A Database for Triticeae and Avena
SIBERIAN INSTITUTE OF PLANT PHYSIOLOGY AND BIOCHEMISTRY
Siberian Division of the Russian Academy of Sciences, Lermontov
str., 132, Irkutsk-33, P.O Box 1243, Russian Federation, 664033.
A.K. Glyanko, N.V. Mironova, and G.G. Vasilieva.
Introduction. Frost during the period of ripening and
maturing of spring wheat grain is a typical natural phenomenon
in many parts of the world (Russian Federation, Scandinavian countries,
Canada, and the USA). The short-term impact of negative temperature
on plants at this time (particularly during milky ripeness) is
a decrease in the technological and sowing qualities of the grain
resulting in the formation of so-called 'frost-beaten grain' with
low germinating capacity, low test weight, and low flour production
(Lamb 1967; Razumovsky and Zabotina 1969; Reimers and Illi 1974).
Wheat from such grain have a low initial intensity of growth and
morphological distortions (Pavlov 1967; Illi et al. 1979). Such
properties of the grain after an early autumn frost are apparently
conditioned by the negative influence of cold on the physiological-biochemical
processes that are responsible for grain ripening and maturing.
Among these processes, those of nitrous substances efflux from
vegetative parts of the plants to reproductive organs in the period
of kernel formation are very important. The present work was aimed
at determining the degree of impact of early autumn frost at milky
ripeness on the nitrogen efflux from leaves, stems, and spike
(chaff) towards the kernel.
Materials and Methods. The spring wheat Skala was grown
in a climatic chamber with the temperature regime of 19 ±
1/1 5 ± 1 C (day/night). Illumination was by incandescent
lamps DRL-700 with a light intensity of 14 ± 0.5 kl and
a 16-hour light period. Infrared radiation of the lamps was precluded
by a water screen. Sandy soil with a low content of total nitrogen
(0.009 %) was used for plant growth. Macroelements from Gelrigel's
mixture and microelements as per Hogland (Grodzinsky and Grodzinsky
1973) were introduced in the enameled vessels with soil (tank
capacity was 9 kg of dry soil). Soil moisture was maintained at
70 % of complete soil moisture capacity (SMC). Ten days prior
to termination of the experiment, soil moisture was reduced to
45-50 % SMC. Each vessel contained five plants at the end of the
The plants were subjected to an artificial frost with the intensity
of -6.5 ± 0.5 C at milky ripeness stage (moisture 55-60
%). The frost was simulated at night, in the dark. All temperature
regulating operations in the freezer were performed automatically
in compliance with the program, which included a gradual reduction
of temperature from 15 to 0 C (1 C/12 min). Further reduction
down to a minimum negative temperature of -6.5 ± 0.5 C
was at 1 C/22 min (Kurets 1974). After 1.5-h exposure at -6.5
± 0.5 C, the temperature was increased to 0°C and then
to 15 C with a speed 1 C/12 min. Total duration of negative temperature
impact on the plants was 6 hours. The plants did not show any
Control plants were placed in the chamber with optimal growth
temperature. One and one-half hours after the termination of extreme
temperature factor impact on the test plants, both sets of plants
were fed via roots with 15N-labeled Ca(NO3)2 at a dose of 100
mg of nitrogen/vessel. Nitrogen enrichment was 95.05 atomic %
15N. Further growth of both control and test plants was conducted
at the temperature conditions preceding the frost.
Samples for analysis were selected after 7 days (beginning
at waxy ripeness phase, grain moisture 38-40 %) and 19 days (end
of waxy ripeness phase, grain moisture 17-19 %). The plants were
divided into leaves, stems, shaft, and grain. After drying at
105°C, the samples were reduced to a powder. Plant material
samples were burned in concentrated sulfuric acid with the catalyst
(selenium). Nitrogen was determined per K'eldal's micromethod
(Ermakov et al. 1987). The samples were enriched with heavy-isotope
nitrogen on an MI-1305 mass-spectrometer (Optical-mechanical Plant,
Sumy, Ukraine). The content of marked nitrogen in the plants samples
was calculated according to the formula of isotope dilution (Korenkov
1977). The test was repeated six times. The results are presented
as average arithmetic means with the standard error identified.
Reliability of differences was assessed by Student criterion.
Results and Discussion. Depending on the state in which
plants overcome the impact of negative temperature, supercooling
or ice formation in the cells, the degree of damage will vary
(Drozdov et al. 1977). Our test wheat plants went through the
artificial frost apparently in the state of supercooling, because
they showed no visible damage. The absolute nitrogen content in
wheat kernel of the control constantly increased until the end
of plant growth (Table 1). In the variant with frost, we observed
a tendency towards reduction of nitrogen content in the kernel,
from the end of the waxy ripeness phase through complete kernel
ripeness. Relative nitrogen content in the kernel (% of dry mass)
increased in both test variants by the end of waxy ripeness phase
and was reduced at complete kernel ripeness. However, this reduction
was statistically reliable only in the variant with frost (P >
Table 1. Change in the nitrogen content (mg/plant)
in different organs of spring wheat plants under the impact of
early autumn frost. * numerator = mg/plant; denominator = % from
dry weight of grain.
With ripening and maturing of kernels, the total nitrogen content
in leaves, stems, and shaft was reduced (Table 1). Nevertheless,
in the period from the end of waxy ripeness until complete kernel
ripeness, the frost variant demonstrate an increase in excessive
15N % and marked nitrogen content in stems and shaft. In the leaves,
analogous changes in these parameters were statistically unreliable.
The rise of marked nitrogen content in the vegetative organs apparently
was related to the absorption of nitrogen introduced in the soil
The highest nitrogen content in the kernel from the stems by
complete kernel ripeness was observed in both control (62 %) and
treated (65 %) plants (Table 2). The leaves had 13 % (control)
and 19 % (treated) and the shaft 25 % (control) and 16 % (treated)
total stored nitrogen. Similar results characterized the period
from the beginning to end of waxy ripeness. We concluded that
during both interphase periods, frost resulted in a reliable reduction
of nitrogen efflux from shaft to kernel (P > 0.99). We also
observed a reliable (P > 0.95) increase in nitrogen efflux
from the leaves during the period from beginning to end of waxy
ripeness. Nevertheless, these changes were unreliable for the
beginning of the waxy ripeness phase.
Table 2. Change in total and marked nitrogen
efflux from vegetative organs of spring wheat under the impact
early autumn frost. Numerator = mg/plant; denominator = % of total
Further data analysis showed that an increase in nitrogen content
in the kernel of the plants subjected to frost by complete ripeness
amounted to 24.7 ± 1.6 mg/plant (range 22.4 ± 0.9-47.1
± 1.3 mg/plant), including marked nitrogen 5.0 ±
0.3 mg/plant (range 3.3 ± 0.1-8.3 ± 0.3 mg/plant)
(Table 1). Consequently, an increase in total and marked nitrogen
content in the kernel exceeded the amount of nitrogen arriving
from the vegetative organs by 24 % (P > 0.95) and 57 % (P >
0,99 for total and marked nitrogen, respectively (the decrease
in total and marked nitrogen from vegetative organs amounted to
18.8 ± 1.0 and 2.2 ± 0.2 mg/plant, respectively).
In other words, plants subjected to frost used both nitrogen accumulated
in the vegetative organs and nitrogen from soil solution for kernel
formation, a considerable part of which was marked nitrogen.
The control plants showed similar regularities but used soil
nitrogen to lesser extent. Thus, the total nitrogen increase in
the kernel exceeded its efflux from vegetative organs by 16 %
(the difference is unreliable) and marked nitrogen increased to
51 % (P > 0.99).
If we calculate the value of parameters considered in relation
to the period beginning to the end of waxy ripeness, we find that
total nitrogen inflow into the kernel will exceed its efflux from
the vegetative organs by 38 % (11.0 ± 2.1 mg/plant; P >
0.99) in the frost variant and 15 % (2.7 ± 18 mg/plant;
the difference is unreliable) in control plants. Consequently,
between the beginning and end of waxy ripeness, nitrogen from
the soil also was used for kernel formation.
In the plants subjected to frost, use of total nitrogen from
soil for kernel formation from the end of waxy ripeness to complete
ripeness declined from 38 % (11.0 ± 2.1 mg/plant) to 24
% (5.9 ± 1.9 mg/plant) (the difference is unreliable) and
that of marked nitrogen increased from 44 (1.9 ± 0.1 mg/plant)
to 57 % (2.8 ± 0.2 mg/plant) (P > 0.95). We conclude
that frost stimulated the use of marked nitrogen for kernel formation.
However, frost did not produce a reliable impact on the total
nitrogen content in the kernel at maturity (Table 1). We note
a reduction in the relative content of total nitrogen in the kernel
from the end of waxy ripeness to complete ripeness (Table 1).
Nitrogen loss from the kernel at maturity has been described (Kumakov
1987; Korovin 1984). Korovin (1984) indicated that this phenomenon
may be accounted for by increased moisture and low environmental
temperature. The concrete physiological-biochemical mechanism
of this fact is unknown.
Our tests on early autumn frosts produced a significant impact
on the efflux of total and marked nitrogen from the vegetative
organs to the kernel. Total nitrogen efflux from the leaves increased
and from the shaft was reduced. We observed an increased of marked
nitrogen efflux from the stems between the beginning and end of
the waxy ripeness stage. Stimulating or hampering physiological
functions after stress may be connected with the ability of the
plant and depend on the character and intensity of the stress
(Drozdov at al. 1977; Al'tergot 1981). For the spike rachis and
the closeness of the nitrogen source used by maturing kernels,
then a reduction in nitrogen after a frost could impact protein
synthesis in the kernel. Earlier investigations showed that an
early autumn frost causes changes in the proportion of vital reserve
proteins of spring wheat kernels (glutenin and gliadin) (Glyanko
and Trufanov 1999). Similar changes may affect baking qualities
of wheat grain.
R.K. Salyaev, L.V. Dudareva, S.V. Lankevich, V.M. Sumtsova,
and E.G. Rudikovcka.
During the last two decades, low-intensity laser radiation
in the visible and near-infrared ranges is finding an increasing
application in biological studies including biotechnological research
(Grishko et al. 1999). However, up to now, the data are scarce
on the mechanism of the effect of laser light on biological subjects
because of difficulties encountered in analyzing light energy
transformations in cells and the complexity of the response of
a complex living system, multilevel in its organization, to laser
effects. At present, we are actively pursued the effect of low-intensity
laser radiation on animal and human tissues (Skobelkin 1997).
At the same time, we emphasize that plants are evolutionary more
adapted to the perception and assimilation of light energy, because
they include a multitude of light-sensitive compounds, such as
phytochromes and photosynthetic pigments. In our opinion, plants
are particularly interesting for investigating the mechanisms
of laser radiation.
Previously, we showed that low-intensity laser radiation stimulates
morphogenetic processes in tissue cultures of wheat and wild grasses,
such as rhizogenesis and the formation of morphogenic calli and
regenerated plants (Salyaev 2001a and b). However, laser light
can be regarded not only as a stimulant, but also as a stress
agent damaging cells and tissues, most notably at the initial
stages of radiation (Rogatkin and Chernyi 1999).
Generation of reactive oxygen species and an enhancement in
lipid peroxidation processes are known to be the primary responses
to many stress agents (Baraboi 1991; Smirnoff 1993; Kurganova
et al. 1997; Kuznetsov 2000). In this connection, establishing
whether or not the low-intensity laser radiation can induce lipid
peroxidation in plant tissues was important. An analysis of accumulation
of the primary and secondary products of lipid peroxidation can
be regarded as one of possible lines of approaching such problem.
Therefore, the objective of this work was to investigate the effect
of low-intensity laser radiation on the process of accumulation
of secondary lipid peroxidation products in wheat tissue culture.
The wheat cultivar Skala (bred in Siberia) tissue culture was
used in this work. A mature embryo with half of endosperm was
used as an explant. Callus formation was induced on a modified
Murashige-Skoog medium complemented with 2 % sucrose and 2 mg/l
2,4-D. The calli were irradiated on the 6th day of the first subculture.
