Plant Physiol. (1984) 74, 1010-10150032-0889/84/74/10 10/06/$0 1.00/0

Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica'

Received for publication October 3, 1983 and in revised form December 14, 1983

IFTIKHAR AHMAD AND JOHAN A. HELLEBUST*Department ofBotany, University of Toronto, Toronto, Ontario MSS JAI Canada

ABSTRACT

Chlorella autotrophica (Clone 580) grows over the external salinityrange of I to 400% artificial sea water (ASW), can photosynthesize overthe range from I to 600% ASW, and survives the complete evaporationof seawater. The alga grown at high salinities shows an increase in cellvolume and a small decrease in cell water content. Measurements of ioncontent were made by neutron activation analysis on cells washed inisoosmotic sorbitol solutions which contained a few millimolar of majorions to prevent ion leakage. Cells grown at various ASW concentrationscontain large quantities of sodium, potassium, and chloride ions. Meas-urements of cations associated with cell wall and intracellular macromol-ecules were made to determine intracellular concentration of free ions.The proline content of cells increases in response to increases in externalsalinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.

Algae living in marine environments such as estuaries, tidepools, and brackish water ponds can tolerate a wide range ofexternal salinities (6, 14, 15). At least some of these euryhalinealgae possess a proper cell wall, thus, so far as the solute andwater relations are concerned, they should respond to salinity ina manner similar to cells of higher plant halophytes.One of the consequences of having a rigid cell wall is that the

cell's capacity to alter the intracellular water volume is ratherlimited. This means that for such a cell osmoregulation is perhapsthe only effective means of adjusting to a changing externalsalinity.

Osmoregulation in angiosperm halophytes has received con-siderable attention over the past 10 years, and the role of bothinorganic ions and organic solutes is now well documented for awide variety of these species (1-3, 9, 27, 28, 30). The generalpattern which emerges from such studies is that in the leaf tissueof vascular halophytes the uptake and accumulation of sodiumand chloride usually accounts for 50 to 80% of the external saltconcentration, and the remainder ofthe cellular osmotic balanceis achieved by the synthesis of low mol wt organic solutes (see27).There is very little reliable information on the osmoregulatory

mechanisms in walled species of microalgae. In view of thedocumented salt sensitivity of isolated enzymes (8, 9, 28), inor-ganic ions are not considered to play a major osmoregulatoryrole in slightly vacuolated microalgae. The data presented byKirst (19) and Setter and Greenway (24) suggest that the growthof microalgae at high salinities occurs with almost completeexclusion of sodium and chloride from the cells. Consequently,

' Supported by Grant A6032 from Natural Sciences and EngineeringResearch Council of Canada.

these cells must osmoregulate primarily by the synthesis andaccumulation oforganic solutes. This appears unlikely, however,as the levels of organic solutes which such algae accumulate athigh salinities are relatively low compared to the external saltconcentrations the cells are exposed to. For example, Stichococ-cus bacillaris contained 520 mm sorbitol and 278 mm prolinewhen grown in 1234 mosm external salinity (7), Platymonassubcordiformis in 1600 mosm NaCI contained 500 mm mannitol(18), and Chlorella emersonii in 675 mosm NaCl contained 190mM proline and 50 mm sucrose (25). Clearly, such cells musthave accumulated some inorganic ions in addition to the organicsolutes for osmotic balance to exist between the cells and themedium. In view of this apparent contradiction in the literature,there appears to be a justifiable need to reassess the role ofinorganic ions in the osmoregulation of marine microalgae.

It can be argued that a complete exclusion of external saltwould be energetically very expensive to maintain for an auto-troph. Ion uptake and accumulation to a certain metabolicallycompatible level, on the other hand, could provide a comple-mentary mechanism of osmoregulation to these algae. As withother eukaryotes, the cytoplasm of marine microalgae can tol-erate the presence of a considerable amount of salt, as a signifi-cant metabolic disruption occurs only when the cytoplasmic saltconcentration exceeds a relatively high level (see 9).The present paper describes the growth and osmoregulation of

a walled, marine euryhaline micro-alga, Chlorella autotrophica.

