1. ISSN 1021 4437, Russian Journal of Plant Physiology, 2015, Vol. 62, No. 4, pp. 499–506. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © Yu.E. Kolupaev, N.I. Ryabchun, A.A. Vayner, T.O. Yastreb, A.I. Oboznyi, 2015, published in Fiziologiya Rastenii, 2015, Vol. 62, No. 4, pp. 533–541.
499
INTRODUCTION
It is known that the adaptation of plants to negative
temperatures is accompanied by expression of a large
number of genes [1] leading to the synthesis of cold
shock proteins (cold regulated proteins, dehydrines,
etc.), changes in membrane lipid composition, cryo
protectant accumulation [2], and activation of the
alternative respiration [3]. However, together with
these specific adaptive responses, universal defense
ones, such as activation of antioxidant defense system
capable to prevent secondary oxidative damages [4],
have a significant importance for survival of plants
under cold conditions. The plant antioxidant system is
known to be represented by the enzyme complex
involving superoxide dismutase (SOD), catalase
(CAT), different peroxidases, the ascorbate–glu
tathione cycle enzymes, and a number of low weight
molecular compounds with antioxidant properties
(ascorbic acid, glutathione, phenol compounds, anti
oxidants soluble in lipids, such as α tocoferol, β car
otene, etc.) [5].
To date, there are some reports demonstrating
that the process of plant cold hardening is accompa
nied by the increase in the activity of antioxidant
enzymes [6, 7]. In addition, there is some evidence
for a certain relation between the activity of antioxi
dant enzymes and the tolerance of different plant
genotypes to hypothermia [8–10]. At the same time,
however, many authors observed the multidirectional
changes in the activity of individual antioxidant
enzymes in response to the action of low tempera
tures that excludes, for this reason, any distinct con
clusion about their role in plant adaptation to hypo
thermia [9–11]. On the other hand, it has been found
that cell oxidative stress significantly contributes to
plant cold damages [11, 12].
In parallel with the changes in plant antioxidant
system functioning during plant cold adaptation, there
also occurs accumulation of low molecular weight
RESEARCH PAPERS
Antioxidant Enzyme Activity and Osmolyte Content in Winter Cereal
Seedlings under Hardening and Cryostress
Yu. E. Kolupaeva, N. I. Ryabchunb, A. A. Vaynera, T. O. Yastreba, and A. I. Oboznyia
a
Dokuchaev National Agrarian University, Kommunist 1, Kharkiv, 62483 Ukraine
e mail: plant_biology@mail.ru
bYur’ev Institute of Plant Breeding, National Academy of Agrarian Sciences of Ukraine, Kharkiv
Received July 14, 2014
Abstract—Activities of antioxidant enzymes and the osmolyte contents in seedlings of winter rye (Secale
cereale L.), soft (Triticum aestivum L.) and durum (T. durum L.) wheat, and barley (Hordeum vulgare L.)
grown at 20°C (control) or after 7 day cold hardening at 2°C and/or 5 hour freezing at –6°C were investi
gated. It was found that nonhardened rye seedlings differed from those of other cereals by their ability to sur
vival after freezing at –6°C and higher activity of guaiacol peroxidase (GPO) and high content of proline.
Hardening induced the increase in the frost tolerance of all cereals under study, and the resistance of rye and
soft wheat was found to be significantly higher than that of durum wheat and barley. Rye and soft wheat exhib
ited more profound tolerance to oxidative damages as well, and it was expressed in lesser increase in the MDA
content after freezing. In the course of hardening, detectable increase in the activities of GPO and catalase
(CAT), as well as the contents of proline and soluble carbohydrates, was observed in seedlings of all cereals
under study. In barley, the activity of superoxide dismutase (SOD) increased to the highest extent under these
conditions. After freezing of both hardened and nonhardened seedlings, higher activities of all tested antiox
idant enzymes were revealed in rye and soft wheat as compared to those in durum wheat and barley. In this
case, hardened rye and soft wheat seedlings after freezing displayed increased content of proline. All these
results lead to the conclusion that the high content of proline and activity of GPO observed in rye seedlings
may determine their increased constitutive frost tolerance, whereas high tolerance of hardened soft wheat
seedlings is primarily associated with accumulation of low molecular weight protectors, such as sugars and
proline, and, to some extent, with the increased activity of antioxidant enzymes.
