Plant Physiol. (1986) 81, 92-960032-0889/86/8 l/0092/05/$0 1.00/0

Determination of Ion Content and Ion Fluxes in the HalotolerantAlga Dunaliella salina

Received for publication August 27, 1985 and in revised form January 24, 1986

URI PICK, LEAH KARNI, AND MOkDHAY AVRON*Department ofBiochemistry, The Weizmann Institute ofScience, Rehovot 76100, Israel

ABSTRACT

A method to determine intracellular cation contents in Dunaliella byseparation on cation-exchange minicolumns is described. The separationefficiency of cells from extracellular cations is over 99"9%; the procedurecauses no apparent perturbation to the cells and can be,applied to measureboth fluxes and internal content of any desired cation. Using this tech-nique it is demonstrated that the intracellular averaged Na', K', andCa2 concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl,5 millimolar K@, and 0.3 millimolar Ca2" are 20 to 100 millimolar, 150to 250 millimolar, and I to 3 millimolar, respectively. The intracellularK' concentration is maintained constant over a wide range of media K'concentrations (0.5-10 millimolar), leading to a ratio of K' in the cellsto K' in the medium of 10 to 1,000. Severe linlitation of external K',induces loss of K' and increase in Na' inside the cells. The resultssuggest that Dunaliella cells possess efficient mechanisms to eliminateNa' and accumulate K' and that intracellular Na and K' concentrationsare carefully regulated. The contribution of the intracellular Na' and K'salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl,is 5 to 20%.

The unicellular green alga Dunaliella has the remarkable ca-pacity to grow and adapt itself to media ranging in salinity fromabout 50 mM up to 5 M NaCl (4). It has been clearly establishedthat the major means of osmoregulation of this wall-less alga isby production of internal glycerol at concentrations which areproportional to the external NaCl concentration (5). However,there is a considerable disagreement as to the contribution ofinorganic ions, in particular for Na+ to the overall osmoticpressure inside the cells. Different groups have determined intra-cellular Na+ concentrations ranging from around 50% (12, 16),to less than 10% (3, 6, 15) and even to about 1% (8) of theexternal NaCl concentration. These differences seem to bemainly due to difficulties in effecting a complete separation ofthe cells from the extracellular Na+; to errors in the determinationof intracellular osmotic volumes with different extracellularmarkers (8, 12, 14, 15); and to possible damage to the cells duringthe separation technique which may result in ion reequilibrationsor losses of internal contents.

This study describes the use of the Dowex-50 cation exchangeminicolumns, adapted from Gasko et al. (1 1), for determinationsof intracellular cation contents in Dunaliella salina. The tech-nique provides an excellent separation efficiency from mediumcations (over 99.9%) and a minimal perturbation to the cellsduring the separation which is brief(about 10-15 s) and is carriedout at 4°C under isoosmotic conditions. With the technique wedetermined the intracellular Na+, K+, and Ca2' content under avariety of conditions, and demonstrate that D. salina cells grown

at 1 to 4 M NaCl maintain low intracellular Na+ concentrations,high intracellular K+ concentrations, and accumulate Ca2l dur-ing normal growth.

MATERIALS AND METHODSGrowth Conditions. D. salina were grown in batch cultures, in

media containing 1 to 4 M NaCl, 50 mm NaHCO3, 5 mM KNO3,5 mM MgSO4, 0.3 mM CaCI2, 0.2 mM KH2PO4, 1.5 /LM FeC13, 6,uM EDTA, 185 ,uM H3BO4, 7 AM MnC12, 0.8 piM ZnC12, 0.02 AMCoCl2, and 0.2 nm CuCl2. Cultures were grown under continuousillimination with white fluorescent lamps at 3,800 lux, 26°C withslow continuous shaking and kept at the logarithmic growthphase. Adaptations to different NaCl concentrations were madeby growth for several days in 1 to 4 M NaCl. For adaptation tolow K+ concentrations the cells were grown for several days in aK+ deficient medium followed by 48 to 72 h growth with thespecified K+ concentrations. For steady state distribution of22Na+, 86Rb+, or 45Ca2 , algae were cultured for 24 to 48 h in thepresence of 5 to 50 ACi/ml 22Na+ or 0.1 uCi/ml 86Rb+ or 45Ca2+.

