LICHEN PHYSIOLOGY IX. CARBOHYDRATE MOVEMENT FROM THE TREBOUXIA OF XANTHORIA AUREOLA TO THE FUNGUS BY D. H. S. RICHARDSON* AND D. C.

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1 New Phytol. (1968) 67, LICHEN PHYSIOLOGY IX. CARBOHYDRATE MOVEMENT FROM THE TREBOUXIA OF XANTHORIA AUREOLA TO THE FUNGUS SYMBIONT BY D. H. S. RICHARDSON* AND D. C. SMITH University Department of Agriculture, Oxford {Received 3 August 1967) SUMMARY The main carbohydrates which incorporate ^*C during photosynthesis in Xanthoria aureola are ribitol, mannitol, arabitol and sucrose. Ribitol is the first product to accumulate ^*C in substantial amounts, and it appears to be produced by the alga. Ribitol is also the main forni in which carbohydrate moves from alga to fungus. Entry of [''*^C]ribitol into the fungus during photosynthesis could be prevented by addition of!/ '^C-pentitols to the medium. In short-term photosynthesis experiments, incorporation of ''*C into mannitol is relatively small, and none is detectable in arahitol. However, in long-term experiments, more '*C eventually accumulates in mannitol than in other carbohydrates, and ['*C]arabitol can also be detected; the delay before appreciable accumulation of '"^C occurs into mannitol and arabitol is consistent with their being fungal products. Sucrose never incorporates much ''*C, but its early appearance in photosynthesis suggests it is an algal product. The rate of movement of a pulse of '*C between the symbionts of X. aureola is much slower than in Peltigeia polvdactyla. INTRODUCTION Xanthoria aureola contains the commonest algal symbiont of lichens, Trebouxia. Knowledge of how carbohydrate moves from alga to fungus in this lichen may therefore be important in understanding some of the wider aspects of the lichen symbiosis. A very brief report on investigations into carbohydrate movement between the symbionts of A', aureola was published by Richardson and Smith (1966). The objects of this present paper are to give a more detailed account of this work, and to amplify and extend the preliminary study on the carbohydrate metabolism of this lichen published by Bednar and Smith (1966). MATERIALS AND METHODS Collection, preparation and sampling of lichefi material As in the experiments of Bednar and Smith (1966), the plants of Xanthoria aureola were collected from a large sloping south-facing tiled roof in the farmyard of the University Field Station, Wytham, Berkshire. In dry weather, water was first thrown on to the roof to moisten thalli, in order to make it easier to collect samples without damage. Marginal lobes, 3-5 mm across, were cut oflf thalli, brought back to the laboratory, washed and surface dried before being weighed into samples of 100 mg fresh weight. Exposure to Samples were placed in 2 x i in. specimen tubes with their upper cortices facing * Present address: Department of Botany, University of Exeter. 61

