Russian Journal of Ecology, Vol. 35, No. 5, 2004, pp. 290295. Translated from Ekologiya, No. 5, 2004, pp.

330335.
Original Russian Text Copyright 2004 by Chupakhina, Maslennikov.

Plant Adaptation to Oil Stress

G. N. Chupakhina and P. V. Maslennikov
Kaliningrad State University, Universitetskaya ul. 2, Kaliningrad, 236040 Russia
Received July 8, 2003

AbstractChanges in the contents of anthocyanins, ascorbic acid, and riboflavin were studied in plants growing under pollution with petroleum products along railroad tracks (Geum urbanum L., Anthriscus sylvestris L.,
Glecoma hederata L., Taraxacum officinalis L., Dactylis glomerata L., and Achillea millefolium L.) and in
seedlings grown in soil containing 510% crude oil (Hordeum vulgare L., D. glomerata, Vicia sativa L., Panicum miliaceum L., and Zea mays L.). In the former case, the plants accumulated ascorbic acid and anthocyanins
(on average, 2 and 5.2 times those in the norm, respectively), and riboflavin (in both reduced and oxidized
forms). In the latter case oil-induced stress also proved to stimulate the accumulation of all test substances in
the seedlings. The content of anthocyanins is proposed as a test parameter reflecting the degree of environmental pollution, which may be useful for prompt bioindication of pollutants in the ecological monitoring of plant
communities.
Key words: petroleum pollutants, anthocyanins, ascorbic acid, riboflavin, bioindication.

To protect the environment from various kinds of

pollution is an urgent problem. As soil resources are
limited, it is especially important to protect the soil
from pollution with oil and petroleum products spilled
in the course of production, transportation, storage,
and utilization. Petroleum pollutants impair hydrologic conditions and physical properties of the soil,
drastically reduce the contents of movable nitrogen
and phosphorus compounds, and have a toxic effect
on plants, causing the destruction of chlorophylls and
carotenoids (Golodyaev and Ivanov, 1988). Flavonoids and phenol acids in the plant cell may play an
important role in plant adaptation to various stress factors (Hoch et al., 2001). Thus, the accumulation of flavonoids is related to the mechanism protecting the
photosynthetic apparatus from extensive oxidative
damage (Yamasaki et al., 1996; Yamasaki, 1997),
which may result from exposure to various pollutants
(Lavola et al., 1994). Unlike other phenolic compounds of plant origin, anthocyanins have not been
studied with respect to the influence of petroleum pollutants on their synthesis, despite an intensive search
for prospective biochemical indicators of environmental pollution among secondary plant metabolites
(Maslennikov, 2001).
Anthocyanins accumulate in the tissues of plants
exposed to stress and change their optical properties,
and the use of methods based on this phenomenon may
be most expedient. The contents of riboflavin and
ascorbic acid are also promising as indicators of the
physiological state of plants. These compounds have a
variety of effects on physiological processes in plant
cells and, according to recent data, are involved in cell
immunity, which accounts for the resistance of plant

cells to stress factors (Mori and Sakurai, 1995; Chupakhina, 1997; Powers, 1999; Silva et al., 1999).
The purpose of this work was to study the accumulation of anthocyanins, ascorbic acid, and riboflavin in
plants growing under conditions of pollution with
petroleum hydrocarbons and crude oil.
MATERIAL AND METHODS
The leaves of Geum urbanum L., Anthriscus
sylvestris L., Glecoma hederata L., Taraxacum officinalis L., Dactylis glomerata L., and Achillea millefolium L. were collected in a zone polluted with petroleum products (motor and transmission oils, grease,
etc.) along railroad tracks. Plants growing 35 m away
from the railroad were used as the control.
In addition, we studied 25-day-old seedlings of
Hordeum vulgare L. (cultivar Roland), D. glomerata L.
(cultivar Asta), Vicia sativa L. (cultivar Orlovskaya),
Panicum miliaceum L. (cultivar Bystroe), and Zea
mays L. (cultivar Ross 144) grown in soil (light loam)
containing the maximum allowable concentrations of
crude oil (510%, depending on the species) from the
Aleshkino field, Kaliningrad oblast (Dedkov and
Fominykh, 1999). The seedlings were grown in a
TKSh-1 unit at constant illumination (LBU-30
fluorescent lamps, 5 J/m2 s) and room temperature
(1822C).
The contents of riboflavin in oxidized and reduced
forms (OR and RR, respectively) and anthocyanins
were determined by means of spectrophotometry
(Muraveva et al., 1987; Chupakhina, 2000), and the
contents of ascorbic acid (AA), by titration (Chupakhina, 2000). The results were recalculated per gram

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291

Dactylis glomerata
Glecoma hederata
Taraxacum officinalis

Clean zone
Polluted zone

Anthriscus sylvestris
Achillea millefolium
Geum urbanum
0

2
3
4
Anthocyanins, mg/g

Fig. 1. Effect of pollution with petroleum products along the railroad on the anthocyanin content in different plant species.

