Eur. J . Biochem. 99, 559-567 (1979)

Biocalcification by the Marine Alga Emiliania huxleyi (Lohmann) Kamptner Elisabeth DE JONG, Lisette VAN RENS, Peter WESTBROEK, and Leendert BOSCH Department of Biochemistry, State University of Leiden (Received April 30/June 25, 1979)

Emiliania huxleyi (Lohmann) Kamptner, belonging to a group of marine unicellular algae, the Coccolithophoridae, possesses a cell wall containing calcified structures called coccoliths. These coccoliths contain a water-soluble acid polysaccharide. The polysaccharide contains ester sulphate and uronic acid groups and binds Ca2+ preferentially from a medium also containing Na' and Mg2+. It is thought to perform a regulatory function in the calcification process. In the present paper we describe a series of studies in vivo intended to give a preliminary characterization of a number of metabolic steps leading to coccolith formation. In view of the putative role of the acid polysaccharide in coccolith synthesis, we have concentrated on the incorporation of polysaccharide precursors by calcifying cells. As a second approach we investigated the differences between calcifying cells and cells of the same species that have lost the capability to produce coccoliths (non-calcifying cells). Calcifying cells were offered radioactive calcium, bicarbonate, galactose and sulphate in the light. Most of the calcium label and a small part of the label of galactose and bicarbonate was incorporated into extracellular coccoliths. The label of galactose was detected in at least seven of the constituent monosaccharides of the coccolith-associated polysaccharide. Radioactive sulphate was also incorporated into the extracellular coccolith polysaccharide. In contrast to the label of bicarbonate and galactose that of calcium reached a steady-state concentration intracellularly within 2 h. A technique was developed for the isolation of intracellular CaC03, presumably coccoliths in statu nascendi. About 10 % of the label of bicarbonate and galactose was incorporated into an intracellular acid polysaccharide, resembling the coccolith-associated macromolecule. Noncalcifying cells fail to take up Ca2+ and galactose. We demonstrated that non-calcifying cells produce an acid polysaccharide, resembling the coccolith-associated macromolecule. The former polysaccharide is shed into the surrounding medium and can be labelled by offering the noncalcifying cells radioactive bicarbonate. The biosynthesis of calcareous skeletons concerns the intimate interaction of two widely divergent processes of material organization : crystallization of inorganic salts and biochemical reactions controlling crystal growth. The crystal units produced in this biochemical environment differ in many respects from those that result from precipitation in a purely inorganic environment : their size, shape and arrangement and also their crystallographic orientation are determined, often to a high degree, by their spatial localization in the skeleton and by the phylogenetic position of the calcifying organism. Moreover, they are closely associated with complex macromolecular compounds that may influence their stability and are thought to have a regulatory function in the crystallization process. In fact they may be regarded as organized crystals with distinct properties. In a way

biocalcification represents an extention of the traditional realm of mineralogy. The biochemical mechanism underlying the formation of these crystals remains essentially unknown. Much attention has been paid to hard tissue formation in higher animals, particularly bone and tooth production and the synthesis of molluscan shells. We concentrate on the calcium carbonate production in Emiliania huxleyi (Lohmann) Kamptner, a marine unicellular alga that belongs to the Coccolithophoridae (Haptophyceae). The cell wall of most coccolithophorids contains elaborate structures of calcite, called coccoliths (Fig. 1). Coccoliths are of particular geological and geochemical importance : many of the widespread limestones of Jurassic and Cretaceous age have largely been formed by their steady accumulation in the oceans

Calcification by a Marine Alga

560

CaZ+

2HCO;

th

on+ co2

Galactose

so’,-

Fig. 1. Coccdiths 01Emiliania huxleyi. Scanning electron microscope picture of coccoliths isolated from cells as described before [lo]. Magnification 14500