Keywords: Secale cereale, Triticum aestivum, T. durum, Hordeum vulgare, frost tolerance, hardening, antiox
idant enzymes, soluble carbohydrates, proline
DOI: 10.1134/S1021443715030115
Abbreviations: GPO—guiaicol peroxidase; CAT—catalase;
SOD—superoxide dismutase; TBA—2 thiobarbituric acid.

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KOLUPAEV et al.
compounds. The most important compounds are
believed to be soluble carbohydrates and proline [2].
In recent years, these compounds are considered to be
not only compatible osmolytes but also the substances
fulfilling more sophisticated functions, including
membrane protection, antioxidant and signal regula
tory ones [12–14]. A direct association between sugar
and proline accumulation and the plant tolerance to
low temperatures is known [15, 16]. However, some
plant objects do not display such an association [9],
which can be explained both by the reasons of a
methodical character (for example, using stresses of
different intensity or monitoring different phases of
plant responses in the experiments in question) and
complex functional interaction of low molecular
weight compounds with other stress protection sys
tems, in particular with the antioxidant enzymes.
Thus, the potato plants transformed with the yeast
invertase gene and accumulating increased amount of
sugars in leaves, as well as the same wild plants grown
on the medium with sucrose, exhibited lower activity
of SOD than the control plants [12]. Here, the authors
suggest that this circumstance is caused by the ability
of sucrose to remove ROS; in other words, SOD and
sugars are considered to be the functionally inter
changeable components of the antioxidant system.
To date, a significant volume of data on both anti
oxidant and prooxidant action of proline in plant cells
had been accumulated [14], and it was found that it
displayed a complex impact on gene expression and
the activity of antioxidant enzymes, including differ
ent forms of SOD [17, 18]. In particular, in the course
of studies of the response of a number of wild plant
species to salinity, reciprocal interrelation between the
changes in SOD activity and proline content was
revealed that, in the authors opinion, can be explained
by the antiradical action of proline [19].
Nevertheless, at present, the results of complex
studies on functioning of the plant antioxidant enzyme
system and the dynamics of osmolyte contents under
the action of hardening and adverse factors, in partic
ular chilling, are extremely limited. There are good
reasons to believe that comparative studies on the spe
cies and varieties of cultivated plants differing by their
tolerance to stressors may be beneficial for develop
ment of suitable approaches for evaluation of their
resistance as well as for more detailed understanding of
functional interaction between individual components
of the stress protection systems and their role in pre
venting oxidative damages upon freeze tolerance
exhibition.
The purpose of the present work was comparative
investigation of role of antioxidant enzymes (SOD,
CAT, and guaiacol peroxidase (GPO)) and also
osmolytes (proline and sugars) in the tolerance of win
ter grain cereal seedlings, such as rye, soft and durum
wheat, and barley to low temperature action.
MATERIALS AND METHODS
In the present work, 3–12 day old etiolated winter
cereal seedlings were used. These were represented by
rye (Secale cereale L., cv. Pamyat Khudoerko), soft
wheat (Triticum aestivum L., cv. Lyutestsens 329),
durum wheat (T. durum L., cv. Alyi Parus), and barley
(Hordeum vulgare L., cv. Zherar) and were obtained
from the collection of the National Center of Genetic
Plant Resources of Ukraine (Kharkiv).
Seeds were subjected to 30 min treatment with
6% hydrogen peroxide solution and, thereafter, germi
nated on purified tap water at 20°C for 3 days. Then,
the seedlings were placed for 7 days into the refrigerate
chamber (Danfoss, Holland) for hardening at 2°С
[20]. In the preliminary experiments, we found that
decreasing the hardening period to 5 days resulted in
repression of freeze tolerance development of the
cereal seedlings under study, while its increase to 9 days
did not favor additional enhancement of the plant tol
erance. After the completion of the hardening, the
temperature in the chamber was decreased at the rate
of 1°C/h; thereafter, the seedlings were subjected to
freezing at –6 or –9°С for 5 h. Further, the tempera
ture was increased at the rate of 1°C/h up to 2°С; then,
the seedlings were germinated at 20°С for 4 days, and
the number of surviving plants was determined taking
into account their ability to grow. The regime of freez
ing the seedlings causing the death of part of the sam
ples was found in the preliminary experiments (data
not shown). The 4 day old seedlings not subjected to
hardening were used as the control because, at low
temperature, the development of the plant seedlings
appeared to slow down and 10 day old hardened
plants corresponded to 4 day old ones grown at 20°С.
