ISSN 10214437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 5, pp. 626–633. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © A.V. Chasov, F.V. Minibayeva, 2014, published in Fiziologiya Rastenii, 2014, Vol. 61, No. 5, pp. 668–675.
The cell surface is first structure to experience the
impact of a stressor. One of fast (from several seconds
to several minutes) plant responses to such action is an
oxidative burst on the cell surface. The investigation of
redox reactions under biotic and abiotic stresses
implies that the conditions of experiment should be
close to natural ones . In this connection it is evi
dent that the application of noninvasive methods for
the analysis of processes taking place on the cell sur
face has definite advantages over other methods . In
particular, when noninvasive methods are used, the
activities of extracellular enzymes are not reduced and
their natural substrates and cofactors are not lost as it
occurs during protein isolation and purification.
The pattern of dynamic changes in the activities of
extracellular enzymes can be correctly assessed at
manipulations with one and the same plant material
(tissue, organ, plant). Earlier we have found that the
solution after root incubation in it and their subse
quent removal (extracellular solution, ECS) manifests
a high redox activity . Stressinduced changes of
this activity are the components of plant stress
response. Indeed, the activity of extracellular peroxi
dase (ECPOX) of detached wheat roots (wounding
stress) was two orders higher than that of intact roots
. The incubation of detached roots in solutions
containing various compounds and substrates allows
manipulations with the enzyme activities in the ECS,
studying processes occurring on the cell surface, and
judging about events taking place inside the cell [5, 6].
In addition, the analysis of ECPOX activity of intact
roots provides an opportunity to study distant signal
transmission from shoots to roots.
The aim of this work was to characterize extracel
lular redox enzymes using a noninvasive approach,
and in particular to study isoenzyme composition and
substrate specificity of ECPOXs of wheat roots follow
ing wounding stress.
MATERIALS AND METHODS
Experiments were performed with
the roots of spring wheat (
Kazanskaya Yubileinaya) seedlings.
Seeds were soaked in tap water for 24 h (135 seeds
per treatment), washed with distilled water, placed on
the glass covered with cheesecloth moistened with
, and germinated under an irradiance
of 100 W/m
with a 12h photoperiod at
Methodological Approaches for Studying Apoplastic Redox Activity:
2. Regulation of Peroxidase Activity
A. V. Chasov and F. V. Minibayeva
Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center, Russian Academy of Sciences,
ul. Lobachevskogo 2/31, Kazan, 420111 Russia;
fax: (843) 2927347; email: firstname.lastname@example.org
Received November 23, 2012
—This work is devoted to the study of mechanisms of substrate regulation of extracellular peroxidase
(ECPOX) activity at a distant stress (wounding) signal transmission from aboveground organ (leaf) of wheat
L., cv. Kazanskaya Yubileinaya) seedlings to the roots. Along with the high dianizidine per
oxidase activity, the extracellular solution manifested 3,4dihydrooxiLphenylalanine peroxidase, ascorbate
peroxidase, and catalase activities. Dianizidine peroxidases were represented by several isoforms and had
broad substrate specificity. It was found that ECPOX was released from the roots into the growing solution
and its activity in the solution increased with root growth. Excision of the apical leaf parts in seedlings induced
a sharp activation of root ECPOX in the growing solution. The interaction between ECPOX substrates at oxi
dation in two and threecomponent systems is demonstrated. The role of ECPOX in the control of ROS bal
ance in the plant cell apoplast might be determined by competitive and complementary interactions between
different peroxidase substrates. Such substrate–substrate regulation of peroxidase activities may be important
for stressinduced oxidative burst in plant cells.
Keywords: Triticum aestivum,
seedlings, reactive oxygen species, wheat roots, peroxidase, extracellular solu
ECPOX—extracellular peroxidase; ECS—extracellular solution;
MGIS—medium for growing intact seedlings.