Apr 10, 1974 - Clean apical portions ... day or it was kept in a cold storage room at 11° C at 100 lux and c. .... Some recovery from the initial effects of sulphite had occurred. ..... The data were equivocal in this matter, it is hard to come to.
New Phytol. (1974) 73, 1193-1205.
SOME EFFECTS OF SULPHITE ON PHOTOSYNTHESIS IN LICHENS BY D. J. H I L L Dept. of Plant Biology, University of Newcastle upon Tyne {Received 10 April 1974) SUMMARY
Details of the effect of sulphite on H'^COj" fixation in the light were studied. The reduction offixationby sulphite was rapid, taking place within 30 min of contact with the lichen. Parmelia physodes recovered in 24 h from almost total reduction offixationcaused by i h treatment with 0.4 mM sulphite but Usnea sp. did not. Studies with [^'S]sulphite indicated that sulphite was taken up nonlinearly with time and that it was bound to protein. Algae isolated from Usnea sp., Parmelia physodes and Lecanora conizaeoides were similar in their response to sulphite, although the intact lichen L. conisaeoides is known to be more resistant. L. cottisaeoides did not seem to be resistant to sulphite by causing rapid oxidation of sulphite to sulphate. There was no marked effect of temperature on the concentration of sulphite required to reducefixationin Usnea sp. INTRODUCTION
There has been much work carried out recently in connection with the effect of air pollution on lichens (Ferry, Baddeley and Hawksworth, 1973). The experimental effect of sulphur dioxide and sulphite on lichens is clearly of interest as sulphur dioxide is a major air pollutant. A number of workers have found that sulphite or sulphur dioxide affect photosynthesis (Hill, 1971; Puckett et al., 1973; Inglis and Hill, 1974) and respiration (Gilbert, 1968b; Showman, 1972; Baddeley, Ferry and Finegan, 1973) in lichens and mosses. Despite these reports there is little detailed information on the effect of sulphite on specific cell processes. This paper aims to contribute some details of the effect of sulphite on photosynthesis in lichens. The results may well be of value in understanding the toxicity of sulphur dioxide. Hill (1971) and Puckett et al. (1973) showed that sulphite and sulphur dioxide in water (respectively) are inhibitory to photosynthesis in lichens ffoating in solutions below pH 5-6. Showman (1972) indicated, in contrast to Rao and LeBlanc (1966) that gaseous sulphur dioxide damage was not associated with chlorophyll breakdown. Puckett et al. (1973) attempted to find out the effects of sulphur dioxide solution on chlorophyll. Although the evidence did not permit definite conclusions, it suggested that chlorophyll may be oxidized. In the case of higher plants. Bell and Clough (1973) confirmed Bleasdale's (1952) finding that SOj can indeed cause the 'invisible' injury in Lolium perenne that Thomas (1951) and his co-workers were unable to show in other plants. Ziegler (1972) on the other hand obtained evidence using isolated spinach chloroplasts which suggested that sulphur dioxide was an inhibitor of ribulose diphosphate carboxylase. The inhibition was apparently competitive at lower concentrations with respect to carbon dioxide and non-competitive at higher concentrations with respect to ribulose diphosphate. However, "93
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these results need confirming with the isolated enzyme. Biscoe, Unsworth and Pinckney (1973) found that very low concentrations of SO2 affected the stomatal movement of Vicia faba. The level of SO2 used was apparently in the same order as that found in urhan air. The SO2 caused a 20% decrease in the stomatal resistance to gaseous exchange, (presumably thus allowing SOj to enter more easily to do damage to the mesophyll). Wellhurn, Majernik and Wellhurn (1972) showed that slightly higher levels of SO2 affected the thylakoid structure of V. faba chloroplasts. The swelling of the thylakoids was, however, reversible. MATERIAL
Details of the collection sites and the sampling of the four lichen species used in this study are given in Table i. All the material was collected in the dry state and remained Table i. Lichens used in experiments Species Usnea sp.*
Source of material On Betula and Alnus, Holystone, Northumberland Parmelia physodes (L.) (a) On Betula, Holystone, Northumberland Ach. (b) On Alnus, Ponteland, Northumberland (c) On Crataegus, Sbotover Hill, Oxford (d) On Pinus, head of Llyn Ogwen, Gwynedd (e) On deciduous trees, Belsay Hall, Northumberland Parmelia saxatilis (L.) On Quercus Ach. Lecanora conizaeoides On dead Betula twigs. Nyl. ex Cromb.