For the biochemical analysis, the shoots of 4 day
old seedlings grown at 20°С (the control), as well as of
nonhardened ones subjected to freezing at –6°С were
used, and the latter were then subjected to gradual
thawing for 8 h up to 2°С. The same analysis was used
in the case of the shoots of 10–11 day old hardened
seedlings, both prior and after their freezing according
to the above regime. Upon investigating the samples
subjected to freezing and thawing, the shoots having
no visible evidence of tissue infiltration were used.
Intensity of the lipid peroxidation in seedling tis
sues was determined by evaluating the amount of the
products interacting with 2 thiobarbiturate acid
(TBA), largely that of MDA [21]. To this end, plant
material was homogenized in 0.1 M Tris–HCl buffer
(pH 7.6); thereafter, the homogenate obtained was
supplied with 0.5% TBA solution in 20% TCA. After
heating the mixture in a boiling water bath for 30 min,
the samples were chilled and centrifuged at 8000 g for
10 min; then, the optical density of the supernatant at
532 nm was measured. Taking into account the pres
ence of pigments in the seedlings tissues, especially in
the case of rye, we subtracted the nonspecific light
absorption in the samples containing the homogenate

3. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 62 No. 4 2015
ANTIOXIDANT ENZYME ACTIVITY AND OSMOLYTE CONTENT 501
prepared as above and 20% TCA without TBA from
the experimental values.
The activity of antioxidant enzymes was deter
mined according to the methods detailed described
earlier [22]. The sample of plant material (200 mg) was
homogenized on the cold in 10 mL of 0.15 M K/Na
phosphate buffer (pH 7.6) supplied with 0.1 mM
EDTA and 1 mM dithiothreitol. For the analysis, the
supernatant prepared by centrifugation of the homo
genate at 8000 g for 10 min at 4°С was used. The activ
ity of the cytosolic SOD (EC 1.15.1.1) represented by
Cu/Zn SOD [23] was determined at pH 7.6 of the
reaction mixture using the method based on an ability
of the enzyme to compete with tetrazolium nitro blue
for the superoxide anions being produced in the course
of aerobic interaction of NADH with phenasine meta
sulphate. The activity of CAT (EC 1.11.1.6) was ana
lyzed at pH 7.0 of the reaction mixture evaluating the
amount of the hydrogen peroxide decomposed in a
unit of time. The activity of GPO (EC 1.11.1.7) was
determined using guaiacol as a hydrogen donor and
hydrogen peroxide as the substrate. The pH of the
reaction mixture was brought up to 6.2 with K/Na–
phosphate buffer.
The protein content in the samples was determined
by the method of Bradford [24] using bovine serum
albumin as the standard.
Total content of sugars in plant materials was deter
mined by the modified method of Morris–Roe based
on the use of antronic reagent [25]. Sugars were
extracted from plant materials with distilled water by
using 10 min heating in a boiling water bath. Clarifica
tion of the extract obtained was carried out by addition
to the reaction tubes of equal solution volumes (0.3–
0.4 mL) of 30% zinc sulphate and 15% yellow blood
salt. The samples were filtered through paper filter,
and, when required, the filtrate obtained prior to the
measurement was diluted several times with distilled
water. Then, 3 mL of antronic reagent and 1 mL of the
filtrate were added to the reaction tubes, while distilled
water instead of the filtrate was added to the control
sample. Thereafter, these samples were boiled for 7 min
in a water bath and further chilled up to room temper
ature. Optical density of the samples was determined
relative to control solution at 610 nm. D glucose was
used as the control.
The content of proline in the roots and above
ground part of wheat seedlings was determined by the
modified method of Bates et al. [26]. The amino acid
was extracted from plant material with distilled water
followed by subsequent 10 min boiling; thereafter, the
extract was filtered, equal volumes of ninhydrin
reagent and ice acetic acid were added to filtrate por
tions, and samples were boiled in a water bath for 1 h.
Optical density of colored reaction product was deter
mined at 520 nm using L proline as the control.
Data are shown as the means ± SD obtained from
three independent experiments with three replicates in
each. Only the differences significant at p ≤ 0.05 are
considered.