Preparation of Ion-Exchange Columns. Columns of Dowex-50Wx8, 50 to 100 mesh (Fluka) were prepared essentially ac-cording to Gasko et al. (11). Conversion of the H-form to theTris form was performed by adding solid Tris to a suspension ofthe protonated resin until the pH was above 8, followed byextensive washing with water. Columns were prepared withinPasteur pipettes (1.7 ml bed volume), or 5 or 10 ml cylinders oftuberculin syringes plugged with either glasswool or Miraclothfilter paper, and washed with 1 to 3 bed volumes of an ice-coldsolution containing isoosmotic glycerol, 10 mm choline-Cl, and10 mm Tris-MOPS,' or 5 mM imidazole, pH 7.0.. Determination of Intracellular Ion Contents. Algae in the log-arithmic phase (1-3 x 106 cells/ml) were concentrated by cen-trifugation (2000g for 5 min) and resuspended either in theoriginal medium or in fresh growth medium to a final concen-tration of 2 to 5 x 108 cells/ml. For some measurements, cellswere suspended in a minimal medium containing 1 M NaCl, 5mM KCI, 5 mM MgCl2, 0.3 mm CaCl2, and 10 mm Tris-MOPS(pH 7), and preincubated for 15 min with gentle stirring beforethe addition of the radioactive tracers. All measurements werecarried out at room temperature. Samples of 75 to 100 ,l of cellswere applied to the columns and the cells were eluted with 1.5ml (Pasteur pipettes), 5.0 ml (5 ml columns), or 10 ml (10 mlcolumns) of the washing solution at 0°C. Radioactive tracerswere determined by scintillation counting whereas Na+ and K+were determined with an Eppendorf flame photometer using K+and Na standards in the eluting medium for calibrations. Var-iation between duplicate samples did not exceed 10%.

Cell number was determined for each sample in a Coultercounter. Calculations of averaged intracellular concentrationswere done by assuming an intracellular osmotic volume of 100

'Abbreviation: MOPS, 4-morpholinepropanesulfonic acid.92 www.plantphysiol.orgon March 29, 2020 - Published by Downloaded from

Copyright © 1986 American Society of Plant Biologists. All rights reserved.

ION CONTENT AND ION FLUXES IN HALOTOLERANT ALGAE

fl/cell ( 15). Chl was extracted with 80% acetone and determinedat 663 nm (1).

RESULTS

Separation of D. salina Cells from Medium Cations on Dowex-50 Minicolumns. Gasko et al. ( 11) have introduced a techniquefor rapid separation of membrane enclosed particles from ionsin the medium on minicolumns of ion exchange resins. Thetechnique has been previously applied for measurements ofuptake and release ofdifferent ions in cells, subcellular organelles,reconstituted proteoliposomes and phospholipid vesicles but notin halophylic or halotolerant organisms. It offers two advantagesover other methods for determination of the intracellular cationcontent in whole cells: an extremely high separation capacity anda short separation time which minimizes damage to the cells.These advantages are particularly important when dealing withhalotolerant organisms, like the unicellular alga D. salina, inwhich an accurate determination of the intracellular sodiumcontent, for example, requires a very efficient elimination of allthe external sodium from the culture medium in a mild proce-dure which will not damage the fragile wall-less cells.

Figure 1 demonstrates the separation efficiency for Na+ ionsof 5 ml Dowex-50 columns to which samples from 1 to 4 M

NaCl solutions (50-200 ul) were applied. Less than 0.05% of theapplied Na+ ions is recovered in the effluent from columns loadedup with up to 300 geq Na+/column. The 1.7 ml bed volumecolumns gave a similar separation capacity for up to 100 ,ueqNa+/column.To estimate the recovery of intact cells from the columns we

have measured the recovery of Chl, and the recovery of K+ ionswhich are actively accumulated inside the cells and provide,therefore, a good criterion for the integrity of the cells. Theinternal K+ content was either measured directly in a flamephotometer or calculated from the distribution of 86Rb+ betweenthe medium and the cells. Figure 2 demonstrates that the contentof K+ recovered with the cells is linearly dependent on the cellconcentration up to about 8 x 108 cells/ml. At higher concentra-tions a significant amount of algae are trapped on the column.Our routine measurements were therefore carried out with cellsuspensions containing 2 to 5 x 108 cells/ml.