Sample Finer branches of thallus Clean apical portions on thallus lobes Clean apical portions on thallus lobes Clean apical portions on thallus lobes Clean apical portions on thallus lobes Clean apical portions on thallus lobes Clean apical portions on thallus lobes Thallus scraped off as powder
Weight SO mg (150 mg)
5° mg 40 mg 50 mg 50 mg 50 mg 150 mg 50 mg
* As Usnea spp. are difficult to identify, the material was left unnamed. It was, however, probably U. subfloridana Stirt.
exposed to the air in unsealed polythene bags. The material was either used the same day or it was kept in a cold storage room at 11° C at 100 lux and c. 65% humidity in the open polythene bags. Carbon dioxide fixation rates did not alter significantly on keeping the material for several weeks. Algal isolates Algae were isolated from Parmelia physodes and Lecanora conizaeoides. Washed thallus fragments were homogenized and single cells were removed with a micropipette, washed several times in sterile distilled water and put on agar slopes containing Bold's basal mineral medium (Ahmadjian, 1967). The alga from Usnea sp. was obtained from a streak of homogenate on the mineral agar medium. The isolates were grown at 11 ° C with a 16 h day. When the colonies had grown to sufficient size, they were used to inoculate liquid Bold's mineral media. These cultures were grown in 50-ml Erlenmyer flasks incubated at 10° C under continuous illumination (10,000 lux) until they had reached maximum density. The growth in all cases was very slow because no organic carbon was included in the media but this was essential in order to ensure that the algae were growing auto-
Effects of sulphite on lichens trophically. The cultures were unialgal but not axenic. When used for an experiment, the algal suspensions were centrifuged and taken up in i ml of 0.02 M phthalate at pH 4.0. Then 0.25 ml of this suspension was added to 9.75 ml of the sulphite solution in the experiment. METHODS
In most experiments the lichen material was first incubated in a solution containing sulphite. Carbon dioxide uptake was then estimated by measuring the amount of ^*C fixed during incubation in NaH^*CO3. Experimental procedure In most of the experiments and unless otherwise stated the lichen material without previous wetting was dropped into 10 ml of a medium containing sulphite and 0.02 M phthalate at pH 4.0 at 15° C under 8000 lux. Then after allowing the sulphite to act, 10 X of a solution containing 5.0 /vC NaH^^COs (20-50 mC/mM) were added. Ten minutes later the lichen was killed and extracted by adding 5.0 ml of glacial acetic acid in a fume r(a)
05
00
00
300
400
500
600
Vfcvelength (nm)
Fig. I. Absorption curve of (a) sulphite complex of cobalamin and (b) hydroxycobalamin.