RESULTS
As follows from Table 1, seedlings of all tested cereal
species except rye practically did not display constitu
tive frost tolerance because, after freezing at –6°С, they
lost their ability to grow and died in 4 days. For all seed
ling species, hardening resulted in significant increase
in their frost tolerance as judged by the fact that, after
freezing at –6°С, relative numbers of survived hardened
seedlings of rye and frost resistant variety of soft wheat
practically did not differ each from other. In other
words, wheat seedlings, despite their absence of the
constitutive resistance in question under the chosen
experimental conditions, developed the freezing toler
ance similar to that of rye possessing a distinct level of
basic resistance to negative temperatures. An extent of
survival for seedlings of durum wheat and barley after
their freezing at –6°С comprised only 32–34%. Also,
in Table 1 it can be seen that, after freezing at –9°С,
hardened seedlings of rye and soft wheat exhibited dis
tinct difference in their frost tolerance that appeared to
be higher in rye, whereas hardened seedlings of durum
wheat and barley in fact completely died.
It is known that one of the indicators of oxidative
damages caused by the action of stressors, including
chilling, is MDA content in the tissues as a product of
lipid peroxidation [12]. Table 2 shows that any relation
between absolute content of MDA and frost tolerance
of nonhardened cereals species under study has not
been observed. At the same time, after hardening,
frost tolerant cereal species, rye and soft wheat, exhib
ited more profound decline in MDA content in their
tissues than nontolerant species, barley and durum
wheat. Freezing of the nonhardened seedlings resulted
in significant increase in MDA content in barley and
durum wheat, whereas only the trend to a little increase
Table 1. Survival (%) of winter cereal seedlings after their
freezing for 5 h at –6°C and –9°C
Species
Freezing temperature, °C
–6 –9
Without hardening
Secale cereale 33.1 ± 2.4 –
Triticum aestivum 0 –
Triticum durum 0 –
Hordeum vulgare 0 –
After hardening
Secale cereale 86.9 ± 3.1 84.0 ± 2.7
Triticum aestivum 86.1 ± 3.3 72.5 ± 3.5
Triticum durum 34.3 ± 4.0 0
Hordeum vulgare 32.3 ± 3.6 2.7 ± 2.1
Dash indicates the absence of assays.

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KOLUPAEV et al.
in the value of this parameter was observed in the case
of soft wheat, MDA content remained practically
unchanged in rye. The hardened seedlings after their
freezing displayed lower absolute content of MDA as
compared to that of the unhardened ones. As this takes
place, the increase in the amount of lipid peroxidation
product observed in the frozen samples as compared to
that of the initial values of the given parameter for the
hardened seedlings was in reciprocal dependence with
the frost tolerance of cereals (Table 2).
Nonhardened seedlings of rye and soft wheat did
not nearly differ by their activity of SOD, which
appeared to be significantly lower in the seedlings of
durum wheat and barley (Fig. 1a). Hardening resulted
in a little enhancement of SOD activity in the seed
lings of rye and both wheat varieties; however, activity
of this enzyme increased significantly in the hardened
barley seedlings. In the freeze tolerant cereal species,
rye and soft wheat freezing did not induce any signifi
cant changes in SOD activity, but the latter somewhat
declined in the case of the nonfrost tolerant species,
durum wheat and barley. In the hardened seedlings of
all tested cereal species, no significant changes in SOD
activity after their freezing and thawing were observed
(Fig. 1a).
Nonhardened seedlings of rye and two wheat spe
cies practically did not differ by their activity of CAT
but this was lower in barley (Fig. 1b). Hardening
resulted in elevation of the activity of the given enzyme
in seedlings of all cereal species and its absolute values
appeared to be higher in rye and soft wheat ones.
Freezing of nonhardened seedlings did not induce pro
found change in CAT activity in rye and both wheat
species but resulted in its decrease in barley. The activ
ity of this enzyme was not substantially changed in
hardened rye and soft wheat seedlings after the action
on them of cryostress but declined markedly after the
same treatment in durum wheat and barley seedlings.