1.600

1.400

1.200

vn 1.000c

X 800

cr 700

600

, 500

400

300

200

100

100 200 300 400 500 600 700 800Na* loaded, p equivalents

FIG. 1. The Na+ separation efficiency ofDowex-50 columns. Samples(50-200 Ml) of growth medium containing 1 to 4 M NaCl were appliedto Dowex-50 columns within 5 ml tuberculin syringe cylinders andtreated as described in "Materials and Methods." The effluent wasanalyzed for Na+ in a flame photometer.

93

OA

E 16

c

P 12

48

Cells x 108 / ml

FIG. 2. Recovery of D. salina cells from Dowex-50 columns of afunction of cell concentration. D. salina cells cultured at I M NaCl weresuspended in the minimal medium at the final indicated concentrationsand incubated for 60 min with 'Rb+ in the dark. Samples (75 Al) wereapplied to Dowex-50 columns within Pasteur pipettes for determinationof intracellular 'Rb+. K+ content was calculated by assuming that K+and Rb+ behaved identically.

-

I'*C

01

E

=L

E14

-

0

L-

01

0.

0

L.

[glycerol] in washing medium, M

FIG. 3. The effect of glycerol concentration in the column washingmedium on the recovery of D. salina cells. D. salina (4 x 108 cells/ml)in the minimal I M NaCl medium were incubated for 30 min with 'Rb+.Samples (75 gl) were transferred through Dowex-50 columns withinPasteur pipettes which were preequilibrated with washing solutions con-taining the indicated glycerol concentrations. Cells were eluted with thesame solutions and the effluent was analyzed for its 'Rb+ and Chlcontent. Internal potassium content was calculated by assuming that K+and Rb+ behaved identically.

The importance of keeping the working solution isoomoticwith an appropriate concentration ofglycerol is shown in Figure3. Complete recovery of both chlorophyll and trapped K+ inalgae cultured in 1 M NaCl necessitates a minimal glycerolconcentration of 1.2 M which is osmotically equivalent to about0.75 M NaCl. A more hypotonic medium led to a partial loss oftrapped K+ probably due to breakage of some of the cells, andof Chl due to binding of broken cell fragments to the column.The routine washing medium contained, therefore, an isoosmoticor a slightly hypertonic glycerol solution. We also found thatincluding a little salt (10 mm choline Cl) and a buffer (10 mMTris-MOPS, or 5 mm imidazole, pH 7) in the washing mediumhelped to prevent cell breakage on the columns without reducingthe separation capacity.Some substances and treatments of D. salina cells interfere in

the assay. For example, extreme pH values (below pH 5 andabove pH 10), higher concentrations of certain divalent cations

II

0

0

0

0

o~~~~

0

A - A lo .. . .. .

www.plantphysiol.orgon March 29, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 81, 1986

(e.g. 1 mm Cd2"), and the presence of detergents causes bindingof cells to the columns and results in incomplete recoveries ofboth Chl and trapped K+.The viability of D. salina cells following separation on the

columns was tested visually under a microscope and by meas-uring their growth rates. The motility ofthe cells and their growthin fresh culture media were essentially unaffected by the treat-ment, suggesting that transfer through the column does not causeany significant damage to the cells.

Determination of Intracellular Concentration of Na+, K+, andCa2". Determinations ofintracellular content ofNa+ in D. salinacells cultured in 1 M NaCl are shown in Table I. The smallamount of cations passing through the columns from a culturemedium without cells were also determined and subtracted fromthe cation content of the cell-containing samples. As shown inTable I, the carry over of Na+ ions from the external mediumamounted to 60% of the total radioactivity in the effluent whenseparated on the smaller columns (Pasteur pipettes), and to about25% on the 5 ml columns. Washes of the recovered cells inisotonic glycerol did not significantly decrease the calculatedcellular content oftrapped Na+ indicating that the Na+ recoveredwith the cells is not easily washed out.

