Physiol. Plant. 47: 223-228. 1979
223
Effects of Sulphur Dioxide on Sugar and Free Amino Acid Content of Pine Seedlings
By
S. S. MALHOTRA and S. K. SARKAR'
Northern Forest Research Centre, Canadian Forestry Service, Fisheries and Environment Canada, 5320 — 122 Street,
Edmonton, Alberta T6H 3S5 Canada
(Received 5 March, 1979; revised 25 June, 1979)
detection of air pollution injury to vegetation prior to visual
Treatment of jack pine (Pinus banksiana Lamb.) seedlings with symptom development of S 0 2 phytotoxicity.
It has been suggested that S 0 2 causes a reduction in plant
gaseous SO, resulted in a shift between the reducing and nonreducing sugars. Increasing concentrations of gaseous S0 2 caused photosynthesis (Ziegler 1973); such a change may cause a
an increase in reducing sugars and a decline in the non-reducing reduction in the formation of serine (Tanaka et al. 1972).
sugars, suggesting a conversion from the latter to the former at high Sulphur dioxide has also been shown to affect glutamic acid
S0 2 concentrations. The total amino acid content of the intact and glutamine content of pea seedlings (Jager and Pahlich
tissues also increased with increasing concentrations of gaseous 1972) and asparagine, glutamine, glutamic acid, serine,
S0 2 . Gas-liquid chromatographic analyses of the amino acids glycine, alanine, arginine, ornithine, and proline content of
indicated that S0 2 (1.34 mg • m~3 for 96 h) resulted in an increase in spruce needles (Jager and Grill 1975). However, the
the content of alanine, valine, glycine, isoleucine, leucine, threonine,
information available on the effect of S0 2 on different
aspartic acid tyrosine, lysine, and arginine, and a decrease in the
content of serine and glutamic acid. The enzymatic and other impli- families of free amino acids is still quite scanty. The present
study was carried out to determine whether different concencations of such changes are discussed.
trations of gaseous S 0 2 (under controlled conditions) have
any effect on the level of pine needle metabolites such as free
Introduction
amino acids and sugars and to explore the possibility of
The growth of plants is the direct result of physiological developing a biochemical technique for the detection of S0 2
and biochemical activities within the tissues. Environmental injury to vegetation prior to visual symptom development.
factors such as temperature, moisture, nutrients, C 0 2 , and
Abbreviation: PVP, polyvinyl pyrrolidone.
light play an important role in determining the rate of
metabolic activity. Air pollutants such as S 0 2 , released from
certain industrial operations, can also affect plant metaMaterials and Methods
bolism either temporarily or permanently depending upon
the concentration and the length of exposure (Brandt and
Growth conditions
Heck 1968, Saunders and Wood 1973, Ziegler 1975,
Jack pine (Pinus banksiana Lamb.) seeds were planted in
Malhotra and Hocking 1976).
the greenhouse as described previously (Malhotra 1976).
It is generally believed that vegetation must exhibit visual
Approximately 5- to 6-month-old seedlings were used as
symptoms for S 0 2 injury to occur, but this may be misexperimental material.
leading. Several controlled environmental studies have
indicated that previsual disturbances such as changes in the
ultrastructural organization (Malhotra 1976) and bioGaseous S02 treatment
chemical functions (Malhotra 1977, Khan and Malhotra
Initially an attempt was made to compare the biochemical
1977) brought about by S0 2 can affect the growth and yield
effects of aqueous S0 2 on excised pine needle tissues with
of vegetation (Bleasdale 1952, Tingey et al. 1971). It is therethose of gaseous S0 2 on intact needles. The excised needles
fore of considerable importance to develop techniques for the
were incubated in an aqueous medium with and without
1
Present address: Department of Food Science, University of dissolved S0 2 . The preliminary results indicated that during
incubation, even in the S02-free aqueous medium, there was
Alberta, Edmonton, Alberta, Canada.
Abstract
0031-9317/79/120223-06 $03.00/0
© 1979 Physiologia Plantarum
224
S. S. MALHOTRA AND S. K. SARKAR
a substantial leakage of low molecular weight compounds
such as sugars out of the excised needles, it was therefore
decided that studies on the effect of S0 2 on low molecular
weight plant metabolites should use only gaseous SO, and
intact tissues.
Fumigations were carried out in small acrylic cuvettes
using a continuous flow-through system. Flow rates for air
and S0 2 were metered and controlled using rotameters and
valves before the gases were mixed and introduced into the
cuvettes. Further mixing within the cuvettes was achieved by
internally mounted fans near the injection ports. The concentrations of S0 2 at the input and output were monitored by
means of a Philips PW9700 S0 2 analyzer.
