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BIOMEDICINOS MOKSLAI
Dinamycs of Superoxide Dismutase and Catalase Activities During Acclimation to
Hypothermia of Wild-Type and Transformed Potato Plants
Maksim Sinkevich, Ilja Demin, Tamara Trunova
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences
Changes in the lipid peroxidation intensity, activities of SOD and catalase in the course of acclimation to hypothermia have been studied in the
leaves of wild-type and transformed potato plants with modified carbon metabolism. All plants were grown in vitro at MS nutrient medium containing
2% sucrose. The response to low temperature was observed during and after 6 days of prolonged exposure to 5oC. Lipid peroxidation intensity and electrolyte leakage intensity were used to estimate the level of stress. The obtained results indicate that, irrespective of the way of changing the level of sugars (due to transformation with foreign invertase gene or via adaptive response) the transformed plants are notable for a higher level of sugars in the
leaves and thus have more active catalase and lower activity of SOD.
Superoxide dismutase, catalase, activities dynamics, leaves of potato plant.
Introduction
by an increase in the content of unsaturated fatty acids (Levitt, 1980), and peroxidation of lipids affects their level in
cell membranes. It is necessary to note that, in the living
cells, there exists a physiologically normal level of free radical processes and peroxidation of lipids, which is regulated
by a complex multicomponent protective system comprising
antioxidant enzymes (superoxide dismutase (SOD, EC
1.15.1.1.), several peroxidases, catalase (EC 1.11.1.6.)) and
low-molecular weight antioxidants (Scandalios, 1990; 1993).
According to conventional view (Kuzniak, 2002),
based mainly on the studies of chilling sensitive plants, the
balance between oxidized and reduced compounds is easily
shifted towards oxidation at the earliest stages of oxidative
stress, while the enzymes are activated, playing key role in
plant protection against active substances. However, as
their capacity is exhausted in plant cells exposed to strong
and prolonged stress, the balance is shifted towards activation of lipid peroxidation. This shift is related to malonic
dialdehyde accumulation due to the activation of freeradical reactions involving the reactive oxygen species that
participate in the peroxidation of unsaturated fatty acids in
membrane lipids; as a result, the amount of membrane material decreases (Baraboi, 1991).
Recent research (Sinkevich et al., 2009) had revealed
an important relation between sugars and antioxidant system so the aim of this work was to study the changes in the
lipid peroxidation intensity, activities of SOD and catalase
in the course of acclimation to hypothermia in transformed
potato plants with modified carbon metabolism.
Acclimation to hypothermia is the most thoroughly investigated area in the group of frost resistant plants, mainly
in winter cereals and trees (Tumanov, 1979; Trunova, 2007).
Among numerous protective processes, an important role
belongs to the intracellular accumulation of sugars caused
by the changes in carbohydrate metabolism, in particular in
the activity of invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) (Sturm et al., 1999). In plants, it is present
in three forms differing in biochemical characteristics and
subcellular location. Invertases with pH optimum of 4.5-5.0
are located in the extracellular space (apoplast) and associated with the cell wall; they also occur as soluble proteins
in the vacuole. Invertases with pH optimum of 7.0-7.8 are
soluble cytoplasmic proteins (Sturm et al., 1999). Extracellular invertase is a key enzyme that catalyzes the cleavage of
transport sucrose (Roitsch et al. 2003).
This study conducted with potato plants where the inserted gene of yeast invertase is controlled by a tuberspecific B33 class I patatin promoter (apoplastic location of
the enzyme) showed that at optimal for growth temperature
of 22оC the expression of yeast invertase gene in the leaves
of transformed potato brought about changes in carbohydrate metabolism manifested in an activation of different
forms of invertase (especially, of acid forms) and increase in
the level of sugars (mainly, of sucrose). It was also found
that potato plants transformed with the yeast invertase gene
were notable for higher constitutive resistance to hypothermia (without cold acclimation) (Deryabin et al., 2003). Accumulation of sugars induced by chilling is considered a
process of protective and adaptive character (Levitt, 1980;
Kasperska-Palach, 1983). The exposure of potato plants to
low temperatures (5oC; 6 days) resulted in the activation of
different forms of invertase (for the most part of acid invertase) and in an increase in intracellular accumulation of sugars (mainly, of sucrose). In the leaves of potato plants expressing the foreign yeast invertase gene, these processes
were much more active (Deryabin et al., 2007).