Table II lists the intracellular concentrations of Na2+, K+, andCa2+ under steady state for cells grown in media containing 1.5M NaCl. As can be seen the correction for K+ or Ca2' due toions carried over from the culture medium is negligible sinceonly low concentrations ofK+ and Ca2" are present in the growthmedium and both ions are accumulated inside the cells. Theaverage intracellular concentrations calculated for Na+, K+, andCa2' are 20 to 60 mm, 100 to 200 mm, and 2 to 3 mM,respectively. Since the external concentrations of these ions inthe culture medium were 1.5 M, 5 mm, and 0.3 mm, respectively,the results suggest that D. salina eliminates Na+ and accumulatesK+ and Ca2+ very effectively.To check whether the distribution of 86Rb+ is a reliable meas-

ure for K+ distribution in D. salina we measured the initial ratesof86Rb+ uptake and its steady state distribution at different Rb+/K+ ratios. Table III demonstrates that both the calculated K+uptake rates and the steady state concentrations are essentiallyunaffected by the Rb+/K+ ratio suggesting that Rb+ is indeed aproper analog for K+. Also, similar intracellular K+ concentra-tions were determined in the absence of Rb+ by flame photom-etry (not shown).Time Course of Equilibration of Na+, K+, and Ca2" in D. salina

in the Dark. The time course of uptake of Na+, K+, and of Ca2+

into D. salina cells in the dark is demonstrated in Figure 4. Therate of equilibration was measured with cells which have beenpreequilibrated in a medium containing I M NaCl, 5 mm KCI,and 0.3 mM CaCl2 and then provided with tracers (22Na', 86Rb+,and 45Ca2+). As can be seen the equilibration of Na+, K+, andCa2l across D. salina cell membranes in the dark is rather slow.The rates of K+ and Ca2+ were similar with half times of about60 min while the equilibration rate ofNa+ was even slower. Theaverage intracellular Na+ and K+ concentrations, after 4 h ofincubation in the dark, were 22 and 215 mm, respectively, similarto the steady state concentrations calculated after 48 h in thelight, indicating essentially complete equilibration. (Table II).Dependence of Intracellular Na+, K+, and Ca2` Contents on

the Extracellular NaCI Concentration. D. salina were adaptedand grown in 1, 2, 3 and 4 M NaCl, and their intracellular Na+,K+, and Ca2+ contents were then determined (Fig. 5). Algaegrown in media containing 1 to 3 M NaCl showed a moderateincrease in K+ content (from 175 to 230 mM) and in Na+ content(from about 20 to 40 mM) and a significant drop in theirintracellular Ca2` content. These results suggest a good regulationofboth intracellular Na+ and K+ concentrations within this rangeof external NaCl concentrations. At 4 M NaCl, K+, and Ca2+contents remained the same but Na+ content increased to 100mM. This may reflect difficulties in maintaining very low Na+content at this high extracellular NaCl concentration. It is ofinterest that the growth rate of D. salina is also markedly de-creased when the algae are grown at media containing more than4M NaCl (4).Dependence of the Intracellular Na+ and K+ Content on the

Extracellular K+ Concentrations. Since D. salina accumulatesconsiderable amounts of K+ from the medium and can thensurvive for several generations without addition of K+, weadapted D. salina cultures to different K+ concentrations for 2weeks before determining their Na+ and K+ content. Figure 6(bottom) demonstrates that below 0.5 mm external K+ there is alarge drop in the intracellular K+ content and a large increase inthe intracellular Na+ content of the cells, while between 1 to 10mm external K+, both intracellular Na+ and K+ are kept ratherconstant. A similar apparent Km[K+]Jout of about 0.15 mm wascalculated for both the drop in [K+Jin and the increase in [Na+]i.As the external K+ concentration drops from 10 to 25 ,uM the[Na+]J/[Na']. increases from 0.001 to 0.1, the [K+]i/[K+]J from10 to 1,000 and the [K+]i/[Na+]i decreases from 15 to 0.3 (Fig.6, top). These results suggest that K+ ions may have a role in theelimination of Na+ in D. salina.