Scheme 1. Merubolic ruutes leading t u coccolith formation iri Emiliania huxleyi

and they are even regarded as the chief binders of calcium and carbonate in the present marine environment [l]. Their architecture varies from one species to another and it is for this reason that they have become powerful tools for the determination of relative ages of fossil strata. Calcification is light-dependent : in the absence of light, coccolith formation nearly comes to a standstill. This phenomenon has been studied by Paasche [2], Crenshaw [ 3 ] , Wilbur and Watabe [4] and others. Interestingly, normal calcifying cells may lose the capacity to form coccoliths. They then become ‘naked’, but the conditions for their cultivation remain the same 151. The morphological and crystallographic architecture of the coccoliths of E. huxleyi has been studied by Watabe [6]. It has been demonstrated that coccolith formation in E. huxleyi is an intracellular process that takes place in specialized vesicles which are derived from the Golgi apparatus [4,7]. Using electron microscopy, Klaveness has shown that the vesicles first attain roughly the form of a coccolith [7]. Then macromolecular material appears inside whereafter CaC03 is precipitated. After completion the coccolith is extruded to the cell surface. So far, the main purpose of our work has been to render this system accessible to biochemical experimentation so that the biosynthesis of the coccoliths can be studied at a molecular level [&lo]. We developed techniques for mass cultivation (up to 1000 1) and for the purification of coccoliths. We then showed that an acid polysaccharide is closely associated with the calcium carbonate of the coccoliths. This poly-

saccharide was characterized in some detail. It contains two types of monobasic anionic groups (carboxyl and ester sulphate) and it binds Ca2+ preferentially from a medium containing Na’ and Mg2+. We postulated that the polysaccharide performs a matrix function in the crystallization process. The composition of the polysaccharide was determined by Fichtinger et al. [ll]. It consists of fourteen different monosaccharide moieties, including two methylated sugars never reported before to be constituents of a natural polysaccharide. The primary structure is under investigation. In the present paper we describe a series of studies in vivo intended to give a preliminary characterization of a number of metabolic steps that lead to coccolith formation. Scheme 1 represents a tentative model where some relevant routes are indicated. The cells were offered radioactive calcium, bicarbonate, galactose and sulphate. Label uptake could be measured in external coccoliths, in cells from which the external coccoliths were removed, in extracellular coccolithassociated polysaccharide, in intracellular coccoliths in statu nascendi and in intracellular acid-soluble material, preliminarily identified as the coccolithassociated macromolecule. Experiments were performed both in the dark and in the light. We assume that an investigation of the difference between calcifying cells of E. huxleyi and cells of the same species that have lost the capability to produce coccoliths (‘naked cells’) may be a fruitful approach to the study of coccolith formation. We have therefore subjected cells of both types to a similar

E. De Jong, L. Van Rens, P. Westbroek, and L. Bosch

series of experiments. The underlying cause of this difference remains unknown, but it is shown in this paper that the uptake of some coccolith precursors is blocked in the naked cells. Moreover, it is demonstrated that a macromolecular species very similar to the coccolith-associated polysaccharide is extruded into the growth medium by the naked cells.

MATERIALS A N D METHODS Cultivation of Coccolithophoridae The calcifying strain ofEmiliania huxleyi(strain F61) was kindly provided by Dr E. Paasche (Oslo University, Oslo, Norway). The non-calcifying strain 451 B was obtained through the courtesy of Dr R. Guillard (Woods Hole Oceanographic Institution, Woods Hole, Mass., U.S.A.). The cultivation of the algae has been described earlier [8, 101. Isolation of Polysaccharides The isolation of coccoliths and the associated polysaccharide from calcifying cells has been described earlier [lo]. Polysaccharide shed into the media by non-calcifying cells was obtained as follows. 3-1 cultures of naked cells were grown until the end of the log phase. They were then centrifuged at 23000xg for 20 min. The supernatants were concentrated on an Amicon PM-10 filter, dialysed against distilled water and lyophilized. About 8- 10 mg of macromolecular material was obtained from a 3-1 culture. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed as described elsewhere [8]. Gels containing I4C-labelled material were cut into 2-mm slices with a razor blade. The slices were oxidized in a Packard Tri-Carb autooxidizer and counted in a liquid scintillation counter (Mark 11, Nuclear Chicago). Cu2+ Uptake by Calcifying and Non-calcifying Cells

of Emiliania huxleyi

3-ml portions of a culture in the log phase were pipetted into 25-ml conical flasks. 2 ml of Eppley's medium [I21 were added. The cells were preincubated at 18 "C in the light for 18 h. 1 pCi of 4sCa2+ (Radiochemical Centre, Amersham) was then added to each vial and the incubation was continued. In order to determine Ca2+ uptake in the dark the vials were wrapped in aluminium foil prior to addition of 4sCa. The 5-ml cultures were then filtered through Millipore