Nonhardened cereal seedlings appeared to be dif
fered substantially by their activity of GPO (Fig. 1c). In
rye it exceeded nearly 2.5 times the values typical for
soft wheat seedlings. In durum wheat seedlings, activity
of GPO was significantly lower as compared to soft
wheat seedlings. At the same time, the activity of this
enzyme in nonhardened barley seedlings was high
enough and only slightly differed from the values
observed in soft wheat. After hardening, this activity
increased in all cereal species under study, most pro
foundly (by a factor of 4.4) in barley but to a lesser
extent in durum wheat. However, the greatest values of
GPO activity were revealed for rye. Freezing of non
hardened cereal seedlings induced elevation of this
activity in all cereal species under study, particularly
profoundly in barley. After freezing of the hardened
seedlings, it increased in the cases of rye and soft wheat
but did not change in durum wheat and barley (Fig. 1c).
As to sugar content in the control, i.e., nonhard
ened seedlings, this was highest in rye plants, while
other cereals under study did not display any signifi
cant differences in this indicative at p ≤ 0.05 (Fig. 2a).
After the impact of hardening temperature, substantial
elevation in the content of soluble carbohydrates was
observed in rye seedlings but especially pronounced in
soft wheat seedlings, whereas it appeared to be lesser
significant in durum wheat and barley seedlings. At the
same time, cryostress did not induce any significant
changes in sugar content in the samples of both hard
ened and nonhardened seedlings.
As follows from Fig. 2b, nonhardened rye seed
lings, unlike other cereals, exhibited high enough con
tent of proline but it was markedly lower in soft wheat
seedlings while its least values were observed in durum
wheat and barley seedlings. Hardening resulted in ele
vation in the content of this amino acid in all tested
cereal seedlings. In this case, unlike rye, the observed
increase in this value appeared to be more significant
for the species (durum wheat and barley) that are char
acterized by low proline content under normal condi
tions. On the other hand, freezing of nonhardened
cereal seedlings of all plant species under study did not
cause the changes in proline content. At the same
time, a substantial elevation of this value was observed
after freezing of hardened rye and soft wheat seedlings,
while this remained in fact unchanged in the case of
durum wheat and barley (Fig. 2b).
DISCUSSION
In soft and durum wheat plants and barley the con
stitutive resistance of nonhardened cereal seedlings
under study to low temperatures, was not practically
revealed and their freezing at –6°С appeared to be
Table 2. Content of MDA (nmol/g dry wt) in winter cereal seedlings
Species Control After hardening
After freezing
of nonhardened seedlings
After freezing
of hardened seedlings
Secale cereale 167.0 ± 4.9 109 ± 3 (65)* 158.0 ± 11.9 (95)* 133.6 ± 5.9 (123)**
Triticum aestivum 100.2 ± 3.3 65.5 ± 2.6 (66)* 112.0 ± 4.2 (112)* 97.5 ± 3.1 (148)**
Triticum durum 134.1 ± 4.0 96.8 ± 3.1 (72)* 186.2 ± 9.1 (139)* 160.3 ± 5.8 (166)**
Hordeum vulgare 92.7 ± 3.6 77.6 ± 2.8 (84)* 157.2 ± 6.8 (170)* 147.4 ± 5.1 (190)**
* In percentage relative to the control.
** In percentage relative to the values after hardening.

5. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 62 No. 4 2015
ANTIOXIDANT ENZYME ACTIVITY AND OSMOLYTE CONTENT 503
lethal, whereas a survival of rye seedlings under the
same conditions achieved more than 30% (Table 1).
Therefore, it is likely that nonhardened rye seedlings,
unlike other cereal species under study, display the
functioning of distinct defense systems providing their
constitutive frost tolerance. Among universal stress
protection systems involved in the formation of plant
resistance to stressors of different origin, including
chilling, is the antioxidant system. It is known that
this, along with the enzyme antioxidants, also includes
a number of low molecular weight compounds [5],
including sugars and proline [12–14]. As was already
noted above, in these compounds, the antioxidant
properties are combined with a number of other ones
participating, for example, in protection of cell mem
branes and proteins against damages, important for
plant adaptation to low temperatures [1, 2]. As judged
by the results obtained, nonhardened rye seedlings dif
fered from nonhardened ones of other cereal species
by higher content of these protectors, such as sugars
and especially proline (Fig. 2).
Despite the fact that proline is long considered as
polyfunctional stress induced metabolite, the avail
able data about its role in plant tolerance to hypother
mia are so far limited. Thus, the increase in proline
content in Arabidopsis plants after their cold harden
ing was found, although any relation between the
dynamics of its accumulation and development of the
frost tolerance was not still revealed [27]. Here,
authors consider this response only as a sequence of
the action on the plants of low temperatures rather
than the reason for their tolerance to this stress factor.