cupboard. The rapid reduction in pH of the medium drove off all the excess H C O , and destroyed the membranes of the lichen, causing all the water-soluble substances to diffuse out into the medium (extract). In the case of experiments using ^'S, the samples were killed and extracted with 5 ml of 80% ethanol containing 10 mM iV-ethylmaleimide (Ellis, 1966). The insoluble fraction in these experiments was treated with i mg/ml protease at pH 7.5 at 37° C for 2 h. When the amount of ^'S in the media containing [^'S]sulphite was going to be measured, 1.0 ml aliquots of the medium were run into 10 ml of 80% ethanol with 10 mM A^-ethylmaleimide to prevent ^'SO^ from escaping into the air. Measurement of ^*C and After allowing sufficient time for extraction (at least 30 min), 10 A aliquots of extract or other fraction were pipetted onto pieces of Whatman No. i filter paper which,
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after drying in the air, were placed into scintillation vials with 10 ml of toluene containing 5 g/1 PPO and 0.3 g/1 POPOP. The vials were counted on a Packard Tricarb Liquid Scintillation Spectrometer. Corrections for quenching were made when results were expressed as dpm. The ^"^C in the insoluble fraction was not counted. The ^'S in insoluble fractions after protease treatment was measured by putting the dry residue directly into the scintillant. Measurement of sulphite
Although there are many methods available for measuring sulphite, the one used here was especially developed because many of the existing methods were not sufficiently sensitive, and it depends on measuring the formation of the sulphite-cobalamin complex spectrophotometrically. Hydroxycobalamin—a form of vitamin Bjj—(kindly provided by Dr R. Williams, Department of Chemistry, University of Oxford and by Dr N. Shaw Department of Chemistry, University of Newcastle upon Tyne) reacts quantitatively with sulphite ions to form a stable complex (Haywood et aL, 1965). The presence of
\ ,
I 03 02
O.I
00
40
80
120
Sulphite (n Moles)
160
200 Sulphite in mediuhn (^moles)
Fig. 2
Fig. 3 Fig. 2. Calibration curve of the loss in absorbance at 350 nm with the addition of sulphite to 500 /ig hydroxycobalamin in 3 ml. Fig. 3. The effect of increasing the amount of sulphite at constant volume and constant concentration onH'*CO3~ fixation in Usnea sp. •.Sulphite at constant concentration (0.1 s mM) increased by volume (5, 10, 20, 40 ml); O, sulphite at constant volume (10 ml) increased by concentration (0.1, 0.2, 0.3, 0.4 mM).
sulphate does not affect the reaction but the presence of thiosulphate and dithionite does. The rate of the reaction is little affected by wide variations in pH. Under acid conditions, the absorption peak at 530 nm was less intense, causing characteristic yellow appearance of the solution, the hydroxycobalamin being pink. The rate of the reaction was such that over 90% took place within 5 min at room temperature. The procedure adopted was as follows: 0.5 ml of hydroxycobalamin (i mg/ml) plus 0.2 ml sulphite solution plus 2.3 ml water were put into a cuvette. The absorption was measured at 350 nm after 10 min (Fig. 2). The virtues of this technique are that it is easy to carry out and it is sensitive (to less
Effects of sulphite on lichens than I ^g SO2 in 2 ml solution). It could be adapted to the measuring of SO2 in air by passing the air through an alkaline solution and then adding hydroxycobalamin and measuring the absorption after 10 min at 350 nm. Measurement of chlorophyll Chlorophyll was extracted and measured using the procedure of Hill and Woolhouse (1966). RESULTS
Although it has already been established that sulphite can stop photosynthesis, the rate at which sulphite acts has not been reported. In an experiment, Usnea sp. was placed in sulphite and the carbon dioxide uptake measured. The fixation of carbon dioxide was rapidly stopped (Table 4); indeed, after 30 min, fixation had been reduced to i /io of the controls. It seems that lengthy incubation in sulphite is not necessary to obtain the Table 2. The effect of sulphite at different temperatures on W*^CO^~ fixation in Usnea sp. Ratio of '*C C Uptake
f "o sulphite 15° C
Concentration of sulphite (mM)
5° C
0.0 O.I
1.00 1.07
1.00 0.71
0.2 0.4
0.17 0.00
o.io 0.00
Samples of lichen were incubated for i h in buffer and sulphite, before the addition of H'*CO3".
Table 3. The effect of increasing the amount of sulphite at constant concentration for 24 h on H^*COi~ fixation by Usnea sp. Moles sulphite o.o O-75 1-5 3.0 6.0
Ratio'*C fixation 1.0
0.74 0.45
e.ox
Samples of lichen were put in 0.15 mM sulphite contained in 5, 10, 20 and 40 ml of buffer and incubated for 24 h. Control sample was incubated in 10 ml of buffer alone.
