UPTAKE AND METABOLISM OF EXOGENOUSLY SUPPLIED SUGARS BY BROWN ALGAE

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1 New PhytoL (1969) 68, UPTAKE AND METABOLISM OF EXOGENOUSLY SUPPLIED SUGARS BY BROWN ALGAE BY EDWARD A. DREW* Botany Department, Leeds University {Received 10 May 1968) SUMMARY In nine of eleven species of brown algae tested, much of the '^''^C-glucose taken up from aqueous media was readily leached out again during subsequent washing in ice-cold seawater. Whilst none of the unleachable fraction was converted to ^''^C-mannitol, a considerable proportion was incorporated into insoluble glucose polymers and some was lost in respiration. Two species Ascophyllum nodosum and Pehetia canaliculata were very different in that very little accumulated ^*C-glucose could be leached out. Most of the ^^C-glucose in the tissues of these two algae was rapidly converted to ^'''C-mannitol, the remainder being either incorporated into insoluble compounds or respired. These two species were also able to convert exogenously supplied ^''^C-mannose to mannitol, and Pehetia incorporated much '^^C from exogenous mannitol into a disaccharide compound. Fucus, as a representative of the other species investigated, was unable to metabolize either exogenous mannose or mannitol. None of the eleven species converted either galactose or fructose, supplied exogenously, to mannitol. INTRODUCTION The major soluble storage carbohydrates in the Phaeophyta are polyols; mannitol is virtually ubiquitous and is found in concentrations of 5-25% of dry weight, whilst volemitol is reported in considerable amounts in addition to mannitol in one species Pehetia canaliculata. The glucosidic derivatives of these polyols are also found but in much smaller quantities. The literature on polyols in brown algae has been reviewed by Lewis and Smith (1967a). The rapid accumulation of mannitol during photosynthesis in these algae is also well documented (Bidwell, 1958; Bidwell, Craigie and Krotkov, 1958; Yamaguchi, Ikawa and Nisizawa, 1966) whilst Drew (unpublished) has shown volemitol as well as mannitol to be formed during photosynthesis in P. canaliculata. If photosynthetic pathways similar to those in other plants operate in the brown algae, it must be assumed that there are present in these algae enzymic systems for reduction of sugars or sugar phosphates to polyols. It would therefore follow that sugars supplied exogenously to the algae would, if taken up into the cells, be converted to and accumulate as polyols. Contrary to such expectations, the report of Bidwell and Ghosh (1963) indicated that when Fucus vesiculosus was incubated on ^^C-glucose media for several hours there was no incorporation of '"^C into mannitol, although it was incorporated to a small extent into a number of other compounds which showed that at least some of the absorbed glucose was being taken into the cells of the tissue. However, in view of the very small * Present address: Gatty Marine Laboratory, St Andrews, Scotland. 35