Calli irradiation was performed during 5 min using a LG-79
helium-neon laser with a radiation wavelength of 632.8 nm and
an intensity of 10 mW at the sample level. Irradiation intensity
was measured using a LM-2 device (Carl Zeiss, Germany). The calli
were irradiated directly in glass test tubes (10 mm in diameter)
used for their culturing, and light-intensity losses in passing
through the glass did not exceed 12 %. Before and after irradiation,
the calli were grown in the dark. In each independent experiment,
100 calli were used; half of them served as a control, and the
other half was treated by the helium-neon laser.
The content of the compounds under investigation in the calli
was determined immediately following radiation and also after
48 h. The lipid peroxidation level was assessed spectrophotometrically
(Kurganova et al. 1997) as an amount of lipid peroxidation products
that react with thiobarbituric acid (TBA-reactive products). To
this end, 0.2 g of callus tissue was ground in a homogenizer with
9 ml of heptane:isopropanol (1:1, v/v) mixture. The homogenate
thus obtained was supplemented with 0.1 ml of saturated TBA solution
and centrifuged at 4000 rev/min for 10 min. The supernatant diluted
with distilled water was shaken; after phase separation, the upper
heptane fraction was collected. This fraction was supplemented
with ethanol (1:5, v/v), and the optical density of the mixture
at 532 nm was determined using an SF-46 spectrophotometer (LOMO,
Russia). In all cases, the amount of secondary lipid peroxidation
products in the samples was calculated from the spectrophotometric
readings using molar extinction coefficient. Means from three
experiments (each including nine biological replications) and
their standard errors are shown in the figure. Each biological
replication included from four to seven calli.
The changes in the accumulation of secondary lipid peroxidation
products in wheat tissue culture induced by low-intensity laser
radiation are sufficiently clear-cut (Figure 1). TBA-reactive product content in
the tissue increased from 0.73 nmol/g fr wt in the control to
1.17 nmol /g fr. wt immediately after irradiation. Within 2 days
after irradiation, this difference somewhat decreased, but nevertheless
remained quite significant: TBA-reactive product contents in the
control and irradiated samples were 1.02 and 1.12 nmol/g fr. wt,
respectively. Thus, after 48 h, the content of TBA-reactive products
in control samples somewhat increased as compared to that observed
immediately after irradiation. Judging from the published evidence
(Smirnoff 1993), this can be related to a cyclic stress-response
pattern in the wheat tissue culture caused by subculturing to
a new culture medium. In this case, the phases of these cycles
in the control and laser-irradiated samples most likely do not
coincide with each other.
The data suggest that low-intensity laser radiation can induce
lipid peroxidation in wheat tissue culture. We showed that the
initial response of a plant tissue to irradiation involved an
increase in the content of secondary lipid peroxidation products
(see Figure 1).
Moreover, we showed that, as an aftereffect, laser light stimulated
the morphogenetic processes in plant tissues (Salyaev et al. 2001a
and b). In our opinion, this stimulation could result from the
metabolic changes induced by the alterations in the contents of
compounds, such as lipid peroxidation products, formed in the
primary photoreactions. The changes in cells produced by the accumulation
of these compounds could serve as a signal triggering not only
respective defense mechanisms, but also some secondary responses
including those on the transcription level (Karu 2001). This suggestion
is indirectly supported by stimulation of the morphogenetic processes
in the tissue cultures of wheat and wild grasses under the effect
of low-intensity coherent radiation (Salyaev et al. 2001a and
Thus, we suggest that a general cell response induced by laser-light
irradiation can be divided into two specific responses, which
do not coincide in time. The first one consists of a rapid stress
effect resulting in an increase in the amount of lipid peroxidation
products, and the second and longer one, are the secondary reactions
related to the adaptive metabolic changes and apparently accompanied
by the stimulation of morphogenetic processes.
A.K. Glyanko, O.A. Pochabova, N.V. Mironova, and G.G. Vasilieva.
Nitrate reductase (NR) is an enzyme well-known to plant biologists,
which acts as a limiting link in the chain of nitrate reduction
to ammonium and catalyzes the first stage of nitrate reduction
to nitrite. Most of the studies of NR physiological and biochemical
properties were published in the 1960s-80s. In particular, plant
physiologists revealed the connection between NR activity and
cultural plants productivity, protein synthesis in crops grain,
as well as intensity of nitrogen assimilation by the plants (Deckard
et al. 1973; Rautou et al. 1977; Peshkova and Khavkin 1980). Pekker
and TOkarev (1984) suggest use of NR activity for diagnosing plants
nitrogen nutrition. In the last few years, interest in NR has
been fostered by the discovery of NR ability to reduce nitrite
to nitric oxide (NO), a molecule that plays an important role
in the vital processes of animals and plants (Yamasaki and Sakihama
2000; Desikan et al. 2002; Garcia-Mota and Lamathina 2003). This
article sums up the results of tests on determination of NR activity
during the vegetation period of spring wheat (Skala) depending
on the dose of nitrogen fertilizer. The work was targeted at finding
the dependence between NR activity and the degree of nitrate nitrogen
availability for the plants.
Materials and Methods. The tests were performed in vegetation
vessels with the capacity of 4 kg of dry soil with low content
of total nitrogen (0.009 %). The mixture with nitrate source of
nitrogen was used as a nutritious mineral mixture (Thomas et al.
1979). The content of mineral nitrogen introduced in the vessels
varied from 0 to 800 mg / vessel. Soil moisture in the vessels
was maintained at the level of 65-70 % of total moisture capacity.
10 plants grown in the vegetation house from June to August were
left in each vessel.
NR activity was determined in anaerobic medium according to
the method described in Mulder et al. (1959) using disks (0.05-0.1
mm) cut out from the leaves. The samples (0.3 g of fresh tissue)
were placed in retorts containing the following: 0.1 M phosphate
buffer with pH 7.8 (5 ml ); neutralized 0.1 M solution of malic
acid (1 ml ); 5 % solution KNO3 (1 ml ) and 3 ml of distilled
water. The retorts were placed in the vacuum-desiccator, with
air pumped out, and kept for 1 h at 27 C. Enzyme reaction was
terminated by adding 1 ml of 10 % acetic acid and 2 ml of saturated
ammonium sulfate solution. In the control sample the enzyme reaction
was terminated prior to incubation of the reaction mixture. NR
activity was determined on the basis of the quantity of nitrites
formed as a result of enzyme reaction; their content was determined
in the filtrate with 0.6 % sulfanilamide in HCI and 0.6 % solution
of N-(l- naphtyl) ethylenediamine hydrochloride. The color intensity
was measured on the photocolorimeter (FEK-56M) at wave length
of 546 nm. The samples for determination of enzyme activity were
taken during the phases of two, three, four leaves, to come into
ear and blooming. The upper growing and lower green leaves were
subjected to analysis. The test was repeated five times; the analytical
repetition is three times. Data are presented as mean ±
Results and Discussion. Table 3 shows that NR activity
on the medium without nitrogen introduction is minimal during
all the phases of plants development as compared to the variants
with nitrogen introduced. The highest enzyme activity in this
option was observed in the phase of two leaves, then with the
plants growth and development it gradually came down, particularly
in the lower leaf. Thus, at anthesis, NR activity in the upper
leaf reduced by 2.6 times, in the lower leaf, by eight times compared
to two-leaf phase.
Table 3. Nitrate reductase activity in spring
wheat leaves in different phases of plant development depending
on the level of nitrogen nutrition 9 nmol NO-2/g (fresh weight)/1
h). Note top values are for the upper leaf; bottom values are
for the lower leaf.
In the options with nitrogen introduction regardless of the
dose, enzyme activity in the upper and lower leaves either did
not differ, or was slightly higher in the lower leaf than in the
upper leaf. Starting from the three-leaf phase, NR activity in
the variants with nitrogen dose 40 and 100 mg/vessel gradually
decreased, and more so in the lower leaf. In the variant with
nitrogen dose 200 mg/vessel the enzyme activity in the upper leaf
remained approximately at the same level up to the blooming phase,
whereas in the lower leaf it decreased. In the variants with high
nitrogen content (400 and 800 mg/vessel), NR activity by the blooming
phase in the upper leaf increased and in the lower leaf did not
change significantly. Thus, the lowest NR activity is characteristic
of the variant without nitrogen fertilizer introduction, and in
the variants with low nitrogen content, NR activity by the blooming
phase decreased significantly both in upper and lower leaves.
A nitrogen dose of 200 mg/vessel is a transient value between
the variants with insufficient level of nitrogen in the medium
and its high level. In the plants of this variant, NR activity
in the upper leaf did not decrease significantly by the blooming
phase, though it fell remarkably in the lower leaf. Apparently,
in this case regulation of nitrogen nutrition by the plant is
directed primarily at providing growing organs with mineral nitrogen,
which causes the flow of newly absorbed nitrates to go to the
growing leaf ensuring fairly high enzyme activity.
In the upper and lower leaves at low nitrogen doses (0, 40,
and 100 mg) there were observed identical regularities of NR activity
change: by the blooming phase NR activity in the upper leaf decreased
in accordance to the doses by 2.6; 3.8, and 3.2 times, and in
the lower leaf by 8.0; 7.1, and 5.4 times. In the variant with
nitrogen dose of 200 mg activity, only insignificantly decreased
in the upper leaf by the blooming phase (approximately by 1.2
times), in the lower leaf the enzyme activity decreased by 2.3
times. At high nitrogen availability (400 and 800 mg/vessel),
NR activity was, as a rule, high in both upper and lower leaves.
The lack of nitrogen results in a decrease of NR activity first
in the lower leaf, whereas in the upper leaf, enzyme activity
remains higher. This effect is well seen particularly in the variant
with nitrogen dose of 200 mg/vessel. Attention should be paid
to the intensification of NR activity during spike formation in
the variants with high nitrogen availability for the plants, which,
apparently, may be accounted for by intense nitrates absorption
by the plants during this vegetation period, as formation of reproduction
organ creates a powerful attracting center and new growth points,
which require inflow of nitrous substances for synthetic processes.
Sufficient nitrogen availability allows the plant to maintain
NR activity at a high level both in the upper and lower leaves.
This, apparently, leads to prolonging of the lower leaves life
via activation of nitrogen assimilation processes, and, consequently,
to a slowdown of the plant ageing process and a prolonging of
the wheat plant's vegetation period.
Thus, the acquired data speak in favor of the connection between
NR activity in the leaves and the level of mineral nitrogen availability
for the plants. These results are of interest for elaboration
and specification of methods of diagnostics of grain-crops nitrogen
nutrition using NR activity. In particular, according to our data
in the course of diagnostics of nitrogen availability for spring
wheat by NR activity, it is efficient to determine enzyme activity
not only in the upper growing leaf, but also in the lower green
leaf, which has completed growth, for the sake of earlier identification
of soil nitrogen identification.
S.V. Osipova, A.V. Permyakov, and T.N. Mitrofanova, and T.N.
Pshenichnikova, M.F. Ermakova, and
A.K. Chistyakova (Institute of Genetics and Cytology, Siberian
Russian Academy of Sciences, Lavrentyev Ave. 10, Novosibirsk).
The technological quality of wheat flour is known to be largely
dependent on highly-molecular subunits of glutenin containing
the biggest amount of SH-groups, capable of forming S-S links
( Payne 1987). Dependence of gluten quality on the content of
disulfide links was experimentally proven by Kretovich et al.
(1978), Vakar et al. (1972 ), Bloksma (1975), and Kaczkowski et
al. (1980). In our laboratory, we extracted the enzyme with thiol:proteindisulfide
oxidoreductase (EC 22.214.171.124) activity from wheat corn (Trufanov
et al. 1999). The enzyme catalyzes dissociation of protein disulfides
using restored glutathione as a cofactor. Our model experiment
has shown that wheat disulfidreductase reduces aggregation ability
of gluten proteins, which is apparently caused by dissociation
of proteins SS-links and consequent weakening of gluten matrix
rigidity (Osipova et al. 2004). The investigation was focused
on the impact of disulfidreductase preparation introduction in
the course of dough kneading on certain technological characteristics
of flour and dough.
Materials and Methods. Disulfidreductase was extracted
and purified from flour of Siberian selection wheat, Tulunskaya
12 variety as per the earlier described methods (Trufanov et al.
