LICHEN PHYSIOLOGY XIII. EFFECTS OF REWETTING DRY LICHENS. Department of Agricultural Science, Oxford University. {Received i November 1972) SUMMARY

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1 New PhytoL (1973) 72, LICHEN PHYSIOLOGY XIII. EFFECTS OF REWETTING DRY LICHENS BY D. C. SMITH AND SUSAN MOLESWORTH Department of Agricultural Science, Oxford University {Received i November 1972) SUMMARY Two distinct phenomena occur when water is added to air-dry lichens: (1) an immediate nonmetabolic release of a substantial volume of gas containing 75-80% COj; and (2) a rapid rise in respiration to rates well above control values. This excess respiration ('resaturation respiration') persisted for ahout 9 hours in Peltigera polydactyla and about 2 in Xanthoria aureola and was, unlike basal respiration, azidc- and cyanide-sensitive. When the mannitol content of Peltigera discs had been doubled by feeding glucose before drying, the initial rate of subsequent resaturation respiration was correspondingly much higher. Cycles of wetting and drying caused progressive reductions both in mannitol content and resaturation respiration. In Peltigera, resaturation respiration occurred if the water content of discs had fallen below approximately 40% of maximum. Xanthoria aureola, which grows in drier habitats than Peltigera polydactyla, was much more tolerant of cycles of wetting and drying and showed much less intense resaturation respiration. INTRODUCTION Ried (i960) showed that when water was added to a dry lichen, the rate of respiration rapidly rose for a time to a value higher than controls, while the rate of CO2 assimilation began at a low level, rising slowly to that of controls. After rewetting there was therefore a period of net carbon loss from the thallus. The magnitude of this rewetting effect was much less in lichens from dry habitats than in those from moist, and it can be inferred from Ried's work that frequency of wetting and drying may be an ecological factor of critical importance in the distribution of lichens. This type of rewetting effect on gas exchange is not confined to lichens and it has been observed in a variety of other tissues such as dried Polypodium leaves (Stuart, 1968), spores oi Polytrichum (Paolillo and Javels, 1969), and dry bryophytes (Hinshiri and Proctor, 1971; Ensgraber, 1954). Earlier observations are reviewed by Stocker (i960) who also discusses effects in higher plants. In the case of lichens, the rewetting effects occur only after resaturation with liquid water, since they have not been observed during the very much slower process of resaturation by water vapour (e.g. Bertsch, 1966). This paper describes more detailed investigations into some of the phenomena which occur when water is added to dried lichens. Most experiments were carried out with Peltigera polydactyla collected from a very moist habitat and which would therefore be expected to be especially sensitive to rewetting. For comparison, a few experiments were carried out with Xanthoria aureola collected from the relatively dry habitat of a farm roof. 525

2 526 D. C. SMITH AND SUSAN MOLESWORTH MATERIALS AND METHODS Healthy thallus lobes of Peltigera polydactyla were collected from Hurst Hill, Cumnor, Berks, cleaned and sampled into 7-mm discs as described by Harley and Smith (1956). Marginal thallus lobes of Xanthoria aureola were collected from the roof of the University Farm, Wytham, Berks and sampled as described by Bednar and Smith (1966) and Richardson and Smith (1968). Immediately after preparation, some samples were surfacedried on filter paper and placed in a desiccator over calcium chloride for at least 20 hours at room temperature, while others were placed on very moist filter paper in Petri dishes, also at room temperature (16-21 C). Respiration rates of lichens were measured by standard Warburg procedures as described by Harley and Smith (1956), with the exception that for studying the effects of rewetting, dried samples were first placed in the annulus of the Warburg flasks without liquid, and 1.5 ml distilled water placed in the side arm. After equilibration (30-40 minutes) the side arms were tipped and the study of effects on gaseous exchange was begun. The methods of extracting lichens in 80% alcohol and measuring photosynthetic ' *C fixation by adding NaH' *C03 to media were the same as those described in previous papers (e.g. Richardson and Smith, 1968). The mannitol content of alcohol extracts of Peltigera polydactyla was measured by the periodate oxidation method described by Lewis and Smith (1967). RESULTS Experiments with Peltigera polydactyla Peltigera discs dried in a desiccator over calcium chloride for 24 hours have a water content of approximately 3-5% of oven-dry weight. When liquid water is added to these discs, two distinct phenomena occur. First, there is an instantaneous release of a substantial amount of gas which is 90% complete after seconds, and 100% complete after 90 seconds. This will be termed the 'wetting burst'. Secondly, after about 4 minutes, respiratory gas exchange can be detected, and this rapidly rises to a relatively high level before declining over a period of hours to rates shown by control, undried discs. This excess respiration will be termed 'resaturation respiration'. The 'wetting burst' The average volume of gas released by a sample of fifteen discs in the wetting burst was 150 /(I. In Warburg flasks with KOH, only 36 /jl was released, suggesting that the remaining 115 /.(I (75%) was COj if so, it would be the equivalent of that produced in about 7 hours of respiration by control undried discs. Since the wetting burst has such a high content of COj (or other gas absorbed by KOH), it cannot be due simply to air trapped in the tissues. The wetting burst was still shown by discs killed before drying by a variety of treatments such as heating to 98 C, or immersion for 5 minutes in either ethanol or chloroform. The gas was therefore presumably released by non-metabolic processes. Progress of 'resaturation respiration' : ' In experiments on resaturation respiration, the taps of Warburg flasks were not closed until 2 minutes after water had been tipped on to dry discs to avoid the large pressure changes of the wetting burst.