It is known that, in addition to many biochemical
processes, plant adaptation to hypothermia is accompanied
Methods
The study was conducted with the potato plants (Solanum tuberosum L., cv. Desiree) transformed with a vector
expressing an yeast invertase gene under the B33 class I patatin promoter and carrying the sequence of proteinase II inhibitor leader peptide for apoplastic location of the enzyme.
Wild-type potato plants, cv. Desiree, served as control. The
plants were obtained from the collection of clones produced
as a result of cooperation between the researchers of the Max
Planck Institute of Molecular Plant Physiology (Golm, Ger-
72
with subsequent incubation for 30 min in white light produced by LB-80 lamps (illuminance of 4 klx). The mixture
without plant extract served as a control sample (in this case,
the formation of formazan was the greatest one). Optical
density was measured against mixture without SOD extract
at SF-46 or Spekol-11 spectrophotometer at the wavelength
of 560 nm using the cells with the optical path length of 0.5
cm. The suppression of formazan production by 50% was
taken as a unit of activity. SOD activity was expressed in the
units/g fr wt of leaves.
Activity of cytosolic catalase was determined using
the method described by Kumar and Knowles (Kumar et
al., 1993). Changes in optical density of H2O2 solution was
measured under UV light with 240 nm wavelength at SF46 spectrophotometer. The catalase activity was calculated
in units per minute, and µmol/(g fr wt) was considered as a
unit of activity.
The results from several replicates were analyzed using the t-test and OriginPro 7.5 program products with embedded statistic analysis (Student’s t-test, P = 0.05). The
figures show the means and their standard errors.
many) and the Chailakhyan Laboratory of Growth and Development (Timiryazev Institute of Plant Physiology, RAS).
The plants were micropropagated in vitro and grown
in a controlled-climate chamber at the Institute of Plant
Physiology, RAS, at 22oC and 16-h photoperiod (illumination of 4 klx) for 5 weeks on the Murashige and Skoog
(Murashige et al., agar medium containing 2% sucrose and
the vitamins (mg/l): thiamine-0.5, pyridoxine-0.5, and mesoinositol-60.0.
Acclimation of potato plants to hypothermia was conducted at 5oC for 6 days in a controlled-climate chamber at
the Institute of Plant Physiology, RAS, under 16-h photoperiod and illumination of 4 klx.
Chilling tolerance of potato genotypes was assessed
by the electric conductivity of water extracts from plant
tissues according to Dexter method (Hepburn et al., 1986).
Middle leaves were sampled, the petioles were detached
and 80 mg of fresh material was placed into glass tube with
10 ml of distilled water. Vacuum infiltration by water was
carried out in the light twice. Closed tubes with samples
were shaken for 1 h. The content of ions leaked from the
tissues was measured in the glass cell with two flat platinum electrodes (1 cm2) arranged 6 mm apart using an R
577 bridge (Russia) at room temperature. When the measurement was over, the tubes were placed into a boiling
water bath for 20 min and then shaken for 30 min. Thereafter, the measurement was repeated. The electric conductivity was calculated as ohm-1 cm-1 · 10-4 using a commonly employed equation (Flint et al., 1967):
Results and discussion
Peroxidation of lipids in the cells is known to intensify
in response to various stresses, including hypothermia; it
results in the accumulation of malonic dialdehyde associated with electrolyte leakage and the activation of free
radical reactions involving reactive oxygen species (Lukatkin, 2002). Both lipid peroxidation and electrolyte leakage serve as indicators of stress.
Under prolonged hypothermia (5oC) a brief decline in
the content of malonic dialdehyde in the leaves of wildtype plants that was observed during the first day was followed by its steady accumulation (Fig. 1a).
Within 3 days of chilling, the shape of the curve describing the level of malonic dialdehyde in the leaves of
transformed plants was the same as that in the plants of
wild type but the absolute level of malonic dialdehyde was
lower and, by the end of the experiment, tended to decline.
