STUDIES IN THE PHYSIOLOGY OF LICHENS

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1 STUDIES IN THE PHYSIOLOGY OF LICHENS V. TRANSLOCATION FROM THE ALGAL LAYER TO THE MEDULLA IN PELTIGERA POLYDACTYLA BY D. C. SMITH AND E. A. DREW Department of Agriculture, University of Oxford {Received 27 July 1964) SUMMARY Carbon fixed by photosynthesis in the algal layer can move into the medulla of Peltigera polydactyla within 30 minutes. This movement continues at a steady rate equivalent to 40-50% of the total rate of carbon fixation in photosynthesis, and it can also continue for a time in darkness. FLxed carbon accumulates initially as soluble compounds, especially mannitol, both in the algal layer and in the medulla. Since translocation in the thallus is relatively rapid it follows that the first stage of this process, namely, the transfer of photosynthetic products within the algal layer from an algal cell to its associated fungal hyphae, is also a rapid process. INTRODUCTION It is usually assumed to be a cardinal feature of the lichen symbiosis that the photosynthetic products of the alga are available to the entire thallus and that this is how the lichen fungus obtains carbohydrates. Direct experimental proof that this occurs has not been attempted hitherto, nor is it known to what extent a lichen fungus normally obtains carbon compounds from sources other than lichen algae. It is also not known if products of photosynthesis tend to be retained within the algal cells, or whether they pass freely out to the rest of the thallus. This paper describes the results of experiments which demonstrate that carbon fixed by photosynthesis moves relatively rapidly from the algal layer to the fungal medulla in the lichen Peltigera polydactyla. This movement of substances is here termed 'translocation', and its rate is measured as the rate at which ^^C accumulates in the medulla in excess of the rate of dark fixation when the thallus is exposed to i*c02. This paper amplifies the very brief, preliminary report which was made earlier (Smith, 1961). MATERIALS AND METHODS The following procedure was adopted in the experiments. Discs of tissue, 7 mm in diameter, cut from the thallus of Peltigera polydactyla, were incubated at 20 C in the light (425 ft-candles) on sodium bicarbonate solution containing the radioactive isotope i^c. At intervals after the start of the experiment, samples of discs were removed, rapidly chilled by washing in distilled water at 0 C, and then the fungal medulla was dissected away from the overlying algal layer and upper cortex. The amount of radioactivity in organic compounds in the two tissue regions was then estimated. 19s

2 196 D. C. SMITH AND E. A. DREW The collection of lichen material, and the preparation and sampling of discs of tissue have been described by Harley and Smith (1956). A detailed discussion of the dissection technique has been given by Smith (i960). In some experiments, the sample size was only five discs. The variability of such small samples is high (see Fig. 3), but the objection to increasing the sample size above this level is that the time required for dissection is increased. Dissection of a five-disc sample takes minutes and, even though discs awaiting dissection are kept at 0 C, the possibility of slow metabolic changes occurring at this low temperature cannot be ruled out. In one experiment, two persons were able to dissect simultaneously, so that ten-disc samples were employed and these showed much less variability (see Fig. i). Two other modifications to the dissection procedure previously described were made in this latter experiment : (a) immediately before dissection, discs were cut into halves, rather than quarters; and (b) there was no selection of particularly thick discs for dissection, so that the samples were the same as those used in other kinds of experiments. In all experiments, the bicarbonate solution was adjusted to ph 6.5. The specific activity ranged from i to 4 JC per ml, and the concentration of the solution ranged from 200 mg to I g NaHC03 per litre; actual values for particular experiments are indicated on the text figures. Alcohol soluble compounds were extracted by heating the tissues in three changes of 80% ethanol in a boiling water bath for periods of 10 minutes each. Most of the radioactivity was extracted in the first change of ethanol and, after the third change, no further radioactivity could be extracted even with prolonged treatment with ethanol. Insoluble compounds were extracted by boiling for 2 hours at 100^ C with 1.5 N sulphuric acid as described previously (Smith, 1963a). In two experiments, radioactivity of extracts was measured as described previously (Smith, 1963^) by a modification of the agar disc method of McCready (1958). In the third experiment (nth December 1963) the procedure was simplified by evaporating aliquots of the extracts directly on to aluminium planchettes (the well of each planchette having been previously ringed with a grease pencil to prevent the migration of the evaporating solution to the edges of the well). One drop of 0.1 N HCl was added to the aliquots of soluble extracts before evaporation so that any residual bicarbonate would be removed as carbon dioxide. The extract of ethanol-insoluble compounds was neutralized with barium carbonate before evaporation on planchettes. In all experiments, control samples were included to determine the extent of dark fixation, and this was found to vary from 4.8 to 9.4% of light fixation, the majority of values lying close to 6 %. The amount of dark fixation was approximately the same in the algal zone as in the medulla. A more detailed discussion of dark fixation and the factors affecting it will be given in a later paper. All results are expressed on a surface area basis. RESULTS When Peltigera discs are fioated in the light on an aqueous solution of NaH^^COs, the rate at which radioactivity appears in organic compounds in the medulla is illustrated in Figs. I and 2. In the experiment shown in Fig. i, dissections were made as rapidly as possible after the start of the experiment in order to determine how soon radioactivity appears in the medulla. Fig. 2 shows that there was good agreement between the results of this experiment and the results of another (part of which is illustrated in Fig. 3). The