3 Lichen physiology. XIII 527 Fig. I illustrates how respiration rates subsequently changed. After 15 minutes, CO2 output was five times the rate in control discs, and oxygen uptake two-and-a-half times. Thereafter, the rates slowly declined but did not finally return to control levels until about 9-10 hours after the start of the experiment. The R.Q. was unity from 60 minutes onwards, but before that time was substantially above unity. The Warburg technique was not appropriate to study how rates changed over the first 15 minutes, so neither the maximum value nor the time when it was achieved is known. During the 9.75 hours of the experiment, the control undried discs lost 121 /d while the dried discs lost 265 /d after wetting (excluding the wetting burst) ^ Minutes Fig. I. Progress of respiration of Peltigera discs after addition of water to dry discs:, O2 uptake; o, CO2 output. Control discs on water:, Oj uptake; D, CO2 output. Effect of inhibitors on resaturation respiration When o.oi M sodium azide was tipped on to dry discs, 'resaturation respiration' (measured as oxygen uptake) was almost completely eliminated, although basal respiration rates were little affected (Fig. 2). Similar results were obtained with 0.01 M potassium cyanide. Both inhibitors also eliminated most of resaturation respiration if this was measured as carbon dioxide output. By contrast, '^ sodium fluoride had no efl^ect on resaturation respiration. These results suggest that resaturation respiration does not simply consist of a stimulation of basal respiration, and may therefore be regarded as a distinct phenomenon. Effect of glucose on resaturation respiration P. polydactyla can absorb substantial amounts of glucose, most of which is immediately converted within the tissues to mannitol (Smith, 1963). Mannitol is the most abundant soluble carbohydrate in the lichen, and it is believed to be the principal respiratory substrate (Smith, 1962). When solutions of 1% (w/v) glucose in distilled water were added to dry discs, the initial rate of resaturation respiration was only slightly greater than when water alone

4 528 D. C. SMITH AND SUSAN MOLESWORTH was added, but these high rates tended to be maintained instead of declining (Fig. 3). The rate of oxygen uptake by discs 4 hours after addition of glucose was approximately 250% of the basal rate, while control undried discs on glucose solutions respired constantly at about 240% of basal. The excess respiration of control undried discs on glucose is, like resaturation respiration, azide and cyanide sensitive. Fig. 3 also shows that if discs are first incubated for 17 hours on glucose solutions and then dried, the initial rate of resaturation respiration is greatly increased when water is subsequently added. The mannitol content of such discs was more than twice that of controls (cf. Table i). This suggests that the initial rate of resaturation respiration may depend partly upon the mannitol content. soo " Hours Fig. 2. Effect of o.oi M sodium azide on respiration of Peltigera discs. A, Azide added to dry discs; #, water added to dry discs; A, azide added to control, undried discs on water; O, control discs on water. Effects of cycles of wetting and drying Discs were exposed to a number of 24-hour cycles each comprising a 2-hour period of soaking in the dark followed by blotting and transfer to a desiccator in the dark for 22 hours; discs became air-dry after about 2 hours in the desiccator. \g. 4 illustrates that the first two cycles caused large progressive reductions in the initial rates of resaturation respiration, but a tbird cycle caused little further fall. Since the loss of carbon during resaturation respiration (and probably also in the wetting burst) was substantial, it could be argued that the effect of cycles of wetting and drying was due primarily to depletion of respiratory substrate. Experiments were therefore carried out in which mannitol content as well as respiration was measured, and also using some discs which had originally been supplied with