In report [9], it was shown that no differences in the
basic content of proline in leaves of both winter and
3
2
1
0
(a)
I
II III
IV
SODactivity
rel.units/(mgproteinmin)
(b)
(c)
CATactivity,
µmolH2O2/(mgproteinmin)
250
200
150
100
50
600
450
300
150
0
Secale cereale Triticum aestivum Triticum durum Hordeum vulgare
GPOactivity,
rel.units/(mgproteinmin)
Fig. 1. Activities of (a) SOD, (b) CAT, and (c) GPO in winter cereal seedlings. (I) control, (II) after hardening at 2°C, (III) after
freezing of nonhardened seedlings at –6°C, (IV) after freezing of hardened seedlings at –6°C.

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RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 62 No. 4 2015
KOLUPAEV et al.
spring soft wheat was observed, but it increased after
cold hardening to the same extent for both these vari
eties. A more significant increase in proline content in
frost tolerant winter wheat variety as compared to that
in the less tolerant one upon long cold hardening these
plants was found [10].
It is possible that increased constitutive proline
content in rye seedlings (Fig. 2b) as compared to that
in other tested cereals is the feature inherent in this
species and related to basic frost tolerance of the latter.
It is not inconceivable that some components of the
plant stress protection systems, such as sugars and
GPO (Figs. 1, 2), also have a distinct contribution to
the tolerance in question. It should be noted that the
activity of key antioxidant enzymes, SOD and CAT, in
nonhardened rye seedlings only slightly differed from
the same parameters in other cereal species. Thus,
after hardening, SOD activity in rye appeared to be
increased, but this response by its intensity did not
exceed those typical for other cereals. In this case,
high content of proline and sugars and increased activ
ity of GPO inherent in rye was combined with its sub
stantial resistance to oxidative damages caused by
freezing. In particular, as follows from Table 2, non
hardened rye seedlings, unlike other cereals, did not
display elevation in MDA content after their freezing.
In this connection, it is believed that the components
of the protection systems, such as proline, sugars, and
possibly peroxidase, have a distinct contribution to the
displaying of rye constitutive frost tolerance. As noted
above, in the literature, there is evidence for functional
replacement of SOD with accumulated sugars [12]
and proline [19] exerting pronounced antioxidant
properties. It is evident that, in order to make a dis
tinct conclusion about the role of proline in the con
stitutive resistance of rye to cryostress induced oxida
tive damages, special investigations on a number of the
varieties of this plant species differing by their toler
ance are required. In addition, the experiments with
the use of the action on the plants in question of other
factors directly resulting in oxidative stress are needed
as well.
In the light of the above phenomena, we need to
note comparative investigations of the functioning of
enzyme and nonenzyme antioxidant (including pro
line) systems of halophytes and glycophytes [28].
Here, the authors believe that high salt tolerance of
halophyte Thellungiella salsuginea may be caused by
its high constitutive proline content, its ability to accu
mulate this amino acid under stress conditions, and
also increased activity of different GPO forms. In con
trast, Plantago major, the plants being lesser salt resis
tant, did not display a high constitutive level of proline
and high GPO activity.
In general, under the conditions used in our exper
iments, hardening of different cereal species induced
all the defense system components under study to a dis
tinct extent. In particular, the hardening induced
increase in SOD activity was observed in seedlings of all
four cereal species, and nonfrost tolerant barley exhib
(a)
I
II
III
IV
Sugarcontent,
mg/gdrywt
(b)
Prolinecontent,
µmol/gdrywt
120
400
Secale cereale Triticum aestivum Triticum durum Hordeum vulgare
300
200
100
0
80
40
0
Fig. 2. Content of (a) sugars and (b) proline in winter cereal seedlings. Symbols as in Fig. 1.

7. RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 62 No. 4 2015
ANTIOXIDANT ENZYME ACTIVITY AND OSMOLYTE CONTENT 505
ited more profound response (Fig. 1a). The activities of
CAT and GPO increased as well (Fig. 1). In addition,
accumulation of sugars and proline under the action of
hardening took place in seedlings of all cereal species,
although this process appeared to be most intensive in
frost tolerant rye and soft wheat (Fig. 2).
