycerol from Dunaliella tertiolecta after Decreasing the External Salinity

M. A. ZIDAN1), M. F. HIPKINS2) and A. D. BONEY

Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.

Received July 4, 1986 . Accepted September 15, 1986

Summary

When subjected to hypotonic shock, Dunaliella tertiolecta cells undergo a decrease in intracellular glycerol. If grown in an artificial seawater medium containing 1.5M NaCl, and transferred to a medium containing 0.1 M NaCl, about 75 % of the intracellular glycerol is lost: twothirds of the glycerol lost from the cells is found in the external medium (<<leakage») whilst the remaining one-third appears to be metabolised to a non-osmotically active storage compound. Loss of intracellular glycerol following hypotonic shocks is essentially light-independent, but both leakage and metabolism are inhibited by the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting a dependence on A TP and that the permeability of the cell membrane to glycerol is low. Low concentrations of CCCP only inhibit loss of intracellular glycerol when cells are incubated in the dark after hypotonic stress: this inhibition is relieved by light when photosynthetic phosphorylation can occur, but loss of intracellular glycerol is then suppressed by the ATPase inhibitor tri-n-butyl tin chloride (TNBT). These data may help resolve the apparent discrepancy between those reports of glycerol leakage from Dunaliella, and those suggesting that the cell membrane of the alga has a very low permeability coefficient for glycerol.

Key words: Dunaliella, hypotonic shock, intracellular glycero~ osmoregulation, uncouplers.

Introduction

Dunaliella tertiolecta is a biflagellate unicellular green alga which lacks a cell wall, but otherwise has a structure typical of members of the order V olvocales (class Chlorophyceae) (Oliveria et aI., 1980). It is a euryhaline marine organism, able to grow in a wide range of salinities. The capacity of Dunaliella for osmoregulation has been attributed to the ability of the cells to synthesise glycerol intracellularly in response to hypertonic stress (Ben Amotz and Avron, 1973). Moreover, glycerol is lost from the cells of Dunaliella when the cells are subjected to hypotonic stress.

There are two particular aspects of osmoregulation in Dunaliella that are at present unclear. The first is the role of substances other than glycerol, particularly NaCl, in osmoregulation. It was proposed by Ben Amotz and Avron (1973) that osmoregula-

I) Permanent address: Department of Botany, Faculty of Science, University of Assiut, Assiut, Egypt.

2) To whom correspondence should be addressed. Abbreviations: CCCP; carbonyl cyanide m-chlorophenyl hydrazone; DCMU; 3-(3',4'-di

chlorophenyl)-l,l-dimethylurea; FCCP; carbonyl cyanide-p-trifluoromethoxyphenyl hydrawne; TNBT: tri-n-butyl tin chloride.

J. Plant. Physiol. Vol. 127. pp. 461-469 (1987)

462 M. A. ZIDAN, M. F. HIPKINS and A. D. BONEY

tion was accounted for by changes in intracellular glycerol alone. It has also been suggested that other substances participate (Latorella and Vadas, 1973; Muller and Wegmann, 1978) with Gimmler and Schirling (1978) suggesting that NaCI must be involved since the uncoupler of phosphorylation carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) inhibited glycerol synthesis, but volume regulation was unaffected. In contrast, Katz and Avron (1985) found that the steady-state intracellular level of NaCI in Dunaliella salina was constant at about 100 mM when the external NaCI concentration was varied from O.5M to 4.0M. Immediately after a hypotonic shock, however, intracellular levels of Na + and CI- do increase transiently (Ginzburg, 1981; M. A. Hajibagheri, D. J. Gilmour, J. C. Collins, and T. J. Flowers, personal communication).

The second unresolved aspect of osmoregulation in Dunaliella concerns the leakage of glycerol from the cells. Glycerol has long been recognised as an extracellular product of Dunaliella (see, for example, Hellebust, 1965; Eppley and Sloan, 1965; Huntsman, 1972), and it has been suggested that glycerol «leaks» from the cells when they are subjected to hypotonic shock (Frank and Wegmann, 1974; Jones and Galloway, 1979; Gilmour et al., 1984). Data have also been presented that show that leakage is only a minor process after hypotonic shock (Kessly and Brown, 1981), or that it only occurs after a certain «threshold» has been exceeded (Ben Amotz and A vron, 1973; Ben Amotz, 1975). In these cases, glycerol is thought to be metabolised to an osmotically inactive form, possibly ex (1-4) glucans (Gimmler and Moller, 1981; Degani et aI., 1985). There is also little agreement on the permeability of the cell membrane of Dunaliella to glycerol. Enhuber and Gimmler (1980) found that the cell membrane of D. parva was not exceptionally impermeable to glycerol, while Brown et al. (1982) using nmr spectroscopy found that the cell membrane of D. salina had a very low permeability to glycerol. Wegmann et al. (1980) found that elevated temperatures caused leakage of glycerol from cells.