561

(BA 85) filters and rinsed three times with 5 ml of sea water. Prior to filtration the cell concentration in each culture was determined by counting at least 400 cells in a haemocytometer. In order to discriminate between Ca2+ uptake by the cells and its incorporation into external coccoliths the latter were dissolved by bubbling C02 gas through the cultures for 30 s. After this treatment the pH had dropped from 8.0 to 4.7 and no external coccoliths were detected with the polarizing microscope. The decalcified cells were filtered immediately and rinsed with sea water. The millipore filters were dried overnight in counting vials. Then 10 ml of scintillation liquid (Hudroluma, Lumac) was added. The filters were counted in a liquid scintillation counter (Nuclear Chicago mark 11). Uptake of Galactose, HCO,, and SO:by Calcijying and Non-calcifying Cells of Emiliania huxleyi Uptake of galactose, SO:-, and H C O j by coccolithophorids was measured in essentially the same way as the uptake of Ca2+. 1 pCi of [l-14C]galactose, 7.5 pCi of [3sS]O:-, or 1 pCi of H[14C]O; was added to each 5-ml culture. The level of intracellular radioactivity was determined after treating the cells with COZ gas. By this procedure the extracellular coccolith-associated polysaccharide is set free and passes the millipore filter. Hydrolysis of 14C-Labelled Polysaccharide from Coccoliths Hydrolysis of the polysaccharide and paper chromatography of the hydrolysis products were performed as described elsewhere [ l l ] . The chromatogram was stained with aniline phthalate (1.69 g phthalic acid, 0.93 g aniline in 100 ml of water-saturated butanol). The radioactivity in the chromatogram was determined after oxidation of the paper in a Packard Tri-Carb autooxidizer. Incorporation of 4sCa2+in Intracellular Coccoliths of Emiliania huxleyi Cultures of 25 ml in the log phase were labelled with 5 pCi of 4sCa2+. After incubation in the light, they were treated with COz gas to remove external coccoliths. The cultures were centrifuged immediately at 5000 x g for 10 min and rinsed three times with 5 ml of seawater. The pellets were resuspended in 2 m l of seawater by ultrasonication for 1 min on an MSE ultrasonic vibrator. The suspension was centrifuged through 10 ml of a 50 % Ludox T M solution as described previously [lo].

562

Calcification by a Marine Alga

The resulting pellets were dissolved in 0.1 ml of 1 M HCl. Subsequently 1 ml of 0.1 M NH4HC03 and 10 ml of scintillation liquid were added.

Incorporation of [l-'4C]Galactose and H['"C]O, in Trichlor oacet ic A c id-Solub le Muter ial from calcifying Cells of Emiliania huxleyi 5-ml cultures were incubated with 1 pCi of [l-'"C]galactose or with 1 pCi of H[14C]O; at 18°C in the light. Subsequently the cells were centrifuged (5000 x g for 10 min), washed three times with 5 ml seawater and resuspended in 1 ml of 5 % trichloroacetic acid. The suspension was allowed to stand in ice for 20 min and was then centrifuged. The pellet was washed with 1 ml of 5 % trichloroacetic acid. The combined supernatants were neutralized with 4 M NaOH and lyophilized. The dry material was dissolved in 0.8 ml of 2.5 "/, trichloroacetic acid and applied to a Biogel P4 column (1 x 20 cm). The column was eluted with distilled water and 1-ml fractions were collected. Half of each fraction was mixed with 10 ml of scintillation liquid and counted. The remainder of those fractions that contained material of high molecular weight were pooled and concentrated for gel electrophoresis.

RESULTS UPTAKE OF RADIOACTIVE PRECURSORS BY CALCIFYING CELLS OF Emiliania huxleyi

Since coccoliths of Emiliania huxleyi are formed in intracellular vesicles [7] the calcification process requires the transport of Ca2+ ions from the surrounding medium into the cell. Paasche [2]. Crenshaw [3], and Wilbur and Watabe [4] found that calcifying cells accumulate Ca2+ in the light, but fail to do so in the dark. We found a similar uptake, as is shown in Fig. 2A. When the external coccoliths were eliminated with C 0 2 gas only a small amount of 45Ca2+ was detected in the cells showing that the 45Ca2 taken up was mainly used in coccolith formation. The intracellular 45Ca2 level reached a steady-state concentration within 2 h. It did not increase during prolonged incubation. The absolute value of the intracellular Ca2 concentration is still somewhat uncertain since it cannot be excluded that a small amount of Ca2+ leaks out of COz-treated cells. Light microscopic observations by Crenshaw [3] failed to reveal any intracellular morphological damage after this treatment. When treated cells were sonicated and centrifuged through Ludox, a small amount of radioactivity could be recovered from the pellet as is shown in Fig.2B. This pellet contains substances with a high specific +