Therefore, the long influence of low above-zero temperature on plants is supposed to raise the physiologically normal level of peroxidation of lipids as compared with its
rate at 22oC. Activation of peroxidation of lipids in the
plants of investigated potato genotypes by the third day of
chilling in the light is also a result of photodynamic injury
of membranes (Wise et al., 1987).
Throughout 6-day-long stay in a climate-controlled
chamber at 5oC (Fig. 1b) few changes of little significance
were observed in membrane permeability of the leaves of
transformed plants. Though their cold tolerance index decreased by 7% after 3 days of chilling, it was almost restored to the 6th day.
Electrolyte leakage revealed that the control plants became somewhat less cold resistant after such a treatment
than untreated plants. This evidence points at the onset of
cellular membrane injury therein. Thus, the transformed
plants were more cold resistant than the control plants, because ion permeability of their membranes was not impaired, apparently, because of the elevated content of sugars and their numerous protective effects under chilling
(Kolupaev et al., 1993).
I = 100(Lt - L0)/(Lk - L0),
here I is the index of tissue injury, %,
L is the electric conductivity of the sample measured
before cold injury (L0), and after it (Lt);
Lk is the electric conductivity of the same sample after boiling, ohm-1 cm-1 · 10-4.
The cold tolerance of the tissue (H) was estimated as
the value complementary to I (H = 100% – I) and also expressed in %. The rate of lipid peroxidation in the leaf tissues was evaluated by the level of malonic dialdehyde determined by means of color reaction with thiobarbituric acid, as described earlier (Deryabin et al., 2003). Optical
density of the samples was measured using an SF-46 spectrophotometer (LOMO, Russia) at the wavelength of 532
nm. The content of malonic dialdehyde was expressed in
µmol/(g fr wt) with the use of coefficient of molar extinction being equal to 1.56·105 cm-1·M-1. Each replicate was a
sample of leaves taken from the middle part of 3-5 plants.
Activity of cytosolic fraction of SOD (Cu,Zn-SOD)
(Bowler et al., 1992; Scandalios, 1993) was determined by a
slightly modified method described by Kumar and Knowles
(Kumar et al., 1993). A sample of leaves (150 mg) from the
middle part of investigated plants was homogenized in 1 ml
of phosphate buffer (pH 7.7). The homogenate was centrifuged for 20 min at 7000 g, and the obtained supernatant
was used as a crude extract containing cytosolic SOD. The
components of the reaction mixture were added in the following order: 1.5% L-methionine, 0.14% Nitro Blue Tetrazolium, and 1% Triton X-100 (3 : 1 : 0.75). The supernatant
(40 µl) was added to 1 ml of the reaction mixture. The reaction was initiated by the addition of 10 µl of 4.4% riboflavin
73
To explore the role of sugars under hypothermia, two
methodological approaches designed to vary their content in
the leaf cells of potato plants were employed in this work.
According to the obtained data constitutive (before cold
treatment) levels of SOD activity were similar in both genotypes (Fig. 2a). Investigation of dynamics of cytosolic SOD
activity in the leaves of tested potato genotypes under prolonged hypothermia showed that during the first day of chilling the plants of wild type displayed a surge of SOD activity; on the third day, it sharply declined, and then the enzyme
activity slightly rose by the end of the experiment.
Activity units / g fr wt
1
5,5
5,0
20
2
15
1
10
5
0
2
0
1
2
3
4
5
6
4,5
4,0
3,5
7
3,0
0
1
2
3
4
5
catalase activity
%
80
2
70
60
1
50
0
1
5
3
2
4
Duration of chilling, days
1
6
6
mcmol Н2О2/(g fr wt per minute)
Content of MDA, µmol / g fr wt
a
6,5
6,0
a
25
6
5
4
2
3
2
1
0
0
1
3
Chilling exposure, days
6
Fig. 2. Activities of cytosolic SOD (a) and catalase (b) in the leaves of
wild-type (1) and transformed with yeast invertase gene (2) potato plants
exposed to 5oC
Fig. 1. Content of malonic dialdehyde (a) and cold tolerance (b) of the
leaves of wild-type (1) and transformed with yeast invertase gene (2) potato plants exposed to 5oC
Firstly, transformed potato plants with modified carbohydrate metabolism induced by the introduction of the gene
for yeast invertase of apoplastic location were used. The
boosted expression of yeast invertase gene caused a retardation of sucrose efflux from the cells because in the apoplast
it was transformed into non transportable hexodes.