Table I. Intracellular Na+Concentration in D. salinaD. salina cells cultured for 24 h in 1 M NaCl growth medium containing 50 uCi/ml Na+, were harvested by

centrifugation and concentrated to 5 x 108 cells/ml in the original growth medium. Samples (75 jul) of cellsuspensions or of medium without cells were applied to columns contained in Pasteur pipettes or in 5 mlsyringe cylinders. Where indicated the cells in the effluent were collected by centrifugation (10 min at 2,000gat 4°C; once washed), resuspended in 5 ml washing medium and collected again (twice washed). Numbers inthe table represent averages of the three samples. Percent in effluent is the percent of the total 22Na' appliedto the column which were recovered in the effluent. Percent extracellular is the calculated percent ofextracellular22Na+ in the effluent of the cell containing samples. [Na+]i values are the averaged intracellular Na+ concentra-tions calculated with or without correction for extracellular Na+ by assuming an intracellular volume of 100 flper cell.

Five Milliliter SyringeParameter Unit Pasteur Pipettes

No wash One wash Two washes

Applied radioactivity cpm 6 x 106 6 x 106 6 x 106 6 x 106Effluent without cells cpm (%) 2,400 (0.04) 570 (0.01)Effluent with cells cpm 4,050 2,340 1,560 1,320Extracellular % 59% 24%[Na+]i (uncorrected) mm 133 75[Nal]i (corrected) mm 54 48 50 42

94 PICK ET AL.

www.plantphysiol.orgon March 29, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

ION CONTENT AND ION FLUXES IN HALOTOLERANT ALGAE

Table II. Steady State Concentrations ofNa+, K+, and Ca"+ inD. salina

D. salina cultures in 1.5 M NaCI were grown for 48 h in the presenceof 22Na+, 86Rb+, or 45Ca2+, harvested and resuspended at 2 x 108 cells/ml in the original growth medium. 22Na+ content was analyzed on 5 mlcolumns and 1Rb+ and Ca2+ contents were analyzed on Pasteur pipettecolumns. K+ concentrations were calculated assuming that Rb+ and K+behaved identically.

Parameter Unit K+ (86Rb+) 22Na+ 45Ca2+Effluent without cells cpm 0 450 45Effluent with cells cpm 32,000 2,400 7,000Extracellular % 0.01 19 0.06[ion]i mm 193 40 3[ion]. mm 5 1,500 0.3[ion]j/[ion]. 39 0.032 10

Table III. Potassium Fluxes and Steady State Internal Concentrationsat Different K+/Rb+ Ratios

D. salina (2 x 108 cells/ml in 1 M NaCl minimal medium) wereincubated with the indicated concentrations of RbCl (including 'Rb4)and KCI. Cell samples (76 zd) were removed after 20 min and 270 minof incubation and assayed for the intracellular 'Rb4 content. The ratesand steady state average concentrations were calculated from the twodeterminations, respectively. Calculation of the values for K4 were basedon the assumption that Rb+ and K+ behaved identically.

Extracellular Intracellular Uptake Rate

[Rbi]. [K+]0 [Rb+]i [K+ + RbWIs Rb+ [K+ + Rb+]mM mM Aeq/J09 cells x h

0.1 4.9 4.3 215 0.025 1.250.3 4.7 12.4 207 0.065 1.081.0 4.0 38.5 194 0.24 1.212.5 2.5 98.0 192 0.85 1.705.0 0.0 182 182 1.60 1.60

0

0

22

C=

(0,

CL

:1.-

2Time of incubation, h

44

40LA

C

30CL,

c

20 6

0C

Cal

10,-

c

FIG. 4. Time course of Na4, K4, and Ca24 uptake by D. salina. D.salina (4 x 108 cells/ml) in the minimal medium were incubated in thedark in the presence of 22Na+, "Rb+, or 45Ca2+. At the indicated timessamples were analyzed for intracellular contents of radioactive tracers as

described in "Materials and Methods."

Dependence of Intracellular Ca2` Content on the ExtracellularCa24 Concentration. The observation that D. salina accumulatesrelatively large amounts of Ca24 from the growth medium (Fig.5) is surprising since most cells contain soluble Ca2+ in the FUMrange. Intracellular Ca2+ content of D. salina is dependent uponthe extracellular Ca24 concentration (Fig. 7). It increased from

r_

X:

E

Im

[NaCt] out, M

FIG. 5. Dependence ofintracellular Na4, K4, and Ca24 concentrationson the extracellular NaCl concentrations. D. salina adapted to I to 4 M

NaCl were cultured for 48 to 72 h at the indicated NaCl concentrationswith or without addition of 'Rb4 or 45Ca2". Intracellular content of Na4was analyzed by a flame photometer and of 'Rb4 and 45Ca2' by scinti-lation counting.

about 0.02 to 0.4 geq per 108 cells (or an averaged intracellularconcentration of 2-4 mM), as the external Ca24 increased from50 ,M to 5 mM.