2 62 D. H. S. RICHARDSON AND D. C. SMITH upwards. Each sample was floated on ml of distilled water containing (5-20 /(Ci), The NaH'"*C03 was supplied by the Radiochemical Centre, Amersham, and was nearly carrier-free. In a few experiments, some carrier sodium bicarbonate was added and the amount used is indicated where appropriate. Each tube was sealed with a i in. diameter glass coverslip held in position by a ring of \'aseline on the lip of the specimen tube. This reduced exchange of '^CO, with the atmosphere without interfering significantly with the incident light. The tubes were clipped in a shaking water-bath at 18 C with an incident light intensity of 500 ft-candles pro\ided by fluorescent tubes. Analytical procedures The samples were killed and extracted in 8o 'o boiling ethanol for 5 minutes. Two further extractions were made and all extracts combined to form the ethanolsoluble fraction. The amount of radioactivity in the media, soluble and insoluble fractions was determined by placing aliquots on planchettes, drying down under infra-red light and counting with a thin end-window automatic geiger counter. Chromatography was carried out at room temperature using Whatman No. i paper and the following solvents: ethyl acetate-acetic acid-water, 14:3:3 (Smith, i960); and ethyl methyl ketone-acetic acid-water saturated with boric acid, 9:1:1 (Rees and Reynolds, 19S8), Reducing sugars were identified with analine phosphate (Erahn and Mills, 19S9), and sugars and sugar alcohols with alkaline silver nitrate (Trevelyan, Proctor and Harrison, 1950) after breaking the boric acid sugar complex (if necessary) with hydrofluoric acid (Britton, 1959), Paper electrophoresis was used to identify the pentitols. The apparatus described by Erahn and Mills (1959) was employed and basic lead acetate (58 g/1 distilled water) proved the most useful electrolyte. Runs were for 3 hours at 800 volts, though in future studies it might be preferable to use a shorter time and a higher voltage. The paper strips were dried at 100 C and the sugar alcohols developed with potassium permanganate-chromium trioxide reagent modified by using 2.^"^ potassium permanganate instead of 0,5" as recommended by Erahn and Mills (1959), RESULTS Products of short term photosynthesis Bednar and Smith (1966) found that most of the ''^C fixed by photosynthesis in Xanthoria aureola initially accumulated in ethanol-soluble compounds, especially polyols. During the first 15 minutes of a photosynthesis experiment, 63.5% of the feed ' *C was found in a compound identified as a 'pentitol', i2.7 o in mannitol and 3.4% in a compound provisionally identified as a '^-glycoside'. These soluble carbohydrates have now been identified more precisely with the following results. Polyols. Xanthoria aureola has now been found to contain two pentitols, ribitol and arabitol. This can be demonstrated by eluting the pentitol region of paper chromatograms of soluble extracts, and separating the two compounds by paper electrophoresis, Ribitol has not previously been reported from lichens although arabitol has been found in numerous species (e,g. Lingberg, Misiorny and Wachmeister, 1953). In photosynthesis experiments extending over periods of up to 3 hours, all the '^C incorporated into pentitols appears in ribitol and none can be detected in arabitol. Thus, the difficulties experienced by Bednar and Smith in making a precise identification of

3 Carbohydrate movement in Xanthoria 63 their ""^C- pentitol' were because it was not appreciated that there were two pentitols in this lichen, only one of which accumulated '^^C during photosynthesis over short periods. Ribitol is the first compound to accumulate large amounts of ' *C during photosynthesis and the experiments of Bednar and Smith suggest that it begins to incorporate ^*C within 30 seconds of exposure of the thalli to NAH''*CO3. In addition to ribitol and arabitol, the presence of mannitol was also confirmed. Sucrose. Bednar and Smith found a carbohydrate which they identified as a '/?- glycoside' because it could be hydrolysed by a /5-glycosidase enzyme to give glucose and another compound with a rather higher RF on paper chromatograms using a phenolwater solvent. In this study it was found that this compound had a chromatographic mobility corresponding to sucrose in a number of solvents and that it could be hydrolysed by dilute acid (0.02 N H2SO4 for 20 minutes at 100 C) to give two compounds corresponding to glucose and fructose. The soluble carbohydrates of the thallus The main soluble carbohydrates of the thallus are thus: ribitol, mannitol, arabitol and sucrose. In studies on the isolated symbionts to be described in the next paper (Richardson and Smith, 1968), it was found that the Trebouxia symbiont of Xanthoria aureola produces ribitol and sucrose during photosynthesis and that the fungus can produce arabitol and mannitol if grown on suitable media. In experiments with intact thalli, the early appearance of ^*C in ribitol and sucrose would be consistent with their production by the algal component. Photosvhthesis over longer periods In experiments carried out over longer periods, the consequences of entry of ' *C into the fungus should become progressively more apparent and the accumulation of ' ^C into fungal products such as mannitol should increase. Thus, Bednar & Smith found that although the proportion of fixed '"^C accumulating in mannitol was initially small, it increased with time relative to the 'pentitol'. This was confirmed here in an experiment which showed that after 24 hours photosynthesis on NAH^'^COj, more '*C appeared in mannitol than in other carbohydrates. In order to study these long-term effects in more detail, an experiment was carried out in which thallus lobes were given an initial short exposure of 2 hours to NaH'^^COj in the light, and then transferred to a non-radioactive medium of distilled water in the light. At intervals during the next 48 hours, samples were removed, killed, extracted in ethanol, and changes in the distribution of ^^C amongst soluble carbohydrates were analysed by paper chromatography. The results are shown in Fig. i. It may be seen that after 4 hours, most of the radioactivity appeared in the pentitol region of chromatograms while there was only a small amount in mannitol. As time progresses, the total amount of ['"^qmannitol rises with a corresponding fall in pentitols. After 36 hours, the activity in both kinds of compound declines, presumably because carbohydrates are respired and '"^C lost to the atmosphere. Fig. I also shows that there was only a slow rise in the amount of activity in insoluble compounds during the experiment. Close examination of the pentitol region of the chromatogram scans indicates that there was a slight change in the position ot the peak, especially noticeable in the 36- and 48-hour samples, indicating incorporation of '"^C into arabitol. The relatively long delay before ^*C begins to accumulate in arabitol was confirmed in another experiment carried out in the same way, and in which distinct E N.P.