Secondly, a prolonged exposure to light at 5°C was
employed, which induced an increase in the content of
intracellular sugars. As it had been shown in previous experiments, the investigated genotypes differed by invertase
activity even at optimal growth conditions (22°C) with the
activity of acid invertases in the leaves of transformed
plants being almost 1.5 times higher than in the wild-type
control plants (Sin'kevich et al., 2008). As it was described
earlier (Deryabin et al., 2003), the activity of acid form
was mostly related to accumulation of sugars (predominantly sucrose and glucose) in leaves.
During the first day of cold acclimation, the activity of
acid invertases in the leaves of both genotypes increased,
and in the transformed plants it was almost 1.5 times greater than in wild-type ones. On the third day of cold acclimation, the activity of acid invertases in the leaves of both
genotypes was the greatest one; by the end of the experiment (on the 6th day), enzyme activity somewhat decreased (Sin'kevich et al., 2008).
Changes in the activity of different forms of invertase
in the leaves of wild-type and transformed plants during 6day-long chilling at 5oC leaded to higher sugar contents.
At the same time, in transformed plants throughout the
entire period of hypothermia, SOD activity did not change
essentially and remained at the level of control plants
(without chilling). The increase in SOD activity observed
in the leaves of wild type potato plants during the first day
of chilling pointed to an elevated reactive oxygen species
production (Merzlyak, 1989). In contrast to the plants of
wild type, such a way of chilling did not activate SOD in
the leaves of transformed plants. The fact that the curves
describing the activity of SOD and the rate of peroxidation
of lipids are reversed suggests that these reactions are activated by different reactive oxygen species.
The activity of SOD and the rate of peroxidation of lipids depended on the level of intracellular sugars in the
leaves of investigated genotypes of potato (Sin'kevich et
al., 2008). When the level of sugars rose, the activity of
SOD and the rate of lipid peroxidation decreased.
Catalase activity (Fig. 2b) acted differently to SOD – it
was about to reduce its level for the first daily period in both
genotypes. After 3 days of exposure at low temperature it
rose greatly but returned to the untreated level on the 6th day
in transformed plants and stayed high in wild-type ones.
And this fact correlates with the sugar content in leaves of
both genotypes – high sugar content serves as background to
increased catalase activity.
74
content of sugars, mmol / g fr w
20
chanisms of protective action of soluble carbohydrates on
the biological membranes have been proposed. One of
them may depend on the formation of bonds between the
oxygen atoms of phosphates within membrane phospholipids and hydroxyls of sugars (Strauss, Hauser, 1986). This
mechanism is consistent with the possibility that in dehydrated cells, water is replaced in biological membranes
with sucrose (Chen et al., 1976; Caffery et al., 1988). Sugars can modify plasma membrane ensuring its homeoviscous adaptation (Uemura, Steponkus, 1997). In addition, sugars participate in antioxidant protection of the cells
acting as interceptors of active forms of oxygen and suppressing destructive oxidative processes (Aver’yanov, Lapikova, 1989; Morelli et al., 2003; Sinkevich et al., 2009).
One may suggest that owing to the accumulation of lowmolecular carbohydrates (sucrose and glucose) in the
course of cold acclimation due to activation of acid invertases cold resistant plants better adapt to temperature drop
occurring in nature.
transformed
monosugars
total sugars
18
wild-type
16
14
transformed
12
10
8
wild-type
6
4
0
1
2
3
4
5
6
Days of chilling
Fig. 3. Sugar content in the leaves of potato plants of wild-type and transformed with yeast invertase gene
The analysis of the content of various sugar forms
(conducted simultaneously to the studies of invertase activity) showed that their greatest accumulation occurred in the
leaves of both genotypes by the third day of chilling. As
compared with the unhardened control, the content of glucose in transformed plants increased twice and that of fructose, more than 5 times; however, as compared with other
sugars, the level of fructose was low. It is important that in
the leaves of both potato genotypes during cold acclimation the content of sucrose increased, especially in the
transformed plants (1.7 times).