1999). In the tests there were used partially purified disulfidreductase
preparation containing admixture of glutathionreductase (N1) and
preparation of disulfidreductase with enzyme purity, that is not
containing admixtures of other thiol-disulfide metabolism (N2)
enzymes. Fifty mg of enzyme preparation containing approximately
3-5 units of disulfidreductase activity and NaCl were added to
50 g of flour of cultivar Mironovskaya 808. A similar portion
(50 mg) of NaCl was added to control. Technical parameters were
analyzed in compliance with the methods of the State Variety Testing
of Agricultures (Methods 1988). The physical properties of dough
were studied using Chopin alveograph, which was used to determine
specific work for dough deformation (strength of flour, W; units
of alveograph, u.a.), tenacity (P, mm) and extensibility (L, mm),
and P/L ratio. Table 4 presents average data of three independent
tests ± standard error.
Results and Discussion. The addition of enzymes only
insignificantly increases dough tenacity (P) (Table 4). Dough
extensibility (L) values increase remarkably, N2 preparation addition
increases extensibility by 7 %, whereas the addition of partially
purified preparation (N1) by approximately 17 %. High efficiency
of N1 preparation may be accounted for by the fact that presence
of glutathionreductase ensures fairly high amount of restored
glutation required for catalytic activity of disulfidreductase.
Flour strength reliably increases with the addition of enzyme
preparations. P/L proportion, however, practically does not change,
so on the whole gluten quality of the Mironovskaya 808 wheat does
not improve. Nevertheless, significant, up to 17 %, increase of
dough extensibility is of interest. These data allow us to infer
that partially cleaned disulfidreductase preparation containing
glutathionreductase activity may be used to improve qualities
of excessively strong, short-tearing gluten.
Table 4. Alveograph parameters with enzyme
additives introduction (Significant at * P > 0.05; ** P >
A.V. Permyakov, T.N. Mitrofanova, and S.V. Osipova.
Glutathione reductase (GR) catalyzes the NADPH-dependent reduction
of oxidized glutathione (Foyer et al. 1997). The primary function
of glutathione reductase is to maintain a high GSH/GSSG ratio
in cells, which is crucial for a variety of cellular functions,
including the biosynthesis of DNA (Noctor et al. 1998). This flavoprotein
oxidoreductase has a central role in maintaining GSH within the
cellular environment, particularly during stress. Most, if not
all, stresses include an oxidative stress component (Prasad et
al. 1994; Wise 1995) that leads to tissue damage if antioxidative
defenses are insufficient.
Wheat leaves contain two charge/mass-separable isoforms of
glutathione reductase, one chloroplastic and the other probably
cytosolic (De Lamotte et al. 2000). The endosperm of durum mature
kernels contained a single form of glutathione reductase; it appeared
about the 18th day after anthesis, whereas another isoform, present
at the early stages of grain development, disappeared between
the 20th and 30th days after flowering (Lascano et al. 2001).
Materials and Methods. Grains of the wheat cultivar
Tulunskaya-12 were used in this research. The soluble, enzymatically
active protein fraction of the wheat grains was extracted with
a 0.1 Tris buffer, pH 7.5, containing 2 mM EDTA, from standard
ground flour in the proportion 1:2 (weight:volume). The extract
was subjected to chromatography on DEAE-Sephadex A-50. The protein
fraction was eluted in a 0-0.6 M NaCl gradient. Fractions obtained
were separated according in 7 % PAAG to the method of Davis (1964)
at the basic pH. Molecular forms of glutathione reductase were
identified immediately on gel slabs by specific coloring (Ye et
al. 1997). Bromophenol-blue was used as the marker for estimating
Rf values, indicating the mobility of enzyme bands relative to
the mobility of the bromophenol-blue front.
Results and Discussion. Selective enzyme coloring after
native electrophoresis in the PAAG slabs manifested its presence
in all the protein fractions acquired through ion-exchange chromatography
of DEAE-sephadex A-50 (Figure
2), revealing that the enzyme is represented by two molecular
forms with different relative electrophoretic mobility (Rf) 0.36
and 0.32 (GR 1 and GR 2, respectively), which split during proteins
desorption from anions by a linear gradient of NaCL concentration.
The initial enzyme extract contained both enzyme isoforms (Figure 2, lane 1).
During ion-exchange chromatography, the first to eluate from the
sorbent was glutathione reductase molecular form (GR 2) with Rf
0.32 (Figure 2,
lanes 25-32), then GR 1, with Rf 0.36 (Figure 2, lanes 33 and 34). Thus, unlike the
grain of complete maturity T. turgidum subsp. durum
(Lascano et al. 2001), T. aestivum subsp. aestivum
grains contain two isoforms of glutathione reductase that may
be split with the help of ion-exchange chromatography.
O.I. Grabelnych, N.Yu. Pivovarova, T.P. Pobezhimova, A.V. Kolesnichenko,
O.N. Sumina, and V.K. Voinikov.
Oxidative phosphorylation is the main ATP source in aerobic
organisms. In certain conditions, the uncoupling of oxidative
phosphorylation (substrate oxidation without phosphorylation)
occurs. Fatty acids are natural uncouplers of oxidative phosphorylation.
Different mechanisms through which fatty acids cause uncoupling
are known (Jezek 1999; Skulachev 1999; Wieckowski et al. 2000;
Kadenbach 2003). Recent studies indicates that fatty acids are
also involved in cell death pathways (Penzo et al. 2002). Fatty
acid uncoupling is inhibited by addition of bovine serum albumin
(BSA), purine nucleotides, and by ATP/ADP antiporter inhibitor,
carboxyatractyloside (CAT) (Skulachev 1991).
As shown previously, unsaturated free fatty acids in winter
wheat mitochondria plays not only a role of uncouplers (oleic,
petrozelinic, linoleic and erucic acids), but also could be the
only oxidation substrate for them (linoleic acid) (Grabelnych
et al. 2004). Although unsaturated fatty acids have more effect
on the mitochondrial membrane potential, the role of saturated
fatty acid in energetic cell metabolism is significant too (Pastore
et al. 2000; Penzo et al. 2002). Lauric and palmitic acids have
caused more significant y decrease rate in durum wheat mitochondria
among the saturated acids (Pastore et al. 2000). Fatty acid-dependent
uncoupling of oxidative phosphorylation plays an adaptive role
during hypothermia and oxidative stress in the plant mitochondria
(Casolo et al. 2000; Pastore et al. 2000).
The aim of the present investigation is to study the influence
of saturated fatty acid on the winter wheat mitochondria function
and determine the ADP/ATP participation in fatty acid-dependent
Materials and Methods. Three-day-old etiolated shoots
of winter wheat cultivar Zalarinka germinated on moist paper at
26 C, were used in this work. Mitochondria were extracted from
winter wheat shoots by differential centrifugation as describes
previously (Pobezhimova et al. 2001). Isolated mitochondria were
resuspended in the following medium: 40 mM MOPS-KOH buffer (pH
7.4), 300 mM sucrose, 10 mM KCl, 5 mM EDTA, and 1 mM MgCl2.
Mitochondrial activity was recorded polarographically at 27
C using a platinum electrode of a closed type in a 1.4 ml volume
cell (Estabrook 1967). The reaction mixture contained 125 mM KCl,
18 mM KH2PO4, 1 mM MgCl2, and 5 mM EDTA, pH 7.4. 10 mM Malate
in the presence of 10 mM glutamate was used as an oxidation substrate.
Lauric (C 12:0), palmitic (C 16:0), stearic (C 18:0), and behenic
(C 22:0) acids were used in concentrations from 0,056 mkM to 10
mM. 1 mkM carboxyatractyloside (Catr) was used as ADP/ATP inhibitor.
Polarograms were used to calculate the rates of phosphorylative
respiration (state 3), nonphosphorylative respiration (state 4),
the rate of respiration after fatty acid addition, the rate of
respiration after Catr addition, respiration control by Chance-Williams,
and the ADP:O ratio (Estabrook 1967). The concentrations of mitochondrial
protein and CSP 310 were analyzed by Lowry method (Lowry et al.
1951). All the experiments were made in three-six preparations.
The data obtained were analyzed statistically, i.e., arithmetic
means and standard errors were determined.
Results and Discussion. To determine saturated fatty
acid uncoupling effect we added different amounts of fatty acid
to state 4 mitochondria, because fatty acid-dependent increase
of state 4 oxygen consumption is one of the main indicators of
fatty acid-dependent uncoupling together with coefficient RC and
ADP:O ratio decrease.
We found that an addition to state-4 mitochondria physiological
concentrations (0.056 and 0.15 mkM) of lauric and palmitic acids
didn't cause any changes in the rate of oxygen consumption but
addition physiological concentrations of stearic and behenic acids
caused even a decrease of oxygen consumption (Figure 3). Behenic acid did not cause any
changes in the rate of oxygen consumption at higher concentrations
added. The most influence of lauric acid on the rate of oxygen
consumption the was found at concentration 50 mkM, which caused
about 54 % stimulation of non-phosphorylative respiration (Figure 3). Palmitic
acid also has the most uncoupling activity at concentration 50
mkM, which cause twofold increase of non-phosphorylative respiration.
The uncoupling effect of stearic acid become apparent only at
high concentrations and didn't exceed 30 % of stimulation of state
4 respiration at concentration 500 mkM (Figure 3). Recoupled effect of Catr (about
39 % decrease of non-phosphorylation respiration rate) in winter
wheat mitochondria shows participation of ADP/ATP-antiporter in
this process. The uncoupling effect of palmitic and stearic acid
was fully associated with participation of ADP/ATP-antiporter.
The uncoupling effect of lauric acid also was partially associated
with participation of ADP/ATP-antiporter.
The other picture of respiration stimulation in fatty acid-treated
mitochondria after ADP addition was observed. In second cycle
of phosphorylation the uncoupling action of all studied saturated
fatty acids, including behenic acid, was found. Figure 4 shows the data about fatty acid influence
on the oxygen consumption at concentrations which cause maximal
uncoupling in the second cycle of phosphorylation. Fatty acids
can be divided into two groups: 1) lauric and palmitic acid (Figure 4A and 4B)
that cause immediate stimulation of state-4 respiration and the
same or smaller stimulation of state-4 respiration in second cycle
of phosphorylation; 2) stearic and behenic acids (Fig\ure 4C and 4D), that did not cause stimulation
of state-4 respiration immediately but cause stimulation of state-4
respiration in second cycle of phosphorylation.
An addition of 50 mkM lauric acid caused the increase in state-4
4A, 3), the subsequent addition of ADP caused the transfer
of mitochondria to phosphorylative state (Figure 4A, 4) and then to nonphosphorylative
state (Figure 4A,
5), whose value was equal to respiration rate in first cycle
of phosphorylation after addition lauric acid. Palmitic acid also
caused the increase in state-4 respiration (Figure 4B, 3) and subsequent addition of ADP
accompanied transfer to state 3 (Figure 4B, 4), but its value
was smaller than in first cycle of phosphorylation. State-4 respiration
was higher (Figure
4B, 5) as compared with state-4 respiration before palmitic
acid addition, but smaller then state-4 respiration after palmitic
acid addition in first cycle of phosphorylation (Figure 4B, 2).
Although 5 mkM stearic acid addition in state 4 did not cause
statistically significant stimulation of respiration (Figure 4C, 3), we observed 58 % increase of
non-phosphorylative rate of respiration in second cycle of phosphorylation
(Figure 4C, 4).
Likewise the influence of stearic acid, behenic acid addition
in state 4 didn't cause stimulation of respiration (Figure 4D, 2), but we observed 56 % increase
of non-phosphorylative rate in the second cycle of phosphorylation
(Figure 4D, 4).
In all cases the increase of non-phosphorylative respiration rate
in second cycle of phosphorylation was accompanied by decrease
of RC coefficient and ADP:O ratio (Table 5). RC coefficient decrease
was 26-49 % and ADP:O ration decrease was 27-33 %. So, all saturated
fatty acids studied cause the uncoupling of oxidative phosphorylation
in second cycle phosphorylation; the increase of non-phosphorylation
respiration and decrease of RC coefficient and ADP:O ratio.
Table 5. The decrease of RC coefficient and
ADP:O ratio in winter wheat mitochondria in presence of fatty
acids during second cycle of phosphorylation. 100 % is RC coefficient
or ADP:O ratio of mitochondria at first cycle of phosphorylation
before fatty acid addition.