Table 4. The rate of the effect of sulphite on H^^COj ~ fixation in Usnea sp. Time in sulphite before adding H'*CO3"' (min)
Ratio of '*C uptake (sulphite/no sulphite)
10
0.29
ao 40 80
0.14 0.09 0.05
Samples of lichen were incubated in 0.2 mM sulphite and then with the W^CO^-.
D. J. HILL 1198 reported effect on carbon dioxide fixation which occurred over 24 h. When Parmeha physodes was dropped into sulphite the effect of sulphite appeared to be immediate (Fig. 4), even after previous soaking. Previously soaked material showed a reduction in photosynthesis to a level similar to that in dry material. It is important to know whether more lengthy incubation in sulphite causes more extensive damage than a short one. The effect of i-h treatment and 24-h treatment were compared in P. physodes and Usnea sp. (Figs. 5 and 6). In both species it was found that at 0.2 mM sulphite i-h treatment with sulphite was apparently more damaging than 24-h treatment. Some recovery from the initial effects of sulphite had occurred. In samples incubated in sulphite for i h followed by 24 h in buffer alone, complete recovery was found in Parmelia physodes. In Usnea sp., however, there was little difference compared with leaving the lichen in sulphite for 24 h. These experiments indicate that recovery from short term effects of sulphite on photosynthesis is possible at least in Parmelia physodes.
It is of interest that P. physodes and Usnea sp. fix a greater amount of '"^COa in these experiments than did Lecanora conizaeoides when the results are expressed on a chlorophyll basis (Table 5). It is conceivable that the resistant species L. conizaeoides has more chlorophyll than is necessary to sustain photosynthesis and could therefore perhaps suffer greater chlorophyll damage before impairment of photosynthesis.
T.me in sulphite before addition of
concentra'ion(mM)
Fig. 4 Fig' 5 Fig. 4. The effect of sulphite treatment immediately before incubation in H'*CO3"" in Parmelia physodes. • , Lichen soaked in buffer for 24 h before experiment; o, dry lichen. Samples of lichen were dropped into 0.02 mM sulphite. Fig. 5. The effect of a recovery period on H' C O j " fixation after treatment of Parmelia physodes with sulphite. • , i h in sulphite; A, i h in sulphite followed by 24 h in buffer alone; O, 24 h in sulphite.
It is possible that the effect of increasing the concentration of sulphite in the medium may be caused by the greater amount of sulphite present rather than the concentration. To test this possibility experimentally, the amount of sulphite present in the medium was increased in two ways. First, by increasing the volume and secondly, by increasing the concentration of the medium. Increasing the volume has much less effect than increasing the concentration in reducing the amount of carbon dioxide fixed (Fig. 3). Even after 24 h, the amount fixed was not drastically reduced by increased volume (Table 3). The experiments of Hill (1971) were conducted at 18° C; those of Puckett et al. (1973)
Effects of sulphite on lichens also at i8° C; those of Showman (1972) at 20-24° C; those of Baddeley et al. at 25° C and those of Hill and Inglis (1974) at 15° C. Apart perhaps from the last, these temperatures are considerably higher than those probably present in the natural habitats especially in winter when the highest levels of pollution usually occur. It is therefore of interest to know whether the toxicity of sulphite was affected by temperature. The concentration of sulphite required to affect photosynthesis in Usnea sp. was at 5° C similar to that at 15° C (Table 2). This would indicate that temperature does not affect the toxicity and
01 Sulphite concentration ( m M I
02
03
04
05
SulDhite concentration (mM)
Fig. 6 Fig. 7 Fig. 6. The effect of a recovery period on H'*CO3~ fixation after treatment of Usnea sp. with sulphite. • , i h in sulphite; A, i h in sulphite followed by 24 h in buffer alone; O, 24 h in sulphite. Fig. 7. The effect of sulphite on H'*CO3" fixation in Parmelia physodes collected from different sites, o, Shotover Hill, Oxford; A, Pontoland, Northumberland; • , Llyn Ogwen, Gwynedd; A, Holystone, Northumberland. Samples of lichen (40 mg for Ponteland material) were incubated in sulphite for i h. They were killed with 2.5 ml glacial acetic acid.