Apparently, yeast invertase in the transformed plants was
responsible not only for accumulation of monosaccharides (at
the expense of sucrose hydrolysis) but also for accumulation
of sucrose due to activation of insoluble acid invertase.
Adaptive changes that occur in the cells of cold resistant
plants in the course of cold acclimation were shown in electron microscopic and morphometric investigations of the
cells of palisade parenchyma in the leaves of potato plants.
According to the obtained data, during this period the investigated genotypes produced xeromorphous ultrastructure
characteristic of cold resistant plants (Trunova et al., 2003).
Ultrastructural and functional transformation of the cells
during cold acclimation largely depends on sugars as a
source of energy and precursors to other metabolites (Trunova, 2007). Therefore, adequate supply of all the plant organs and tissues with water-soluble carbohydrates is necessary for the adaptation to prolonged chilling. In the course of
cold acclimation of plants, water-soluble carbohydrates are
used for the production of new ultrastructural elements of
the cell and for the synthesis of more reduced chemical
compounds that bring about the changes in the composition
of cellular membranes (elevation of the ratios lipids/ proteins, unsaturated FA/saturated FA, phospholipids/sterols,
digalactosyl diacylglycerols/ monogalactosyl diacylglycerols, phosphatidyl-cholin / phosphatidylethanolamine (Palta
et al., 1993; Uemura, Steponkus, 1997). As a result, the lipid
bilayer becomes capable of preserving liquid properties at
low temperatures (homeoviscous adaptation of membranes)
ensuring cellular functions that depend on its physical state.
Sugars also are known to protect protein-lipid components of the cells, especially in membranes. For instance,
under hypothermia, sucrose participates in the preservation
of the structural integrity of plasma membrane in the cells
of wheat leaves (Savitch et al., 2000). Several possible me-
Conclusions
The obtained results indicate that, irrespective to the
way of changing the level of sugars the transformed plants
are notable for higher level of sugars in the leaves and thus
have more active catalase and lower activity of SOD and
intensity of lipid peroxidation. It is an evidence for several
potent protective actions of sugars: membrane stabilization, dehydration and enhanced antioxidant scavenging.
Acknowledgments
This work was supported by the Russian Foundation
for Basic Research, project no. 07-04-00601.
List of Literature
1.
AVER’YANOV, A.A.; LAPIKOVA, V. P. 1989. Interaction between Sugars and Hydroxyl Radical as Related to Fungal Toxicity
of Leaf Excretions. Biokhimiya, vol. 54, p. 1646–1651.
2. BOWLER, C.; van MONTAGU, M.; INZE D. 1992. Superoxide
Dismutase and Stress Tolerance. Annual Review of Plant Physiology
and Plant Molecular Biology, vol. 42, p. 83–116.
3. CAFFERY, M.; TONSECA, V.; LEOPOLD, A.C. 1988. LipidSugar Interactions Relevance to Anhydrous Biology. Plant Physiology, vol. 86, p. 754–758.
4. CHEN, P.M.; BURKE, M.J.; LI P.H. 1976. The Frost Hardiness of
Several Solanum Species in Relation to the Freezing of Water, Melting Point Depression and Tissue Water Content. Botanical Gazette
(Chicago), vol. 137, p. 313–317.
5. DERYABIN, A.N., et al. 2003. Chilling Tolerance of Potato Plants
Transformed with a Yeast-Derived Invertase Gene under the Control
of the B33 Patatin Promoter. Russian Journal of Plant Physiology,
vol. 50, p. 449–454.
6. DERYABIN, A.N., et al. 2005. Influence Expressing YeastDerived Invertase Gene in Potato Plants on Membranes Lipid Peroxidation at Low Temperature. Journal of Thermal Biology, vol.
30, p. 73–77.
7. FLINT, H.L.; BOYCE, B.R.; BEATTIE, D.J. 1967. Index of Injury
– a Useful Expression of Freezing Injury to Plant Tissues as Determined by the Electrolytic Method. Canadian Journal of Plant
Science, vol. 47, p. 229–230.
8. HEPBURN, H.A.; NAYLOR, F.L.; STOKES D.I. 1986. Electrolyte
Leakage from Winter Barley Tissue as Indicator of Winterhardiness.