+

10

2

0 0 Time (h)

1

2

3

4

Fig.2. 4sCu2+ uptuke by culcdying cells of Emiliania huxleyi. Cells were preincubated in Eppley's medium [12] in the light for 18 h. 4sCaZ+ At 0 h 0.2 pCi of 45Ca2+/mlculture was added. (A) (0) uptake by calcified cells in the light; ( 0 ) 4sCa2+ uptake by illuminated cells decalcified prior to sampling; (A) 45Ca2+uptake by 0 ) as A ; (0) 45CaZ+incorporation calcified cells in the dark.(B) (0, into intracellular high density material (see text)

gravity (> 1.38 g/cm3). We assume that they represent intracellular CaC03. The amount of Ca2+ in this material reached a steady-state concentration after 1 h. Cells incubated with 45Ca2+in the dark did not yield a radioactive pellet after centrifugation through Ludox.

Uptake o f [ l-'4CJGalactose, H [ '"C/O;, and [ 3 5 S10:by Calcifying Cells of Emiliania huxleyi

45Cu Uptake

+

0

Since the acid polysaccharide associated with coccoliths is thought to perform a matrix function in calcification, its synthesis may be intimately connected with coccolith formation. In order to get more insight in the polysaccharide synthesis we studied the incorporation of the label of [1-'"C]galactose, H[I4C]O; and [35S]Oi-.Bicarbonate is a precursor of both the calcium carbonate and the polysaccharide. We have mainly concentrated on the metabolic routes leading to the polysaccharide. The acid polysaccharide contains ester sulphate groups [ l l ] and therefore some preliminary experiments were performed on the uptake of radioactive SO:-.

Uptake of (l-'4CjGalactose In Fig.3A it is shown that the radioactivity increased in cells which had been incubated with [I-'"C]galactose in the light. In the dark the uptake of galactose was strongly inhibited. Unlike the Ca2+ uptake, the light-dependent galactose uptake exhibited a lag phase of about 4 h. The lag phase was not displayed by cells preincubated in Eppley's medium con-

563

E. Dc Jong, L. Van Rens, P. Westbroek, and L. Bosch 0

A

-

. C ._ E

100

E

3

8 1 .o

3

5.0

c

50

r.

c ._ > ._ c

%

0

m

0 ._

f P n

U

IY

4

2.5

0.5

0

10

20 0 Time (h)

10

20

Fig. 3. (I-L4CJCulactoseuptake by calcifying cells of Emiliania huxleyi. Cells were preincubated in the light for 18 h in Eppley's medium (A) or in Eppley's medium containing 1 pM non-radioactive galctose (B). At 0 h, 0.2 pCi of [l-'4C]galactose/ml culture was added. (0) [l-'4C]Galactose uptake by calcified cells in the light; (0) radioactivity in illuminated cells decalcified with COz prior to sampling; (A) [l-14C]galactose uptake by calcified cells in the dark; (A) radioactivity in cells incubated in the dark and decalcified prior to sampling. For further experimental details see Materials and Methods

taining 1 pM of non-radioactive galactose (Fig. 3 B). Further experiments will be needed to determine whether the lag phase shown in Fig. 3A represents an induction phenomenon. The level of radioactivity in cells decalcified with COZwas lower than that in cells plus external coccoliths. Cells which had been incubated in the dark displayed no such difference. Apparently a small amount of the galactose taken up by illuminated cells was used for coccolith formation, most likely for the synthesis of the polysaccharide associated with the coccoliths. This possibility was investigated by labelling a 25-ml culture of calcifying cells with 5 pCi [l-'4C]galactose for 24 h. The culture was then mixed with 100 ml of an unlabelled culture and centrifuged. The coccoliths were separated from the cell walls, isolated as described before [lo], and the polysaccharide was prepared from the isolated coccoliths. The polysaccharide was applied to a 5 % polyacrylamide gel. After electrophoresis a peak of radioactivity was detected which coincided with the absorbance peak of the stained polysaccharide (Fig. 4). In a control experiment the possibility of adsorption of [l -'4C]galactose to coccoliths and/or to the associated polysaccharide was ruled out. Moreover, radioactive polysaccharide was hydrolyzed and the hydrolysis products were separated by paper chromatography (Fig. 5). Radioactivity was detected in all spots of the chromatogram. Fichtinger et al. [ l l ] have shown that the coccolith-associated polysaccharide is built up from at least

..~ .....-~_.