2 36 EDWARD A. DREW amount of glucose actually metabolized (approximately 7 /^g/ioo mg dry weight of tissue in 15 hours) it is possible that this metabolism occurred within contaminating microbial cells in the thallus rather than in cells of the alga itself. Lin and Hassid (1966) have also shown that F. gardneri does not convert exogenously supplied mannose or glucose to mannitol. It was therefore decided to investigate the uptake and metabolism of various exogenously supplied sugars by Fucus and other brown algae in detail; the results of experiments carried out are given in this paper. MATERIALS AND METHODS Plant material Algae were collected from the shore on the day before use in experiments and kept in darkness at 5 C. Experimental samples usually consisted of 2 cm sections of sterile thallus with as little branching as possible; it was necessary to use 2.5 cm discs of the lamina of Laminaria species, 1 cm squares of Colpomenia and small bundles of filaments of Pylaiella. Experimental procedures Samples were incubated in the dark, floating on 2.5 ml of medium contained in 50 ml beakers, at 15 C in a shaking incubator bath. Media consisted of natural seawater (from the Marine Biological Association Laboratories, Plymouth) containing io~*m concentrations of appropriate carbohydrates plus approximately i ^Ci of the ^''^C-labelled compound per 2.5 ml; speciflc activity approximately 4000 cpm//^g of carbohydrate. When ^'^COj was to be collected, small phials containing 2 ml of 10% KOH were stood in the beakers which were themselves sealed with rubber bungs. At the end of the experiment the contents of the phials were added quantitatively to excess saturated Ba(0H)2 solution in centrifuge tubes and the precipitate then washed twice with hot water. Washing procedures after incubation were carried out in 2 x i in. specimen tubes containing 5 ml seawater and standing in an ice-bath in the refrigerator; washing water was not stirred. Samples of algae were briefly rinsed in distilled water after incubation or washing periods, stored overnight in 80% ethanol, and further extracted with two changes of near-boiling alcohol. All the extracts were combined and made up to 10 ml. Alcoholinsoluble material was dried at 80 C and weighed. Radioactivity assay. Aliquots of the soluble extracts were dried down on aluminium planchets and radioactivity determined with a Nuclear Chicago automated gas-flow geiger counter. Insoluble material was counted directly as fragments of intact tissue on planchets using the same equipment. Corrections for self-absorption in the tissue during counting were calculated for Fucus, Ascophyllum and Pelvetia by hydrolysing (in i N H2SO4 for 3 hours at 100 C) certain samples after the initial intact count and then recounting aliquots of the neutralized hydrolysates and also the material remaining unhydrolysed. Correction factors are shown in Table i and are considerably lower than would be expected if ^'^C was considered to be homogeneously distributed throughout the samples. The Bai'iCOa precipitates prepared from ^^COj absorbed in KOH (see 'Experimental procedures' above) were resuspended in small amounts of water, transferred to aluminium planchets and then dried down. The weight of BaCO3 was determined by weighing the

3 Sugar utilization by brown algae 37 previously tared planchets plus precipitates, so that self-absorption corrections could be applied to the values obtained by counting them in the same equipment used for soluble and insoluble material. Chromatography. Paper chromatographic separation of carbohydrates in soluble extracts and neutralized hydrolysates of insoluble material was carried out using Whatman No. I filter paper and two different solvent systems. Chromatograms were first developed with the picric acid/tertiary butanol solvent of Hanes and Isherwood (1949) which is relatively insensitive to the presence of inorganic ionic material in the extracts. The carbohydrate portions of these chromatograms, now free from ionic material, were eluted and re-chromatographed with the ethyl methyl ketone solvent of Rees and Reynolds (1958) which separates sugars and the corresponding polyols extremely well (see Lewis and Smith, 1967b). Table i. Correction factors for self-absorption during solid counting of alcohol-insoluble algal material Density of Self-absorption Species Correction solid sample correction factor factor (mg/cm^) expected Fucus vesiculosus Ascophyllum nodosum Pelvetia canaliculata io.o Distribution of ^'^C along the chromatograms was determined using an Actigraph 471 strip scanner. Expression of results. Results of uptake experiments are expressed as counts/minute radioactivity/100 mg dry extracted tissue; no weighing of samples was carried out until after they had been killed and extracted in 80% ethanol. RESULTS Uptake of glucose Fucus. The results shown in Fig. i(a) indicate that the absorption of glucose by the tissue of F. vesiculosus is a two-phase process in which the rate of uptake becomes extremely slow after an initial rapid phase during the first 2 or 3 hours. Most of this absorbed ^*C was readily leached out of the tissues when washed in ice-cold seawater for i hour after incubation. The ^*C leached out was still in the form of glucose, even after a 23-hour incubation period. However, about 45% of that remaining in the tissues had been incorporated into insoluble compounds mainly glucose polymers and another 30% had been respired away during that time. Chromatographic analysis of the soluble extract of washed tissue showed that more than 90% of the ^"^C in this fraction was still in the form of glucose after 23 hours incubation, although the only soluble carbohydrate present in quantity was the polyol mannitol. The actual quantity of glucose of exogenous origin in this fraction was very small, being of the order of i fig/ioo mg dry tissue, whereas about 5 mg of mannitol would be expected in the same amount of tissue (assuming a minimal content for brown algaeof 5% dry weight).