Annals of Applied Biology, vol. 108, p. 164–165.
9. KASPERSKA-PALACH, A. 1983. Mechanism of Hardening of
Herbaceous Plants. Moscow: Kolos.
10. KOLUPAEV, Yu.V.; TRUNOVA, T.I. 1993. Specific Features of
Invertase Activity under Hypothermia as Related to Winter Cereal
75
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Frost Tolerance. Физиология и биохимия культурных растений,
vol. 25, p. 378–393.
KUMAR, G.N.; KNOWLES, N.R. 1993. Changes in Lipid Peroxidation and Lipolytic and Free-Radical Scavenging Enzymes during
Aging and Sprouting of Potato (Solanum tuberosum L.) SeedTubers. Plant Physiology, vol. 102, p. 115–124.
KUZNIAK, E. 2002, Transgenic Plants: An Insight into Oxidative
Stress Tolerance Mechanisms. Acta Physiologiae Plantarum, vol.
24, p. 97–113.
LEVITT, J. 1980. Responses of Plants to Environmental Stresses.
New York: Academic Press.
MORELLI, R.; RUSSO-VOLPE, S.; BRUNO, N.; LO SCALZO, R.
2003. Fenton-Dependent Damage to Carbohydrates: Free Radical
Scavenging Activity of Some Simple Sugars. Journal of Agricultural and Food Chemistry, vol. 51, p. 7418–7425.
MURASHIGE, T.; SKOOG, F. 1962. A Revised Medium for Rapid
Growth and Bioassays with Tobacco Tissue Cultures. Physiologia
Plantarum, vol. 15, no. 3, p. 473–497.
PALTA, J.P.; WHITAKER, B.D.; WEISS L.S. 1993. Plasma Membrane Lipids Associated with Genetic Variability in Freezing Tolerance and Cold Acclimation of Solanum Species. Plant Physiology,
vol. 103, p. 793–803.
ROITSCH, T.; BALIBREA, M.E.; HOFMANN M.; PROELS R.;
SINHA, A.K. 2003. Extracellular Invertase: Key Metabolic Enzyme
and PR Protein. Journal of Experimental Botany, vol. 54, p. 513–524.
SAVITCH, L.V.; HARNEY T.; HUNER N.R.A. 2000. Sucrose Metabolism in Spring and Winter Wheat in Response to High Irradiance, Cold Stress and Cold Acclimation. Plant Physiology, vol.
108, p. 270–278.
SCANDALIOS, J.G. 1993. Oxygen Stress and Superoxide Dismutases. Plant physiology, vol. 101, p. 7–12.
SCANDALIOS, J.G. 1990. Response of plant antioxidant defense to
environmental stress. Advances in Genetics, vol. 28, p. 1–41.
SIN'KEVICH, M.S., et al. 2008. The Changes in Invertase Activity
and the Content of Sugars in the Course of Adaptation of Potato
Plants to Hypothermia. Russian Journal of Plant Physiology, vol.
55, p. 449–454.
22. SIN’KEVICH, M.S.; DERYABIN, A.N.; TRUNOVA, T.I. 2009.
Characteristics of oxidative stress in potato plants with modified
carbohydrate metabolism. Russian Journal of Plant Physiology, vol.
56, p. 168–174.
23. STRAUSS, G.; HAUSER, H. 1986. Stabilization of Lipid Bilayer
Vesicles by Sucrose during Freezing. Proceedings of the National
Academy of Sciences of the United States of America, vol. 83, p.
2422–2426.
24. STURM, A.; TANG, G.-Q. 1999. The Sucrose-Cleaving Enzymes
of Plants Are Crucial for Development, Growth and Carbon Partitioning. Trends in Plant Science, vol. 4, p. 401–407.
25. WISE, R.R.; NAYLOR, A.W. 1987. Chilling-Enchanced Photooxidation. The Peroxidative Destruction of Lipids during Chilling Injury to Photosynthesis and Ultrastructure. Plant Physiology, vol. 83,
p. 272–277.
26. TRUNOVA, T.I.; ASTAKHOVA, N.V.; DERYABIN, A.N.; SABEL’NIKOVA, E.P. 2003. Ultrastructure of Chloroplasts in Leaves
of Potato Plants Transformed with the Yeast Invertase Gene under
Normal Conditions and Hypothermia. Doklady Akademii nauk, vol.