5 Lichen physiology. XIII r 500 o Hours Fig- 3- Effect of 1% (vv/v) glucose on respiration of Peltigera discs. L, Discs incubated for 17 hours on 1% glucose before drying, then distilled water added to the dry discs., Glucose added to dry discs (not previously incubated on glucose). O, Water added to dry discs.. Control discs on i% glucose;, control discs on water. Table i. Effect of cycles of wetting and drying on mannitol content and resaturation respiration of Peltigera discs with and without prior incubation in glucose No. of previous cycles of wetting and drying 0 I 2 3 Wa Mannitol content of dry discs before adding water (mg/15 discs) 3-7S Incubation of discs before start of experiment ter 1% glucose O2 absorbed Mannitol content of dry in first hour after discs before rewetting adding water (/(I/15 discs) (mg/15 discs) S O2 absorbed in first hour after rewetting (ml/15 discs) Discs incubated on distilled water or 1 % (w/v) glucose for 17 hours at room temperature before start of experiment. Each cycle consisted of 2 hours' soaking on distilled water followed by 22 hours' drying in a desiccator over CaClj, all in the dark at room temperature. Respiration measured at 25 C

6 530 D. C. SMITH AND SUSAN MOLESWORTH glucose so that their mannitol content was approximately doubled before the start of the wetting/drying cycles. The results showed a close correlation between mannitol content of a dry disc and its subsequent rate of resaturation respiration (Table i), providing further evidence for the importance of mannitol as a respiratory substrate. In all experiments, the mean loss of mannitol between the first and second desiccations was 0.77 mg per fifteen discs. This would more than account for the amount of CO2 lost, even if the initial 'wetting burst' is also taken into account. Moisture content In all experiments so far described, rewetting phenomena were studied on discs dried in a desiccator, and when the water content was only 3-5% of oven-dry weight. The effect of the water content of 'dry' discs upon resaturation respiration was studied 300 r Hours Fig. 4. Effects of previous cycles of wetting and drying upon respiration oi Peltigera discs after rewetting., d; o, d/s/d;, d/s/d/s/d; A, AjslAlsjAjsId; where d = desiccator for 22 hours, and s = soaking on distilled water for 2 hours., Respiration of control, undried discs. by allowing saturated discs to dry out for various lengths of time on the laboratory bench, and then after weighing, putting them into Warburg flasks. The flasks were allowed to equilibrate in the water bath for 30 minutes before water was tipped on to these discs. It was assumed that there was only a negligible change in water content during the equilibration period, since dry lichens take several days to absorb water from a saturated atmosphere (Smith, 1962). The results (Fig. 5) show that there appears to be a critical water content in the region of approximately 40-50% of saturation below which marked resaturation respiration wiil occur when discs are remoistened. Resaturation respiration is not therefore a

7 Lichen physiology. XIII 531 Table 2. Comparison of respiratiofi after rewetting between Peltigera polydactyla awjxanthoria aureola Peltigera polydactyla Xanthoria aureola Respiration over first hour after re-wetting (as % water controls) O2 uptake CO2 output Respiration measured at 25 after distilled water tipped on to dry lichen material. 250r- -iloo % o 50 $. 100 Hours of drying Fig. 5. Effect of water content of Peltigera discs on their subsequent respiration rates after resoaking., Water content before resoaking; O, O2 uptake at 25 C during first hour after resoaking. Table 3. Effect of cycles of wetting and drying upon resaturation respiration of Xanthoria aureola No. of previous cycles of wetting and drying o I Water controls Respiration during first hour after rewetting CO mg fresh weight) Oz 59 4a Respiration rate of samples (100 mg fresh weight thallus lobes) measured at 25 C. Each cycle = 2 hours' soaking on distilled water in the dark followed by 22 hours' drj'ing in a desiccator over CaCU, all at room temperature (18 C).

8 532 D. C. SMITH AND SUSAN MOLESWORTH phenomenon resulting only from the extreme dryness induced by desiccation over calcium chloride, and presumably occurs even after moderate drying in the field. Experiments with Xanthoria aureola As with Peltigera polydactyla, there was a substantial 'wetting burst' when water was added to dry material, equivalent to 82 /d per roo mg dry weight. Approximately 80% of it could be absorbed by KOH. No further investigation of this phenomenon was made. Xanthoria aureola also showed the phenomenon of resaturation respiration, but to a much lesser degree than Peltigera polydactyla. In particular, CO2 output was proportionately much less stimulated (Table 2). In most experiments, resaturation respiration was complete 2 hours after addition of water to dry material. As might be expected of a lichen from a dry habitat, Xanthoria aureola was much more resistant to cycles of wetting and drying, and even after four cycles, there was little reduction in respiration rates (Table 3). DISCUSSION Addition of liquid water to dry Peltigera discs results in a substantial loss of CO 2. A balance sheet for the 9 hours after rewetting would be as follows. Wetting burst: 115 /'I COj per fifteen discs Resaturation respiration: 114 Basal respiration: I2i Total: 380 (Control samples: 121) Thus, dry discs lost more than three times the CO2 of control undried discs during this period. An important component of this loss is the 'wetting burst'. Since it was shown by discs previously killed by a number of treatments it was unlikely to be the immediate consequence of some metabolic phenomenon. On the other hand, since 75-80% of the burst was absorbed by KOH in both lichens, it clearly does not result simply from air occluded in the tissues. It could well arise, for example, from the formation of carbonate and organic acid crystals during the drying process which then become mixed upon addition of water. But whatever the cause, it is important to establish whether the burst really does contain CO2, and whether it originates ultimately from metabolic sources. The phenomenon clearly merits further investigation. It may perhaps be of marginal significance that the wetting burst in P. polydactyla was of a similar order to Xanthoria aureola, although there were marked differences in the intensity of resaturation respiration. Unlike basal respiration, resaturation respiration in Peltigera polydactyla was azideand cyanide-sensitive. It is therefore not simply the result of a stimulation of basal respiration, but involves some additional pathways. These pathways are evidently brought into play when the moisture content of the lichen had dropped below a certain critical level before rewetting. The large drop in mannitol content during resaturation respiration implies that this is the substrate, although the changes in R.Q., especially during the early stages, are complex. It is not clear whether resaturation respiration is a mechanism of advantage to the lichen, or a deleterious but inevitable consequence of its ability to withstand desiccation