4 38 EDWARD A. DREW Other species. Data in Table 2 show that many other genera of brown algae showed a pattern of glucose absorption and metabolism similar to that of Fucus, with much remaining in a leachable form and a considerable proportion of the remainder incorporated into insoluble compounds. The only ^"^C-lahelled carbohydrate found in either the soluble or insoluble fractions of these algae was chromatographically similar to glucose. Table 2. Uptake of glucose by eleven species of brown algae {tissues incubated on ^'^Cglucose media for 3 hours, then washed for 1 hour) Species Fucus vesiculosus Himanthalia elongata Chorda fllum Cystoseira granulata Halidrys siliquosa Laminaria digitata L. saccharina Pylaiella littoralis Colpomenia sinuosa Ascophyllum nodosum Pelvetia canaliculata Total >*C in tissues (cpm X 10-3) Unwashed 73-O O , , O 33-O 73-5 Washed 18,3 53-O 32,5 43-O 6, % of initial ^*C washed out % of retained ^*C in insoluble compounds 12, O 68,0 18,0 6, o Nature of soluble ^*C Mannitol Mannitol There were, however, two notable exceptions to this pattern and those algae will now be considered. Ascophyllum and Pelvetia. Fig. 1 (b and c) demonstrates that in these algae uptake of glucose was a continuous process, with little ^^C remaining leachable and the amounts ot C accumulated m the washed tissues considerably exceeding that in Fucus (Fig la)

5 Sugar utilization by brown algae 39 Furthermore, chromatographic analysis showed that after 3 hours incubation more than 90% of the soluble ^'''C was in mannitol in both species, with only traces of ^*C-glucose. Radiochromatograms of the soluble extracts of these algae are compared, and contrasted with that of Fucus, in Fig. 2. (a) 1 (b) Mannitol Fig. 2. Radiochromatograms of soluble carbohydrate fractions of (a) Fucus vesiculosus, (b) Ascophyllum nodosum and (c) Pelvetia canaliculata after incubation on '*C-glucose medium for 3 hours followed by i hour washing. Uptake of mannitol The rates of uptake of exogenous ^'^C-mannitol were compared in Fucus and Pelvetia (Figs. 3a and b) and were found to be very different. These data refer only to ^*C remain- 800r 700 Z fe 400 O r Hours Fig. 3. Uptake and metabolism of i*c-mannitol by (a) Fucus vesiculosus and (b) Pelvetia canaliculata over a peiiod oi 2^ hours. O, Total (unwashed);, insoluble (washed);, COj.

6 4 EDWARD A. DREW ing in the tissues after i hour washing subsequent to incubation; the data in Table 3 show that, in another experiment, over 80% of the initially absorbed ^^C could be leached out of Fucus during the washing period following uptake from ^'^C-mannitol media, but only 36% from Pelvetia and none at all from Ascophyllum. Investigation of the fate of the unleachable ^*C within the tissues showed considerable differences in these experiments. Thus, incorporation of ^*C into insoluble compounds occurred to a much greater extent in Pelvetia and Ascophyllum than in Fucus (see Table 3). In the soluble fraction the ^*C-mannitol remained unchanged in both Fucus and Ascophyllum, but in Pelvetia about 30% of the soluble ^*C was incorporated into another compound with the chromatographic characteristics of a disaccharide. Although critical identification of this compound has not yet been made, it may be a glucose-polyol glycoside since such compounds are the only disaccharides known to occur in brown algae. Table 3. Loss of ^"^C from tissues during washing after incubation of Fucus, Ascophyllum and Pelvetia for 3 hours on ^'^Cmannitol medium % of initially cpm X io "' in tissue after washing Species absorbed '"C (per 100 mg dry weight) lost in I hour Total % insoluble Fucus vesiculosus 81,5 82,5 7 Ascophyllum nodosum Pelvetia canaliculata Other sugars The absorption and metabolism of other hexoses (mannose, fructose and galactose) were investigated in Fucus, Ascophyllum and Pelvetia. Samples were not washed after incubation so that no data on the proportion of accumulated ^'^C which remained leachable are available and data for total uptake are, therefore, not relevant. However, data are given in Table 4 for incorporation of ^^C into insoluble compounds in these Table 4. Incorporation of ^'^C from exogenous mannose, fructose and galactose into insoluble compounds during 3 hours incubation Species Mannose Fructose Galactose Fucus vesiculosus 3% (60) 3% (63) 2 / (j-i) Ascophyllum nodosum 35% (68) 24% (26) 14% (28) Pelvetia canaliculata 13% (99) 35% (69) 14% (46) Tissues not washed after incubation. Figures in parentheses mdicate total cpmxio^^ actually absorbed. Percentage incorporation expressed as percentage of total cpm absorbed. experiments and it can be deduced that, since so little is converted into insoluble compounds m Fucus as compared with the other two algae, very little of these sugars had been taken up into the cells in that species. Paper chromatographic analysis of the soluble extracts showed that Fucus converted none of these sugars to other compounds, whereas both Ascophyllum and Pelvetia converted considerable amounts of ^^C-mannose into mannitol, although they too had little eftect on either fructose or galactose which remained unchanged.