MATERIALS AND METHODSChlorella autotrophica Shihira and Krauss (clone 580 obtained

from Dr. R. R. L. Guillard, Woods Hole Oceanographic InstituteWoods Hole, MA; Culture Collections) was grown axenically inan ASW2 medium described previously (7, 14). Culture condi-tions and cell counting were as described by Liu and Hellebust(20).Measurement of Cell Volume and Water Content. One to two

L of exponentially grown culture at each salinity were harvestedby centrifugation at 7000g for 5 min at 10 to 15C. Cells wereresuspended to 5 ml in fresh isoosmotic ASW medium in a finelygraduated centrifuge tube and centrifuged at 2000g for 5 min todetermine the pellet volume. Then the cells were resuspended to1 ml in isoosmotic ASW medium which contained 0.5 iCi3H2Oml-', and the pellet water content (3H20 space) was calculatedfrom the dilution of 3H20 in the supernatant.To determine extracellular space, a fresh cell pellet was pre-

pared as described above, suspended to 1 ml in isoosmotic ASWwhich contained 0.5 ;Ci['4C]mannitol ml-', and then centri-fuged immediately at 2000g for 5 min. The supernatant wasdiscarded, the sides of the centrifuge tube were wiped dry, and['4C]mannitol trapped in the pellet was recovered by suspending

2 Abbreviations: ASW, artificial sea water, MCW, metha-nol:chloroform:water.

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OSMOREGULATION IN CHLORELLA AUTOTROPHICA

16

T

,W20

. I

-

_ 048I-'

000x 0-439

D 100 200 300 0,25, 50 100 200

SALINITY (% ASW) SALINITY (% ASW )

1011

0

50 x

T40 E

30

20 0

-i

10 '

0

FIG. 1. Effect of salinity on growth rate and cell yield. NH41-grown cells (a); N03--grown cells (b). Growth rate (0); cell yield (vertical bars).

Table I. Effect ofGradually Increasing Salinity by Evaporation on CellVolume and Survival Rate ofCells Preadapted in 300% ASW

NH4CI NaNO3Salinitya Cell Survivalc Cell survivalc

volume volumeb%ASW fl % f %500 38.4 100 27.0 100600 50.6 95 34.6 98750 46.7 93 34.6 92Saturated 42.0 85 35.7 88'Time course of salinity increases from 300 to 750% ASW is shown

in Figure 3. Saturation ofASW was achieved in 120 h.b The ratio between the geometrically determined cell volume and the

known intracellular volume (Table II) at 300% ASW was used to calculateintracellular volume from geometric measurements of the cells at highersalinities.

I Determined by a modification of Evans blue staining techniquedescribed by Kanai and Edwards (17).

Table II. Cell Volume and Water Content

Nitrogen Source

Salinitya NH4CI NaNO3Cell Water Cell Water

volumeb content volume content

%ASW fi % f%10 20.7 71.0 15.7 69.750 19.5 70.3 16.5 69.6100 20.4 71.5 17.3 69.1200 25.7 67.4 19.5 65.6300 30.0 62.5 23.0 60.5

'Cells grown for several generations at each salinity before analysis.b As ['4C]mannitol space (see "Materials and Methods") includes the

cell wall area, the values quoted in this table are essentially ofintracellularspace.

the cells to 1 ml in nonradioactive isoosmotic ASW medium.The radioactivity ofthe supernatant was determined to calculatethe pellet extracellular volume (['4C]mannitol space).The average cell size was calculated by dividing the difference

between the pellet volume and extracellular volume by the totalnumber of cells in the cell pellet. Cell water content was basedon the difference between the pellet water volume and extracel-lular volume.

4

I3

E

t-

2

0

01

E

4%

0 50 100 200 300

SALINITY ( % ASW)

FIG. 2. Effect of salinity on photoassimilation of H'4C03-. NH4+-grown cells (0); N03-grown cells (0).

15II*

E

1-0

01

0*5

=

E'4%

0

750

500

In41

250

0

0 24 48 72 96 120

TIME (h)

FIG. 3. Effect of gradually increasing salinity by evaportion on pho-toassimilation of H'4CO3- of cells preadapted in 300% ASW. NH4'-grown cells (0); N03-grown cells (0); external salinity (A).

Bicarbonate Photoassimilation. To determine the net rate ofphotoassimilation, four ml of culture growing exponentially ateach salinity was incubated at 500 ft-c with 2.5 gCi ofNaH'4CO3for 20 min. Cells were harvested and assayed for 14C content asdescribed previously (14).