9 Lichen physiology. XIII 533 and its effects on cell structure and the control of metabolism. Thus, 'resaturation respiration' could be a symptom of a temporary loss of control over metabolic rates during recovery from drying, or it could supply energy vital to the rapid re-establishment of cell function after wetting. Thus, it would be particularly important for lichens to reestablish permeability barriers as rapidly as possible after rewetting so that intermediary metabolites and nutrients are not leached out during the initial soaking, especially since they typically live in nutrient-poor habitats. It would also be very advantageous to them if properties such as active nutrient uptake and transport of carbohydrates from alga to fungus could re-commence as soon as possible. The azide-sensitivity of resaturation respiration might prove a useful tool in the investigation of its role. The marked difference in the intensity of resaturation respiration and in tolerance of wetting/drying cycles between P. polydactyla and Xanthoria aureola is correlated with the marked difference in the moistness of their habitats, and hence supports the important conclusions which can be drawn from the exhaustive study of Ried (i960). It highlights still further the importance of the frequency of drying out as an environmental factor in the distribution of lichens, and one which ecologists ought to attempt to measure. Finally, the phenomena which occur on rewetting should be borne in mind by those who conduct laboratory experiments with lichens which have been allowed to dry out since collection. In particular, the 'wetting burst' should be eliminated from any measurements of respiration. REFERENCES BEDNAR, T. W. & SMITH, D. C. (1966). Studies in the physiology of lichens. VI. Preliminary studies of photosynthesis and carbohydrate metabolism of the lichen Xanthoria aureola. New PhytoL, 65, 211. BERTSCH, A. (1966). Uber den CO2-Gaswechsel einiger Flechten nach Wasserdampfaufnahmne. Planta, 68, 157- ENSGRABER, A. (1954). Uher den Einfiuss der Antrocknung auf die Assimilation und Atmung von Moosen und Flechten. Flora, Jena, 141, 432. HARLEY, J. L. & SMITH, D. C. (1956). Sugar absorption and surface carbohydrase activity of Peltigera polydactyla (Neck.) Hoffm. Ann. Bot., N.s. 20, 513. HINSHIRI, H. M. & PROCTOR, M. C. F. (1971). The effect of desiccation on subsequent assimilation and respiration of the hryophytes Anomodon viticulosus and Porella platyphylla. New PhytoL, 70, 527. LEWIS, D. H. & SMITH, D. C. (1967). Sugar alcohols (polyols) in fungi and green plants. II. Methods of detection and quantitative estimation in plant extracts. New PhytoL, 66, 185. PAOLILLO, D. J. & JAGELS, R. H. (1969). Photosynthesis and respiration in germinating spores of P0/3'- trichum. Bryologist, 72, 444. RICHARDSON, D. H. S. & SMITH, D. C. (1968). I^ichen physiology. IX. Carbohydrate movement from the Trebouxia symhiont of Xanthoria aureola to the fungus. Neu) PhytoL, 67, 6i. RIED, A. (i960). Nachwirkungen der Entquellung auf den Gaswechsel von KrustenHechten. Biol. ZbL, SMITH, D. C. (1962). The biology of lichen thalli. Biol. Rev., 37, 537. SMITH, D. C. (1963). Studies in the physiology of lichens. IV. Carbohydrates in Peltigera polydactyla and the utilization of absorbed glucose. New PhytoL, 62, 205. STOCKER, O. (i960). Physiological and morphological changes in plants due to water deficiency. UNESCO Arid Zone Res., 15, 63. STUART, T. S. (1968). Revival of respiration and photosynthesis in dried leaves o( Polypodium polypodioides. Planta, 83, 185.

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