Download Effects of Sulphur Dioxide on Biochemical Activity and

New Phytol. (I976) 76, 239-245.
EFFECTS OF SULPHUR DIOXIDE ON BIOCHEMICAL
ACTIVITY AND ULTRASTRUCTURAL ORGANIZATION OF PINE NEEDLE CHOROPLASTS
BY S. S. MALHOTRA
Northern Forest Research Centre, Canadian Forestry Service, EnvironmentCanada 5320I22 Street, Edmonton, Alberta, T6H 3S5, Canada
(Received I5 August I975)
SUMMARY
The effects of aqueous SO2 on the ultrastructuralorganizationof pine (Pinus contortaDougl.
var. latifoliaEngelm.) chloroplastsand on their photosyntheticactivity were determinedunder
laboratoryconditions. At aqueous concentrationsof ioo and 500 ppm, SO2 caused swelling of
thylakoid discs and disintegratedother intrachloroplastmembranes,resulting in the formation
of small vesicles (in older, maturedtissues). Chloroplaststructuralinjurywas more pronounced
in old tissues than in younger and more metabolicallyactive tissues. The biochemicalobservations (Hill reaction activity) made on chloroplasts isolated from SO2-treated pine needle
segments are in good agreementwith the cytological observations.
INTRODUCTION
The histological approach to determine various types of injury to plant tissue has been
used by many workers (Bobrov, I952; Solberg and Adams, I956; Treshow, I957;
Thompson, Dugger and Palmer, I965, i966; Tingey, Heck and Reinert, I97I;
Evans and Miller, I972; Stewart, Treshow and Harner, I973). Electron microscopy has
been used to demonstrate the extent of ultrastructural injury caused by air pollutants
such as peroxyacetyl nitrate and ozone (Thompson et al., I965, I966). However, very
little is known about SO2 effects on ultrastructural organization and metabolic activities
of cells, and most of the cytological work has been done using light microscopes (Solberg
and Adams, I956; Stewart et al., I973). The latter reported that SO2 causes breakdown
of mesophyll tissue below stomata, occlusion of resin ducts, and abnormality of phloem
tissue in conifer needles.
Exposure of most types of vegetation foliage to SO2 results in changes such as discoloration of leaves (Rao and LeBlanc, I965). These foliar colour changes have been
attributed to impaired pigment metabolism (Rao and LeBlanc, I965; Coker, I967),
and since plant pigments are located within the chloroplasts, studying the effects of SO2
on the chloroplast structural organization should illuminate the mode of SO2 action.
Recently, Wellburn, Majernik and Wellburn (1972) reported the effects of SO2 on the
ultrastructure of broad bean chloroplasts, which are swelling of the stroma and granum
thylakoids. So far there has been no report on the subcellular effects of SO2 on forest
vegetation. Therefore electron microscopy was employed in this study to detect ultrastructural changes within chloroplasts from distal, midsection and basal segments of
pine needles affected by various concentrations of SO2. The results thus obtained were
then related to the biochemical activity (Hill reaction) of chloroplasts isolated from the
different pine needle segments.
239
240
S.
S. MALHOTRA
MATERIALS AND METHODS
Growth conditions
Lodgepole pine seeds (Pinus contorta Dougl. var. latifolia Engelm.) were treated
with 3% H202 for io min, washed with distilled water, and planted in Styroblock trays
containing peat (each cavity contained two to three seeds). The seeds were covered with
No. i granite grit and irrigated. The trays were placed in the glasshouse and covered
with polyethylene until the seeds started to germinate (approx. 7 days). Starting 4 weeks
after germination the seedlings were fettilized once a week with nutrient solution (Hocking, 1971) and watered as required. Fully developed needles 2-3-months-old were
harvested from seedlings 5-6-months-old for use as experimental material.
Sulphur dioxide treatment
The pine needles were washed with distilled water and cut into distal, middle and
basal portions. The basal portion included the younger, actively growing cells; the midportion included the middle-aged and semimature tissue; and the distal portion was
composed of older, fully matured cells (Linzon, i967). About i-cm sections were cut
from each of these zones and used for the biochemical and electron microscopy experiments.