Taraxacum officinalis
Anthriscus sylvestris
Dactylis glomerata

Clean zone
Polluted zone

Glecoma hederata
Achillea millefolium
Geum urbanum
0

50

100
150
Ascorbic acid, g/g

200

Fig. 2. Effect of pollution with petroleum products along the railroad on the ascorbic acid content in different plant species.

dry tissue weight. The values shown in the figures

(means and mean square deviations) were obtained in
four to six independent experiments, each performed in
three biological replications.
RESULTS AND DISCUSSION
Pollution along the railroad proved to stimulate the
accumulation of anthocyanins in all plant species
(Fig. 1). The contents of these pigments were markedly higher than in the control plants (on average, by
a factor of 5.2). The greatest and smallest differences
from the control were observed in the cases of
G. urbanum and G. hederata: by factors of 16.9 and
1.4, respectively.
The contents of AA in the leaves of plants from the
polluted zone also proved to exceed the control level by
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factors of 2.3 (G. urbanum), approximately 2 (A. millefolium, A. sylvestris, T. officinalis, and G. hederata),
and 1.6 (D. glomerata) (Fig. 2).
A similar result was obtained when analyzing the
contents of riboflavin: the accumulation of both RR and
OR proved to be activated in the polluted zone. The
contents of OR in the leaves of G. urbanum, A. millefolium, A. sylvestris, T. officinalis, G. hederata, and
D. glomerata exceeded the control level by a factor of
1.21.5 (Fig. 3a). The contents of RR in the leaves of
G. urbanum, G. hederata, and D. glomerata exceeded
the control level by a factor of 1.3; and in the leaves of
A. millefolium, A. sylvestris, and T. officinalis, by a factor of 1.52 (Fig. 3b).
A positive correlation was observed between the
contents of anthocyanins and AC. This correlation was
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CHUPAKHINA, MASLENNIKOV
()

Taraxacum officinalis
Geum urbanum
Anthriscus sylvestris
Glecoma hederata
Achillea millefolium
Dactylis glomerata
0

0.5

1.0

1.5

2.0

2.5

(b)

Taraxacum officinalis
Geum urbanum

Clean zone
Anthriscus sylvestris

Polluted zone

Glecoma hederata
Achillea millefolium
Dactylis glomerata
0

2
3
Riboflavin, mg/g

Fig. 3. Effect of pollution with petroleum products along the railroad on the contents of (a) oxidized and (b) reduced forms of riboflavin in different plant species.

of medium strength in the plants from the control

(clean) zone (r = 0.540.68) and strong (r = 0.870.98)
in the plants from the polluted zone. Likewise, the correlation between the contents of riboflavin and anthocyanins was stronger in the latter (r = 0.690.81) than in
the former (r = 0.120.34).
To study the effects of petroleum products on plant
growth and the synthesis of test substances, seedlings
of H. vulgare, D. glomerata, P. miliaceum, and
Z. mays were grown in soil containing the maximum
allowable concentrations of crude oil: 10% for H. vulgare, Z. mays, P. miliaceum, and V. sativa and 5% for
the less tolerant D. glomerata (Dedkov and Fominykh, 1999). At this concentration of oil in the soil,
the emergence of D. glomerata seedlings was retarded
by two to seven days, the first true leaves appeared one
to three days later, and the height of two-week-old
plants grown in the soil contained 5% crude oil was
29% smaller than in the control. The growth and
development of V. sativa, H. vulgare, Z. mays, and
P. miliaceum seedlings in the soil containing 10% oil
were retarded. In H. vulgare, seedlings appeared one