-, .- 5

04 0

0

.......... 0

5 10 Distance from origin (cm)

Fig. 4. Polyacrylumide gel electrophoresis of polysacchuride from coccoliths of Emiliania huxleyi after labelling with (I-'4C]g~laciose. Cells were labelled with [l-*4C]galactose for 24 h whereafter 100 ml of an unlabelled culture was added. The polysaccharide was isolated as described elsewhere [lo]. Gels were scanned at 620 nm after Then they were cut into 2-mm slices staining with Alcian blue (-). and the radioactivity in each slice was determined (-- --). For further experimental details see Materials and Methods

14 different monosaccharides, including two methylated sugars never reported before to be constituents of a polysaccharide. One of these two, 2,3-di-0methyl-L-rhamnose, was also detected in the chromatogram (spot 10 in Fig.5), and contained radioactivity. Since only seven of the constituent monosaccharides were separated by paper chromatography (see legend to Fig. 5) the possibility exists that the label of galactose did not appear in all the sugar components of the polysaccharide. In order to study the metabolic fate of the galactose label, cells were incubated with [l-'4C]galactose for 3 h in the light. Subsequently the cells were treated with 5 o/, trichloroacetic acid which dissolves intracellular polysaccharides as well as the polysaccharide contained in external coccoliths. As is shown in Fig. 3A a negligible amount of [l-14C]galactose is used for extracellular coccolith formation after 3 h. About three times as much radioactivity was found in the pellet than in the supernatant after treatment of the cells with trichloroacetic acid and centrifugation of the mixture. When the supernatant was passed through a Biogel P4 column it appeared to contain material of high and low molecular weight (Fig. 6). The material collected from the void volume contained radioactive macromolecules which comigrated with the polysaccharide from coccoliths in polyacryl-

564

Calcification by a Marine Alga

a -

0

b

7 3

h

9 0

100

200

i

300

Radioactivity (counts/rnin)

0

10

20

Time (h)

Fig. 5. Paper chromatogrc~phyof a hydrol~suteof the polysnccharide isolated from coccoliths of Emiliania huxleyi clfter labelling cells with [l-'4C]galactose ,for 24 h. The polysaccharide was isolated from the coccoliths and hydrolyzed as described in Materials and Methods. The chromatogram is represented at the left. At a, A mixture of galacturonic acid (3), mannose ( 5 ) and xylose (6) was applied, and at b, the hydrolysate. The spots were identified in a separate study [ I l l by gas-liquid chromatography (1, 2) oligosaccharides ; (3) galacturonic acid ; (4) galactose and/or glucose; ( 5 ) mannose; (6) xylose; (7) rhamnose and/or 6-0-methyl-mannose; (8) 3-0-methyl xylose; (9) unidentified and (10) 2,3-di-O-methylrhamnose. At the right the radioactivity of each spot is shown. For further experimental details see Materials and Methods

Fig. 7. H['4C']0< uptuke b , c~nlc(/j.r/ig ~ cc,//.\ of Emiliania huxleyi. Cells were preincubated in Eppley's medium 1121 in the light for 18 h. At 0 h, 0.2 pCi of H['4C]Or/ml culture was added. (0) H['4C]O; uptake by calcified cells in the light; (0) H['4C]O< uptake by illuminated cells decalcified prior to sampling; (A) H['4C]Os uptake by calcified cells in the dark. For further experimental details see Materials and Methods

amide gel electrophoresis (not shown). It represented about 10 % of the macromolecular material soluble in trichloroacetic acid. Attempts to chase the radioactive label after a 3 h [l-'4C]galactose pulse into coccolith-associated polysaccharide or its precursors failed, probably due to the bulk of intracellular polysaccharide. These experiments seem to indicate that biosynthesis of any intracellular polysaccharide very similar if not identical to the coccolith-associated polysaccharide can be demonstrated. Although intermediate stages in this process have not been detected as yet, further labelling studies particularly over longer periods of time probably will be more revealing. Uptake of H('4C]OJ