Pine seedlings were gently pulled out of the styrofoam
trays and inserted into vertically slitted neoprene stoppers so
that the foliage extended through the top of the stopper and
the sphagnum plugs containing the roots extended through
the bottom. The stoppers holding the seedlings were inserted
through holes cut in the bottom of the cuvette so that the slit
closed firmly around the stem and sealed the hole. The
cuvette was placed over a tank filled with enough water to
keep the sphagnum plugs moist during the experiment. The
access ports of the cuvettes were sealed to avoid any leakage.
The air flow was set at 10 1 • min~'; S 0 2 flow was adjusted to
give the desired concentration at the output of the cuvette.
The cuvettes were placed inside a controlled environmental chamber to maintain uniform light and temperature
conditions. Fumigations were carried out for 96 h at a temperature of 22°C under 0.80 W-m^ 2 light source (Sylvania
high-pressure sodium and mercury halide lamps). Relative
humidity measured in the output gas stream varied between
60 and 80%.
Extraction of the plant material
After treatment with S 0 2 , the needles were washed with
distilled water, dried, and transferred to 100 ml of boiling
95% ethanol. After boiling for 5 min, the liquid was allowed
to cool and was filtered into a large flask. The tissue was
transferred to 50 ml of 60% ethanol and ground for 5 min
(Virtis 23 homogenizer). The homogenate was filtered
through a Biichner funnel under vacuum, and the residue
was washed twice with 60% ethanol. The pigments were
removed from the combined 60 and 95% ethanol extracts by
double extraction with petroleum ether. The pigment-free
extract was evaporated to dryness using a rotary evaporator.
The dried residue was then dissolved in about 50 ml of
distilled water and centrifuged for 10 min at 27,000 g. The
residue was washed in 10 ml of distilled water and centrifuged again. The supernatant layers from the two centrifugations were combined, centrifuged once more to obtain a
clear supernatant layer, evaporated to dryness, and dissolved
in 10 ml of distilled water (crude extract). The phenolic
compounds were then removed from the crude extract by
treatment with purified insoluble PVP, which was purified
according to McFarlane and Vader (1962). The mixture was
Physiol. Plant. 47. 1979
filtered through a 0.8 ,um Millipore filter under vacuum to
obtain a clear solution.
Purification of amino acids and sugars
The above clear solution was passed through a Dowex
50W x 8, H + form (20-50 mesh) cation exchange resin
column. Before the column was loaded with the sample, the
resin in the column was thoroughly washed with water until
the pH of the effluent was close to neutrality. The flow rate
was adjusted to approximately 20 drops per minute. For
easier operation, the cation exchange resin column was fixed
on top of another column with anion exchange resin
(Dowex 1 x 8, CI" form, 20-50 mesh) so that every drop
flowing from the former would fall into the latter. The
organic acids were held by the anion exchange column, and
the effluent which passed through the anion column was the
neutral fraction containing sugars. After the cation exchange
column was washed with water, the elution of amino acids
was carried out with 50 ml of 2 M NH 4 OH. The eluate was
evaporated to dryness until no NH 3 odor could be detected.
The residue was dissolved in 5 ml of distilled water, and the
pH of the solution was adjusted to 2.5 with 4 M formic acid
to release any remaining ammonia. The solution was once
again evaporated to dryness, and the residue was dissolved in
water to give a final volume of 5 ml.
The neutral fraction containing sugars was reduced to a
volume of 10 ml on a rotary evaporator.
Amino acid determination
The amino acid content of the purified amino acid fraction
was determined by ninhydrin assay as described by Rosen
(1957).
Determination of reducing and non-reducing sugars
The reducing sugar content in the neutral fraction was
determined according to Bell (1955). For non-reducing
sugars, an appropriate aliquot of the neutral fraction was
mixed with formic acid to a final concentration of 6 M formic
acid. The mixture was then refluxed for 2 h to convert
non-reducing sugars to their monosaccharide components
(reducing sugars) before quantitation according to the above
method. The difference between the total sugars and the
reducing sugars thus corresponds to the non-reducing
sugars.
Preparation of amino acid derivatives for gas-liquid
chromatography
The derivatization was carried out as suggested by
Cancalon and Klingman (1974) with a slight modification.