An appropriate amount of the excised i-cm sections from each zone was placed in 50ml Erlenmeyer flasks containing one of the aqueous SO2 solutions (IO, 25, 50, IOO, 250,
5oo and iooo ppm). Aqueous SO2 stock solution (5000 ppm) was prepared by bubbling
anhydrous SO2 through a known quantity of O.I M tris buffer (pH 7.2) until a desired
gain in weight was reached (Puckett et al., I973). The various concentrations of aqueous
S02 were then prepared by diluting the stock solution. Concentrations of SO2 in air bear
a relationship to aqueous solutions that has not been fully explored. Other workers
(Saunders, I966; Puckett et al., I973), on the basis of limited experimental evidence, have
proposed that aqueous solutions of SO2 are equivalent to iooo-fold lower concentrations
in air. The relationship may not be linear through all concentrations and is, of course,
strongly temperature-dependent.
The flasks were then covered with glass cover slips, sealed with silicone grease, and
placed on a wrist action shaker under 64,000 lux source (reflection flood lamps).
The needle segments were allowed to photosynthesize for 22 h at 23?C and then were
thoroughly washed with distilled water and used for various experiments.
Electron microscopy
The pine needle segments that had been treated with io-iooo ppm aqueous SO2
concentrations were fixed overnight at 40C in 3 % glutaraldehyde made up in o. i M phosphate buffer (pH 7.o). After fixation, the material was washed twice with the buffer
(I5 min each time) and then post-fixed for 2-4 h in 2% OS04 mixed in the phosphate
buffer. After rinsing the material with the phosphate buffer it was dehydrated through a
graded series of ethanol (50, 70, 85, 95, ioo%). The material was left in each for I5 min,
removed and replaced in fresh alcohol of the same concentration for I5 min, and then
transferred to the next higher concentration.
The plant material was then infiltrated (under vacuum) with propylene oxide-araldite
mixture (I: i, v/v) for 36 h in open containers. After infiltration, the sections were embedded in araldite and placed in a 650C oven for 36 h to promote polymerization. After
cutting, thin sections were picked up with 200-mesh formvar coated grids. The sections
Effects of SO2 on pine needlechloroplasts
241
were stained in 2% aqueous uranylacetate for 2 h followed by staining in 0.2% lead
citrate for 2-3 min and examined under a Phillips 300 electron microscope at 8o Kv. The
electron micrographs were taken from the mesophyll tissue of treated and untreated
needle segments.
Isolationof chloroplasts
Chloroplasts were isolated from 2.5 g of pine needle tissue using a slightly modified
method of Oku and Tomita (I97I). The needle segments (distal, mid-, and basal portions)
that had been treated with aqueous SO2 were washed with cold water and then ground
gently with a pestle and mortar for I min in the following isolation medium: o.5 M
sucrose, o.oi M NaCl, 0.05 M tris-HCl (pH 7.8), o.5s% bovine serum albumin, I2.5%
polyethylene glycol-4000, and IO mM cysteine. The homogenate was squeezed through a
double layer of cheesecloth. The filtrate was centrifuged at 3000 rpm for I min. The
supernatant layer was then centrifuged at 5000 rpm for I min. The resultant pellet was
suspended in a washing medium that contained o.5 M sucrose, o.oi M NaCl, O.OI M trisHCI (pH 7.8), and O.I % bovine serum albumin and centrifuged at 5ooo rpm for I min.
After washing, the chloroplast fraction was resuspended in o.5 ml of the following suspending medium: o.I m sucrose, o.oi M NaCl, O.OI tris-HCl (pH 7.8), and o.I % bovine
serum albumin. All steps were carried out at o-40C.
Determinationof Hill reactionactivity
The Hill reaction activity was determined by measuring oxygen evolution. The
amount of oxygen evolved was determined polarographically with a Yellow Springs
Instrument Co. Model 53 oxygen monitor equipped with a Clarke probe. Data were
recorded on a Beckman model I oo5 I 00 mV potentiometric recorder. The final volume of
the reaction mixture was 3.2 ml and had the following composition: 6o ,umoles potassium
chloride, I0 ,umoles magnesium chloride, 3 ,umoles potassium ferricyanide, I00 ,umoles
tris-HCl (pH 7.8), 0.75 m moles sucrose, and 0.2 ml of chloroplast preparation. The
reaction chamber was maintained at 250C and illuminated with 500 W projection lamp
(47,000' lux). The oxygen evolution results were based on milligram chlorophyll as
determinedby the method of Arnon (I949).