0

0

5

10 Fraction number

15

Fig. 6. Frwtionation of' triclrloroac~eric-acitl-.solubl~~mutrrial from calcqjing cells. Cells were preincubated in Eppley's medium containing 1 pM non-radioactive galactose for 18 h in the light. At 0 h 0.2 pCi of [l-'4C]galactose/ml culture was added. The trichloroacetic acid-soluble-fraction was prepared and passed through a Biogel P4 column (20 x 1 cm, void volume 5 ml). 1-ml fractions were collected and counted. Elution patterns of trichloroaceticacid-soluble material from cells incubated in the light for 0 h (A); 1 h (A); 2 h ( 0 )and 4 h (0)

H['4C]O; uptake by calcifying cells is very similar to that of 45Ca2+,both in the.light and in the dark (Fig. 7). About 10% of the HCO; taken up is used in coccolith synthesis as judged from the level of radioactivity in decalcified cells (Fig. 7). The radioactivity set free by decalcification may represent incorporation into C a C 0 3 and into the polysaccharide of extracellular coccoliths. The percentage of HCO; used for coccolith synthesis is relatively low as compared to that found in other studies [2,13], indicating that the strain used here has a low rate of calcification.

565

E. De Jong, L. Van Rens, P. Westbroek, and L. Bosch ~

A

B

1 I

I

-

I I I I I I

.

C .-

E

u)

c t

I

0.50

s .> .-

._ c

s

.U c? 0.25 ?*

0

/ 0

0

10 Time (h)

20

0

5

10 Distance from origin (cm)

Fig. 8. Uptake 0 f ' ( ~ ~ S / 0 by 2 - ccdc(/ying ce//.\. (A) Uptake by cells plus coccoliths in the light. (B) Polyacrylamide gel electrophoresis of the polysaccharide from extracellular coccoliths after labelling cells with [35S]O:-(-) or with [l-14C]galactose (----). Gel slices containing 35S-labelled material were incubated overnight in 0.5 M HCI at 70 'C, whereafter scintillation liquid was added. For further experimental details see Materials and Methods, and text

An intracellular polysaccharide with the same electrophoretic mobility as the polysaccharide associated with extracellular coccoliths was isolated from cells labelled with radioactive HCOJ. To this aim labelled cells were decalcified with COZ, centrifuged and the sediment treated with 5 % trichloroacetic acid. From the radioactivity incorporated into the intracellular polysaccharide H['4C]0; proved to be a more efficient precursor than [l-'4C]galactose.

Uptake Of("S]O, When cells were incubated in the light with [3'S]0, (1.5 pCi/ml culture) for 24 h label was readily taken up as illustrated in Fig. 8A. That this label was incorporated into polysaccharide of external coccoliths was demonstrated by dissolving the latter with C02. After centrifugation of the decalcified cells a radioactive acid polysaccharide could be isolated from the supernatant with the same electrophoretic mobility as the coccolith polysaccharide labelled under similar conditions with [l-'4C]galactose (Fig. 8A). The COZ treatment also liberated labelled material with remained at the origin of the gels during electrophoresis. The nature of this material remains to be investigated. UPTAKE OF 45Ca21,[1-14C]GALACTOSE A N D H[14C]0< BY NON-CALCIFYING CELLS

As was demonstrated by Paasche and Klaveness [5] calcifying cells may lose their capacity to form coccoliths. Here we show that these cells shed a polysaccharide into the surrounding medium. Naked cells were centrifuged down and the supernatant was con-

centrated and dialyzed. The solution was then analyzed by ion-exchange chromatography on DEAE-cellulose (Fig. 9 A) and polyacrylamide gel electrophoresis (Fig. 9 B). In separate experiments polysaccharide isolated from coccoliths was submitted to similar analysis. The results obtained with both types of polysaccharide were plotted in the same figures (Fig.9A and B). The identical behaviour of the two substances during ion-exchange chromatography and polyacrylamide gel electrophoresis suggests that they are quite similar if not identical. This is supported by the finding (A. M. F. Fichtinger, personal communication, cf. [I l]), that they have virtually the same monosaccharide composition. Both polysaccharides bind Ca2+. Equilibrium dialysis experiments [lo] showed that the maximum binding capacity of Ca2+ binding is the same for both polysaccharides (0.92 pmol Ca2'/mg coccolith-polysaccharide and 0.94 pmol CaZf/mgpolysaccharide from non-calcifying cells). Some polysaccharide can also be detected in the medium of calcifying cells, albeit in much lower amounts than found with naked cells. Dissolution of coccoliths detached from calcifying cells may be responsible for this finding. Non-calcifying cells take up H[14C]0; in the light, but they fail to take up 45Ca2+ or [l-'4C]galactose under these conditions (Fig. 10). Calcifying and noncalcifying cells take up HCO, to virtually the same extent at the light intensity used (5000 lux, cf. Fig. 10 and 7). Part of the H C O j taken up by non-calcifying cells is used for the synthesis of the acid polysaccharide. This was shown by incubating the cells with H[14C]O; for 18 h. Subsequently a radioactive polysaccharide with the same electrophoretic mobility as the cocco-