An aliquot of an aqueous sample containing 0.5 mg amino
acids was evaporated to dryness in a silylation tube under a
stream of dry nitrogen at 60° C. Any trace of moisture was
Physiol Plant 47 1979
SO, EFFECTS ON PINE SUGARS AND AMINO ACIDS
removed azeotropically with methylene chlonde. The residue
was dissolved in i 5 ml ot 3 M butanohc HC1 by sonication
for 30 mm. For the butylation reaction, tubes were capped
and heated in an oil bath at 100° C for 15 min, after which
the caps were removed and the contents evaporated to
dryness at 60° C under a stream of dry nitrogen. Methylene
chloride was added to the residue and evaporated to dryness
to ensure the complete removal of moisture. The residue was
dissolved m 0.9 ml methylene chlonde and 0.1 ml tnfluoroacetic anhydride by sonication. The tubes were cooled, and
the solution was analyzed by gas-liquid chromatography.
Known amounts of standard amino acids were treated in
the same manner as above. Butyl stearate was used as the
internal standard.
Gas liquid chromatography
The complete separation of 20 amino acids was carried
out on two glass columns (1.8 x 2 mm) with different
packings: 'Tabsorb' and -Tabsorb H A C (both obtained
from Regis Chemical Co., Chicago, Illinois). 'Tabsorb H A C
facilitated the separation of histidme, arginine, and cystine;
'Tabsorb' separated the remainder of the ammo acids. Regis
Chemical Co. has used these packings for successful
separation of all 20 ammo acids. The columns were mounted
in the gas chromatograph oven and conditioned for 24 h at
200°C with a N 2 flow of 15 ml-min-'
Amino acid analyses were carried out with a Hewlett
Packard Model 5834A gas chromatograph equipped with
dual flame ionization detector and built in data processor.
The N, carrier flow rate was adjusted to 30 ml •mm - 1 . The
H 2 and air pressure were set at 140 and 190 kPa,
respectively.
High pressure liquid chromatography
The analysis of ammo acids was carried out using a
standard Durrum column on a D-500 amino acid analyzer
according to Benson (1973). Neither the gas-liquid
chromatography nor the high pressure liquid chromatography was versatile enough to analyze and estimate the
levels of glutamine and asparagine in pine needle extracts.
The limitations of these methods were, however, imposed by
different factors. The column and the buffer system used in
the high pressure liquid chromatography were not capable of
resolving the mixture of threonine, serine, glutamine, and
asparagine. Although the method provided a measure of
glutamic and aspartic acids, no measurement of glutamine
and asparagine could be made. On the other hand, the gasliquid chromatography method was unable to resolve
glutamic acid from glutamine and aspartic acid from
asparagine. This occurred because of hydrolysis during
denvatization of glutamine and asparagine to glutamic acid
and aspartic acid, respectively. Paper chromatography was
therefore employed for the estimation of glutamine and
asparagine.
225
Paper chromatography
The quantitative estimation of glutamine and asparagine in
ammo acid fractions from control and S0 2 treated plant
tissues was achieved by applying the sample as a band on
3 mm Whatman paper and developing it in H-butanol acetic
acid-H,0 (12 3 5, v/v) as described by Smith (1969)
Standard ammo acids were used for identification and
quantitation After development, the paper was dried and
sprayed with a reagent containing 0.2 g nmhydnn, 5 ml
acetic acid, and 95 ml rc-butanol saturated with water. The
paper was then heated at 75 °C for 30 mm. The bands corre
spondmg to glutamine, asparagine, glutamic acid, and
aspartic acid were cut out, and the color was eluted in 5 ml
of methanol as described by Jager et al. (1972). The absorbance at 500 nm for each eluent was measured by a spectrophotometer.
Results and Discussion
All experiments reported herein were repeated at least
three times with qualitatively similar results.
Effects ofS02 on sugars
Increasing concentration of gaseous S 0 2 resulted in an
increase (p < 0.05) in reducing sugars (Table 1), the increase
was much more pronounced at 1 34 mg-m~ 3 S0 2 concentration than at 0.89 mg m~3 At the same time, S0 2 at 1 34
mg m~3 concentration produced a considerable drop (p <
0.05) in non reducing sugars (Table 1). Another pollutant.