RESULTS AND DISCUSSION
Saunders and Wood (1973) have reported that even though gaseous SO2 can penetrate
into plants, its absorption into the water film and formation of H2S03 between individual
cells and on cell walls are prerequisite for any activity relating to metabolic disturbance
or physical damage. Our present work on ultrastructural and biochemical aspects was
therefore carried out with aqueous SO2 (H2SO3).
Ultrastructural
changes
Distal.tissue. The electron microscopy of the older, fully matured tissue of fresh
pine needles produced normal chloroplasts and mitochondria (Plate I, No. I). The
fresh tissue was not incubated in the buffer of SO2 solution. However, the tissue incubated in the buffer alone also produced normal chloroplasts and mitochondria similar
to those from the fresh tissue (preliminary results).
The older, fully matured distal tissue of pine needles that had been treated with SO2
ranging from I Oto 50 ppm showed no detrimental effect on the chloroplast ultrastructure
242
S. S. MALHOTRA
(Plate i, No. 3), which was similar to that present in fresh tissue (Plate i, No. i). The
chloroplasts from ioo-ppm-treated tissue showed some disorganization while those from
5oo-ppm-treated tissue showed a significant change in the ultrastructure such as disruption of inner structure and formation of numerous vesicular bodies (Plate i, No. 4).
The thylakoids started to become swollen and deformed after exposure to IOO ppm S02,
and this swelling became more pronounced at 500 ppm, forming large vesicles (Plate i,
No. 4).
Since 500 ppm S02 lowered the pH of the incubation medium from 7.2 to 3.95, it was
thought desirable to prepare an HCI control in order to separate direct SO2 effects from
indirect ones such as increased acidity. The HCI control incubation medium containing a
tris-buffer solution, adjusted to pH 3.95 with HCI, resulted in no detectable damage to
the ultrastructure of the chloroplasts or the mitochondria (Plate i, No. 2). The granum
thylakoids which are the essential centres of photosynthetic activity appeared quite
normal. These results strongly suggest that the swelling and disintegration of thylakoid
membranes are not due to increased acidity even at the very high concentration of 500
ppm S02 (Plate i, Nos 2 and 4). Sulphur dioxide, therefore, must have specific direct
effects on the inner membranes of chloroplasts. As well, chloroplasts in the control tissues
(Plate i, Nos i and 2) and in the tissues treated in low S02 concentrations (50 ppm,
Plate i, No. 3) were aligned very close to the cell periphery. However, in 500-ppm S02treated tissue they were positioned considerably away from the cell periphery. This indicates that S02 causes either plasmolysis of cells or destroys the structure that holds the
chloroplasts in close proximity to the cell wall.
Mitochondria appeared normal in control, Io-ppm, and 5o-ppm tissues but were absent
in ioo-ppm and 500-ppm-treated tissues, suggesting their dissolution at high concentrations of S02. However, pine needle cells have very few mitochondria and it is possible
that the treated tissue examined did not contain detectable mitochondria.
Midtissue. The subcellular structures from fresh control and Io-ppm S02-treated
midtissues from pine needles appeared normal (Plate 2, Nos 5 and 7). Midtissue incubated with the buffer alone (pH 7.2) contained chloroplasts and mitochondria similar to
those in the fresh untreated tissue (preliminary results). There was a slight swelling of
the thylakoid membranes in the chloroplasts from HCI control tissue (Plate 2, No. 6)
which was probably due to increased acidity in the incubation medium.
Injury to the chloroplast structure was severe at ioo ppm and 500 ppm S02 and the
identity of grana membranes was completely lost (Plate 2, No. 8) as in older, fully matured
tissue (Plate i, No. 4). The mitochondria were not observed in 500-ppm S02-treated
tissues (Plate 2, No. 8). This indicates that the ultrastructure of old and middle-aged
pine needle tissue is highly sensitive to S02 injury at ioo ppm or higher concentrations.
