1021-4437/04/5104- © 2004
Russian Journal of Plant Physiology, Vol. 51, No. 4, 2004, pp. 480–485. Translated from Fiziologiya Rastenii, Vol. 51, No. 4, 2004, pp. 534–540.
Original Russian Text Copyright © 2004 by Maksimov, Cherepanova, Surina, Sakhabutdinova.
At present, salicylic acid (SA) is one of the most
studied compounds. SA is known to induce the sys-
temic acquired resistance with reactive oxygen species
as an important component [1, 2]. In addition, SA can
change (increase or reduce depending on H
tration, cited after ) catalase and peroxidase activi-
ties [4, 5]. It is supposed that SA modulates these activ-
ities via its direct interaction with these enzymes  or
by controlling their synthesis [5, 7].
Activation of peroxidase in response to stress is one
of the key processes in the plant-cell defense responses
. It occurs after treatments with pathogens and elici-
tors , wounding , or changing the temperature
regime . Peroxidases can be involved in the control
of the level and activity of endogenous and exogenous
signaling molecules in plants . For example, they
affect the synthesis and degradation of some phytohor-
mones, peroxides, and phenols.
Since we have demonstrated a reduced resistance of
wheat cells cocultured with the fungus
, we expected that this model system could be suc-
cessfully used in the studies of plant-cell resistance to
the bunt pathogen. In this connection, it was of interest
to examine the changes in the activities of particular
peroxidases in the cultured wheat cells under the effects
of SA and a phytopathogenic biotrophic fungus
MATERIALS AND METHODS
Experiments were performed with
calli of soft wheat
L., cv. Zhnitsa.
Calli were grown at 26
C in darkness on the MS
medium in the presence or absence of 0.05 mM SA.
After 5 days, some calli were infected with
(DC.) Tul. teliospores (100 spores per callus). Con-
trol calli were neither infected nor treated with SA.
Plant material was weighed and ﬁxed in liquid nitrogen
3, 6, 10, and 20 days after infection. Each sample com-
prised four calli.
Isolation of the cytoplasmic peroxidase.
were homogenized in three volumes of 0.01 M sodium
phosphate buffer (PB), pH 6.0, and the enzyme was
extracted at 4
C for 1 h. The extract was centrifuged at
14000 rpm (type 310b centrifuge, Mechanica pre-
cyzyjna, Poland) for 15 min. The isoelectric pattern of
cytoplasmic peroxidases was analyzed in the superna-
Isolation of the ionically bound peroxidase.
walls of wheat calli were washed ten times with ten vol-
umes of 0.01 M PB containing 1% Triton X-100 (ICN,
United States). Thereafter, peroxidases were extracted
with 2 M NaCl in 0.01 M PB (1 : 1, w/v). The mixture
was centrifuged at 14000 rpm (type 310b centrifuge)
for 15 min, and the supernatant was used for assays.
Isolation of the covalently bound peroxidase.
washing off the ionically bound peroxidases, cell walls
were treated with 0.01 M PB containing 0.25% cellu-
lase and 0.25% pectinase (Onozyka, Japan).
The secreted peroxidases
were isolated from 20 ml
of the agar-solidiﬁed culture medium after 20-day-long
The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli
Cocultured with the Bunt Pathogen
I. V. Maksimov, E. A. Cherepanova, O. B. Surina, and A. R. Sakhabutdinova
Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences,
pr. Oktyabrya 69, Ufa, 450 054 Russia;
Received April 24, 2003
—The effect of salicylic acid (SA) on peroxidase activity in wheat (
L.) calli cocul-
tured with the bunt pathogen
was studied. Fungal infection was shown to activate cytoplasmic
peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI ~9.8. A novel
chitin-speciﬁc peroxidase with pI ~3.5 appeared after the SA treatment. The infection of SA-treated cells with
activated the isoenzymes with pI ~3.5, ~4.8, and ~7.5 and stimulated their secretion into the cul-
ture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The
important role of this control in plant defense responses to the bunt pathogen is emphasized.
Key words: Triticum aestivum - Tilletia caries - calli - coculturing - salicylic acid - isoperoxidases
: IEF—isoelectrofocusing; MS—Murashige and
Skoog nutrient medium; PB—phosphate buffer; SA—salicylic