In this paper we report our investigations on the fate of glycerol in cells of D. tertio· lecta grown in 1.5 M NaCI and subjected to various hypotonic shocks. We have specifically investigated the roles of glycerol leakage and metabolism.

Materials and Methods Dunaliella tertiolecta strain 19/6A was obtained from the Culture Centre of Algae and Proto

zoa, Cambridge, U.K. The cells were cultured in 250 ml conical flasks using an artificial sea water medium containing either 1.5M, 1.0M or 0.5M NaCI (Bruggemann et al., 1978). They were illuminated with irradiation from high-pressure mercury lamps (radiant flux density 11 W m - 2, 12 h photoperiod) at a temperature of 25°C. Algae were used at the late log phase of growth.

Cells were harvested by centrifugation at 3500 x gfor 5 min, and were resuspended with minimal agitation at ten times the original cell density in 16 mM phosphate buffer, pH 7.5, containing 2 mM MgCh and the same concentration of NaCI as in the growth medium. Chlorophyll was determined from methanolic extracts of the cells (Holden, 1976).

Cells were hypotonically stressed by resuspending harvested cells (chlorophyll content about 150/Lg) in 20 ml of incubation medium which contained 16 mM phosphate buffer and the appropriate concentration of NaCI. Tubes were incubated in the growth room; those for dark treatments were covered with aluminium foil. At the end of the incubation the cells were


Intracellular glycerol in Dunaliella 463

harvested by centrifugation at 3500 x g for 10 min. The reaction was stopped by the addition of 3 % trichloroacetic acid (final concentration). Glycerol in the cells and the external medium was assayed by the method of Lambert and Neish (1950).

The photosynthetic inhibitors 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and tri-n-butyl tin chloride (TNBT) were dis~ solved in ethanol. The final concentration of solvent did not exceed 0.2 % (v/v).

Results

Fig.l shows a time-course for intracellular and extracellular glycerol for cells which were grown in artificial sea water containing 1.5 M NaCl, and then transferred to a medium containing 0.5 M NaCI at the start of the experiment. The data indicate that the amount of glycerol in the cells decreased by about half in the first 30 min after the hypotonic shock; the sharp decrease was then followed by a much slower decline over the next 90 min. The glycerol in the external medium showed a reciprocal trend, whilst the total glycerol declined steadily over the 2 h incubation period by about 13 %.

Fig. 2 shows the results of an experiment where the cells were incubated in the light for 2 h after hypotonic shocks of various magnitudes. In each case the glycerol content of the cells decreased during the incubation period, with the intracellular glycerol reaching a level dictated by the external NaCl concentration, not the salinity in which the cells were grown. Moreover, the cells responded to hypotonicity by a combination of leakage of glycerol to the external medium, and metabolism of glycerol to some other substance inside the cell, leading to a decrease in the aggregate amount of

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0 30 60 90 120

TIME (min)

Fig. 1: The effect of decreasing the external salinity from 1.5 M to 0.5 M NaCl on the intracellular glycerol content of D. tertiolecta. Total glycerol (--e--), glycerol in the cells (-11--) and glycerol in the medium (--A--). The bars show the standard errors of at least three determinations.



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0.5 0.3 0.' 1.0 0.5 0.' 1.5 1·0 0.5 0.'

(FINAL Noel) (MOLAR)

Fig.2: The effect of decreasing the external salinity to values between: A, 0.5 M and 0.1 M NaCI; B, 1.0 M and 0.1 M NaCI and C, 1.5 and 0.1 M NaCI on the intracellular glycerol content of D. tertiolecta. The algae were incubated in the light for 2 hours. The symbols and the bars are as described in Fig. 1.

Table 1a: Percentage of total glycerol lost aher hypotonic shock.