Basal tissue. The younger, actively growing tissue treated with pH 7.2 buffer alone
(buffer control) produced normal chloroplasts (Plate 3, No. 9) similar to those observed
in the fresh tissue. The chloroplasts from HCI control tissue also looked normal except
for some slight swelling of grana membranes (Plate 3, No. io). In the basal tissue, S02
produced only a little change in the structural organization of chloroplasts and mitochondria even at a concentration of ioo ppm S02 (Plate 3, No. i i). At 500 ppm S02 the
chloroplast granum was slightly swollen but most of the structure was still undamaged
(Plate 3, No. i2). The mitothondria also appeared intact.
In general, electron microscopy has shown that older, fully matured tissue is more
sensitive to S02 injury than the younger, actively growing tissue. This is shown by disorientation of chloroplast structure, dislocation of chloroplasts, and absence of mito-
Effects of SO2 on pine needlechloroplasts
243
chondria in the older tissues treated with high concentrations of SO2. The detrimental
effects of SO2 on the ultrastructural organization of pine needle cells appear to be significantly different from the effects due to low pH alone (HCI control).
Hill reaction activity
Possible relationships between structural disorganization and disruption of biochemical activities of chloroplasts from SO2-treated tissue were determined by measuring
the Hill reaction activity of isolated chloroplasts. The chloroplasts were isolated from the
old mature, middle-aged semimature, and young actively growing tissues that had been
treated with various concentrations of aqueous S02. Chloroplasts from the old tissue
5 -
0o 4
E
'E
3
E
2_
-7--
"\
.
"\
0
C) 50 100
Fig.
I.
needle
I,
3500
250
1000
Aqueous S02 concn. (ppm)
10
The effect of various concentrations of aqueous SO2 on Hill reaction activity of pine
segments.
, Basal tissue;
, mid-tissue;
-.-.,
distal
tissue.
0,
Controls,
basal tissue; 2, distal tissue; 3, mid-tissue.
exhibited similar activity up to a concentration of IOO ppm S02, ranging between 2.30
and 2.64 imoles 02 released/min/mg chlorophyll; activity then dropped to I.9I, 0.I4
and o, at 250 ppm, 500 ppm, and iooo ppm respectively(Fig. i). The concentrationof
IOO ppm S02 that resulted in partial disorientation of chloroplast inner structure had no
inhibitory effect on the Hill reaction activity (Fig. i). At 500 ppm S02, the ultrastructure
of chloroplasts was completely disorganized and the grana membranes had lost their
identity (Plate i, No. 4). When the tissue was incubated in the buffer medium without
SO2, the pH of which was adjusted to 3.95 with HCI (that is, the same pH as that obtained by 500 ppm + SO2), the Hill reaction activity was I .70 ,umoles 02 liberated/min/mg
chlorophyll compared to 2.30 for the control and 0.I4 for 500 ppm SO2. These results
clearly demonstrate that the inhibition of Hill reaction activity by SO2 is not a result
of low pH alone.
The chloroplasts isolated from the mid-tissue that had been treated with various
concentrations of aqueous SO2 exhibited a pattern of Hill reaction activity very similar
to that of the older tissue except that the basal activity in the mid-tissue was higher than
that of the distal tissue (Fig. i). In contrast, the young, actively growing tissue maintained
fairly active chloroplasts even at high concentrations of SO2. The Hill reaction activity
244
S.
S. MALHOTRA
of chloroplasts from the control and io ppm treated tissue was similar (Fig. I). This
activity increased from 4.03 (control) to 4.54 (50 ppm S02), then showed a continuous
decline at the higher concentrations and eventually reached a value of o at iooo ppm
S02. Even though the ultrastructure of the chloroplasts from 5oo-ppm-treated tissue
(Plate 3, No. I2) showed some signs of S02 injury, most of the grana membranes still
appeared intact, which accounts for the relatively high Hill reaction activity (3.IO pmoles
02/min/mg chlorophyll). The basal Hill reaction activity from the young or basal tissue
was much higher than that from the mid-tissue or distal tissue (Fig. I), showing that
younger tissue with a high rate of metabolic activity is capable of incorporating more
S02 into its metabolism than older tissue with lower metabolic activity.