The stimulation of respiration by lauric acid was fully associated
with ADP/ATP-antiporter participation (Figure 4A, 6). At the same time, the stimulation
of respiration by palmitic, stearic and behenic acids decreased
after addition of Catr only about 50 % (Figure 4B, C, D, 6). Therefore, we conclude
that different carrier proteins participate in these fatty acid-dependent
All saturated fatty acids studied were able to cause the increase
of winter wheat mitochondria respiration. Lauric, palmitic, and
stearic acids caused oxidative phosphorylation uncoupling immediately,
at the same time the uncoupling effect of behenic acid started
after subsequent ADP stimulation. In our experiments with T.
aestuvum subsp. aestivum, it is shown that palmitic
acid have the most uncoupling effect, whereas Pastore with coauthors
(2000) found using T. turgidum subsp. durum mitochondria
that lauric acid have the most uncoupling effect. ADP/ATP-antiporter
participated in fatty acid-dependent uncoupling the all studied
Acknowledgments. The work has been performed, in part,
with the support of the Russian Science Support Foundation, Russian
Foundation of Basic Research (projects 03-04-48151 and 05-04-97231)
and Siberian Division of Russian Academy of Sciences Youth Grant
I.V. Stupnikova, G.B. Borovskii, A.I. Antipina, and V.K. Voinikov.
Cold stress adaptation has been established to induce dehydrin
synthesis and accumulation (Close 1996). These proteins belonging
to the family of the late embryogenesis proteins are characterized
by the presence of highly conserved Y, S, and K segments. A peculiarity
of dehydrin amino-acid composition provides for their functions.
Amphiphilic-helices found in dehydrins are considered to bind
to intracellular molecules, especially membranes and proteins,
mainly by hydrophobic interactions (Close 1997). These associations
may protect the functions of membranes and proteins by preventing
coagulation during environmental stress and maintain water in
dehydrated cells (Hoekstra et al. 2001). A correlation was observed
between the endogenous concentration of dehydrins and frost tolerance
of plant species, organs, or tissues (Sarhan et al. 1997). The
patterns of dehydrin accumulation in trees coincided with seasonal
fluctuations in their frost resistance (Wisniewski et al. 1996).
In tissues of leaves (deciduous species), flower buds, blueberry
bark, peach, black current, poplar, willow, and acacia, the accumulation
of dehydrin-like proteins was reported in autumn and winter. However,
the seasonal changes in the composition and content of these proteins
in wintering grasses have not been studied. The presence of dehydrins
in the nucleus, cytoplasm, cytoskeletal elements, and plasma membrane
of various cells in seedlings and adult plants was demonstrated
by immunolocalization and subcellular fractionation (Houde et
al. 1995). Earlier, we detected two dehydrin-like proteins, which
accumulated in mitochondria of the freezing-resistant wheat seedlings
during cold hardening under laboratory conditions (Borovskii et
al. 2000). Taking into account all facts above the objective of
the study was to follow possible seasonal fluctuations in amount
and spectra of dehydrins in winter wheat crowns being the vitally
important winter wheat part, which determines winter survival.
Materials and Methods. Experiments were performed on
field-grown winter wheat plants cultivar Irkutskaya ozimaya hardened
under natural conditions. Mitochondria were isolated by differential
centrifugation, as was described in (Borovskii et al. 2000). The
crowns were ground with a mortar and pestle in the medium containing
0.3 M sucrose, 40 mM Mops (Sigma, United States), 2 mM EDTA, 10
mM KCl, 1 mM MgCl2, 0.05 % polyvinylpyrrolidon, 0.5 % cysteine,
and 0.5 % BSA (ICN, United States), pH 7.5. All other procedures
were performed similarly as during mitochondria isolation from
seedlings. Mitochondria from the crowns were purified using a
discontinuous Percoll gradient comprising 21 % and 35 % Percoll.
Purified mitochondria were used for protein isolation. SDS-PAGE
was run in 11 % acrylamide gel in the modified Laemmli system
(Laemmli 1970). Immunodetection was performed by the method of
Timmons and Dunbar (1990). Polyclonal antibody against K fragment
of dehydrins was kindly presented by T.J. Close (University of
Results and Discussion. All our previous experiments
concerning the detection of mitochondrial dehydrins were carried
out with seedlings. Therefore, elucidating whether or not similar
accumulation of dehydrins occurred in mitochondria of overwintering
adult plants was important. Mitochondria were isolated from winter
wheat crowns during autumnal hardening, in winter, and during
spring deadaptation. The mitochondria from crowns contained proteins
with molecular weights being similar to proteins from seedlings,
63 and 52 kD (Figure
5). Any other proteins related to dehydrins were not detected.
The content of these proteins increased substantially during autumnal
hardening. Peshkova et al. (1998) showed that the crowns of this
winter wheat cultivar lost up to 67-69 % of water during hardening.
The highest content of mitochondrial dehydrins was in December,
and thereafter it began to decline gradually. In spring, the content
of dehydrins in mitochondria from wheat crowns decreased to their
level in autumn. In April, dehydrin content was similar to that
in September; in May, it was even lower (Figure 5). We know that winter wheat cryotolerance
increases during cold acclimation starting from early autumn and
achieves its highest value by the beginning of winter, indicating
the interrelation between dehydrin content and winter wheat freezing
tolerance, what is in agreement with the results of other researchers
(Sarhan et al. 1997). Earlier we showed that in contrast to mitochondrial
dehydrins, the spectra of those in total cell water-soluble fraction
contained much more protein groups. In the total water-soluble
protein fraction, both the amount of dehydrins in each group and
the number of dehydrin groups of a particular molecular weight
increased with autumnal hardening and attained the highest values
by the end of January. At this time, dehydrins with molecular
weights of 209, 196, 66, 50, 41, 24, 22, 17, 15, and 12 kD were
detected among soluble proteins (Stupnikova et al. 2004). Two
protein groups (66 and 50 kD) had molecular weights close to those
of mitochondrial dehydrins (63 and 52 kD). The methods used did
not permit us to decide whether these proteins with close molecular
weights were similar or different proteins.
Adaptation process is known to include two stages: unspecific
stress-response and specific adaptation (Kuznetsov et al. 1987).
During the first stage, mobilization and development of plant
defensive systems occur, which provide for organism short-term
surviving. During the second stage, specific adaptation mechanisms
develop, which are responsible for plant life under the conditions
of long-term stress (Kuznetsov et al. 1987). The accumulation
of these dehydrins during cold adaptation of field-grown plants
and the absence of their further changes in autumn and winter
indicate evidently that these proteins operate like cryoprotectors
during both stages of adaptation.
A.A. Truhin, E.L. Tauson, and G.B. Borovskii.
The different stress effectors are able to create similar damages
in cell: the increased viscidity of membranes, formation of gel
phases, degradation, and destruction of proteins, phospholypids
and other macromolecules. An identical phenomenon was shown during
treatment of cells by superoxide (Vanlerberghe et al. 1992). Accumulation
of active oxygen and its derivatives also have been shown in plants
in response to various environmental stresses. Oxidizing stress
can be considered as secondary answer to damage of cell organelles
and activation of redox-enzyme system in the cell (Vanlerberghe
et al. 1997). Recent reports show the existence of various pathways
of this process leading to similar results - decreasing of transmembrane
potential on inner membrane of mitochondria and reduction of mitochondrial
reactive oxygen species (ROS) generation. Protein participation
in this type of regulation depends on plant species, stage of
the development, type of stress, and some other factors. Alternative
oxidase (AOX) and plant uncoupling mitochondrial proteins (PUMP)
seems to play a significant role in ROS production regulation
processes. These proteins decrease transmembrane potential in
different ways. Activation of the AOX pathway leads to uncoupling
of oxidation and phosphorylation in three of four respiratory
complexes. On the other hand, PUMP (like other members of UCP
(uncoupling protein) family) does not block phosphorylation, but
stimulates the spreading of potential via transport of fatty acids
ions (Umbach 1993). The correlation of these two ways in decreasing
of transmembrane potential is of great interest. We note that
AOX and PUMP can work as antagonists because fatty acids that
necessary for PUMP functioning depresses AOX activity (Jezek et
al. 2000). The aim of the present study was to demonstrate the
amounts of AOX and PUMP expression patterns in winter wheat under
different stressing conditions using multiplex RT-PCR method.
Materials and methods. After growing for 3 days, wheat
seedlings were subjected to environmental stress treatments. Low
temperature heat and dehydration treatments were conducted by
transferring of etiolated seedlings to separate conditions: 4
or 39 C, 30 % H2O2 solution for 1.5 h, polyethylene glycol (PEG
MW8000) solution treatment for 3 h, and dehydration in air for
24 and 48 h. Samples were immediately frozen in liquid nitrogen
and stored at -70 C until processed for RNA extraction. Transcript
levels of aox and pump was also controlled under unstressed conditions.
Primers were designed from published sequence data of genes
encoding AOX, PUMP (EMBL database) and RDF (Monstein et al. 2002)
to amplify 415-bp, 505-bp, and 890-bp fragments of corresponding
transcripts from T. aestivum cv. Irkutskaya ozimaya. Using
a SV Total Isolation System (Promega, Madison, WI), 200 ng of
total RNA was isolated and used for the first-strand cDNA synthesis
with REVERTA kit (Amplisense, Moscow) and 3'-primer. The amount
of total RNA in reactions was identical in experiments which results
we compared. The transcript levels of the constitutively expressed
rdf gene were also monitored on corresponding gels to confirm
that amount of RNA in compared experiments was equal.
Results and Discussion. The early events of plant adaptation
to environmental stresses are perception and subsequent stress-signal
transduction that lead to the activation of various physiological
and metabolic responses, including the development of oxidation
stress. Therefore, they can cause induction of aox and pump genes
expression since AOX and PUMP have been shown efficiently reduce
the generation of mitochondrial reactive oxygen species (Kowaltowski
et al. 1998; Umbach 1993).
The expression patterns of aox and pump were analyzed under
various stresses. Expression of aox and pump were not included
under high temperature treatment (Figure 6) or short-time cold stress (Figure 7), but aox expression was induced
after 24 hour of low temperature treatment whereas pump gene expression
was not affected (Figure
7). Expression of aox was induced after 48 h of dehydration
The strongest and fastest accumulation of aox and pump mRNAs within
1,5 h was observed when wheat seedlings were subjected to high
oxidative stress with hydrogen peroxide (Figure 9).
These results demonstrate that expression of pump was induced
in winter wheat seedlings mainly by strong oxidative stress. Previously,
Maia et al. (1998) reported that cold-inducible AtPUMP protein
may play a role in heat-requiring physiological events in Arabidopsis.
Although, two cDNAs encoding UCP-like proteins have been isolated
from potato (StUCP) (Laloi et al. 1997). Because the expression
of StUCP was detected mainly in flowers and fruits, it has been
hypothesized that StUCP can be associated with burst of respiration
in flowering and fruit ripening in combination with AOX (Laloi
et al. 1997). The cold treatment at 4 C for 12 h of skunk cabbage
plants (Symplocarpus foetidus) was enough for expression of two
UCP-like genes SfUCP and SfUCP encoding uncoupling proteins (Ito
Only powerful oxidative stress with hydrogen peroxide could
induce strong and simultaneous expression of aox and pump genes
in winter wheat seedlings (Figure
9). We propose from our results that aox induction most likely
and mainly occurs as part of the acclimation process and is necessary
for adaptation to stress conditions. aox, therefore, is a gene
of adaptation. This possibility is supported by other investigators
(Djajanegara et al. 2002). On the other hand, it may be that two
energy-dissipating systems, both PUMP and AOX are involved in
the regulation of the oxidative stress development in extreme
situation of oxidative burst as it was in our experiment with
hydrogen peroxide treatment.