or two days later; in V. sativa, P. miliaceum, and

Z. mays, six to seven days later than in the control.
The emergence of the first and second true leaves in
V. sativa was retarded by seven and four days; in
P. miliaceum, by six and seven days; and in
H. vulgare, by four and six days; plant height on day
14 was 56, 83, and 45% smaller than in the control
plants of the same species, respectively. Higher concentrations of oil in the soil prevented seed germination.
Under conditions of oil pollution, the contents of
anthocyanins in the seedlings of all species increased
by a factor of 2.4, on average (Fig. 4). The contents of
AA increased by a factor of 22.4 in P. miliaceum,
D. glomerata, and H. vulgare and by a factor of 1.71.8
in Z. mays and V. sativa (Fig. 5). The riboflavin pool
proved to increase in the seedlings of all species, with
OR exceeding the control level by a factor of 1.82.5
(Fig. 6a), and RR, by a factor of 1.62.2 (Fig. 6b). In
both experimental and control plants, a direct correlation of medium strength between the contents of anthocyanins and total riboflavin (r = 0.290.64) and a strong

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PLANT ADAPTATION TO OIL STRESS

293

Zea mays
Hordeum vulgare
Panicum miliaceum
Experiment
Control

Dactylis glomerata
Vicia sativa
0

2
3
Anthocyanins, mg/g

Fig. 4. Effect of oil pollution on the accumulation of anthocyanins in the leaves of test plants.

Zea mays
Panicum miliaceum
Experiment
Vicia sativa

Control

Dactylis glomerata
Hordeum vulgare
0

50

100
150
Ascorbic acid, g/g

200

250

Fig. 5. Effect of oil pollution on the accumulation of ascorbic acid in the leaves of test plants.

correlation between the contents of anthocyanins and

AA (r = 0.890.99) were revealed.
Thus, soil pollution with petroleum products and
crude oil promoted the accumulation of anthocyanins,
AA, and riboflavin (both OR and RR) in G. urbanum,
A. sylvestris, G. hederata, T. officinalis, D. glomerata,
A. millefolium, H. vulgare, V. sativa, P. miliaceum, and
Z. mays plants.
The accumulation of anthocyanins and riboflavin
under the effect of soil pollution with crude oil (in
V. sativa, D. glomerata, H. vulgare, Z. mays, and
P. miliaceum) and petroleum products along the railroad (in G. urbanum, A. sylvestris, G. hederata, T. officinalis, D. glomerata, and A. millefolium) may be due
to numerous disturbances of soil conditions: impaired
aeration resulting from air displacement by oil; activation of anaerobic microorganisms; a disturbed water
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balance in the soilplant system; poisoning with sulfides and excess manganese released upon decomposition of some hydrocarbons; impaired hydrologic
conditions; and changes in the physical, morphological, and agrochemical soil properties entailing a sharp
decrease in the contents and accessibility of movable
nitrogen, phosphorus, and calcium compounds
(Maslennikov and Chernova, 2001). In addition, oil
pollution leads to sodium chloride salinization of the
soil, and its absorption capacity decreases (Golodyaev
and Ivanov, 1988).
The parallel accumulation of anthocyanins and
ascorbic acidcompounds differing in functional compartmentalization within the cellin the plants growing under conditions of oil pollution may contribute to
the efficiency of the antioxidant system in neutralizing
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CHUPAKHINA, MASLENNIKOV
()

Zea mays
Panicum miliaceum

Experiment
Control

Vicia sativa
Dactylis glomerata
Hordeum vulgare
0

0.2

0.4

0.6

0.8

1.0

(b)

Zea mays
Panicum miliaceum

Experiment
Control

Vicia sativa
Dactylis glomerata
Hordeum vulgare
0

0.5

1.0
1.5
Riboflavin, g/g

2.0

2.5

Fig. 6. Effect of oil pollution on the accumulation of (a) oxidized and (b) reduced forms of riboflavin in the leaves of test plants.

the products of oxidative stress and, hence, to plant

resistance against the effect of pollutants.
The content of endogenous anthocyanins may be
used as an indicator of the physiological state of plant
cells, tissues, and ecosystems. It is a promising test
parameter characterizing the degree of environmental
impact of various pollutants, which may be useful for
their prompt bioindication in the ecological monitoring of plant communities. Changes in the optical properties of plant tissues caused by the accumulation of
anthocyanins under stress can be monitored by
remote-sensing methods. Their use will allow specialists to reveal changes in individual components of an
ecosystem, and on this basis predict the course of its
evolution.
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