4 64 D. H. S. RICHARDSON AND D. C. SMITH arabitol spots were not visible on autoradiographs of chromatograms until 9 hours after the start of the experiment. The results of the experiment illustrated in Fig. i can be explained in terms of photosynthetically produced [''*C]carbohydrate moving from the alga to the fungus where it is converted to mannitol and arabitol. However, this experiment does not show which of the carbohydrates produced by the alga moves to the fungus. Distribution of'*c on chromatograms of soi extract % fixed Hours insoi i 0% i-0% % 2-5% 3G 2'7% % Sucrose Mannitoi A H Pentitols Fig. I. Chromatogram scans illustrating redistribution of a pulse of fixed '''C in the soluble fraction of Xanthoria aureola during 48 hours photosynthesis on distilled water. O, Origin; A, arabitol; R, ribitol. Sample size = 200 mg fresh weight; incubated on 3 ml distilled water at 18" C; light intensity 500 ft-candles. Preliminary incubation of each sample before start of experiment for 2 hours in 20 fjci NaH'^COj (carrier-free). Solvent system = ethyl methyl ketone-acetic acid-water saturated with boric acid (g: i: i). Inhibition of carbohydrate movement between the svmbionts Drew and Smith (1967) describe how, during photosynthesis of '"^C by polydactyla, the ['"^Cjglucose released by the algal cells was prevented from entering the fungus if ['^CJglucose was present in sufficient concentration in the medium. The radioactive glucose appeared to be unable to compete with the large amount of the nonradioactive form at the fungal uptake sites, and so diffused out into the medium. The

5 Carbohydrate movement in Xanthoria 65 amount of radioactivity appearing in the medium was taken as a measure of the amount of glucose available to move between the symbionts. It appeared as if this inhibition effect was specific to glucose and some of its derivatives, since other ['*C]hexoses added to the medium could not prevent '"*C moving between the symbionts. A series of experiments with Xanthoria aureola was therefore carried out in which a range of different [' ^Cjcarbohydrates were added to media of samples during photosynthesis in NaH''^C03 solutions. Table i summarizes the results of these experiments and it shows that appreciable amounts of ^"^C are released from the tissues only in the presence of pentitols. Examination of the pentitol media by paper chromatography and paper electrophoresis showed that in each case, all the radioactivity released from the tissues was as [^'''C]ribitol. This demonstrates that ribitol moves between the symbionts during photosynthesis. Table i also shows that addition of ['^C]sucrose to the medium had no effect, indicating that although Trebouxia can produce sucrose, it is not released from the cells in detectable amounts. Table, i. Eff release of fixed t of externally supplied 1% \^ C]carbohydrates on the Cfrom Xanthoria ^tureolnphotosynthesizing in NaH^'^CO^ solutions "o total fixed ''*C released to medium in 24 Medium hours Water Sucrose Trehalose Glucose Ribose Mannitol Arabitol Ribitol Xylitol I.I Results represent mean values from a series of experiments. In each experiment: sample size = 100 mg fresh weight, incubated on 3 ml distilled water with 10 //Ci carrier-free NaH'^'COs at 18" C and 500 ft-candles for 24 hours. In some samples, carrier NaH'^COj added at 100 //g per ml. In all cases, ['-^CJcarbohydrates in medium at 1% (w/v). The distribution of radioactivity amongst carbohydrates in the soluble extracts of tissues (Fig. 2), shows that in the presence of pentitols, no '"^C is incorporated into mannitol, confirming that ['*C]ribitol was prevented from entering the fungus. Comparison between Xanthoria aureola and Peltigera polydactyla It appears as if '"^C moves more slowly between the symbionts of Xanthoria aureola than it does in Peltigera polydactyla. In the latter lichen. Drew and Smith (1967) found that addition of ['^CJglucose to the medium resulted in nearly 50% of the total fixed ' *C being released from the tissues in 3 hours. By contrast, in Xanthoria aureola much longer periods were required for a similar proportion of total fixed '"*C to be released when ['^CJribitol is present. In order to obtain a direct comparison between the two lichens, experiments were carried out under identical conditions to measure the rate at which a pulse of photosynthetically fixed '"^C moved from alga to fungus. The experimental procedure was as