3 Studies in the physiology of lichens 197 values in Figs, i and 2 have been corrected for dark fixation, and thus they reflect only the movement of photosynthetically fixed carbon. It can be concluded from Figs, i and 2 that significant amounts of photosynthetically fixed carbon can move into the fungal medulla within 30 minutes. This rate of movement is even more rapid than that found in an earlier experiment (Smith, 1961). This may be partly due to differences in experimental conditions and improvements in experimental techniques, and some of the factors which have been found to influence the rate of translocation will be described in a subsequent paper. Fig. I. Movement of photosynthetic products from algal layer (:) to medulla (_) in Peltigera discs (total = C). Sample size ten discs; 200 mg NaH^CO:; per ml; ph 6.5; light intensity 425 ft-candles; temperature 20'C. Results corrected for dark fixation. Experiment 11 December Once it has begun, the movement of fixed carbon into the medulla continues in the light at a steady rate. This rate can be compared with the total rate of carbon fixation by calculating values for the ratio of the increases in radioactivity in the medulla to those in the whole disc over the 4 hour period commencing 2 hours after the start of an experiment a time when approximately 'steady' conditions prevail: Experiment 12 March May 1963 II Dec Increase in ^'^C in medulla in 4 hours Increase in ^''C in whole disc m 4 hours Thus, the flow of carbon into the medulla is not only relatively rapid, but it also corresponds to an appreciable fraction (40-50 %) of the total fiow of carbon into the disc.

4 198 D. C. SMITH AND E. A. DREW It was shown in the preliminary report (Smith, 1961) that when discs were incubated on NaHi4CO3, the medulla continues to show a net gain in i'*c for about 4 hours after the light is switched off, suggesting that the flow of carbon from the algal zone continues 30 "10 Hours Fig. 2. Increase in percentage of the total '^''C fixed in photosynthesis which appears in medulla during 6 hours. Combined results of two experiments: O, experiment 14 May 1963 (see Fig. 3 for details);, experiment 11 December 1963 (see Fig. 1 for details). 20- Fig. 3. Re-distribution of radioactivity in discs after initial 2 hours incubation in the light on NaH"CO3 (a), followed by 4 hours in the light (b), and 4 hours in the dark on nonradioactive media (c). Solid symbols = incubated on moist filter paper after initial exposure period; open symbols = incubated on NaHi^COs after initial exposure period; x = values for medullae of discs kept on NaHi-^COs throughout (but in dark like other samples for last 4 hours of experiment)..o = Algal zone; A,A = medulla;,d = total. Sample size five discs; i g NaH"CO3 per litre, 2 MC per ml; light intensity 425 ft-candles; temperature 20 C. Results not corrected for dark fixation. Experiment 14 May for a time in darkness. Two values inserted (x) at 6 and 10 hours on Fig. 3 illustrate that this effect was again found here, as it also was in later experiments yet to be described. The main object of the experiment illustrated in Fig. 3 was to determine if, after an