Calcification by a Marine Alga

566 A

s

E

8 W

.c0 \f

1 .0

1.0

8

8

m

0 n

$n

b

9 0.5

0.5

0

0 0

50

100

Fraction number

0

5 10 Distance from origin (cm)

Fig. 9. Chromatographic und c~lcctruphorericunulysis of the polysucchnride shed into the nic4liurn by nun-calcifying cells. (A) Ion-exchange chromatography. 1 ml of a solution containing 4 mg of polysaccharide in 0.01 M Tris buffer pH 7.5 was applied to a DEAE-cellulose column (1 x 20 cm). The column was eluted with a linear NaCl gradient (0-0.5 M). The eluant was tested for the presence of polysaccharide with the indole test [ 3 5 ] . (B) Polyacrylamide gel electrophoresis. Gels were scanned at 620 nm after staining with Alcian blue. (--) Polysaccharide produced by non-calcifying cells; (-----) polysaccharide isolated from coccoliths. For further experimental details see Materials and Methods

0

0

10 Time (h)

20

Fig. 10. Uptakr hj' non-culcifjing cells of'H['"C]OF ( O ) , [I-'4C]gulactose (a),4 5 ~ a 2(0) + in the light and of H('4C]O< in the dark ( A ) . For further experimental details see Materials and Methods

lith-associated polysaccharide could be isolated from the surrounding medium.

DISCUSSION The main purpose of the present investigation is to study the biosynthetic pathways leading to the synthesis of the polysaccharide associated with coccoliths of Emiliuniu huxleyi and to its incorporation into these calcified structures. Furthermore we hope to shed

more light on the putative regulatory role of the polysaccharide in the calcification by the algae. A generalized scheme presumed to represent these pathways is given in Scheme 1. Some of the pathways, viz. the uptake of Ca2+ and HCO; by the cells and the incorporation of the latter into the CaC03 of the coccoliths were already explored by Crenshaw [3], Paasche [2], Wilbur and Watabe [4], Steeman Nielsen [14], and others. In general, our results are in keeping with those previous investigations (Fig. 2, 7 and 10). Radioactive calcium and bicarbonate are taken up by calcifying cells in the light. In contrast naked cells only take up bicarbonate but fail to take up calcium. In the dark the uptake of the two precursors is blocked in both types of cells. The techniques developed in our laboratory permitted a more detailed analysis of these reactions. After elimination of exterior coccoliths from cells labelled with 45Ca2+ a radioactive fraction with a high density could be obtained which tentatively was identified as intracellular calcified material (see also below). Labelling of this material with 45Ca2' reached a steady state, 1 h after addition of the isotope to the medium. The total intracellular 45Ca2+concentration became constant within 2 h, whereas the45Ca2+incorporation into exterior coccoliths continued during the entire run of the experiment (20 h, Fig. 2A). Biosynthesis of the coccolith-associated polysaccharide was studied by incubating the cells with H[l4C]0;, ~ - [ l - ' ~ C ] g a l a c t o s and e [35S]Oi-. The radioactive polysaccharide isolated from the exterior coccoliths was further characterized by polyacrylamide gel electrophoresis (Fig. 4 and 9) and compositional