Extraction and Determination of Organic Solutes. One L ofexponentially growing culture was harvested and washed in N-free isoosmotic ASW by centrifugation. Cells were extracted inmethanol:chloroform:water (12:5:3, v/v) as described previously

I

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AHMAD AND HELLEBUST

I

0

0M

0

E

E

0 -s | I

10 50 100 200 300 10 50 100 200 300

SALINITY (%ASW) SALINITY (%ASW)

FIG. 4. Effect of salinity on organic solute content of cells. NH44-grown cells (a); NO3-grown cells (b). Proline (0); amino acids (0);soluble carbohydrates (U); sucrose (A). Inset, change in sucrose concen-

tration with external salinity. Units are the same as for main figure.

Table III. Amino Acid Content ofNH44-Grown CellsCells were grown exponentially over the external salinity range of 10

to 300% ASW. Amino acids were extracted in MCW and determined onan amino acid analyzer. Data are the average values of amino acidcontent at low (10-50% ASW) and high (100-300% ASW) salinities.Other amino acids remained at a level below 5 Mmol g-' cell H20 atvarious salinities.

Concentration

Low salinity High salinity

Amol g-' cell H20

Aspartic acid 2.5 9.7Asparagine 8.1 10.4Glutamic acid 27.9 41.5Alanine 21.8 43.2

Table IV. Distribution ofRadioactivity in the 80% Ethanol-SolubleFraction of'4C-Uniformity Labeled 200% ASW Cells

Cells were grown for 4 d in 4 ml 200% ASW medium containing 5gCi of NaH'4C03 (2 mM) at 500 ft-c and 20°C. Metabolites soluble inhot 80% aqueous ethanol were chromatographed and subjected to au-

toradiography for identification, and their '4C content determined byscintillation counting. Pro, proline; Glu, glutamic acid; Ala, alanine; Suc,sucrose; Glc, glucose; U, unknowns.

Radioactivity Pro Glu Ala Suc Glc U Total

dpm x 10-3 37.1 0.19 0.27 0.14 0.17 2.2 40.1% of total 92.7 0.5 0.7 0.3 0.4 5.4 100

(1, 2). Amino acids, the imino acid proline, and soluble carbo-hydrates were determined as described previously (1-3). Individ-ual amino acids were separated and quantified on a Beckmanamino acid analyzer model 121 using lithium citrate buffers.Sucrose and glucose were determined by a modification of the

8

-6

E 4%4%

2

0

010 50 100SALINITY (% ASW)

200

FIG. 5. Effect of salinity on ion content of NH44-grown cells.

method described by Bergmeyer (5).Uniform "C Labeling. Cells were grown for 96 h in ASW

containing NaH 14C03 (1.25 gCi ml-' culture), and extractedwith 80% aqueous ethanol. The extracts were analyzed for pro-line, amino acids, and soluble carbohydrates as described previ-ously (14).

Inorganic Ions. One L of exponentially growing cultures washarvested by centrifugation. Cells were suspended to 1.5 ml inisoosmotic sorbitol solution that contained 2.5 mm NaCl, 1 mMKCI, 1 mm CaSO4, and 1 mM MgSO4, and rapidly centrifuged at9000g for 30 s using a Beckman Microfuge B. This washingprocedure was repeated twice in less than 5 min. The sides ofthe microcentrifuge tube were wiped dry with paper tissue, andthe cells were suspended to 2.5 ml in ion-free isoosmotic sorbitolsolution. Fifty-Ml fractions of the resulting cell suspension weretransferred to each 25- x 1-mm ion free polyethylene tubing,dried at 70°C, and then sealed. Standard solutions of Na+, K+,Ca2,Mg2', and Cl-, each containing 1 ,umol ml-', were prepared,and 50-Al fractions were transferred to polyethylene tubing asdescribed above.Neutron Activation Analysis. The polyethylene vials were

sealed in irradiation capsules and then irradiated in the low-fluxSLOWPOKE-1 nuclear reactor at the University of Toronto.Long-lived radioisotopes of sodium, potassium, and magnesiumwere prepared by 16 h irradiation at a neutron flux of 2.5 x 10"neutronscm 2 s-', and the measurements of y-ray activities weremade as described previously (16). Short-lived radioisotopes ofcalcium and chloride were prepared by 5-min irradiation at aneutron flux of 1 x 1012 neutrons cm-2 s ', and y-ray activitieswere measured exactly after 1 min as described above.