ozone, has also been shown to increase the reducing sugar
content and decrease the starch content in rough lemon
seedlings (Dugger and Palmer 1969). Recently, Koziol and
Jordan (1978) reported that there is an increase in free sugar
levels after exposure of red kidney bean seedlings, at least up
to a concentration of 2 mg • ITT3 S0 2 . Our results on pine
needle tissues are in good agreement with theirs. However,
Koziol and Jordan (1978) had to use a much higher S0 2
concentration than the 1 34 mg m~3 used in our experiments to observe an appreciable reduction in starch content
The increased concentration of reducing sugars after
Table 1 The effect of gaseous S02 on reducing and non reducing
sugar content of jack pine seedlings Each reading is from one
experiment with triplicate extractions (12-15 plants) Values bearing
the same letter do not differ significantly (p < 0 05) in Duncan's
multiple range test (Steel and Tome 1960)
Reducing sugar
content
SO,
mg m
3
Control
0 89
134
Non reducing sugar
content
umol g '
fr wt
,umol g '
dry wt
fimo\ g '
fr wt
36 0
48 8
68 8
116 9a
163 5 b
187 0 c
68
74
10
fjmoi g_1
dry wt
22 1 a
25 0 a
2 8b
226
S S MALHOTRA AND S K SARKAR
Physiol Plant 47 1979
fumigation with S0 2 (Table 1) may be due either to their
increased biosynthesis or to the breakdown of reserve polysaccharides rich in reducing sugars The decline in non
reducing sugars may be an inhibitory effect of S 0 2 on their
biosynthesis. Another plausible explanation for such a
response would be the partial breakdown of non-reducing
carbohydrates into simple reducing sugars.
Fumigation of seedlings with 0.89 mg • m~3 S 0 2 concentration produced no change in the non-reducing sugar
content (Table 1) and no visual symptoms of toxicity. However, at the higher concentration of 1.34 mg • m~3 S 0 2 there
was a pronounced drop in non-reducing sugar content, and
needle tips of older foliage exhibited chlorosis. This suggests
that extreme changes in cellular metabolites may have a
direct beanng on visual symptom expression.
The content of alanine, glycine, threonine, lysine, and
methionine in pine seedlings increased with increasing con
centration of gaseous S0 2 (Table 3). A maximum increase
was observed for alanine, followed by a somewhat smaller
increase in lysine, glycine, and threonine at a concentration
of 1.34 mg-m~ 3 S 0 2 (p < 0.05). It is suggested that S0 2
caused an increase in the amount of these amino acids by
stimulating the breakdown of membrane and other proteins.
Sulphur dioxide has previously been shown to cause breakdown of chloroplast membranes in pine needles (Malhotra
1976).
At 0.89 mg • irr 3 concentration, S 0 2 caused a decrease
(p < 0.05) in the levels of valine and leucine, but at 1.34
mg • m~3 (p < 0.05) an increase. It appears that S0 2 at a
low concentration (0.89 mg-m~ 3 ) inhibits the synthesis of
valine and leucine, and at a higher concentration of 1.34
mg • m~3 may bring about protein breakdown. If the protein
Effect ofS02 on totalfree amino acids
hydrolysis hypothesis is correct, one would expect to find an
The data in Table 2 show an increase ( p < 0.05) in the increase in amino acids such as proline, serine, and phenyltotal free amino acid content of pine needles after treatment alanine; instead, their content declines at 1.34 mg • m~3 S0 .
2
with gaseous SO z similar to that presented for reducing Under these conditions, such a response can be explained by
sugars m Table 1
increased conversion of proline, serine, and phenylalanine
The initial increase in ammo acid content preceded visible into related amino acids within their families; for example,
symptoms of injury, which only occurred after fumigation at serine may have been converted into glycine.
1.34 mg • m~3 S 0 2 concentration. The increase in free amino
The ammo acids related to the aspartate family, namely
acid content at the higher S 0 2 concentration was probably
lysine, methionine, threonine, and isoleucine, either increased
brought about by protein hydrolysis, which can eventually
or remained unchanged with S 0 2 (Table 3). This may be a
lead to tissue senescence (Fischer 1971).
result of S 0 2 effect on increased biosynthesis or on breakWorking with Phaseolus vulgaris, Trifoltum repens, and a
down of membrane and other proteins, or both. Since plants
mixture of grasses, Arndt (1970) also found that S 0 2
exposed to 1.34 mg m~~3 S 0 2 for 96 h showed some
fumigations at 0.63 mg-m" 3 caused an increase in the
content of free ammo acids. Similar changes in total ammo
acid content of spruce needles from high industrial emission Table 3 The effect of gaseous S02 on jack pine ammo acids
_1
regions have been reported by Jager and Grill (1975). In separated by gas liquid chromatography Data expressed in p% g
dry
wt
Each
reading
is
from
one
experiment
with
triplicate
order to explain such S0 2 effects on total free ammo acids,
extractions (12-15 plants) Values bearing the same letter do not
we studied changes in the levels of individual amino acids by differ significantly (p < 0 05) in Duncan's multiple range test
means of gas-liquid chromatography.
(Steel and Tome 1960)
Gas-liquid chromatographic analysis of ammo acids
All amino acid fractions from the control and S 0 2 treated
tissues were also analyzed by high pressure liquid chromatography Because the results were similar to those obtained by
gas-liquid chromotography, only the latter are discussed.