After freezing, SOD activity in hardened seedlings
of all cereals under study did not significantly change,
while CAT activity in rye and soft wheat remained
practically unchanged but decreased in durum wheat
and barley. In this case, GPO activity increased in rye
and soft wheat seedlings but did not change in durum
wheat and barley (Fig. 1). In nonhardened rye and soft
wheat seedlings as more frost tolerant plants, SOD
activity did not undergo any changes after their freez
ing, whereas decline in this activity was observed in
durum wheat and barley. It is not excluded that this
may be associated with oxidative damages that are pos
sible as a result of the action of different origin stres
sors, including chilling [29]. Significant elevation in
MDA, the product of lipid peroxidation in barley and
durum wheat seedlings after their freezing, argues in
favor of this suggestion (Table 2).
On the other hand, in nonhardened seedlings of all
cereals species after their freezing, the increase in PO
activity was observed. However, the results of our
experiments do not allow us to answer the question
when namely this occurred, during freezing or after
thawing, because thawed plants were used for the anal
ysis. In this connection, it should be pointed out that
the phenomenon of PO activity elevation after freez
ing was revealed earlier on other plant objects [12].
Sugar content after freezing both in nonhardened
and hardened seedlings of all four cereal species was
not significantly changed. In the case of nonhardened
seedlings of all cereals under study, proline content did
not change either. At the same time, after freezing of
hardened seedlings, distinct differences in proline
content changes were revealed. Proline content
increased in resistant species, rye and soft wheat,
whereas it did not change nonresistant ones, durum
wheat and barley (Fig. 2). We cannot exclude the pos
sibility that, in the case of the frost tolerant species,
unlike the nontolerant ones, activity of the enzymes
involved in the synthesis of proline increased due to
influence of negative temperatures or immediately
after thawing the samples. It is probable that a similar
mechanism of the elevation in the activity of the bio
synthetic enzymes in question was induced only in the
case of preliminary hardened cereal seedlings. There is
evidence of a distinct relation between the activity of
the key enzyme in the biosynthesis of proline, Δ1 pyr
roline 5 carboxylatesynthase, and plant resistance to
stressors. Thus, in the tobacco lines transformed with
genes of this enzyme from Vigna aconifolia and Arabi
dopsis thaliana, excess accumulation of proline and
enhancement in their frost tolerance were revealed
[30].
The results of the present work, taken together,
allow us to conclude that the functioning of the stress
protective systems in the winter cereals differing by
their frost tolerance is characterized by distinct differ
ences, including qualitative ones. In particular, a con
stitutive frost tolerance of rye revealed without harden
ing may be explained by both high basic content of low
molecular weight protectors, sugars and, especially
proline, and increased activity of GPO, one of the anti
oxidant enzymes. In addition, nonhardened rye seed
lings exhibited a distinct tolerance to freezing induced
oxidative damages as well, which was expressed in the
absence of the increase in the content of MDA, prod
uct of lipid peroxidation, the effect typical, however,
for other cereal species. Sugars and proline likely play a
distinct role in development of the frost tolerance of
soft wheat seedlings as well, although the increase in
their content required for achieving a proper level of
such a tolerance occurs under the action of hardening
temperatures. Changes in the activity of antioxidant
enzymes (SOD, CAT, and GPO) contribute to a vary
ing extent to development of hardening induced frost
tolerance of all four winter cereal species under study.
An expression of high enough activity of these enzymes
in response to freezing appeared to be typical for frost
tolerant rye and soft wheat.
Another important feature of frost tolerant cereal
species is their ability to accumulate proline under the
action of negative temperatures. It is possible that pro
line exhibits the properties of molecular shaperone
[14] and, thereby, is involved in the maintenance of the
activity of antioxidant enzymes under the conditions
of cryostress. In addition, the freezing induced pro
line accumulation typical only for hardened rye and
soft wheat seedlings can be considered as an additional
polyfunctional defense response triggered already at
the stage of damage action of cryostress. In this con
nection, it is remarkable that the observed increase in
the amount of MDA under cryostress was less signifi
cant namely in the cereal species (rye and soft wheat)
accumulating additional quantity of proline under the
action of negative temperatures as compared to that in
barley and durum wheat, which did not display any
increase in the content of proline under these condi
tions (Fig. 2, Table 2).
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Translated by I. Andreev

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