Most of our results are in good agreement with those of Brandt and Heck (I968) and
Wellburn et al. (I972) who found that phytotoxic concentrations of S02 result in plasmolysis coupled with swelling of chloroplasts. Stewart et al. (I973) found that S02 breaks
down mesophyll tissue below stomata and upon prolonged exposure to S02, the entire
mesophyll can collapse. The cytological approach coupled with the biochemical analyses,
with certain modifications, can be used to determine the extent of damage by air pollutants under field conditions. Because of the importance of various environmental conditions on the vegetation responses to S02 pollution, a study involving these factors is
currently underway.
ACKNOWLEDGMENTS
The author thanks Drs S. K. Malhotra and M. Tu of the Department of Biology,
University of Alberta, Edmonton for their valuable help in electron microscopy work;
Dr D. Hocking and Mr R. Blauel of the Northern Forest Research Centre, Edmonton
for valuable discussions on this work; and Mr J. Shuya and Mr P. Bihuniak for technical
assistance.
REFERENCES
ARNON, D. I.
Physiol.,
Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant
(I949).
24,
I.
B3OBROV,R. A. (I952).
The effect of smog on the anatomy of oak leaves. Phytopathology, 42, 558.
BRANDT, C. S. & HECK,W. W. (I968). Effects of air pollutants on vegetation. In: Air Pollution (Ed. by
A. C. Stern), p.
40I.
Academic Press, New York.
COKER, P. D. (I967). The effects of SO2 pollution on bark epiphytes. Trans. Br. Bryol. Soc., 5, 341.
EVANS, L. S. & MILLER, P. R. (1972).
Ozone damage to ponderosa pine: a histological and histochemical
appraisal. Am. Y'. Bot., 59, 297.
HOCKING, D. (I971).
Preparation and use of a nutrient solution for culturing seedlings of lodgepole pine
and white spruce; with selected bibliography. Environ. Can., For. Serv., North. For. Res. Cent.
Edmonton, Canada. Inf. Rep. NOR-X-i, i.
LINZON,S. N. (I967). Histological 'studies of symptoms in semimature-tissue needle blight of eastern white
pine. Can.Y. Bot., 45, I33.
The preparation of photoactive chloroplasts from pine leaves. PhotoOKU, T. & TOMITA,G. (197I).
synthetica, 5, 28.
Sulphur-dioxide: Its effect on
PUCKETT, K. J., NIEBOR, E., FLORA,W. P. & RICHARDSON, D. H. S. (I973).
photosynthetic-14C fixation in lichens and suggested mechanism of phytotoxicity. New Phytol., 72,
I4O.
RAO,D. N. & LEBLANC, F. (I965). Effects of SO2 on the lichen algae with special reference to chlorophyll.
Byrologist, 69, 69.
SAUNDERS,
P. J. W. (I966). The toxicity of sulphur dioxide to Diplocarpon rosae Wolf causing blackspot of
roses. Ann. Appl. Biol., 58, I03.
SAUNDERS,
P. J. W. & WOOD,C. M. (I973). S02 in the environment, its production, dispersal, and fate. In:
Air Pollution and Lichens (Ed. by B. W. Ferry, M. S. Baddeley, and D. L. Hawksworth), p. 6. Athlone
Press, London.
SOLBERG,A. & ADAMS, D. F. (1956). Histological responses of some plant leaves to hydrogen fluoride and
sulfur dioxide. Am. J. Bot., 43, 755.
THE NEW PHYTOLOGIST,
i
w
..
PLATE I
76, 2
:'
.......
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
._~~~~~~~~~
_.. .
L
=~~~~~~~~~~~~~~~~~fcn
pag 244
...
K_
THE NEW PHYTOLOGIST, 76, 2
PLATE 2
4~~~~~~~~~~~
S.