Preparation of Cell Wall Fraction and Permeabilized Cells.Cells from 6 x 1 L of exponentially growing cultures wereharvested by centrifugation and suspended to 8 ml in superna-tant. The resulting cell suspension was divided into two homog-enous fractions of 4 ml each. Cells collected from one suchfraction were permeabilized by four cycles of freeze-thaw usingliquid N2, and then suspended to 1.5 ml in H20.

Cells from the other 4-ml fraction were disrupted by twopassages through a French Pressure Cell (American InstrumentCo., Silver Spring, MD) at 0 to 5°C and a cell pressure of 7 KPa.Microscopic examination of the resulting suspension showed acomplete breakage of cells. The disrupted suspension was centri-fuged at 6000g for 5 min at 5°C, and the pellet was suspendedto 1.5 ml in H2).

Dialyses. The cell wall and the permeabilized cell preparations

No'_

~~~~~~~~~K4

Mg2'

p p r Co2+I I -I

10).

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OSMOREGULATION IN CHLORELLA AUTOTROPHICA

Table V. Distribulion ofIntracellular Solutes and Cell Osmotic Balance

Salinity Cell Ion Conc. Osmotic Potential PressurePotential

Monovalent cations Divalent cations Cl-'Total Bound Free Total Bound Free (Free) ng g a

% ASW MPa mmol kg-' cell H20 MPa10 -0.27 501 82 419 79 50 29 295 1.68 0.27 -1.95 1.6850 -1.07 664 88 576 81 53 28 454 2.32 0.46 -2.78 1.71100 -2.00 711 83 628 60 50 10 509 2.49 0.74 -3.23 1.23200 -3.93 666 72 594 54 42 12 497 2.37 2.04 -4.41 0.48

, = external osmotic potential; '(w = cell osmotic potential (rinorg + worg); winorg calculated fromintracellular ion concentration using an osmotic coefficient of 0.9 for free ions, 0.8 for monovalent boundcations, and 0.2 for divalent bound cations; I MPa = 445 mosm at 20C; rorg = organic solutes; calculatedaccording to Smith and Smith (26) from the results shown in Figure 4a; i,,= pressure potential (6Xrj Xt - 0.10.

were separately sealed in short lengths of 12-mm wide dialyzertubing (12,000 mol wt cut-off; Fisher Scientific), and dialyzedagainst deionized H20 for 12 h. Water in the 2 L dialyzer tankwas changed at least four times during this period.

Extraction of Bound Cations. The content of the dialyzer tubewas suspended to 4.5 ml in H20 to which 0.5 ml concentratedHNO3 was added, and left to stand overnight at room tempera-ture. Then the suspension was centrifuged at 2000g for 10 min,and appropriate dilutions of the supernatant were analyzed forNa+, K+, Ca2+, and Mg2' content on a Perkin-Elmer atomicabsorption spectrophotometer model 4000.

RESULTSThe growth of C. autotrophica was studied as a function of

external salinities with 2 mM ammonium chloride or sodiumnitrate as the nitrogen source in the culture medium. With eitherN source, cells growing exponentially divided most rapidly andshowed maximum yield over the salinity range of 1 to 50% ASW(Fig. 1). It is interesting to note from Figure 1 that over thissalinity range the presence of ammonia in the culture mediumsupported a faster division rate and a higher cell yield than thatof nitrate.At salinities higher than that of 50% ASW the cell division

rate and cell yield of both ammonia- and nitrate-grown culturesdeclined. At 400% ASW, the cells were still dividing, but at arate too slow for accurate analysis. Cell division ceased com-pletely above 400% ASW. Nonetheless, the cells of C. autotroph-ica were able to survive extremely high salt concentrations. Whencells grown at 300% ASW were subjected to increasing salinityby evaporating the culture solution at room temperature, morethan 80% of the cells remained green and viable in saturatedseawater (Table I). Such cells grew normally when the culturesalinity was brought back from saturation to 300% ASW level.Cells isolated from dried salt crystals started dividing after a lagperiod of less than 3 d.At salinities higher than that of 100% ASW, the cell water

content decreased with an increase in external salt concentration(Table II). At 300% ASW, the water content of both ammonia-and nitrate-grown cells showed a decrease of about 15%.The response of cell volume to external salinity was, however,

different. There was an increase in cell size with increasingexternal salt concentration, and consequently the cells at 600%ASW were about twice as large as the cells at 10% ASW (TablesI and II).The alga exhibited changes in photoassimilation rate with

increases in external salt concentration (Fig. 2) which parallel itspattern ofgrowth response to salinity (Fig. 1). At salinities higherthan 10% ASW, the photoassimilation rate declined with anincrease in external salinity. At 300% ASW, the photoassimila-