389, p. 842–845.
27. UEMURA, M.; STEPONKUS P.L. 1997. Artificial Manipulation of
the Intracellular Sucrose Content Alters the Incidence of FreezeInduced Membrane Lesions of Isolated Protoplasts of Arabidopsis
thaliana. Cryobiology, vol. 35, p. 336.
28. БАРАБОЙ, В. А. 1991. Механизмы стресса и перекисное окисление липидов. Успехи современной биологии, т. 111, с. 923–931.
29. ЛУКАТКИН А. С. 2002. Холодовое повреждение теплолюбивых растений и окислительный стресс. Саранск: Изд-во Мордов. ун-та.
30. МЕРЗЛЯК, М. Н. 1989. Активированный кислород и окислительные процессы в мембранах растительной клетки. Итоги
науки и техники. Серия Физиология растений. Т. 6.
31. ТРУНОГА Т. И. 2007. Растение и низкотемпературный
стресс. Москва: Наука.
32. ТУМАНОВ, И. И. 1979. Физиология закаливания и морозоустойчивости растений. Москва: Наука.
M. S. Sinkevič, I. N. Demin, T. I. Trunova
Superoksido dismutazės ir katalazės dinamika laukinių ir transformuotų bulvių augalų hipoterminės aklimatizacijos eigoje
Santrauka
Darbe tiriama laukinių ir modifikuotos anglies metabolizmu transformuotų bulvių augalų lapuose vykstantys lipidų peroksidacijos intensyvumo,
SOD veiklos ir katalazės pokyčiai hipotermijos aklimatizacijos eigoje. Visi augalai auginami in vitro MS maistinėje terpėje su 2% sacharozės. Reakcija į
žemą temperatūrą stebima laikant 5oC temperatūroje 6 dienų laikotarpiu ir po jo. Streso lygis vertinamas lipidų peroksidacijos intensyvumu ir elektrolitų
nuotėkio intensyvumu. Rezultatai rodo, kad nepriklausomai nuo cukrų lygio pakeitimo būdo, transformuoti augalai išsiskiria aukštesniu cukrų lygiu lapuose, taigi, aktyvesne katalaze ir silpnesne SOD veikla.
Superoksido dismutazė, katalazė, veiklos dinamika, bulvių lapai.
М. С. Синкевич, И. Н. Демин, Т. И. Трунова
Динамика активностей супероксиддисмутазы и каталазы в процессе акклиматизации к гипотермии в листьях растений дикого картофеля и трансформантов
Резюме
Изучались изменения интенсивности перекисного окисления липидов, а также активностей супероксиддисмутазы и каталазы в процессе акклиматизации к гипотермии в листьях растений картофеля дикого типа и трансформантов с измененным углеводным метаболизмом, выращенных в
условиях in vitro в среде Мурасиге и Скуга с добавлением 2% сахарозы. Изменения в интенсивности перекисного окисления липидов и выхода
электролитов (использовались как маркеры уровня стресса) наблюдались в ходе длительной холодовой экспозиции в течение 6 дней при температуре 5oС. Полученные результаты показывают, что независимо от способа изменения содержания сахаров (путем введения чужеродного гена или
адаптивного ответа) у обогащенных сахарами растений наблюдалось снижение активности супероксиддисмутазы при более активной каталазе.
Супероксиддисмутаза, каталаза, динамика активности, листья картофеля.
Gauta 2009 m. vasario atiduota spaudai 2009 m. birželio mėn.
Maxim SINKEVICH. Ph.d (biochemistry and plant physiology), Laboratory of Frost Resistance, Timiryazev Institute of Plant Physiology, Russian
Academy of Sciences. Address: Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail: [email protected]
Ilya DEMIN. Post-graduate student, Laboratory of Frost Resistance, Timiryazev Institute of Plant Physiology, Russian Academy of Sciences. Address:
Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018.
Tamara TRUNOVA. Doctor of biological sciences, professor; Laboratory of Frost Resistance, Timiryazev Institute of Plant Physiology, Russian Academy of Sciences. Address: Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail: [email protected]
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