ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 4, pp. 456–462. © MAIK “Nauka /Interperiodica” (Russia), 2006.
Original Russian Text © D.I. Chrikishvili, G.V. Zaalishvili, T I. Mitaishvili, E.P. Lomidze, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 511–517.
In recent years, it was established that plants are efﬁ-
cient remediators of an ecosystem polluted by various
organic xenobiotics [1, 2], which include benzene, tol-
uene, and other components of oil fuel [3, 4].
It was shown that, in higher plants, benzene and tol-
uene were mainly absorbed by leaves. The aromatic
ring of these compounds was disrupted via oxidation,
and the aliphatic fragments thus formed were involved
in the general cell metabolism [5–7]. It was also shown
that the aromatic xenobiotics exerted a considerable
effect on the plant cell ultrastructure [2, 8].
The objectives of this work were to investigate ben-
zene and toluene biotransformation by means of their
conjugation with the cell constituents, to determine the
life time of the conjugates after removing plants from
the benzene- and toluene-containing atmosphere, and
to analyze the effect of benzene on the cell ultrastruc-
ture in the English ryegrass seedlings.
MATERIALS AND METHODS
English ryegrass (
seedlings grown on a Knop nutrient solution under
axenic conditions were used. [
]toluene preparations (Isotop, St. Petersburg)
with a speciﬁc activity of 1.76 and 1.50 mBq/mmol,
respectively, were applied in the experiments.
The plants were grown in a desiccator separated into
upper and lower parts by a screen with small openings.
A lower part of the desiccator contained sterile Knop
solution, and an upper one, a 20% KOH solution and a
compound under investigation. In the course of incuba-
tion, seedling leaves absorbed benzene and toluene
vapors. The seedlings were incubated for 24 h at
and under natural illumination.
After a 24-h-long incubation with labeled benzene
and toluene, one third of seedlings were washed with
distilled water, the seedlings were separated into leaves
and roots and ﬁxed in boiling 80% ethanol. Other seed-
lings were placed for 48 and 120 h into an atmosphere
devoid of benzene and toluene, and
seedlings was continuously absorbed by 20% KOH. In
order to remove labeled benzene and toluene, the KOH
solution was washed with petroleum ether. KOH solu-
tion radioactivity was estimated after 24, 48, and 120 h.
The 80% ethanol-soluble low-molecular-weight
metabolites were separated into individual components
by paper chromatography using FN-6 paper (Germany)
and a butanol : formic acid : water (6 : 1 : 5) solvent sys-
Peptide Conjugates of Benzene and Toluene Metabolites
in English Ryegrass
D. I. Chrikishvili
, G. V. Zaalishvili
, T. I. Mitaishvili
, and E. P. Lomidze
Durmishidze Institute of Biochemistry and Biotechnology, Academy of Sciences of Georgia, David Agmashenebeli alley,
10th km, Tbilisi, 0159 Georgia;
fax: (99532) 52-0618; e-mail: email@example.com
State Agricultural University of Georgia, Tbilisi
Received May 30, 2005
—Biotransformation of [
]benzene and [
]toluene in English ryegrass (
seedlings was investigated. Vapors of these compounds were absorbed by the leaves of this plant. Benzene and
toluene were oxidized, forming phenol and benzoic acid, respectively. A portion of phenol and benzoic acid
was bound by low-molecular-weight peptides forming conjugates. A qualitative amino acid composition of the
peptides involved in the conjugation was determined. After removing plants from the atmosphere containing
]benzene and [
]toluene, the radioactivity of the conjugates gradually decreased. This process was
accompanied by the evolution of
, indicating the breakdown of these conjugates. Radioactive compounds
thus formed were oxidized, yielding carbon dioxide. A portion of phenol and benzoic acid, along with peptide
conjugation, was subjected to further oxidative transformations up to disruption of the aromatic ring. By this
pathway, nonvolatile carboxylic acids, such as muconic, fumaric, succinic, malic, malonic, glycolic, and gly-
oxylic, were formed. Using electron microscopy, a damaging effect of benzene on the cell ultrastructure of
English ryegrass leaves was shown, and this toxic effect depended on the benzene concentration.
Key words: Lolium perenne - benzene - toluene - peptide - conjugation - xenobiotic - ultrastructure
: IR—infrared; T