tion rate of both ammonia- and nitrate-grown cells was less than40% of its maximum value. When such cells at 300% ASW weresubjected to further increases in the external salt concentrationby allowing the culture to evaporate at 20°C in a growth chamber,the photoassimilation rate continued to decline with increases inexternal salinity, and photosynthesis ceased completely when theculture salinity exceeded 600% ASW (Fig. 3).

Figure 4, a and b, shows that the imino acid proline is themajor osmoregulatory organic solute in this alga. The prolineconcentration of the cells increased with an increase in externalsalinity, particularly at levels higher than 100% ASW. At 300%ASW, the proline concentration of both ammonia- and nitrate-grown cells was in excess of 1500 mm on a cell-water basis. Withammonia as the N source, cells showed a small but consistentincrease in the levels of amino acids, which was mostly due toan accumulation of aspartic acid, asparagine, glutamic acid, andalanine (Table III). Soluble carbohydrates were also present insignificant amounts both in ammonia- and nitrate-grown cells.However, their concentrations remained largely unchanged overthe wide range of external salinity used in the experiment.Sucrose was present in detectable amounts only in cells grownwith nitrate, and showed a small initial increase with increasingsalinity, then a decline at salinities above 200% ASW (Fig. 4b,inset).

Table IV confirmed that proline was by far the most importantorganic solute present in this alga at high salinities.

Figure 5 shows the neutron activation analysis of the cellrapidly washed in isoosmotic solutions of sorbitol to which 1 to3.5 mm of major ions were added to prevent a possible ionleakage from the cells (see "Discussion"). According to ourknowledge of the extracellular volume of the cell pellets, theamounts of ions in the trapped washing solution ofthe cell pelletwere quantitatively insignificant compared to the cell ion con-tent; therefore, no correction of the cell ion content was consid-ered necessary. The data in Figure 5, however, include the cationsassociated with the cell wall, which, being outside the plasma-membrane, do not contribute to the osmotic potential of thecell. Therefore, to determine the intracellular cation content,measurements were made of the cations bound to the isolatedcell wall, and a correction was made accordingly.The concentrations of cations and chloride based on intracel-

lular water content are shown in Table V. It is evident fromthese results that the alga possesses a marked capacity to take upand accumulate inorganic ions from the medium to make os-motic adjustments. However, in order to make a reasonableassessment of the osmotic contribution of the accumulated ions,it may be necessary to consider the ionic environment of theintracellular space. Table V shows a large disparity between theconcentrations of cations and chloride in the intracellular space,

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AHMAD AND HELLEBUST

suggesting that cations may not be balanced entirely by negativecharges of the inorganic anions. A requirement for extra positiveinorganic charges to counterbalance the negative charges of thenucleic acids and proteins has been shown for a number ofmicroorganisms (see e.g. 21). Consistent with this was our obser-vation that permeabilized cells, when dialyzed to remove freeions, retained cations in amounts at least 2-fold greater thanthose found associated with the isolated cell wall, indicatingcation-binding in the intracellular space. Such levels of intracel-lular bound cations in cells grown at various salinities were foundto be similar, and an average bound content of 1.2 fmol cell-'monovalent cation and 0.54 fmol cell-' divalent cation wasdetermined for these cells. Such cations bound to macromole-cules are likely to be less active osmotically than the free ions;therefore, an osmotic coefficient of 0.9 for free ion (22), 0.8 formonovalent bound cations (4), and 0.2 for divalent bound cation(see 29) was assumed to estimate the cell inorganic osmolarity(Table V). It is evident from these results that over the salinityrange of 10 to 100% ASW, the accumulation of inorganic ionsis the primary source of osmoregulatory solutes in this alga. At200% ASW, however, the cells do not accumulate enough ionsto account for the external salt concentration.

Table V also shows the pressure potential of cells calculatedfrom the difference between intracellular and external osmolari-ties. These values of pressure potential for cells at differentsalinities are similar to those reported for other marine microal-gae (see 12).