Among proteins related to cold acclimation and cold stress
tolerance, the dehydrin family presents peculiar interest. This
family, known as a group-2 late embryogenesis abundant (LEA) proteins,
is one of the ubiquitous water-stress-responsive proteins in plants
(Close 1997). These proteins are hydrophilic, thermostable and
glycine-rich and possess unique repeated sequences that are believed
to form putative amphiphilic a-helices. The putative multiple
functions of dehydrins have been described from the viewpoint
of deduced secondary structures. They were hypothesized to function
by stabilizing large-scale hydrophobic interactions such as membrane
structures or hydrophobic patches of proteins. Highly-conserved
polar regions of dehydrins have been suggested to hydrogen-bond
with polar regions of macromolecules, acting essentially as a
surfactant, to prevent coagulation under conditions of cellular
dehydration or low temperatures (Ismail et al. 1999). Immunolocalization
and subcellular fractionation results have showed that members
of the dehydrin family are present in the nucleus, cytoplasm,
and plasma membrane (Danyluk et al. 1998). We have found that
two dehydrin-like proteins (dlps) accumulate in mitochondria of
cereals in response to cold (Borovskii et al. 2000). We have also
demonstrated a positive correlation between accumulation of the
two mitochondrial dlps and cold-tolerance of the species studied.
The aim of the study was to elucidate whether dehydrins penetrate
mitochondria or they associate with the surface of the organelles.
Materials and Methods. Experiments were performed on
etiolated winter wheat seedlings of the cultivar Irkutskaya ozimaya.
Seedlings were grown on moistened filter paper at 22 C for 4 days.
Mitochondria were isolated by differential centrifugation, as
was described in (Welin et al. 1994). Mitochondria from wheat
shoots were purified using a discontinuous Percoll gradient (De
Virville et al. 1994). This gradient comprised 18, 23, and 35
% Percoll in the medium containing 0.3 mM sucrose, 40 mM Mops-KOH,
pH 7.4, and 0.1 % BSA. Mitochondria were collected from the interface
between 23 and 35 % Percoll. Purified mitochondria were used for
protein isolation or they were treated with pronase E (1 mg/ml)
at 37 C for 1 h in order to determine proteins localized at the
outer membrane. Purified mitochondria were placed in the sample
buffer, and proteins were extracted at 60 C for 15 min. Similar
procedure was applied to the proteins from membrane fraction.
SDS-PAGE was run in 10-12 % acrylamide gel in the modified Laemmli
system (Laemmli 1970). Immunodetection was performed by the method
of Timmons and Dunbar (1990). Polyclonal antibody against K fragment
of dehydrins was kindly presented by T.J. Close (University of
Results and Discussion. Earlier we found that dehydrins
are able to associate with mitochondria very rapidly, as it was
detected during seedling freezing (Borovskii et al. 2002). We
supposed that these proteins did not penetrate inside the mitochondria
but associated with their outer membrane. To verify this assumption,
we used seedlings acclimated to 4°C for a week. Such a term
for acclimation was chosen to permit dehydrins to be transported
into mitochondria if such a transport could take place in the
cell. In order to elucidate dehydrin localization, we treated
isolated and purified mitochondria with pronase E. The results
obtained permitted us to conclude that, during seedling cold acclimation,
two dehydrins accumulated in mitochondria being localized at their
outer membrane because they were accessible for protease (Figure 10). Earlier,
outer localization of other protective proteins, maize low-molecular-weight
heat-shock proteins, was detected in mitochondria in the test
with protease (Borovskii and Voiniko 1993).
Dehydrins evidently fulfill a defensive function in the mitochondria.
Understanding the mechanism of this defense is difficult, because
it is unknown whether membrane lipids or proteins are the targets
for dehydrin action. A dehydrin WCOR410 is known to associate
with the plasma membrane. This dehydrin is supposed to protect
this very frost-sensitive structure (Danyluk et al. 1998). Thomashow
(1999) believes that dehydrin can suppress lipid phase transition
from lamellar to hexogonal state, which implies its interaction
with membrane lipids. Hara et al. (2003), relying on their data,
suggested that dehydrin facilitates plant cold acclimation by
acting as a radical-scavenging protein to protect membrane systems
under cold stress. Such type of protection is especially important
for mitochondria because reactive oxygen species arise therein
inevitably during respiration. Mitochondria convert about 2 %
of consumed oxygen into hydrogen peroxide, which could damage
membranes (Scandalios et al. 1997). On the other hand, dehydrin
protective effects on proteins are not less significant. Dehydrin
protection of some enzymes against denaturing was demonstrated
in experiments in vitro (Wisniewski et al. 1999).
Thus, our results showed that the mitochondrial dehydrins are
peripheral proteins and associated with the surface of the outer
mitochondrial membrane. We suppose that dehydrins interact with
membrane lipids and their stabilizing effect during cell dehydration
is exerted via the retardation of lipid layer phase transition
or due to reduction of lipid peroxidation. It seems likely that
dehydrin association with mitochondria is an important mechanism
of cold stress resistance.
Primary sites of cold injury in plants are cell membrane systems
(Thomashow 1999). Therefore, protecting membrane structures is
one of the important parts of efficient cold adaptation and tolerance
mechanisms, in which cold-regulated (COR) proteins play significant
role, functioning as cryoprotectors or regulators. Among COR-proteins,
presumably protecting cell membranes the dehydrin family presents
particular interest. Danyluk et al. (1998) found acidic dehydrins
WCOR 410, a subtype of the dehydrin family, which are largely
hydrophilic and have high content of charged residues and hydroxylated
residues. WCOR 410 accumulates in the vicinity of the plasma membrane
of cells in the sensitive vascular transition area where freezing-induced
dehydration is likely to be more severe (Danyluk et al. 1998).
Hara et al. (2003) showed that a dehydrin protein, purified from
E. coli expressing citrus dehydrin cDNA prevented peroxidation
of soybean liposomes in vitro. They suggested that the dehydrin
facilitates plant cold acclimation by acting as a radical-scavenging
protein to protect membrane systems under cold stress (Hara et
al. 2003). We also found total cell dehydrin-like proteins with
molecular weights of 209, 196, 169, 66, 50, and 41 kD, which are
characteristic for hardening state of winter wheat plants (Stupnikova
et al. 2000; Borovskii et al. 2002a), and mitochondrial proteins
ranging from 52 to 63 kD depending on the species of cereals,
which accumulated during cold adaptation (Borovskii et al. 2002b).
Our next task was to elucidate whether and which dehydrins associate
with other (nonmitochondrial) cell membranes at low temperature.
Materials and Methods. Experiments were performed on etiolated
winter wheat seedlings of the cultivar Irkutskaya ozimaya. Seedlings
were grown on moistened filter paper at 22 C for 4 days. For hardening,
3-day-old seedlings grown under optimal conditions were exposed
to low temperature for 7 days at 4 C. Mitochondria were isolated
by differential centrifugation, as was described in (Welin et
al. 1994). Mitochondria from wheat shoots were purified using
a discontinuous Percoll gradient (De Virville et al. 1994). Purified
mitochondria were used for membrane protein isolation. Membranes
were sedimented by ultracentrifugation at 105,000 g for 90 min.
Membrane proteins were extracted with sample buffer at 50-60 C
for 20 min and separated in 12 % SDS-PAAG. Proteins were transferred
to the nitrocellulose membrane and detected with antibody against
dehydrins. Immunodetection was performed by the method of Timmons
and Dunbar (1990). A polyclonal antibody against the K fragment
of dehydrin was kindly provided by T.J. Close (University of California,
Results and Discussion. To study dehydrins associated with
other (nonmitochondrial) cell membranes at low temperature, we
isolated the fractions of such cell membranes as tonoplast and
plasma membrane from wheat seedlings exposed to low temperature
for 7 days and from seedlings grown at 22 C (control). Among wheat
membrane proteins, we detected four dehydrins with molecular weights
of 209, 196, 66, and 50 kD (Figure
11). Dehydrin-like proteins with similar molecular weights
were found earlier among total cell water-soluble dehydrins, isolated
from winter wheat seedlings and crowns (Borovskii et al. 2002a;
b). The main part of these proteins has membrane localization.
Both total cell and membrane dehydrins accumulated during cold
11), what taking together with their hydrophilic and heat-stable
nature allows suggesting that they protect and stabilize membranes
structures of wheat cells during cold stress. We also believe
that a limited number of dehydrin types could exert stabilization
of diverse membrane structures, which could be injured by cold.
Other dehydrin-like proteins (Borovskii et al. 2002a) revealed
in total cell fraction possibly are water-soluble proteins protecting
possibly cold-sensitive cell proteins. Two membrane dehydrin groups
(66 and 50 kD) had molecular weights close to those of mitochondrial
dehydrins (63 and 52 kD). The methods used did not permit us to
decide whether these proteins with close molecular weights were
similar or different proteins. Therefore, so far we can not answer
the question whether dehydrins associated with mitochondria can
be detected in other cell compartments. On the basis of the data
we may conclude that dehydrins with molecular weights 209, 196,
66, and 50 kD could interact with lipids of cell membrane structures
during cold treatment and stabilize them preventing lipid phase
E.V. Berezovskaya, V.A. Trufanov, T.N. Mitrofanova, and L.S.
Wheat reserve proteins gliadin and glutenin are the key components
of gluten, which are responsible for rheological and baking qualities
of wheat flour. Glutenins belong to the class of polymeric proteins,
whose individual polypeptides are known as high- and low-molecular
weight subunits (HMW- and LMW- glutenin subunits). Subunits of
these two groups differ in amino-acid composition, molecular mass
(from 23 to 68 kDa for LMW-glutenin subunits and from 77 to 160
kDa for HMW-glutenin subunits), and structure (Kasarda 1999).
HMW-glutenin subunit genes are located on the long arms of chromosomes
1A, 1B, and 1D, loci Glu-A1, Glu-B1, and Glu-D1,
respectively (Payne 1987). LMW-glutenin subunit genes are on the
short arms of chromosomes 1AS, 1BS, and 1DS (Singh et al. 1988).
Many researchers have shown that allele variations of HMW- and
LMW-glutenin subunits are associated with differences in technological
parameters of flour and dough (Payne 1987; Autran et al. 1987;
Gupta et al. 1989). Both LMW- and HMW-glutenin subunits, whose
number is normally three-four times that of LMW-glutenin subunits,
make a significant impact on viscose-elastic properties of dough
and other technological parameters of flour (Pogna et al. 1990;
Metakovsky et al. 1997). Variations in the composition of these
subunits among different genotypes are in fact the major factor,
which determines differences in baking qualities of dough and
This work summarizes the results of complex investigation of
grain reserve protein, glutenin, in connection with productivity
and quality of gluten of two unrelated cultivars of soft spring
wheat and four hybrid F9 and F10 from their interbreeding, establishment
of the impact of various alleles coding glutenins, and the results
of quantitative determination of HMW- and LMW-glutenin subunits
and their correlation and role in determination of rheological
Materials and Methods. The objects of investigation
were the varieties of soft spring wheat Rollo (R) and Drott (D)
and four constant hybrid forms from their interbreeding: RxD-I,
RxD-II, RxD-III, and RxD-IV in F9 and F10 generations. The spring
wheat cultivar Drott (Fylgia II/Sviöf 0990) has high resistance
to fungal diseases, it is a highly productive variety with fairly
strong straw. The Early Tyumenskaya cultivar was selected by mass
selection method from the cultivar Rollo (K-45657, Norway).
For glutenin extraction, we used a Tris-HCl buffer, pH 6.8
containing sodium dodecylsulphate and 10 % b-mercaptoethanol following
complete gliadin extraction by 70 % ethanol. Restoration of S-S-bonds
was conducted for 15 hours at 40 C. SDS-PAAG electrophoresis was
performed according to Laemmli (1970), overnight, at constant
current strength and stable voltage. Concentrating and separating
gels (3 and 10 %, respectively), containing acrylamide, methylenbisachrilamide,
Tris-HCl (pH 6,8), TEMED, ammonium persulphate, and 10 % SDS.
The gels were scanned and density-measured. A standard protein
mixture was used to determine molecular mass (Mr) of glutenin
subunits. HMW-glutenin subunits were identified according to Payne
et al. (1983). Aggregation ability of gluten proteins, as one
of the major characteristics of gluten rheological properties,
was determined by the modified method of Arakawa and Yonezawa
(1975), which is widely used for classifying cultivars by gluten
quality. The tests were conducted directly with flour and gluten
washed in 1 % NaCl solution and in distilled water. Washed gluten
was extracted by 0.01 M acetic acid for 16-20 h at 4 C with constant
agitation. The extracts then were centrifuged at 7,000 g for 15
min. Protein concentration was determined by spectrophotometric
Calcar's method and calculated as per the following formula:
1,45 D280 - 0,74 D260 = C mg/ml,
where 0.74 and 1.45 = recalculation ratios,
C = protein concentration in the solution,
D280 and D260 = optical solution density.