6 66 D. H. S. RICHARDSON AND D. C. SMITH follows. Samples of each lichen were given a preliminary short exposure to j in the light. They were then washed with distilled water and allowed to continue photosynthesis on distilled water without radioactive bicarbonate. At increasing time intervals, samples were then transferred to i % solutions of either glucose (for Peltigera polydactyla) or ribitol (for Xanlhoria aureola) and replaced in the light. It was assumed that the residual Water Mannitol Sugars Monosaccharides + Disaccharides Pentitois Mannitoi A R, Pentitois Fig. 2. Chromatogram scans showing effects of externally supplied i/o (w/v) ['^C]carbohydrates on the distribution of fi.xed '''C in soluble e.xtracts of Xanthoria aureola after 24 hours photosynthesis on NaH''*CO3. Experimental details as in Table i (and media of all samples here had 100 /lglml carrier bicarbonate). Solvent = ethyl methyl ketone-acetic acid-water saturated with boric acid (g: i: i). O, Origin;.\, arabitol; R, ribitol. ^"^C available for transfer would then diffuse out of the tissues. The total '"^C available for transfer was assumed to be equivalent to the amount released by lichens transferred directly from ['"^Cjbicarbonate to ['^Cjcarbohydrate solutions. The amount of - moving from alga to fungus during a particular time on distilled water will be equivalent to the total amount available for transfer minus the amount released subsequently 11

7 Carbohydrate movement in Xanthoria 67 ['^C] carbohydrate. From these measurements it was possible to estimate the rate at whieh an initial pulse moved between the symbionts (Fig. 3). It appears that the pulse of fixed '"^C passes more rapidly between the symbionts ot Peltigera polvdactvla than between those of Xanthoria aureola. However, it must be stressed that only the rate of movement of a pulse of ''^C has been measured, and that this is not a measure of the absolute quantity of carbohydrate tiioving between the symbionts. The significance of this differenee is dealt with in the discussion. It is also important to consider the experimental conditions which were used before placing ecological interpretations on these results. The light intensity, 500 ft-candles, was relatively low but corresponds to that in the shaded habitat of Peltigera polydactyla during damp conditions when it is able to photosynthesize (Drew, 1966). By contrast, Xanthoria aureola lives in an unshaded habitat on a farm roof. However, the light I Time (hours) Fig. 3. Comparison of the rate of transfer of a pulse of ' *C from alga to fungus in Xanthoria aureola and Peltigera polydactyla durmg photosynthesis. Sample size = 100 mg fresh weight for Xanthoria, ten discs (7 mm diam.) for Peltigera..^11 material incubated at 18 C at 500 ftcandles light intensity on 3 ml media per sample. Preliminar\' exposure to NaH'^'COs (10 /;Ci earrier-free per sample) = 10 minutes for Peltigera, 2 hours for Xanthoria. Percentage of total fixed ''*C subsequently mo\ing to fungus in 24 hours = 4i 'o Peltigera, 23 o for Xanthoria. Other details in text. intensity at which this latter species normally photosynthesizes is not known. In periods of full sunlight, the thalli rapidly dry out. Photosynthesis can only occur when the thalli are wet, and this would be most often in dull cloudy weather or when the plants are moistened by dew in the early morning. Nevertheless, this lichen may be adapted to photosynthesis at higher light intensities than Peltigera polydactyla. DISCUSSION The pattern of carbohydrate metabolism during photosynthesis in Xanthoria aureola appears to be as follows. Carbon is fixed by the algal component mainly into ribitol but with a smaller proportion into sucrose and other substances. Some of the ribitol is released to the fungus where it is converted to mannitol and arabitol. There is no evidence of any movement of sucrose between the symbionts.