5 Studies in the physiology of lichens initial 2 hour exposure to radioactive bicarbonate in the light, there was any subsequent redistribution of radioactivity in the tissues when discs were incubated on non-radioactive media during a period of light followed by a period of darkness. It was also hoped to ascertam whether such re-distribution would be affected by the conditions under ioo Fig- 4- Proportion of ^*C fixed into soluble (A) and insoluble x io (D) compounds in whoje discs (total = O). Discs incubated on NaHi^COa in light for 6 hours (a) (experimental details as in Fig. i), and then transferred to distilled water at 20 C in the dark for a further 48 hours (b). Fig. 5. Proportion of i*c fixed into soluble and insoluble compounds in algal zones and medullae. Other details as in Fig. 4. Algal zone, soluble, ; algal zone, insoluble x 10, O; medulla, soluble. A; medulla, insoluble x 10, A. which discs were incubated after their exposure to radioactive bicarbonate. Because of the large variability of the five disc samples used in the experiment, only very broad conclusions can be drawn from the results. Thus, it appears that there is no major redistribution of radioactivity in the discs although again, the medulla shows a distinct net

6 ..M 2OO D. C. SMITH AND E. A. DREW gain in ^*C for a period after exposure to radioactive bicarbonate. The amount of i*c in the algal zone tends to decline, especially in darkness, and this may be due at least in part to respiratory losses. Throughout the experiment, the percentage of the total ^^C in the disc which is in the medulla gradually rises: it is 25 % immediately after the initial 2 hour exposure period and increases to % at the end of the final dark period. There appeared to be no marked differences between the two methods of incubating discs, although the possibility that minor differences exist cannot be excluded. When intact thalli are incubated on NaHi^COa in the light, most of the ^^C fixed in photosynthesis initially accumulates in mannitol. The first detectable radioactive compound to accumulate in the medulla in the experiments reported here is also mannitol, but this is not yet conclusive proof that mannitol is the mobile compound in the thallus. A detailed discussion of the path of carbon in photosynthesis in the lichen will be given in a later paper, but the general point can be made here that during photosynthesis, fixed carbon initially tends to accumulate in soluble compounds and insoluble compounds are formed much more slowly (Fig. 4). Fig. 5 illustrates that this tendency holds true for both the medulla and algal regions of the thallus. Figs. 4 and 5 show that although movement of carbon into insoluble compounds is restricted, it continues to occur for quite a long time after discs are placed in the dark. DISCUSSION At first sight, it might seem surprising that in a slow growing plant such as a lichen, products of photosynthesis move relatively rapidly in the thallus. If significant quantities of them can move out of the algal layer into the medulla within 30 minutes, it implies that the transfer from algal cell to fungal hypha in the algal layer is at least as rapid. Since the rate of fiow of carbon into the medulla is approximately % of the total rate of carbon fixation by photosynthesis in the algal layer, it follows that the algal cells are efficient at supplying the thallus with carbon compounds. After carbon has been fixed it remains in a soluble, and therefore a potentially mobile, form for a relatively long time. Formation of insoluble reserves proceeds only very slowly. The following hypothesis has been developed to explain the efficiency and rapidity with which products of photosynthesis become available to the thallus. In the lichen, the growth of the algal cells is severely restricted, but the ability to photosynthesize much less so. Since most of the carbon fixed in photosynthesis cannot be directed to growth processes (as it would be in a free-living alga), as soon as the limited capacity of the algal cell to store fixed carbon is saturated, it is inevitable that carbon compounds should move out of the algal cells to the surrounding thallus tissue. This is, therefore, one more reason for supposing that the slow growth of lichens is a significant aspect of their physiology (cf. Smith, 1963^). REFERENCES HARLEY, J. L. & SMITH, D. C. (1956). Sugar absorption and surface carbohydrase activity of Peltigera polydactyla (Neck.) Hoffm. Ann. Bot., N.s., 20, 513. MCCREADY, C. C. (1958). A direct-plating method for the precise assay of carbon-14 in small liquid samples. Nature, Lond., 181, SMITH, D. C. (i960). Studies in the physiology of lichens. 3. Experiments with dissected discs of Peltigera polydactyia. Ann. Bot., N.S., 24, 186..SMITH, D. C. (1961). The physiology oi Peltigera polydactyla (Neck.) HofTm. Lichenologist, i, 209. SMITH, D. C. (1963a). Studies in the physiology of lichens. IV. Carbohydrates in Peltigera polydactyla and the utilization of absorbed glucose. New PhytoL, 62, 205. SMITH, D. C. (1963*). Experimental studies of lichen physiology. Sth Symp. Soc. gen. MicrobioL, 13, 31.

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