1,45 D280 - 0,74 D260 = C mg/ml,
where 0.74 and 1.45 = recalculation ratios,
C = protein concentration in the solution,
D280 and D260 = optical solution density.
Aggregating ability was determined by the change of absorption
at turbidity increase, for 10 min with the interval of 30 sec,
protein solution + 0.2 M phosphate buffer, pH 5.65, (Arakawa and
Yonezawa 1975) or pH 9.5
Trufanov 1994) containing 2 M NaCl. The constant of aggregation
speed K was calculated at the first stages of aggregation by the
turbidity change using the following equations:
K = r/C4, r = t3/3t, where
K = constant of the initial aggregation stage;
t = solution turbidity index;
t = time in sec;
C = protein concentration, %; and
t10/C = aggregation constant after 10 min of processing, calculated
per unit of protein concentration in the solution.
K = r/C4, r = t3/3t, where
K = constant of the initial aggregation stage;
t = solution turbidity index;
t = time in sec;
C = protein concentration, %; and
t10/C = aggregation constant after 10 min of processing, calculated
per unit of protein concentration in the solution.
Result and Discussion. Electrophoresis of proteins from
standard cultivars (Hope, Bezostaya (unawned) spring, Novosibirskaya
67, and Chinese Spring) allowed the identification of high-molecular-weight
subunits according to Payne (1983). The genes encoding glutenin
HMW-glutenin subunits are distributed on the long chromosome arms
1A, 1B, and 1D. Three loci, Glu-A1, Glu-B1, and
Glu-D1, jointly make up a locus i and encode HMW subunits.
Two subunits of loci Glu-B1 and Glu-D1 have two
subgroups x and y, subunits differing in mobility and amino-acid
composition, including cystein content. Standard cultivars with
identified subunits (Table 6) allowed us to establish the position
of each subunit via comparison of densitograms (Figure 12) and Rf in the course of electrophoresis
in SDS-PAAG. We also used alternative identification system using
molecular masses (Ng and Bushuk 1989) (Table 7).
Table 6. Genotype of high-molecular-weight
glutenin subunits of some standard wheat cultivars.
Table 7. Molecular weight of the glutenin
subunits of some standard wheat cultivars.
The data on the composition and molecular weight of functional
glutenin subunits and density-measuring scanning of electrophoretic
spectra of the standard wheat cultivars show that there were identified
hypothetic genotypic formulas for HMW-glutenin subunits of the
cultivars Rollo and Drott and their four hybrids. We demonstrated
redistribution of inherited information between hybrids (Table
Table 8. Allelic variation in glutenin subunits
in the cultivars Rollo and Drott and their hybrids.
All the four hybrids produced by crossing of two parental cultivars
Rollo and Drott have significant differences in their HMW-glutenin
subunits. Thus, the I hybrid completely correspond to the Drott
HMW-subunits, but manifests differences in the realm of LMW-glutenin
12). In the other three hybrids, parental HMW-glutenin subunits
were redistributed: the 'Rolo/Drott' hybrid II inherited from
Rollo subunits 2+12 of locus Glu-D1 and nonexpressing subunit
of null-locus Glu-A1, and from Drott subunits 6+8 of locus
Glu-B1; 'Rolo/Drott' hybrid III is closer by component
composition to Drott , but is different from it by the single
subunit 7, typical of Rollo and belonging to locus Glu-B1;
'Rolo/Drott' hybrid IV is closer to Rollo, differing by one subunit
(1) encoded by locus Glu-A1 (Table 8).
Subunit 1 is associated with high baking quality in contrast
to the null allele. This subunit might possess a unique primary
structure, which participates in the formation of stable gluten
aggregates. Subunits 5+10 also correlate with good baking quality,
in contrast to subunits 2+12. Differences in amino-acid sequence
and secondary structure between allele subunits 5+10 and 2+12
also were revealed (Anderson 1996). We identified genotypic formulas
that contain both 'good', 5+10 and 1 (in Drott), and 'bad' subunits,
2+12 (in Rollo).
Apparently, Drott is likely to produce higher dough quality
than Rollo, which is confirmed by aggregation and technological
qualities parameters. 'Rolo/Drott' hybrids I and III might also
yield good parameters, as their spectrum contains HMW-glutenin
subunits 5+10 obtained from Drott. We assumed that 'Rolo/Drott'
hybrid IVwill manifest poor baking qualities, because it inherited
subunits 2+12 from both parents, which do not have good parameters
of technological properties.
High-molecular-weight subunits represent only 10 % of all the
gluten proteins, but their role in determining baking quality
in accordance with different subunit types is immense. Another
important factor in determination and more precise definition
of various technological parameters is the second group of polymeric
proteins LMW-glutenin subunits. HMW- and LMW-glutenin subunits
differ in the values of amino-acid composition, molecular weight
(from 23 to 68 kDa) and structure (Kasarda 1999).
The quantity of LMW-glutenin subunits exceeds that of the HMW-glutenin
subunits by 3-4 times (Kasarda 1999). Their characterization is
complicated, because they are controlled by a large number of
genes and their subunits are often insoluble after restoration
of intermolecular disulfide bonds.
To investigate their participation in the characterization
of the cultivars, we determined their relative quantity and calculated
the proportion between the quantities of LMW- and HMW-glutenin
subunits. According to Hou et al. (1996), this proportion in flour
proteins may act as an important parameter in the evaluation of
rheological and baking qualities of wheat.
Quantitative determination of the LMW- and HMW glutenin subunits
was conducted with the help of density-measuring analysis DDS-PAAG
of electrophoretogram (Figure
13). Quantitative content of HMW- and LMW-glutenin subunits
was calculated with the help of software; the results are presented
in relative units (Table 9). The data demonstrate the fact that
LMW-glutenin/HMW-glutenin subunit proportion is significantly
higher in Rollo (2.95) compared to Drott and lower in their hybrids
(3.0-3.23, Table 9). This value has a positive correlation with
the data on the flour sedimentation analysis: The higher the LMW-glutenin/HMW-glutenin
subunit index, the higher the sedimentation parameters. Rollo
has a much higher index (2.95) than that of Drott, with a high
sedimentation index (42 ml). So, we see a tight correlation of
LMW-glutenin/HMW-glutenin subunit proportion and parameters of
sedimentation flour analysis. Hybrids do not manifest significant
differences in these parameters (Table 10).
Table 9. Percentage of high-molecular-weight
(HMW) and low-molecular-weight (LMW) glutenin subunits in the
cultivars Rollo and Drott and their hybrids.
Table 10. Technological properties and aggregation
parameters of Rollo and Drott and four 'Rollo/Drott' hybrids.
Data is from 2001; t10/C - aggregation constant at the 10th minute
of the process.
Comparative investigation of aggregating ability of the vinegar-soluble
fraction of flour reserve proteins of hybrids in the F9 and F10
with various types of electrophoretic spectra showed that among
the biotypes under study, the ratio of initial aggregation stage
(K) ranges between 4.84-13.6. Genotypes, whose electrophoretic
spectrum contains glutenin subunits 6+8 (Drott and hybrids I and
II), regardless of the presence of other subunits, had aggregation
ratio 12.63, 8.91, and 13.6, respectively; with the final aggregation
stage index (t10/C) equaling 26.11, 28.68, and 34.82, respectively.
Thus, we performed a complex investigation of the grain-reserve
protein glutenin. We identified correlations between the composition
of HMW-glutenin subunits, the proportion of HMW- and LMW-glutenin
subunits, and a number of technological properties. HMW-glutenin
subunit 1 was shown to be associated with high baking quality,
in contrast to the null allele. Subunits 5+10 also correlate with
high baking qualities in contrast to subunits 2+12.
E.V. Berezovskaya, V.A. Trufanov, T.N. Mitrofanova, and T.A. Pshenichnikova.
Native wheat glutenin is a complex set of biochemically diverse
polypeptides connected by different forces of protein-protein
interactions. The interest in identifying peculiarities in the
genetically determined composition of functional glutenin subfractions
is because of their immediate participation in the formation of
a gluten-protein complex that is responsible for baking properties
in flour. High-molecular-weight glutenin subunits of the majority
of industrial wheat cultivars of many countries have been well-studied.
The participation of these structural elements in the formation
of compositionally complicated, multicomponent protein-gluten
complex responsible for dough and bread quality are known.
Glutenin is known to consist of at least 15-17 subunits with
different molecular mass, amino-acid composition, and primary
and secondary structure are associated into a single permolecular
protein complex via intermolecular disulfide bonds stabilizing
a three-dimensional structural gluten matrix. The number of these
bonds is a genotypically determined trait of a genetically specific
character that determines gluten physical properties and dough
rheological characteristics (Trufanov 1994).
To find favorable alleles, we were interested in the impact
of chromosomes of homoeologous groups 1 and 6, which control storage
protein synthesis, in wheat lines with intervarietal substitutions.
These lines have contrasting baking qualities based on the quantitative
content of HMW-glutenin subunits present.
Materials and Methods. Soft wheat lines with intervarietal
substitutions of chromosomes 1A, 1B, 1D, 6A, 6B, and 6D of the
cultivar Novosibirskaya 67 (N67), a strong wheat, and the high-protein
recipient cultivar Diamant 1 (Dm) with low technological quality
were used (Obukhova et al. 1997; Maystrenko et al. 1993).
Glutenin subfractions (GN) were obtained from freshly ground
flour after removal of albumins, globulins, and gliadins by successive
extraction with 0.05 M acetic acid (subfraction GN-1), 4 M urea
(subfraction GN-2), and 4 M urea in the presence of 2-mercaptoethanol
(subfraction GN-3) (Trufanov 1994). Protein fractions were dialyzed
against 0.01 M acetic acid and lyophilized. Subunits composition
in the subfractions GN-1, GN-2, and GN-3 was studied after SDS
electrophoresis in 9 % polyacrylamide gels according to Laemmli
(1970). Evaluation of quantitative content of PAGE zones in glutenin
subfractions of each substituted line fraction was by densitometric
analysis of electrophoretograms (Figure 13). Quantitative content
of five HMW-glutenin subunits was calculated with the help of
a computer program. The results were expressed in units and in
% relative to the recipient cultivar Dm (Figure 14).
Result and Discussion. The donor cultivar N67 exceeds
the recipient Dm in relative content of subunits 2 and 3 in the
easily soluble GN-1 fraction (by 10-12 %); in sparingly soluble
GN-2 fraction, subunits 4 and 5 by 13 and 45 %, respectively;
and in the insoluble (without restoration of SS bonds) GN-3 fraction,
subunits 1 and 2 by 25 and 10 %, respectively) (Figure 14). Simultaneously, the donor N67
is inferior to the recipient Dm in terms of GN-1 content of subunits
1 and 4 by 11 and 20 %, respectively; in GN-2, subunits 1 and
3 by 17 and 7 %, respectively; and in GN-3, subunits 3, 4, and
5 by 5, 20, and 36 %, respectively.
Obukhova et al. (1997) and Maystrenko et al. (1993) showed
that in Dm and N67, subunits 1 and 2 are controlled by genes on
chromosomes 1A, 1B, and 1D, whereas subunit 3, which contains
three components, is controlled by genes on chromosomes 1B and
1D. One component of subunits 3 belongs to N67 and is controlled
by genes on chromosome 1B; the second belongs to Dm and also is
controlled by genes on 1B; and the third component, common for
both cultivars, is controlled by genes on chromosome 1D. Several
authors (Obukhova et al. 1997; Maystrenko et al. 1993; Payne 1979,
1997) assume that chromosomes of different homoeologous groups
participate in genetic control of compositionally complicated
subunits 4 and 5 (Figure
Compared to Dm, subunit 1 prevails in the subfraction GN-1
in N67-Dm DS1A DS6A, and DS6B; in subfraction GN-2 in Dm-N67 DS1B;
and in subfraction GN-3 in Dm-N67 DS1B, DS1D, DS6A, and DS6B.