8 68 D. H. S. RICHARDSON AND D. C. SMITH The rate at which a pulse of '*C moves from alga to fungus is much slower in X. aureola than in Peltigera polydactyla. It is most important to note that this difference in rates refers only to '"^C and not to total carbohydrates. It is possible that the size of the ribitol pool which becomes labelled with '^C in the Trebouxia symbiont of Xanthoria aureola is much greater than the corresponding glucose pool in the Nostoc symbiont of Peltioera polydactyla. Similar rates of total carbohydrate movement in the two lichens might thus be accompanied by quite different rates of ''*C movement. These comments likewise apply to the differences in the rate of '"^C movement observed between the species of Lobaria containing blue-green and green phycobionts (Richardson, Smith and Lewis, 1967). It is therefore not possible at the moment to compare the efficiencies of Nostoc and Trebou.xia at supplying their fungal partners with carbohydrates. This would require the development of methods for measuring the absolute size of the pool of mobile carbohydrates in the lichen alga and for determining the specific activity of the [' *C]carbohydrates moving between the symbionts. REFERENCES BEDN.^R, T. W. & SMITH, D. C. (IQ66). Studies in the physiology- of lichens. \l. Preliminary studies of photosynthesis and carbohydrate metabolism in the lichen Xanthoria aureola. Nezv Phytol., 65, 211. BRUTON, H. G. (1959). The detection of carbohydrates with silver in the presence of borate. Biochem. J., 73. ly ' DREW, E. A. (1966). Some aspects of the carbohydrate metabolism of lichens. D.Phil. thesis, Universit\' of Oxford. DREW, E. A. & SMITH, D. C. (1967). Studies in tbe ph\siology of lichens. VIII. Movement of glucose from alga to fungus during photosynthesis m the thallus of Peltigera polydactyla. New Phytol., 65, 389. ER.^HN, J. L. & MILLS, J. A. (1959). Paper ionophoresis of carbohydrates, i. Procedures and results for four electrolytes. Aust.J. Chem., 12, 65. LiNDHERC, B., MisiORNY, A. & W.ACHMEISTKR, C..A. (i953). Studies in the chemistry of lichens. IV. In\'estigation of the low-molecular weight carbohydrate constituents of different lichens. Acta chem. scand., 7, 591. REES, W. R. & REYNOLDS, T. (1958). A solvent for the paper chromatographic separation of glucose and sorbitol. Nature, Loud., 181, 767. RiCH.XRDSON, D. H. S. & SMITH, D. C. (1966). The physiology of the symbiosis in Xanthoria aureola. Ltchenologist, 3, 202. RiCH.^RUsoN, D. H. S. & SMITH, D. C. (1968). Lichen physiology. X. The isolated algal and fungal symbionts of Xanthoria aureola. Ne2v Phytol., 67, 69. RICHARDSON, D. H. S., SMITH, D. C. & LEWIS, D. H. (1967). Carbohydrate movement between the symbionts of lichens. Nature, Lond., 214, 879. SMITH, I. (Ed.) (i960). Chromatographic and Electrophoretic Techniques. Vol. i. Chromatography. London. TREVELYAN, \\. E., PROCTER, R. D. P. & H.ARRISON, J. S. (1950). Detection of sugars on paper chromatogranis. Nature, Lond., 166, 444.

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