7 Sugar utilization by brown algae 41 Chloroform treatment Quillet and Legrand (1952) showed that the permeability of fungal cell membranes to simple sugars could be increased by pre-treatment of the tissues with chloroform vapour. This procedure has been used in this investigation with brown algae in an attempt to get ^'''C-glucose from the medium into contact with a mannitol-synthesizing system in Fucus. However, exposure to chloroform vapour for periods of 5-90 minutes, followed by 3 hours incubation on ^"^C-glucose media, had no effect on the chemical nature of the ^"" C accumulated in the tissues, other than depressing the formation of insoluble compounds. Table 5. Effect of pretreatment of Fucus and Ascopyllum with chloroform vapour on absorption of ^'^Cfrom ^'*'Cglucose medium during 3 hours incubation {tissues not subsequently washed) Duration of Fucus vesiculosus Ascophyllum nodosum pretreatment Total uptake % Total uptake % (minutes) (cpm X io~^) insoluble (cpm X io ~^) insoluble o i.o O.I 88 2 Samples of Ascophyllum were treated similarly to act as controls for the effect of the chloroform pretreatment on the monosaccharide-reducing system itself, since this had previously been shown to be active in this alga. Five minutes exposure to the chloroform vapour did not damage the system substantially, but 30 minutes inactivated it completely. Data given Table 5 show that this treatment also had the apparent effect of increasing accumulation of ^*C in both species, but samples were not washed after incubation so that the proportion remaining leachable was not determined. The gross reduction of incorporation of ^*C into the respective insoluble fractions is also illustrated there. DISCUSSION The pattern of glucose absorption and metabolism reported by Bidwell and Ghosh (1963) for Fucus vesiculosus is very similar to that established in this paper for 'unwashed tissue' of that species and it seems likely that in their experiments, as in those reported here, most of the glucose apparently taken up by the tissues was actually retained extracellularly in a readily leachable state. Thus the small percentage of glucose actually metabolized by this alga would be explained. However the overall effect, that much of the soluble ^*C retained in the soluble fraction, even after washing out the extracellular fraction, was ^"^C-glucose, is shown in both sets of data and the total lack of ^"^C-mannitol synthesis is substantiated. These features of glucose uptake have now been established in another seven genera (eight species) of common brown algae and it is probable that their metabohsm in this respect differs only in degree. The two genera Ascophyllum and Pehetia show a completely different pattern and clearly indicate that a hexose-mannitol conversion pathway occurs in these algae. Their taxonomic position cannot explain the differences observed since they are members of the Fucales and closely related to four of the other algae tested. Thus, the