Subunit 2 prevails in Dm-N67 DS6A in the GN-1 subfraction, in
all substituted lines of the GN-2 subfraction, and in Dm-N67 DS1A,
DS1B, and DS6A in subfraction GN-3. Subunit 3 content was higher
only in Dm-N67 DS6A in subfraction GN-1 and in Dm-N67 DS6B and
Dm-N67 DS6D. Subunit 4 content was higher in all the lines of
subfractions GN-1 and GN-3. Subunit 5 prevailed only in the subfraction
GN-1 in substituted lines with chromosomes 1D and 6A and was slightly
higher in Dm-N67 DS1A in subfraction GN-3.
The positive influence of the subunit content on glutenin in
the recipient cultivar Dm was to varying degrees caused by chromosomes
from the donor cultivar N67. A considerable substitution effect
was evident in the case of chromosome 6A. Substitution of this
chromosome resulted in intensifying synthesis of all the subunits
in soluble subfraction GN-1, two in the sparingly soluble subfraction
GN-2, and two in the insoluble fraction GN-3. Extraction of GN-3
subunits is possible only after complete restoration of inter-
and intramolecular SS links that stabilize the gluten structural
matrix. Simultaneously, the content of individual subunits in
these substitution lines was lower than in the recipient, particularly
that of subunits 3, 4, and 5.
Differences in solubility of subfractions GN-1, GN-2, and GN-3
are known to be associated with the density of their spacial structure,
which determines the ability of these proteins to form permolecular
protein associates, characteristic of gluten (Trufanov 1994).
Formation of protein glutenin macroassociates in the grains in
vivo happens with the participation of various intermolecular
forces: ion-electrostatic interactions conditioned by acidic and
basic amino acids (AA), hydrophobic contacts (hydrophobic AA),
and SS links (cystine). Consequently, the combined impact of nonvalent
and covalent forces are determined by the quantity and biochemical
properties of individual polypeptides (subunits), primarily by
the content and location in polypeptide chains of reactive SH
groups that are able to form intermolecular SS bonds. The folding
of proteins of various origin and the chaperon-dependent assembling
of protein macroassociates in the cell in vivo are known to happen
in cotranslation and/or posttranslation periods and to be catalyzed
by the system of SH/SS-metabolism enzymes, in particular, by protein
disulfide isomerase (Chi-Chen Yong 1998; Marusich 1998; Fisher
1998) responsible for formation, splitting, and isomerization
of SS bonds in the proteins. The significant changes observed
in the substitution lines in the quantitative content and proportion
of HMW-glutenin subunits in functional glutenin fractions of contrasting
in technological properties wheat varieties may play an important
role in the formation (assembling) and stabilization of functionally
important glutenin complexes as a structural basis of gluten.
M.D. Permyakova, V.A. Trufanov, A.V. Permyakov, and T.A. Pshenichnikova
(Institute of Cytology and Genetics, Siberian Division of the
Russian Academy of Sciences, Novosibirsk, Russian Federation).
Lipoxygenase (linoleat:oxygen oxidoreductase, EC 1.13.12, Lpx)
is known to catalyze the oxidation of unsaturated fatty acids
resulting in formation of peroxide and hydroperoxide compounds.
This enzyme is widely distributed in plant cells. Lipoxygenase
reactions may initiate the synthesis of signaling molecule or
be involved in inducing structural or metabolic changes in the
cell (Siedow 1991; Grechkin 1999).
Our research involved T. aestivum subsp. aestivum
/ Ae. speltoides introgression lines. Species belonging
to the genus Aegilops, a wild relative of wheat, are widely
used for introducing agriculturally important traits, such as
resistance to fungal diseases and pests and tolerance to high
salinity and temperature, into the genome of common wheat. The
common spring wheat Rodina was used as the female parent in producing
the introgression lines. This line is part of the 'Arsenal' collection
produced by I.F. Lapochkina (Institute of Agriculture of Central
Regions in Non-Chernozem Zone of Russia, Nemchinovka and Lapochkina
Lipoxygenase activity was analyzed in Tris-soluble flour extracts
according to Doderer et al. (1992). The average values of specific
Lpx activity in the parental cultivars and eight introgression
lines for 2 years are in Figure
16. Enzyme activity is a level lower in wild cereals than
that in wheat lines. This difference possibly is because Lpx
genes are in triplicate in hexaploid wheat, although this point
requires further study. Introgression causes a significant increase
in Lpx values in lines 32W, 75W, 84W, 178i, and 170i in a genetic
background of Rodina. Lipoxygenase activity in lines 167i, 182i,
255i, and 176i significantly decreased in comparison with Rodina.
Previously, the same introgression lines was characterized
by means subtelomeric repeats Spelt 1 and Spelt 52 being specific
markers Ae. speltoides (Salina et al. 2001). We have found
that values of specific Lpx activity are directly connected to
Spelt 1 amount (Figure
17). Any connection Lpx with Spelt 52 was not revealed.
Alien introgression also can lead to change of Lpx activity,
which is connected with physiological processes in plants. Lines
with alien chromosomes may be a source of certain disease-resistance
genes and other traits that change a plants adaptive potential.
These transfers into common wheat cultivars may be useful for
M.D. Permyakova, V.A. Trufanov, A.V. Permyakov, and T.A. Pshenichnikova
(Institute of Cytology and Genetics, Siberian Division of the
Russian Academy of Sciences, Novosibirsk, Russian Federation).
Lipoxygenases (Lpx, EC 126.96.36.199) are nonheme, iron-containing
dioxygenases widely distributed in plants, fungi, and animals.
Lipoxygenase catalyzes the addition of molecular oxygen to polyunsaturated
fatty acids to produce an unsaturated fatty acid hydroperoxide.
Products of the Lpx pathway are involved in inducing structural
or metabolic changes in the cell. In plants, Lpx has been associated
with some processes in number of developmental stages (Siedow
1991) and with mobilization of storage lipids during germination
(Feussner et al. 2001). Lipoxygenase also is used as a storage
protein during vegetative growth (Tranbarger et al. 1991). Lipoxygenase
also influences quality parameters of wheat gluten (Mc Donald
1979; Shiba et al. 1991).
Lipoxygenase expression is regulated by different effectors
such as the source/sink status (Feussner et al. 2001), jasmine
acid (Park et al. 1994) and abscisic acid (Melan et al. 1993),
and also by different forms of stress, such as wounding, water
deficiency, or pathogen attack (Porta et al. 1999; Melan et al.
Lines with substitutions of individual chromosome pairs of
the homoeologous group in wheat are convenient genetic material
for the study of donor and recipient gene effects on their expression
and enzyme specific activity. The objective of our research is
the substitution lines of Saratovskaya 29-Janetskis Probat (S29-JP)
for all chromosomes excluding 1. These lines were developed at
the Institute of Cytology and Genetics, Novosibirsk, Russian Federation.
In order to measure Lpx activity, proteins were extracted from
the flour by Tris buffer 1:1 (w:v) at 4 C. Lipoxygenase activity
was assayed spectrophotometrically under the wavelength 234 nm
(Doderer et al. 1992). One unit of activity was defined as the
change in optical density on 0.001/min. Specific activity was
expressed by ratio of activity units to 1 mg of protein in 1 ml
of incubation media. Protein concentration was determined by according
to Lowry et al. (1951).
Results of Lpx activity measurement in all investigated lines
are shown in Figure
18. The diagrams present average data of the three biological
and three analytical replicates. The donor JP exceeded the recipient
S29 by 60 % for the specific activity of Lpx. In all lines excluding
3D and homeologous group 5, this trait is significantly higher
than that of the donor parent JP.
The genes responsible Lpx synthesis are located on the chromosomes
of the homeologous groups 4 and 5 (Hart and Langstone 1977). Judging
from our results, chromosomes of different homoeologous groups
participate in control of this character. We have shown that intervarietal
substitution of the homeologous groups 1 and 6 chromosomes affect
the functional activity of this enzyme (Trufanov et al. 2001).
From this data, we concluded that there are genes or regulators
of Lpx activity along with structural genes.
The data from 3 years of Lpx activity measurements are in Figure 19. Levels
of Lpx change over different years, but Lpx activity inheritance
in the parental cultivars and the substitution lines is similar
during the experimental period. Average data for the 3-year experiment
with this set are given in Figure
20. The most significant influence on enzyme specific activity
is from the substitution of chromosome 4A. Substitution of chromosomes
from homeologous group 5 does not change Lpx activity. Probably,
chromosomes from JP have such Lpx alleles that cannot compensate
for the activity of corresponding alleles of S29. The chromosomal
location of Lpx structural genes is known, but the regulation
of its activity may be under separate genetic control. Taking
into account the important physiological role of Lpx, further
genetic studies will be extremely important.
VAVILOV INSTITUTE OF GENERAL GENETICS,
RUSSIAN ACADEMY OF SCIENCES
Gubkin str. 3, 119991 Moscow, Russian Federation.
SHEMYAKIN AND OVCHINNIKOV INSTITUTE OF BIOORGANIC CHEMISTRY,
RUSSIAN ACADEMY OF SCIENCES2
Ul. Miklukho-Maklaya 16/10, Moscow, Russian Federation.
T.I. Odintsova and V.A. Pukhalskiy (Vavilov Institute of General
Genetics) and A.K. Musolyamov and Ts.A. Egorov (Shemyakin and
Ovchinnikov Institute of Bioorganic Chemistry).
Peptides with antimicrobial activity are present in most plant
species. Most plant antimicrobial peptides isolated so far contain
even numbers of cysteine residues (4, 6, or 8) forming disulfide
bridges, thus providing high stability to the peptides. Based
on homologies at the primary structure level and cysteine motifs,
plant antimicrobial peptides can be classified into distinct families
including thionins, plant defensins, lipid transfer proteins,
and hevein- and knottin-type antimicrobial peptides (Garcia-Olmedo
et al. 1998). All antimicrobial peptides studied so far exert
their antimicrobial activity at the level of the plasma membrane
of the target microorganism, but different peptide types are likely
to operate via different mechanisms. In a number of plant species,
a considerable induction of genes expressing antimicrobial peptides
has been observed upon infection with pathogens suggesting their
role in inducible defense response of plants. Constitutive expression
of heterologous antimicrobial peptide genes, which has resulted
in enhanced resistance to particular microbial pathogens, has
been achieved for several plant species. New potent antimicrobial
peptides are of considerable interest for production of transgenic
resistant crops, they also have a considerable potential as novel
therapeutic agents, disinfectants and food preservatives. Considerable
progress has been made recently in the identification of new antimicrobial
peptides in different plant species. However, wild relatives of
wheat and related species highly resistant to pathogenic microorganisms
are poorly studied. In this work, we analyzed the peptide composition
of seeds of Triticum kiharae, a synthetic allopolyploid
produced by crossing T. timopheevii subsp. timopheevii
with Ae. tauschii, which is highly resistant to most fungal pathogens.
Materials and Methods. The peptide fraction was extracted
from T. kiharae flour with acids (1 % trifluoroacetic acid,
1 M HCl, and 5 % HCOOH) for 1 h at room temperature and precipitated
overnight with cold acetone, redissolved, and subjected to chromatography
on Heparin Sepharose. Proteins and peptides were eluted with a
stepwise NaCl gradient. The unbound, 100- and 500-mM NaCl fractions
were desalted on a C8 cartridge, dried on a Speedvac concentrator,
and separated by size-exclusion chromatography on a Superdex Peptide
HR 10/30 column (Amersham-Pharmacia, Biotech, Uppsala, Sweden).
Proteins and peptides were eluted with 0.05 % TFA, containing
5 % acetonitrile at a flow rate of 250 l/min, and monitored by
absorbance at 214 nm. The peptide fraction was further separated
by RP-HPLC on a Vydac C18 column (4.6 x 250 mm, particle size
5 m) with a linear acetonitrile gradient (10-50 %) for 1 h at
a flow rate of 1 ml/min and 40 C. Peptides were detected at 214
nm. The chromatographic fractions were tested for the antifungal
activity against Helminthosporium sativum and characterized by
mass spectrometry (MS) and N-terminal sequencing. Mass spectra
were acquired on a model Reflex III mass spectrometer (Bruker
Daltonics, Bremen, Germany). Dehydrobenzoic acid was used as matrix.
