Artikel04.indd Journal of Applied Botany and Food Quality 81, 126 - 131 (2007) 1 Agricultural University of Cracow, Faculty of Agriculture and Economics, Department of Plant Physiology, Kraków Poland; 2 Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland Impact of cold-induced antioxidant activity on frost resistance in androgenic Festulolium genotypes E. Pociecha1, A. Płażek1, Z. Zwierzykowski2 (Received March 22, 2007) Summary The aim of the study was: (i) to state if selected in the field conditions androgenic Festulolium genotypes are diverse in frost tolerance, and (ii) to investigate if changes in anitoxidant activity could be recognized as a physiological marker of this type tolerance. Antioxidative system induced by prehardening (12°C) and hardening (2°C) temperatures was investigated in 6 androgenic genotypes generated from a Festuca pratensis × Lolium multiflorum (2n = 4x = 28) amphidiploid hybrid (four genotypes derived from the F 1 hybrids, and two genotypes derived from the Festulolium cultivar ‘Rakopan’). The electrolyte leakage, frost resistance expressed as the values of temperature causes 50% damages (LT 50 ), the activities of superoxide dismutase (SOD), catalase (CAT), non-specific peroxidase (PX) and ascorbate peroxidase (APX) were measured. The results obtained indicated weak diversity of frost tolerance among studied androgenic genotypes. Only the one genotype was chosen as the most resistant to frost, while the other genotypes demonstrated no significant differences in values of LT 50 coefficient recorded after hardening. Prehardening temperature of 12°C caused an increase in cell membrane permeability in all genotypes studied. After hardening ion leakage from cells declined up to the control level. Generally, cold activated SOD, PX and APX in leaves of the genotypes studied, and inhibited strongly CAT activity. The most frost tolerant genotype was characterized by high PX activity after hardening process. Abbreviations: APX – ascorbate peroxidase, CAT – catalase, EDTA – ethylenediaminetatraacetic acid, LT 50 – letal temperature causing 50% membrane cell damages, NBT – nitroblue tetrazolium, PPFD – photosynthetic photon flux density [µmol m-2 s-1], PX – non-specific peroxidase, RH – relative humidity, ROS – reactive oxygen species, SE – standard error, SOD – superoxide dismutase Introduction Grasses are a great important plant group to sustainable agriculture. Especially at present, according to European Union (EU) guidelines relating fodder quality the culture of forage grasses comes into promi- nence. There is a challenge of a creation of new cultivars characterized by high quality of biochemical composition and resistance to environ- mental stresses (HUMPHREYS et al., 1998). Species within the Lolium- Festuca complex belong to the most important forage grasses. They offer many valuable and complementary traits, including the high productivity and forage quality of Lolium, and the persistency and resistance to environmental stresses of Festuca (THOMAS and HUM- PHREYS, 1991). So, Festuca species can be used as sources of genes for winter hardiness, drought tolerance as well as disease resistance. Lolium and Festuca species hybridise and their chromosomes pair and recombine in hybrids, thus, it is possible to transfer stress resistance genes from Festuca into Lolium by backcross breeding procedures (ZWIERZYKOWSKI et al., 1999; KOSMALA et al., 2006). Whilst conven- tional plant breeding programmes provide many opportunities for combining useful traits, pre- and post zygotic selections limit access to all potentially useful gene combinations. In addition, many useful gene combinations in breeding programmes may remain ‘hidden’ due to the non-expression of recessive alleles, epistasis, or pleiotropy (THOMAS et al., 2003). Androgenesis (in vitro anther culture of microspores) is a frequent component of plant breeding methodology for several crop species to a rapid achievement uniform and homogeneous genotypes. In grasses within the Lolium-Festuca complex androgenesis was found to be an effective way to select new gene combinations which have rarely or never been recovered by conventional backcross breeding programmes (HUMPHREYS et al., 1998; LEŚNIEWSKA et al., 2001). In some experi- ments androgenesis enhanced gene expression for complex traits, including freezing-tolerance (RAPACZ et al., 2005), in excess of that found in the parent genotypes. Overwinter grasses should be characterized by good winter hardiness including tolerance to frost and other factors occurring during winter. Frost can damage cells, tissues, whole organs, and finally lead to plant death. Tolerance to low temperature depends on tolerance to dehydra- tation of the cell