[NaCl] in Incubation growth medium, Dark Light M

0.5 1.0 1.5

11.1 17.4 21.8

19.6 25.8 24.2

Cells were incubated for 2 h aher the hypotonic shock; final [NaCl] = 0.1 M in each case.

Table Ib: Percentage of intracellular glycerol found outside the cells aher hypotonic shock.

[NaCl] in Incubation growth medium, Dark Light M

0.5 1.0 1.5

53.7 46.7 51.0

61.7 53.9 54.0

Cells were incubated for 2 h aher the hypotonic shock; final [NaCl] = 0.1 M in each case.

glycerol found inside and outside the cells. There was no «threshold» effect: glycerol leakage and metabolism both occurred even for relatively small hypotonic stresses, although the proportion of the glycerol lost by each route varied (Table 1).

J. Plant. Physiol. Vol. 127. pp. 461-469 {1987}


70

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60

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~ £ 40

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~ -l

~ lJJ

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, \ I , , I 0.5 0.3 0.\ \.0 0.5 0.\ 1.5 1.0 OS 0.\

(FINAL NoCI) (MOLAR)

Fig.3: The effect of decreasing the external salinity to values between: A, 0.5 M and 0.1 M NaCl; B, 1.0M and O.lM NaCl and C, 1.SM and O.lM NaCl on the intracellular glycerol content of D. tertiolecta. The algae were incubated in the dark for 2 hours. The symbols and the bars are as described in Fig. 1.

The results of a corresponding experiment where the cells were incubated in the dark following hypotonic shock are shown in Fig.3. The data broadly correspond with those of Fig. 2; the detailed differences are given in Table 1.

DCMU is an inhibitor of non-cyclic photosynthetic electron transfer (Izawa and Good, 1972). When cells were shocked hypotonically and incubated in the light, the addition of 10 I'M DCMU gave results which closely resembled those when the cells were incubated in the dark (Fig. 4 A). In contrast, the uncoupler of photosynthetic and oxidative phosphorylation CCCP (Izawa and Good, 1972) almost completely suppressed the ability of the cells to respond to hypotonic shock. When added to a final concentration of 20 I'M to cells incubated in the light only a few percent of the glycerol leaked from the cells and none was metabolised (Fig. 4 B).

Fig. 5 shows the results of an experiment where sub-optimal concentrations of CCCP were used. In full incubating light (11 W m- 2

), 10 I'M CCCP had only a slight inhibitory effect on glycerol leakage and metabolism (Fig. 5 A), whilst when light intensity was reduced to 5.sW m- 2 the inhibition was more marked (Fig. 5B). Both glycerol leakage and metabolism were affected about equally. When cells were incubated in the dark after hypotonic shock, 10 I'M CCCP almost completely inhibited glycerol leakage and metabolism (Fig. 5 C).

TNBT is an inhibitor of the chloroplast ATPase (coupling factor) (Kahn, 1968). When added to light-incubated cells together with a sub-optimal concentration of CCCP (10 ~), the responses of the cell to a hypotonic shock were eliminated (Fig. 6). The concentration of TNBT used (4.5 I'M) inhibited COrdependent oxygen

]. Plant. Physiol. Vol. 127. pp. 461-469 (1987)


60

50

~ -540 01 E

~ 30 E ~

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o a: w (.) 10

~ o

o , I , , I!

1.5 1.0 0.5 0.1 1.5 1.0 o.s 0.1

(FINAL NaCI)(MOLAR)

Fig. 4: The effect of decreasing the external salinity from 1.5 M to 0.1 M NaCI on the intracellular glycerol content of D. tertiolecta. The algae were incubated in the light for 2 hours with the addition of A: DCMU (lOpM) and B: CCCP (20pM). The symbols and the bars are as described in the Fig. 1.

60

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oJ 20 o a:: w <:> 10 ~ (!)

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ts 0.5 0.1 1.5 0.5 0.1 1.5 0.5 0.1

(FINAL NoCI) (MOLAR)

Fig. 5: The effect of decreasing the external salinity from 1.5 M to 0.1 M NaCI on the intracellular glycerol content of D. tertiolecta. The algae were incubated for 2 hours with the addition of CCCP (10 pM) under the following conditions: A, in full light (11 W m -2); B, in medium light (5.5 W m - 2) and C, in the dark. The symbols and the bars are as described in Fig. 1.

evolution and appreciably decelerated the rate of decay of the flash-induced field-indicating absorption change at 520nm (data not shown; Hipkins and Musto, 1985).