N-terminal amino acid sequences were determined by automated Edman
degradation on a model 492 Procise sequencer (Applied Biosystems)
according to the manufacturer's protocol.
Results and Discussion. Previous analysis showed that
the seeds of T. kiharae contained several families of antimicrobial
peptides with molecular masses from 3 to 6 kD. I n this work,
we focused on the glycine-rich peptides. We discovered least eight
glycine-rich peptides. Their molecular masses varied from 4,295
to 4,750 Da. N-terminal sequencing (from 10 to 50 amino acid residues)
showed that they were homologous, extremely glycine-rich, and
cysteine-free. In sequenced regions, glycine constituted 70-80
% of amino acids, and only few other amino-acid residues were
found (Tyr, His, Pro, and Ala). A remarkable feature of these
peptides was the presence of different repeat motifs, such as
GnYPGH, GnYP or GnYPGR, where n is a variable number. Similar
repeat motifs were discovered in glycine-rich proteins involved
in plant defense against biotic and abiotic stress. The glycine-rich
peptides are low-abundant constituents of T. kiharae seeds,
their yields were as follows: 0.4 g/g dry weight for Tk-AMP-G1,
0.6 g/g for Tk-AMP-G2, 0.7 g/g for peptides Tk-AMP-G3 and Tk-AMP-G4.
Preliminary antifungal tests with H. sativum showed that
the Tk-AMP-G peptides caused morphological changes in the fungus
at concentrations of 100-150 mg/ml in a dose-dependent manner.
These data indicate that T. kiharae is a valuable source
of glycine-rich peptides, whose biological activities will be
investigated in more detail.
T.V. Korostyleva, G.V. Kozlovskaya, and V.A. Pukhalskiy.
During the last years, knockout technology based on molecular
mechanisms of silencing (RNA interference) is actively used in
experiments to study of gene functions. Now this approach has
become a tool for wheat research. The method includes transformation
of plant with the genetic construct providing the synthesis or
endogenous formation in the cells of double-stranded RNA homologous
to any transcribed region of the target gene. The appearance in
cells of such dsRNA triggers specific defensive mechanisms of
restriction of dsRNA and mRNA with homology to it, which results
in silencing of target gene. This process was believed to be very
specific. However, recently it was reported that dsRNA might exert
unspecific effects on expression of the genome and the genes of
the transgenic construct.
Earlier in laboratory the transgenic plants of spring, common
wheat cultivar Khakasskaya were obtained using Agrobacterium transformation
in planta. T-DNA of these plants contains inverted repeat of 350-bp
fragment of tetracycline resistance gene from pBR322 under the
control of 35S CaMV promoter and nptII gene as marker. This construct
provides the synthesis of untranslatable dsRNA in wheat cells.
It is supposed that its nucleotide sequence has no homology to
the wheat genome.
In the present study we have investigated morphological stability
of 18 independent transgenic lines on T1 selfed progeny. The presence
of T-DNA in individual plant has been confirmed by PCR-analyses
with primer to inverted repeat sequence from pBR322. Segregation
of T-DNA in progeny was observed only in 2 from 18 transgenic
lines (with non -Mendelian ratio), in other lines all offspring
plants had transgenic insertion. In contrast to T1 plants with
normal phenotype, T2 progeny of some lines displayed alteration
in several traits in field conditions. Using analysis of variance
by statistical program AGROS 2.10 we have estimated a significance
of differences between transgenic plants from individual lines
and control group of non-transgenic wheat plants of Khakasskaya.
The established changes manifested in the decrease of spike length
(a significant decrease in 13 from 18 lines, i.e., 16.6 % of transformants),
a spikelet number per spike (16.6 %), kernel weight (a significant
decrease in 13 from 18 lines, i.e., 72 %), and in the increasing
of productive tillering in wheat (11 %).
The reason of these changes might be in the shifts in the hormonal
balance in transformants; however, this assumption demands additional
investigations. We are studying inheritance of these alterations.
With regard to the nature of observed changes, we can conclude
that a high frequency of similar-type changes in independent lines
cannot result of either insertion mutagenesis or somaclonal variability.
We believe that the insertion expressing dsRNA induces unspecific
pleiotropic effect on the recipient wheat genome.
N.I. VAVILOV RESEARCH INSTITUTE
OF PLANT INDUSTRY
42, B. Morskaya Str., St. Petersburg, 190000, Russian Federation.
S.P. Martynov and T.V. Dobrotvorskaya.
We studied winter bread wheat cultivars from southern Russian
Federation (Krasnodar, Stavropol, and Rostov regions) and the
Ukraine. For the analysis, cultivars with known pedigrees and
were used not conflicting with estimates of reaction to Fusarium
spp. Among the lines were 40 resistant and moderately resistant
cultivars (see Table
5) and 61 susceptible cultivars from southern Russia including
Bezostaya 1 (Borojevic and Dencic 1988, Buerstmayr et al. 1996),
Avrora (Borojevic and Dencic 1988), Lgovskaya 167 (Anonymous 1990),
Bystritsa, Delta, Podarok Donu, Donskoj Mayak, Tarasovskaya Ostistaya,
Zernogradka 10, Zarnitsa, Stanichnaya, Bat'ko, Donskoj Syurpriz,
Zernogradka 11, Rodnik Tarasovskij (Anonymous 1994-2003), Sfera
(Puchkov et al. 1996), Spartanka (Terekhina 1993), Dialog 1061-10,
KN-1221-k-7-2-14, KN-201-90-k-2, KN-2503-h-112-6-11, KN-2503-h-112-6-3,
KN-3071-h-16-16, KN-3303-h-117, KN-3385-h-756, KN-4594-h-370-41,
KN-5835-h-427, Massiv 809-1016, Rannyaya 47, Zamena, Krasnodarskaya
70, Olimpiya 2, Skifyanka, Soratnitsa, Ejka, Novokubanka, Otrada,
Yugtina, Krasnodarskaya 90, Nika Kubani, Ofeliya, Polovchanka,
Azau, Aliza, Zimorodok, Nak, Umanka, Kupava, Uskoryanka, Krasota,
Selyanka, Krasnodarskaya 99, Yashkulyanka, Fisht (Puchkov et al.
2001); and from Ukraine: Albatros Odesskij, Donetskaya 46 (Anonymous
1989), Kiyanka (Anonymous 1981), Lutescens 7 (Anonymous 1990),
Mironovskaya Yubilejnaya (Mesterhazy 2001), Odesskaya 132 (Anonymous
1976-1995), and Odesskaya Polukarlikovaya (Babayants et al. 2001).
For each of cultivars a genetic profile was constructed and
transfer of scab resistance from ancestors to the descendants
was traced on genealogical trees with the help of the Genetic
Resources Information and Analysis System GRIS3.5 (Martynov and
Dobrotvorskaya 2000). All cultivars of the analyzed set are the
descendants of the most popular cultivars Bezostaya 1 (frequency
of presence in pedigrees 94.5 %), Mironovskaya 808 (62.6 %), and/or
Odesskaya 16 (65.9 %). To reveal differences in the contributions
of these parents in groups of resistant and susceptible cultivars
the two-way ANOVA of the parentage coefficients for the randomized
design was used (Table 1). The investigated factors were cultivar
groups (factor A) with two gradations (resistant and susceptible)
and dominant ancestors (factor B) with number of gradations b
Table 1. Two-way ANOVA of the parentage coefficients
of the three most important ancestors of winter bread wheat cultivars
from southern Russian Federation and the Ukraine (original data
are transformed through arcsinx) . Items with an * are significant
at P < 0.001.
The ANOVA (Table 1) has shown the significance of differences
of groups of resistance/susceptibility (factor A) and ancestor
contributions (factor B). The greatest interest represents interaction
of the investigated factors. The significant interaction (A´B)
specifies differences in distribution of the contributions of
the same ancestors in groups of resistant and susceptible cultivars.
The comparison of average coefficients of parentage in cultivar
groups (Table 2) shows, that the contributions of cultivars Bezostaya
1 and Mironovskaya 808 are identical in both groups, and the contribution
of Odesskaya 16 is significant higher in group of resistant cultivars.
It is known, that Bezostaya 1 is susceptible to FHB (Borojevic
and Dencic 1988, Buerstmayr et al. 1996), and the estimates of
reaction to Fusarium spp. for Mironovskaya 808 are conflicting,
from moderately resistant (Javor et al. 1997) up to susceptible
(Buerstmayr et al. 1996). The results of our analysis show that
most likely Mironovskaya 808, as well as Bezostaya 1, does not
carry genes of resistance to FHB. Moderately resistant cultivar
Odesskaya 16 (Kazmin and Shindin 1997) most frequently was the
donor of resistance of scab-resistance genes in cultivars from
southern Russian Federation and the Ukraine. Odesskaya 16 was
selected from cultivar Odesskaya 12 (Hostianum 237/Zemka, where
Hostianum 237 is selection from Kharkovskaya, a landrace of Kharkov
region, and Zemka is local variety from Odessa region). Odesskaya
16 is absent in pedigrees of some resistant cultivars (Dakha,
Russa, Echo, and Zimdar 4), but there are other derived cultivars
of Hostianum 237. Therefore, we assume that a source of resistance
to FHB in cultivars from southern Russian Federation and the Ukraine
was the landrace Kharkovskaya via Hostianum 237.
Table 2. Average coefficients of parentage
of important ancestors in groups of resistant to scab and susceptible
winter bread wheat cultivars from southern Russian Federation
and the Ukraine. Items with an * are significantly different at
P < 0.01.
To understand the role of other sources of resistance, we used
ANOVA of the contributions of some known donors of resistance,
which are in pedigrees of the investigated cultivar set (Table
3). The investigated factors were groups of resistant/susceptible
cultivars (factor A) and donors of resistance genes (factor B)
with the number of gradation b = 9. The ANOVA (Table 3) shows
significant differences in the average parentage coefficients
of groups of resistant/susceptible cultivars (factor A) and donors
(factor B). The significant 'A x B' interaction specifies differences
in distribution of the contributions of the donors in groups of
resistant and susceptible cultivars.
Table 3. The two-way ANOVA of the parentage
coefficients of donors of resistance to scab in pedigrees of bread
wheat cultivars from Southern Russia and Ukraine (original data
are transformed through arcsinx). Values with an * are significant
at P < 0.001.
Comparing average coefficients of parentage of the donors (Table
4) shows that in addition to Odesskaya 16 in the group of resistant
cultivars are the significant cultivars Redcoat, Frontana, and
Cheyenne. These donor cultivars are in pedigrees of the southern
Russian and Ukrainian cultivars via Biserka (Redcoat), Red River
68 (Frontana), and Colt (Cheyenne).
Table 4. Average coefficients of parentage
of the donors of scab-resistance genes in sets of resistant and
susceptible winter wheat cultivars from southern Russian Federation
and the Ukraine. Values with an * are significantly different
at P < 0.05.
Established with the help of the pedigree analysis, the probable
donors of resistance genes are given in Table 5. Of the 43 resistant and moderately
resistant lines, three of the pedigrees are unknown and two cultivars
(Maslovchanka 90 and Kharkovskaya 20) were not established. Most
frequently, Odesskaya 16 and its derivatives (Odesskaya 51, Obrij)
(55 %) and Frontana (28 %) were donors of resistance genes. Resistance
to FHB in the older cultivars Kooperatorka, Novokrymka 102 (Ukraine),
and Cheyenne (USA) were selected from the landrace Crimean, which
probably was heterogenous for reaction to Fusarium spp.
The Canadian cultivar Canus (Marquis/Kanred) also most likely
received resistance genes from Crimean via Kanred, because Marquis
is susceptible to scab.
This analysis has shown that despite of the rather large number
of known sources of resistance to FHB in the Russian and Ukrainian
wheat breeding programs, they are practically unused. Our analysis
was made on the basis of the information about resistance or susceptibility
of bread winter wheat cultivars received by the different authors
in different time. Therefore, we consider the data about source
of resistance and statistical estimations made by comparison of
sets of resistant and susceptible cultivars, as preliminary. Nevertheless,
the approach, based on the genealogical information, can be useful
for the analysis of the comparable data.