Amino acid
Alanine
Valine
Glycine
Isoleucine
Table 2 The effect of gaseous S02 on the total amino acid contentLeucine
of jack pine seedlings Each reading is from one experiment with Proline
triplicate extractions (12-15 plants) Values bearing the same letter Threonine
do not differ significantly (p < 0 05) in Duncan's multiple range test Serine
(Steel and Tome 1960)
Cysteine
Methionine
Ammo acid
Ammo acid
Phenylalanine
_1
S0 2
//mol g
fimol g '
Aspartic acid
3
mg m~
fr wt
dry wt
Glutamic acid
Tyrosine
Control
40
13 0 a
Lysine
0 89
58
19 5 b
Tryptophan
1 34
74
20 2 b
Arginine
Control
0 89mg m"3
1 34 mg m~3
984 a
67 a
23 a
75 a
90 a
144 a
88 a
360 a
0
14 a
40 a
191a
849 a
32 a
18a
1837 b
35 b
30 a
64 a
13b
74 b
108 a
325 a
16
17a
26 a
374 b
1091b
26 a
28 a
191
611b
3248 c
107 c
54 b
96 b
169 c
123 a
177b
266 b
16
21a
34 a
244 c
721c
86 b
49 b
0
558 b
0a
436 a
Physiol. Plant. 47. 1979
SO, EFFECTS ON PINE SUGARS AND AMINO ACIDS
227
symptoms of S0 2 phytotoxicity, it is possible that the cant (p < 0.05) change in phenylalanine. The tyrosine level,
-3
increase in these amino acids was mainly due to protein on the other hand, increased dramatically at 1.34 mg-m
(p
<
0.05);
again
this
may
be
due
to
protein
hydrolysis.
hydrolysis. Fischer (1971) reported that chlorosis of Vicia
The amino acids related to glycerate-3-phosphate, namely
faba and Nicotiana tabacum leaves caused by exposure to
gaseous S 0 2 resulted in a substantial reduction of the protein serine and glycine, responded to S0 2 differently. The serine
content declined (p < 0.05) after S 0 2 fumigation at 1.34
content of these leaves.
3
Aspartic acid also exhibited a considerable increase at mg-m~ concentration, whereas glycine increased (p <
3
0.89 m g - n r S 0 2 ( p < 0.05): however, this increase was 0.05) under the same conditions. While studying the effects
substantially reduced at 1.34 mg • m~3 (p < 0.05), suggesting of S 0 2 on wheat plants, Tanaka et al. (1972) observed a
a conversion of aspartic acid to asparagine at higher similar decline in serine content, but failed to detect any
S0 2 concentrations. Paper chromatographic analyses of glycine in their preparation. Possibly a conversion of serine
asparagine in the above fractions confirmed that S 0 2 at 1.34 into glycine accounts for the decline in serine vis-a-vis an
mg-mr 3 concentration produced more asparagine than increase in glycine.
Even at a low concentration (0.89 mg • m~3) that did not
aspartic acid, indicating a conversion of aspartic acid to
asparagine, probably mediated through asparagine syn- produce any visual symptoms on the foliage, S 0 2 had a
thetase. Changes similar to these were also observed for drastic effect on the levels of the most essential metabolites,
glutamic acid and glutamine. Studies on the effect of S 0 2 on such as sugars and amino acids. The analyses of such metabolites may provide an effective tool to determine the extent
glutamine and asparagine synthetase are under way.
Jager and Grill (1975) reported a change in the glutamic of hidden S 0 2 injury to vegetation. The mechanisms by
acid content of spruce similar to the one demonstrated in this which some of the changes in metabolites are brought about
paper. At the same time, they showed a decline in the glycine cannot yet be fully explained. Some of the enzymatic procontent under the influence of S0 2 , which is contrary to our cesses that we speculate are involved in these changes and
findings. However, Jager and Grill (1975) worked with are currently under investigation.
spruce tissues collected from a region damaged by industrial
We thank Mr. J. Shuya and Miss E. Hargesheimer for technical
exhaust, which may have contained a variety of emission
assistance
and Mr. P. Hurdle for construction and operation of S0 2
elements in addition to S 0 2 , whereas all our work was
fumigation facilities. We are grateful to the Alberta Oil Sands
carried out under controlled environmental and S 0 2 Environmental Research Program forfinancialassistance.
fumigation conditions.
The effects of S 0 2 on amino acids belonging to the
glutamate family, such as arginine and proline, were not
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228
S S MALHOTRA AND S K SARKAR
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