8 42 EDWARD A. DREW possibility remains that the system for interconversion is present in the other algae but is inaccessible. Despite the fact that at least some ^"^C-glucose must have entered their cells in order to be incorporated into insoluble material and for respiration of ^^COj to occur. An experiment using chloroform vapour to break down any interfering barrier in Fitcus was unsuccessful in bringing about conversion of glucose to mannitol, as also was another experiment, not described here in detail, in which the effect of increased intracellular reducing power, provided by illumination and consequent photosynthesis, was tested. Investigation of the uptake and metabolism of exogenously supplied mannitol by these brown algae is complicated by the large amounts of this compound already present in the tissues. Bidwell and Ghosh (1962) reported that little conversion of this compound to others occurred during experiments in which ^"^C-mannitol was supplied to F. vesiculosus. Although this is surprising in view of the recent report that a considerable portion of the mannitol pool accumulated during photosynthesis by another brown alga Fisenia is rapidly utilized during subsequent starvation in the dark (Yamaguchi et al., 1966), it is clearly explained by the observation in this present investigation that most of the mannitol apparently accumulated in 'unwashed tissue' was in fact readily leached out from Fucus in just the same way as glucose. This would also explain the observation of Bidwell and Ghosh that mannitol accumulation in this alga was apparently a process of passive diffusion, despite the high, opposing concentration gradient due to the internal concentration of this compound, as pointed out by Drew (1966). Mannitol could, indeed, diffuse passively into the extracellular spaces of the tissue where it would remain leachable until actively transported into the cells, a process which seems to be very slow in Fucus but not so in Ascophyllum and Pelvetia. The experiments reported in this paper indicate that many brown algae possess very slow mechanisms for the uptake of exogenous sugars. The inability of most to convert exogenous glucose into their usual soluble carbohydrate, mannitol, may be attributed, in the main, to this factor, since the small amounts which are taken up could be accounted for by contaminating microorganisms in the experimental material collected from the shore. However, intracellular inaccessibility of the reducing system must also be considered, since the mannitol-synthesizing system is probably located in the chloroplasts whereas the pathways for synthesis of insoluble carbohydrates and for respiration, to both of which exogenous glucose apparently contributed, will be elsewhere in the cell. ACKNOWLEDGMENTS The investigations reported in this paper were supported by the Science Research Council (Grant-in-aid B/SR/477). Dr D. H. Lewis kindly offered much constructive criticism of the manuscript. REFERENCES BIDWELL, R. G. S. (1958). Photosynthesis and metabolism of marine algae. II. A survey of rates and products of photosynthesis in ''''COa. Can. y. Bot., 36, 337. BIDWELL, R. G. S., CRAIGIE, J. S. & KKOTKOV, G. (1958). Photosynthesis and metabolism in marine algae III. Distribution of photosynthctic carbon from '*CO2 in Fucui z;es2cu/oius. Can. 7 fioi -xs <;8i BIDWELL, R. G. S. & GHOSH, N. R. (1962). Photosynthesis and metabolism in marine algae. IV.'The fate' of '*C-mannitol in Fucus vesiculosus. Can. y. Bot., 40, 803. BIDWELL, R. G. S. & GHOSH, N. R. (1963). Photosynthesis and metabolism in marine algae. V. Respiration and metabolism of '*C-labelled glucose and organic acids supplied to Fucus vesiculosus Can If Bot 41. iss-

9 Sugar utilization by brown algae 43 DREW, E. A. (1966). Some aspects of the carbohydrate metabolism of lichens. D.Phil, thesis, University of Oxford, HANES, C. S. & ISHERWOOD, F, A. (1949). Separation of phosphoric esters on filter paper chromatograms. Nature, Lond., 164, LEWIS, D. H, & SMITH, D. C. (1967a), Sugar alcohols (polyols) in fungi and green plants. I, Distribution, physiology and metabolism. New Phytol., 66, 143, LEWIS, D, H. & SMITH, D. C. (1967b), Sugar alcohols (polyols) in fungi and green plants, IL Methods of detection and quantitative estimation in plant extracts. New Phytol., 66, 185. LIN, T.-Y. & HASSID, W. Z. (1966), Pathways of alginic acid synthesis in the Marine Brown Alga Fucus gardneri Silva. j^. biol. Chem., 241, QUILLET, M, & LEGRAND, M. (1952), Sur la metabolisme glucidique des champignons superieurs, IIL Le fructose, glucide intermediare du metabolisme du mannitol chez A. campestris var, bispora. C.r. hebd. Seanc. Acad. Sci., Paris, 23s, 628, REES, W. R. & REYNOLDS, T, (1958), A solvent for the paper chromatographic separation of glucose and sorbitol. Nature, Lond., 181, 767, YAMAGUCHI, T,, IKAWA, T, & NISIZAWA, K, (1966), Incorporation of Radioactive carbon from H'^COs ~ into sugar constituents by a brown alga, Eisenia bicyclis, during photosynthesis and its fate in the dark, PI. Cell Physiol, Tokyo, 7, 217.

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