567

E. De Jong, L. Van Rens, P. Westbroek, and L. Bosch

analysis (Fig. 5). Intracellular polysaccharide, comigrating with coccolith-associated polysaccharide during electrophoresis, became labelled after incubation of E. huxleyi with [l-'4C]galactose and H[14C]O;. This would mean that steps 4 , 4 a and 5 (of Scheme 1) are in principle amenable to more detailed investigation. Preliminary experiments with H[l4C]O;, [I -14C]galactose and [35S]O:- indicated that intracellular calcified material with a high density (see above) contained a small part of the label after incubation. These results, together with those obtained after labelling with 45Ca2+ (Fig.2B) suggest that this material represents intracellular coccoliths, possibly in .statu nascendi. Experiments to study macromolecular intermediates in polysaccharide synthesis are in progress. Fichtinger et al. [ l l ] demonstrated that the polysaccharide in the coccoliths contain mainly L-galactose rather than the D-form sugar. In the present study the cells were offered D-galactose. Since the galactose label was detected in at least seven of the constituent sugars (Fig. 5 ) isomerization, primary conversion and metabolic recycling of breakdown products must have occurred. The light dependence of the uptake of radioactive Ca2+, HCO; and galactose may be due to the fact that photophosphorylation is needed for the active transport of the substances across the cell membrane. Furthermore photosynthetically generated ATP is required in later steps of coccolith biosynthesis (e.g. steps 4a, 4 and/or 5 and 10). In the case of the galactose uptake there are two reasons to assume the presence of a transport carrier. The lag phase in the uptake (Fig. 3 A and B) and the specificity of the latter: it is inhibited by the addition of excess non-radioactive galactose, not of non-radioactive glucose. Glucose itself is taken up by calcifying cells (unpublished results). The question arises why Ca2+ and galactose are not taken up by non-calcifying cells. Steemann Nielsen [ 141 has suggested that non-calcifying cells cannot utilize HCO, for photosynthesis, because step 2 and/ or 2 b is blocked. Consequently in naked cells no COiis generated and Ca2+ uptake would be blocked since

it is not precipitated into CaC03. However, this explanation does not account for the lack of galactose uptake. A change in membrane permeability as the cause of the non-calcifying state of E. huxleyi may thus also be envisaged. The finding that the acid polysaccharide is extruded into the medium by naked cells shows that its synthesis is not affected in the non-calcifying state (Fig. 9). It is not yet certain whether the rate of polysaccharide production is the same in calcifying and non-calcifying cells. Since in both cases the polysaccharide could be labelled by offering the cells H[14C]O;, it is now possible to compare the rates of systesis. We like to thank M. A. Crenshaw for his valuable suggestion to analyze the medium of non-calcifying cells for acid polysaccharides.

REFERENCES 1. Lowenstam, H. A . (1974) in The Seu (Goldberg. E. D., ed.) vol. 5, Marine Chemistry, pp. 71 5 - 796, New York. 2. Paasche, E. (1964) Phjsiol. p h i . Suppl. 3 , 1-82. 3. Crenshaw, M. A. (1964) Ph. D. The Duke University, Durham, North Carolina, U.S.A. 4. Wilbur. K. M. Sr Watabe, N. (1963) N . Y . Acud. Sc.i. I O Y , 82-113. 5. Paasche, E. 9( Klaveness, D. (1970) A r c h . Mikrohiol. 73. 143-152. 6. Watabe, N. (1967) Cukif: Tissue Res. I , 114-121. 7. Klaveness, D. (1972) Protisiologicu, 8, 335 346. 8. Westbroek, P., De Jong, E. W., Dam. W. Sr Bosch, L. (1973) Culcic Tissue Re.c.. 12, 227-238. 9. De Jong, E. W. (1975) Ph. D. Thesis, University of Leiden. 10. De Jong, E. W., Bosch, L. SC Westbroek. P. (1976) Eur. J . Biochem. 70, 61 1 - 621, 11. Fichtinger, A . M . J., Kamerling, J. P., Vliegenthart. J . F. G., De Jong, E. W., Bosch, L. s( Westbroek, P. (1979) Ctrrhoh~~tlr. Res. 69, 181 189. 12. Eppley, R. W., Holmes, R . W. Sr Strickland, J. D. H. (1967) J . L k p . Mar. Biol. Ecol. I , 191 -208. 13. Sikcs, C. S., Roer, R. D. Sr Wilbur, K. M . (1978) Ahstr. 4 l t h Ann. kfeef. Am. Sot,. L~nmol.Occ.uno,qr. 14. Steeinann Nielsen. E. (1966) Plz>,siol.Plunt 19. 232-240. 15. Dische, 2. (1955) in Meihods of' Biochemrcal Ai/u/j..sr.r (Click. C. D., ed.) vol. 2. pp. 313-358. Interscience Press, New York. ~

~

E. De .long, L. van Rens, P. Westbroek, and L. Bosch, Biochemisch Laboratorium, Rijksuniversiteit Leiden. Postbus 9505, NL-2300-RA Leiden, The Netherlands