MALHOTRA-EFFECTS
S
OF
SO
ON : PINE
NEEDLE
OF
S0
ON
NEDECLRPAT
t+
s ..,%
>nJ...
:HS......
-1_.
IF1 Sk
......
.
.'i
S.
S~~~.
MAHOR..ET
PIN
CHLOROPLA
THE NEW PHYTOLOGIST, 76, 2
PLATE3
1!
_
.
*
~~~~r
K
_
*
.
..
-
.*
a.t.
't
-
;
?:'
,
o
*,
w_J
ll
id
-
|
i
'
;
7
.-l.
.
.
#l
.
u_
\
\
Sg
K
1B
E
\ w_ffffid;
ffi;t' . a~~~~i
S MALHOTR-EFFECTSO
SO2 ON PNE NEEL
S~~~~~~~~
0i
00;
CHORPLST
_=
~1w.A4
..:11r....s.t
11(~~~~~~~~~~~~~~~~~~~~
Effects of SO2 on pine needlechloroplasts
245
D., TRESHOW, M. & HARNER, F. M. (I973). Pathological anatomy of conifer needle necrosis.
Can. Y. Bot., 51, 983.
W. W., DUGGER, W. M. & PALMER, R. L. (I965). Effects of peroxyacetyl nitrate on ultraTHOMPSON,
structure of chloroplasts. Bot. Gaz., I26, 66.
THOMPSON, W. W., DUGGER, W. M. & PALMER, R. L. (I966). Effects of ozone on the fine structure of the
palisade parenchyma cells of bean leaves. Can. Y. Bot., 44, I677.
Effect of low concentrations of ozone and sulfur
TINGEY, D. T., HECK, W. W. & REINERT, R. A. (1971).
dioxide on foliage growth and yield of radish. Y. Am. Soc. Hort. Sci., 96, 369.
TRESHOW, M. (I957). The effects of fluoride 6n the anatomy of chinese apricot leaves. Phytopathology, 46,
649.
Effects of SO2 and NO2 polluted air upon
WELLBURN, A. R., MAJERNIK, 0. & WELLBURN, F. A. M. (1972).
the ultrastructure of chloroplasts. Environ. Pollut., 3, 37.
STEWART,
EXPLANATION
OF PLATES
PLATE I
Ultrastructure of chloroplasts and mitochondria from the distal tissue of pine needles as
affected by SO2.
No. i. Fresh needle cell (control tissue) showing normal chloroplast and mitochondria.
X 36,300.
No. 2. Normal ultrastructure of cell from tissue incubated in tris-buffer solution, the pH of
which was adjusted to 3.95 with HCI (HCI control). x 46,200.
No. 3. Normal chloroplast structure in the tissue treated with 50 ppm aqueous SO2- x 72,600.
No. 4. Chloroplast in the tissue treated with 500 ppm aqueous SO2 showing swelling and
disintegration of thylakoid membranes. x 59,400:
PLATE 2
Ultrastructure of chloroplasts and mitochondria from the mid-tissue of pine needles as affected
by S02No. 5. Fresh needle cell (control tissue) showing normal chloroplast and mitochondria.
X 72,600.
No. 6. Ultrastructure of cell from tissue incubated in tris-buffer solution, the pH of which
was adjusted to 3.95 with HCI (HCI control). x 36,300.
No. 7. Normal chloroplast in the tissue treated with io ppm aqueous SO2- x I9,800.
No. 8. Chloroplast in the tissue treated with 500 ppm aqueous SO2 showing total disintegration of its inner structure. x 46,200.
PLATE 3.
Ultrastructure of chloroplasts and mitochondria from the basal tissue of pine needles as
affected by SO2.
No. 9. Ultrastructure of cell from tissue incubated in tris-buffer solution (buffer control).
X 59,400.
No. io. Ultrastructure of cell from tissue incubated in Tris-buffer solution, the pH of which
was adjusted to 3.95 with HCI (HCI control). x 59,400.
No. i i. Normal chloroplast in the tissue treated with ioo ppm aqueous SO2. X 92,400.
No. i2. Ultrastructure of chloroplast and mitochondria from the tissue treated with 500 ppm
aqueous SO2. x 72,600.