DISCUSSION

The method described herein to measure the intracellularconcentrations of different cations in D. salina offers severaladvantages over previously used methods: (a) Very good resolu-tion from extracellular ions (over 99.9%) which is essential foran accurate determination of intracellular Na4, (b) rapid sepa-ration of cells from medium cations, and (c) lack of any notice-able damage to the cells or losses of intracellular content duringthe separation.The possibility that part of the ion content recovered in the

column effluent is externally bound to the cells rather thanintracellular, seems unlikely for the following reasons: (a) themaintenance of high external ionic strength throughout theprocedure, (b) the time course of equilibration which suggest alinear monophasic uptake into the cells, and (c) the specificeffects of inhibitors and ionophores on the uptake of differentions (to be described elsewhere). The results clearly indicate thatthe intracellular Na4 concentration in D. salina is very muchlower than the extracellular one, when cultured in 1 to 4 M NaCl.Previous measurements of intracellular Na4 concentrations inDunaliella gave varying results ranging from below 10%. (3, 6,15) up to 50% (12, 16) of the external Na4 concentration. Theinconsistencies seem to be mainly due to inherent methodologi-cal inaccuracies. In retrospect it is clear why any distributiontechnique of Na4 between pelleted cells, or cells separatedthrough a silicone layer, and the supernatant would necessarilyfail to give an accurate measure of the intracellular Na4 concen-tration. The trapped osmotic volume of pelleted cells is around30% (15) but their intracellular Na4 concentrations are as low as1% of the external solution. Thus, an underestimate of 5% in

95

www.plantphysiol.orgon March 29, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

Plant Physiol. Vol. 81, 1986

0

0

'U

C-

0

E

'I

100.01 0.1 1[Kf] . mM

FIG. 6. Dependence of the intracellular Na+ and K+ concentrationson the extracellular KCI concentration. D. salina cells were grown for 2weeks in media containing I M NaCl and different KCI concentrationsand were then analyzed for intracellular Na+ and K+ contents and forthe final external K+ concentrations, [K+]o, in the growth medium, byflame photometry.

40

70,l

30H

0.01 0.1 10

[Ca2?]o, mM

FIG. 7. Dependence of intracellular Ca2+ concentration on the extra-cellular one. D. salina cells were cultured for 48 h at the indicated Ca2+concentrations in the presence of 45Ca2+, harvested, and analyzed forinternal 45Ca2+ content.

the determination ofthe nonosmotic volume would lead to a 10-fold overestimation of the intracellular Na+ content. By consid-ering the large variation in the determinations of osmotic spacewith different markers (8, 12, 14, 15) and the existence ofnonosmotic spaces which may be inaccessible to large macro-molecules like polyethyleneglycol (14) or Dextran 70 (8), it isreasonable to expect rather large variability in determinations ofintracellular sodium concentrations in Dunaliella. Recently, Eh-renfeld and Cousin (8) determined very low intracellular sodium

concentrations in D. tertiolecta, similar or lower than the valuesreported here, when employing a rapid washing technique. Theirsomewhat lower calculated intracellular concentrations for Na+(about 5 mM) and K+ (about 100 mM) may be due to anoverestimation of the osmotic volume (determined by Dextran70), to losses during the washing procedure, and/or to differencesin the culture technique or algal species.The observation that K+ is accumulated in Dunaliella is in

agreement with previous determinations. Averaged intracellularconcentrations of 100 to 200 mm have been reported (2, 8, 12,14). Also our observations that the intracellular K+ content iskept fairly constant over a wide range of salinities and thatstrongly limiting external K+ causes an increase of intracellularNa+ and a decrease of intracellular K+ are consistent with similarobservations in D. tertiolecta (6, 8, 9).The finding that Ca2" is accumulated by D. salina may suggest

a specific yet unknown role of Ca2" in the algae. Dunaliella cangrow in the presence ofa wide range of Ca2" concentrations froma few gM to over 10 mM. The Ca2" content dependence onexternal Ca2+ concentration indicates that the cellular Ca2+ is notas strictly regulated as the intracellular Na+ or K+ in Dunaliella.Since most living organisms sustain very low cytoplasmic Ca2`concentrations it may be expected that most of the accumulatedCa2e is precipitated in a specific target organelle inside the cells.