! Plant. Physiol. Vol. 127. pp. 461-469 {1987}


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::- 50 1 :c <.)

01 40 E

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...J 20 0 a: w <.) 10 >-...J t-O

0

,I 1.5 1.0 0.5 0.1

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Fig. 6: The effect of decreasing the external salinity from 1.5 M to 0.1 M N aCI on the intracellular glycerol content of D. tertiolecta. The algae were incubated in the light (11 W m -2) for 2 hours with the addition of a combination of CCCP (10~) and TNBT (4.5 ~). The symbols and the bars are as described in Fig. 1.

Discussion

When subjected to hypotonic stress, Dunaliella tertiolecta lost intracellular glycerol (Figs. 1-3), confirming previous findings for this species (Frank and Wegmann, 1974; Jones and Galloway, 1979; Gilmour et aI., 1984), D. parva (Ben Amotz and Avron, 1973; Ben Amotz, 1975; Gimmler and Moller, 1981) and D. salina (Degani et al., 1985). Our data indicate, however, that under all conditions used in the present study the decrease in intracellular glycerol was due to two processes: firstly, the loss of glycerol to the external medium (<<leakage»), and secondly, loss of intracellular glycerol without a corresponding change in extracellular glycerol (<<metabolism»), a process which presumably involves the metabolic conversion of glycerol to an osmotically inactive storage carbohydrate (Gimmler and Moller, 1981; Degani et al., 1985). Most of the intracellular glycerol was lost from the cells by leakage, but proportionately more was metabolised when the magnitude of the hypotonic shock was greater (Table 1). Both leakage and metabolism are to a certain extent dependent on photosynthesis, since incubation of the cells in the dark after the shock (Fig. 3, Table 1), or addition of DCMU to cells incubated in the light (Fig. 4) decreased both leakage and metabolism, although metabolism was more markedly inhibited when the extent of the hypotonic shock was small. If the processes involved in loss of intracellular glycerol require ATP, presumably this can be furnished by oxidative phosphorylation.

High concentration of the uncoupler CCCP, however, inhibited both glycerol leakage and metabolism, suggesting that both require A TP. That the metabolic conversion of glycerol to a storage polysaccharide requires ATP is not surprising (see,

J Plant. Physiol. Vol. 127. pp. 461-469 (1987)


for example, Degani et aI., 1985), and inhibition of the metabolic decrease in intracellular glycerol by the uncoupler FCCP has already been reported for D. parva (Ben Amotz and Avron, 1973). But the inhibition of glycerol leakage by an uncoupler of phosphorylation suggests that the leakage process is not a simple passive one, but requires metabolic energy. It also suggests that the passive permeability of the cell membrane to glycerol is rather low. This observation may resolve the apparently conflicting reports of a low passive permeability of the plasmalemma of Dunaliella to glycerol (Brown et al., 1982), and of glycerol leakage following hypotonic shock (Frank and Wegmann, 1974; Jones and Galloway, 1979; Gilmour et aI., 1984) if metabolic energy is used to move glycerol across a membrane that is otherwise essentially impermeable.

Lower concentrations (10 pM) of CCCP inhibited the decrease in intracellular glycerol only when the cells were incubated in the dark: oxidative phosphorylation is presumably unable to furnish the necessary ATP. But 10pM CCCP only inhibits photosynthetic phosphorylation to a certain extent, as judged by the rate of decay of the flash-induced field-indicating absorption change in Chlorella emersonii (Hipkins and Musto, 1985), so that when illuminated, sufficient photosynthetically derived A TP is available both for glycerol leakage and metabolism. When photosynthetic phosphorylation was inhibited by TNBT, the decrease in intracellular glycerol ceases, again suggesting an absolute requirement for ATP.

Our data do not allow us to draw precise conclusions about the nature of any mechanism which promotes trans-membrane glycerol movements at the expense of metabolic energy, except that photosynthetic electron transfer is not required, and the sensitivity to CCCP and TNBT suggest an absolute requirement for ATP.

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Loss of Intracellular Glycerol from Dunaliella tertiolecta after Decreasing the External Salinity