Table 5. The
Species Usnea sp. Parmelia physodes Lecanora conizaeoides
fixation content Chlorophyll content (jUg) 6.1 5.1 IO.I
in relation to chlorophyll ; fixed dpm in 10* s dpm in 10's /Jg~ chlorophyll 16.3 12.6 S-4
2.67 2.47 0.54
Samples of lichen were treated for 3 hours before the addition of H' *CO3 ~ for I h. The lichen was then killed and extracted by dropping the lichen in 10 ml of methanol at room temperature. The chlorophyll content was measured on separate comparable samples.
that the temperature selected for the experiments in the present study (15° C) is not likely to cause spurious results. From the foregoing experiments, it is not clear whether the sulphite in the medium remains constant during incubation with the lichen. Sulphite could be rapidly absorbed, adsorbed or oxidized io sulphate, or lost to the air as sulphur dioxide. When the concentration of sulphite was measured with hydroxycobalamin, there was little sulphite lost from the medium during incubation of Usnea sp. or Lecanora conizaeoides (Table 6). The use of p'S]sulphite enabled the uptake of sulphite to be measured. The uptake of
D.
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HILL
sulphur did not proceed linearly with time in Usnea sp. (Table 7). This result agrees with previous experiments on the uptake of H^*SO3~ by Usnea (Hill, unpublished). This fact could well have been due to the penetration of the free space in the lichen tissues. It could also have been due to adsorption of sulphite on to cell walls and cell surfaces. Interestingly, however, the amount of ^'S found in the insoluble fractions remained constant with time. The use of protease on the insoluble fraction resulted in nearly all the •''S being solubilized (Table 7). This suggests that there was an immediate uptake of ^*S into protein which did not increase much with time. The use of two different specific activities [^"^SJsulphite resulted in the uptake of a similar amount of ^*S. This indicates that the uptake was proportional to sulphite concentration. The ^'S in the protein fraction, however, was higher at the higher specific activity. This suggests that uptake into protein was not proportional to sulphite concentration. Rather, it was as if it had been saturated at a concentration lower than 0.7 mM sulphite. Table 6. The uptake of [^^SJsulphite by Usnea and Lecanora conizaeoides Usnea sp. Before After
Sulphite in medium (mM)
" S in medium (cpm in 10*)
" S in extract (cpm in 10")
O-4S O-37
69.3 50-4
o 5-7
O-45 0.30
54-a
o 2.8
Lecanora conizaeoides
Before After
Samples of lichen were incubated for go min with 0.7 fiC'i mC/mM) in phthalate at pH 4.0.
(1.4
Table 7. The uptake of [^^S]sulphite by Usnea Time in sulphite (min) (A)
5 15 30
(B)
S 15 30
Sulphite cone. (mM)
Before 0.78 0.72 0.71 0.46 0.45 0.40
After 0.74 0.64 0.61 0.42 0.41 0.38
Total " S uptake (cpm in 1000s) 1266 224s ao88 1389 aiiS 3263
%m
insol fraction 1-45 1.26 1.83 a.73 a.07 1.96
% of insol "Sin protein 83 83 84 87 88 90
Samples of 50 mg were incubated in 0.7 fiCi H"SO3~ (1.4 mCi/mM) in phthalate at pH 4.0. Non-radioactive sulphite was added to the medium of the first three samples.