Finally, the contribution of intracellular inorganic ions to theoverall osmotic balance in Dunaliella can be reevaluated. Thesum of Nae and K+ concentrations under different conditions,assuming that the ions are soluble and evenly distributed insidethe cell, ranges from 150 to 300 mM. At an external NaClconcentration of 1 to 4 M, their contribution to the overallosmotic balance is in the range of 5 to 20%. These results areconsistent with the close to isoosmotic intracellular glycerolconcentrations in Dunaliella (4, 5, 7) and with the normalsusceptibility of the intracellular enzymic systems of this alga tohigh salt concentrations (3, 6, 10, 13).

LITERATURE CITED

1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidasein Beta vulgaris. Plant Physiol 24: 1-15

2. BALNOKIN YV, AV MEDVEDER, IV BODNA 1984 Potassium transport systemsin cells of the halophylic alga Dunaliella. Sov Plant Physiol 30: 718-726

3. BEN-AMoTz A, M AVRON 1972 Photosynthetic activities of the halophylic algaDunaliella parva. Plant Physiol 49: 240-243

4. BEN-AMoTz A, M AVRON 1980 Glycerol and carotene metabolism in thehalotolerant alga Dunaliella: a model system for biosolar energy conversion.Trends Biochem Sci 6: 297-299

5. BEN-AMoTz A, M AVRON 1983 Accumulation of metabolites by halotolerantalgae and its industrial potential. Annu Rev Microbiol 37: 95-119

6. BOROWIrZKA L, AD BROWN 1974 The salt relations of marine and halophylicspecies of the unicellular green alga Dunaliella. Arch Microbiol 96: 37-55

7. DEGANI H, I SUSSMAN, GA PESCHEK, M AVRON 1985 '3C and 'H NMR studiesof osmoregulation in Dunaliella. Biochim Biophys Acta. 846:313-323

8. EHRENFELD J, JL COUSIN 1982 Ionic regulation of the unicellular green algaDunaliella tertiolecia. J Membr Biol 70: 47-57

9. EHRENFELD J, JL COUSIN 1984 Ionic regulation of the unicellular green algaD. tertiolecta: response to hypertonic shock. J Membr Biol 77: 45-55

10. FINEL M, U PICK, S SELMAN-REIMER, BR SELMAN 1984 Purification andcharacterization of a glycerol-resistant CF0-CF, and CF,-ATPase from thehalotolerant alga Dunaliella bardawil. Plant Physiol 74: 766-772

1 1. GASKo DO, AF KNOWLES, HG SHERTZER, EM SOULINNA, E RACKER 1976The use ion-exchange resins for studying ion transport in biological systems.Anal Biochem 72: 57-65

12. GIMMLER H, R SCHIRLING 1978 Cation permeability of the plasmalemma ofthe halotolerant alga Dunaliella parva. Cation content and glycerol concen-tration of the cells as dependent upon external NaCI concentration. ZPflanzenphysiol 87: 435-444

13. GIMMLER H, R KAADEN, U KIRCHNER 1984 The chloride sensitivity ofDunaliella parva enzymes. Z Pflanzenphysiol 1 14: 113-150

14. GINZBURG M 1981 Measurements of ionic concentrations and fluxes inDunaliella parva. J Exp Bot 32: 321-332

15. KATZ A, M AVRON 1985 Cellular volume and sodium concentrations inDunaliella. Plant Physiol 78: 817-820

16. ZMIRi A, BZ GINZBURG 1983 Extracellular space and cellular sodium contentin pellets of Dunaliella parva. Plant Sci Lett 30: 211-218

[Ca2-][caca2

20r._

' '10

I

96 PICK ET AL.

l

www.plantphysiol.orgon March 29, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.