DISCUSSIONC. autotrophica (clone 580) is exceptionally well adapted to

environments of extreme salinities. As the alga exhibits maxi-mum cell division and photoassimilation rates at external salin-ities as low as 1% of normal seawater, there is little evidence tosuggest that this strain, originally isolated from a marine envi-ronment, has a true salt requirement. Nonetheless, it can grow,although at reduced rates, at salinities as high as 400% of sea-water, and it can photosynthesize at even higher salinities. Itsurvives the saturation, and even complete evaporation of sea-water.The very small change in the cell water content exhibited by

the alga over a wide range of external salinities indicates itsexceptional capacity to osmoregulate. The accumulation of pro-line clearly plays an essential role in the osmoregulatory mecha-nisms of this alga. Cells grown at 300% ASW (2575 mosM)contained about 1600 mm proline. To our knowledge such alevel of proline in growing cells with normal turgidity is thehighest observed for any plant or microorganism species. How-ever, even-with such exceptional capacity for proline accumula-tion, the alga does not appear to make exclusive use ofthis iminoacid for osmotic adjustment. Our data show that cells grown atseawater osmolarity of up to 890 mosM (100% ASW) accumu-lated little proline (Fig. 4). Furthermore, even at 300% ASW(2575 mosM), an accumulation of 1600 mm (1750 mosm accord-ing to Smith and Smith [26]) proline, though large, equals only68% of the external osmolarity. Other amino acids and solublecarbohydrates including sucrose were present in relatively smallamounts and changed very little with increasing salinity. Con-sistent with this were the data obtained from uniform labeling ofNaH'4C03 of the cells, which showed more than 90% of theorganic carbon in the soluble fraction of the cell being confinedto proline at high salinities. This clearly excludes a quantitativelyimportant presence of other osmoregulatory organic solutes inthese cells. It is, therefore, evident that organic solute accumu-lation can not be the sole osmoregulatory response in this alga.From the results of our present studies, it is clear that C.

autotrophica possesses a marked capacity to take up and accu-mulate ions from the external medium. This accumulation of

ions appears to be almost suffilcient to counterbalance the exter-nal salt concentration in cells grown at salinity levels of up to100% seawater. At higher salinities, however, the alga's capacityto accumulate ions was insufficient to effect the required osmoticadjustment, and rapid accumulation-of proline took place.As the data for inorganic ion content of C. autotrophica

presented in this paper differ significantly from the ion contentreported for C. emersonii (24) and C. salina (19), it may bepertinent here to evaluate the methodology used. One of themajor reasons for the reports in literature showing the growth ofmicroalgae at high salinities with biophysically impossible lowlevels of intracellular solutes appears to be the fact that, ingeneral, prolonged washings in ion-free isoosmotic solutions ofnonpenetrating sucrose and polyols have been used prior to theinorganic analysis of the cells. It has been shown previously thatisoosmotic solutions of sucrose can induce plasmolysis ( 11) andleakage of ions from the algal cells (23). Such a loss of ions canbe prevented by an addition of some inorganic ions to thewashing solutions (R. F. Davis, Rutgers University, personalcommunication). It was, therefore, decided to add small amountsof major ions to the isoosmotic solutions of sorbitol used forwashing in our present studies (see "Materials and Methods").As was pointed out earlier, there is no danger of a quantitativelysignificant contamination of the centrifuged cell pellet, if theseions in the washing solution are kept at a few mm concentration.Using this washing technique, we measured ion levels consider-ably higher than those reported previously for other species ofChlorella (19, 24).The question arises as to the metabolic compatibility of the