As Usnea sp. and Lecanora conizaeoides differ so widely in the response of photosynthesis to sulphite (Hill, 1971), these two species were compared in their ability to oxidize sulphite to sulphate and to take up sulphur from sulphite solutions. As already pointed out, the sulphite concentration did not change much during incubation of up to 1.5 h nor was there great change in the amount of sulphur in the medium. There was a slight indication that more sulphite disappeared in the medium containing L. conizaeoides and that L. conizaeoides took up less ^'S from the sulphite than did Usnea sp. (Table 6). However, it must be stressed that it is very difficult to make proper comparisons between different species in this type of experiment especially when they are so different morphologically. In the present experiment, Usnea sp. and Lecanora conizaeoides were compared
Effects of sulphite on lichens
1201
on an air-dry weight basis. Had another basis been chosen, a different picture may have arisen. Moreover, uptake by a dual organism is difficult to interpret as it is not clear to what extent each partner is contributing to the uptake. If sulphite is oxidized to sulphate, even at a slow rate compared with the rapid effect of sulphite, it is conceivable that this could be a mechanism by which resistant lichens could avoid to some extent the effects of sulphite. Indeed, prolonged exposure to sulphur dioxide in the air, however, can lead to quite high levels of sulphate in lichens (Gilbert, 1969). Therefore, although Hill (1971) found that low levels of sulphate did not effect photosynthesis, it is important to know whether these high levels of sulphate can be toxic. In a study aimed at finding out the levels of sulphate tolerated by photosynthesis in lichens, S. Jaggard (unpublished) found that Parmelia saxatilis and Usnea sp. could, at Table 8. The effect of drying in the presence of sulphate on //''^COj" fixation in Parmelia saxatilis {Jaggard, unpublished) Carbon dioxide uptake (cpm in iooos) 72 h sulphate Cone, sulphate 72 h sulphate with two io-h (mM) drying periods in air 5 2397 I173 as 1813 l68o 50 1783 ia8a 500 1044 Carbon dioxide uptake by controls without sulphate ranged from 1326 to 2170 iooos cpm. Samples of 150 mg lichen were incubated in 10 ml of 0,02 M phthalate buffer at pH 4, under 10,000 lux illumination in a bath at 15° C. Then 5 //Ci of NaH'^CO^ were added in lok of solution to the medium and, after 30 min. 5 ml of glacial acetic acid were added.
. „ .r.
Table 9. The effect of sulphate on H^^CO^ ~ fixation on Usnea sp. and Parmelia saxatilis {Jaggard, unpublished) .':
,
Cone. SO4 (mM) o S 50 • 500
'"C carbon dioxide uptake (cpm in iooos) p. saxatilis Usnea 61 133 ia6 154 XI7 loi to 117
Samples of 150 mg lichen were incubated for 3 h in phthalate at pH 4.0 containing sulphate. Conditions were otherwise as Table 8.
least to a certain extent, tolerate 0.5 M sulphate and Parmelia saxatilis could tolerate to a similar extent 0.5 M sulphate even over a period of 72 h which included two drying periods (Tables 8 and 9). As far as photosynthesis is concerned then, sulphate at pH 4.0 can be regarded as harmless. While it has been shown that alga in Lecanora conizaeoides appears to be less sensitive to sulphite than those of other lichens tested (Hill, 1971)' it is not clear whether this sensitivity is due to the alga being a more resistant strain. When the algae of Usnea sp., Parmelia physodes and Lecanora conizaeoides were isolated and grown in culture, they
D. J. HILL
1202
showed a similar response to sulphite with regard to carbon dioxide uptake (Table io). While the alga of L. conizaeoides seemed slightly more resistant, the differences involved were not sufficient to explain the very much greater resistance of the alga in the intact lichen. In conducting the type of experiments carried out in this project it is valuable to know whether lichens collected from different places showed the same response to sulphite, Parmelia physodes was collected from Wales, Oxford and two sites in Northumberland. The effect of increasing sulphite concentration on carbon dioxide uptake was compared in the four lots of material. Table io. The effect of sulphite on tP^CO^-fixation in algae isolated from Usnea, Parmelia physodes aw