intracellular levels of ions exhibited by C. autotrophica at highsalinities. The alga is only slightly vacuolated, therefore, thecytoplasmic ion concentrations of these cells are not likely to besignificantly different from the reported intracellular levels. Inview of the documented sensitivity of the isolated enzymes tosalt inhibition, Flowers et al. (9) postulated that an accumulationof ions in the cell cytoplasm in excess of 200 mm salt (400 mosmions) would cause growth inhibition by disrupting the metabolicfunctions of the cell. No growth inhibition was found, however,in C. autotrophica at 50% ASW although the cells were found tohave intracellular ion concentrations of 750 mm cation and 460mm chloride. A similarly high monovalent cation concentrationof 900 mM has been reported by Gimmler and Schirling (10) innormally growing cells of wall-less micro-alga Dunaliella parva.Unfortunately, a preoccupation of these workers with monova-lent cations has limited the usefulness of their data, as themeasurement ofchloride ion was ignored. It is becoming increas-ingly evident that the salt sensitivity of the enzymes is due to theinhibitory effects of chloride ion (9). Our present study showsthat the intracellular ionic environment is such that it allowsinorganic anion to be presented at a level lower than that ofinorganic cations. However, even with this 'restricted' presence,the concentration of chloride exhibited by cells at high salinities,including those growing optimally at 50% ASW, ranged between460 and 510 mm, which is considerably higher than levels of saltfound inhibitory to the activities of the enzymes in vitro. Itappears, therefore, that the intact metabolic machinery of thealga may be more resistant to salt than the evidence obtainedfrom the isolated enzymes would suggest. Glucose-6-P dehydrog-enase, an enzyme involved in the oxidative pentose-P cycle ofthe chloroplast, when isolated from the leaves of Suaeda mari-tima was strongly inhibited by 167 mm NaCl (8). The chloroplastsin the leaves of this angiosperm halophyte growing under opti-mum growth conditions may, however, have chloride concentra-tions of up to 210 mm (13). These studies, therefore, point outthe limits to which reliance can be placed on in vitro studies ofenzymes to determine the salt tolerance ofhighly organized intactcytoplasmic machinery of plant cells.

1014 Plant Physiol. Vol. 74, 1984

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OSMOREGULATION IN CHLORELLA AUTOTROPHICA

In C. emersonii a severe growth inhibition at 200 mM NaClwas attributed by Setter and Greenway (25) to the presence of100 to 120 mm proline in the cells under these conditions. Theseworkers have argued that, if an amino acid is present in highconcentration, the cell's normal pattern of protein synthesis canbe disrupted by its erronously high incorporation into protein.The results ofour present studies, however, show that the growthof C. autotrophica at 50% ASW with an intracellular prolinelevel of 105 mm was normal. Furthermore, the alga was able togrow well even with internal proline concentration of 1600 mM.These results, therefore, appear to support the role of proline asa compatible cytoplasmic solute in plant cells (2, 3, 7, 9, 15, 27,28). In our present studies, the inhibition of growth in C. auto-trophica at high salinities was found to be associated with adecline in the cells' apparent turgor pressure under these condi-tions.

Acknowledgments-We are grateful to Dr. R. G. V. Hancock (Institute forEnvironmental Studies, University of Toronto) for making available neutronactivation analysis facilities and for his expert technical assistance. We also wouldlike to thank Dr. J. Dainty for helpful discussions about ion activities and distri-bution.

LITERATURE CITED

1. AHMAD 1, F LARHER, GR STEWART 1979 Sorbitol, a compatible osmotic solutein Plantago maritima. New Phytol 82: 671478

2. AHMAD I, F LARHER, GR STEWART 1981 The accumulation of A'-acetylorni-thine and other solutes in the salt marsh grass Puccinellia maritima. Phyto-chemistry 20: 1501-1504

3. AHMAD I, SJ WAINWRIGHT, GR STEWART 1981 The solute and water relationsof Agrostis stolonifera ecotypes differing in their salt tolerance. New Phytol87: 615-629

4. ALEXANDROWICZ Z 1962 Osmotic and Donnan equilibria in polyacrylic acid-sodium bromide solutions. J Polymer Sci 56: 115-132

5. BERGMEYER HU 1965 Methods of Enzymatic Analysis. Academic Press, NewYork

6. BIEBL R 1962 Seaweeds. In R Lewin, ed, Physiology and Biochemistry ofAlgae. Academic Press, New York, pp 799-815

7. BROWN LM, JA HELLEBUSr 1978 Sorbitol and proline as intracellular osmoticsolutes in the green alga Stichococcus bacillaris. Can J Bot 56: 676-679

8. FLOWERS TJ 1972 The effect of sodium chloride on enzyme activities fromfour halophyte species of Chenopodiaceae. Phytochemistry I1: 1881-1886

9. FLOWERS TJ, PF TROKE, AR YEo 1977 The mechanism of salt tolerance inhalophytes. Annu Rev Plant Physiol 28: 89-121

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