and damages of cell membranes. Accumulation of low molecular compounds decreasing osmotic potential and its freezing temperature is the most common mechanism initiated during plant hardening at cold (CROW et al., 1984). Hardening to frost is preceded by prehardening. Prehardening proceeds at day temperature of about 12°C. Night temperature and light intensity during this period do not influence so significantly plant acclimation to cold. Prehardening effects the frost tolerance via stimulation of photosynthesis at hardening temperature and increasing energy amount for biochemical processes by simultaneous inhibition of elongation growth. During plant hardening proceeding usually at 1-5°C the accumulation of soluble carbohydrates, a decrease in gibberellin content and an increase in growth inhibitor level were indicated (RAPACZ and JANOWIAK, 1998). After cold-hardening in cell membranes many structural changes are observed. Mainly there should be mentioned changes in proportion between saturated and non-saturated fatty acids, an increase of synthesis of phospholipids and a decrease in sterol content. These changes influence a decrease in membrane permeability, which protects cells against unfavourable electrolyte leakage (YOSHIDA and UEMURA, 1990; SKOCZOWSKI, 1999). High correlation between frost tolerance and membrane permeability in hardened plants of barley, meadow fescue and winter rape was found (PŁAŻEK and ŻUR, 2003). During cold conditions also activity of antioxidant enzymes is changed, as an effect of generation of reactive oxygen species (ROS). ROS traditionally considered as toxic products play also a positive role, i.e. they control programmed cell death, participate in defence response to abiotic stress and pathogens and are mediators in signal transduction pathways. So, a balance between ROS accumulation and antioxidant activity is important for plant reaction to stresses (ALSCHER et al., 1997). ROS are generated as a result of disturbances on different metabolic pathways. Chloroplasts and mitochondria are the most important sources generating superoxide radical (O 2 · –). At low temperature high- energy state electron is not utilised by photosynthetic CO 2 fixation but is converted to reactive O 2 form. Increase of ROS concentration due to cold could be a result of over-reduction of the electron transport chain in mitochondria. ROS cause oxidative damages to protein, nucleic acid and lipid peroxidation, and membrane deterioration (BESTWICK, 1998; PASTORI, 2000). Plants are equipped with complex antioxidant systems composed by low molecular mass antioxidants and enzymes protecting against oxidative damages. Essential for ROS detoxification are such ROS- scavenging enzymes as superoxide dismutase (SOD), which converts O 2 · – to H 2 O 2 , catalase (CAT), ascorbic peroxidase (APX) and other peroxidases (PX; mainly wall-bound ones), which decompose H 2 O 2 to H 2 O and O 2 . APX is probably responsible for the fine modulation of ROS because of its low concentration. Similarly, low concentration of hydrogen peroxide takes part in signal transduction involved in re- sistance mechanisms (MITLER, 2002; VRANOVÁ et al., 2002). The aim of the study was to investigate the relationship between changes in antioxidant enzyme activities during prehardening and hardening and frost tolerance of studied plants. Obtained results could serve as physiological markers for breeding of new cultivars charac- terized by higher winter hardiness. Material and methods The study was performed on six androgenic genotypes generated from the amphidiploid Festuca pratensis × Lolium multiflorum (2n = 4x = 28) hybrid (named Festulolium) (ZWIERZYKOWSKI et al., 2001). Four genotypes, nos. 715, 716, 729 and 768, derived from F 1 hybrid plants, and two genotypes, nos. 561 and 621, derived from the Festulolium cultivar ‘Rakopan’. The genotypes were chosen in the previous study conducted in the field conditions in Polish Breeding Station in Lo- puszna on the basis of visual scale estimated physiological state and regrowth after two winter seasons 2002/2003 and 2003/2004 (RAPACZ et al., 2005). Selected plants were cloned and grown in glasshouse condition at 18°C (day/night) and day light for 5 weeks (September), then were prehardened for 2 weeks at 12°C (day/night) and 10h- photoperiod at photosynthetic photon flux density (PPFD) of 250 µmol m-2 s-1 and 80% RH and next hardened for 3 weeks at constant tem- perature of 2°C at 8h-photoperiod at the same light intensity. The plants were fertilized weekly with Hoagland’s liquid medium. Analyses were performed at 18°C (control), after prehardening and hardening. Electrolyte leakage Five leaf disks cut to a size of 5 mm in diameter were placed in a vial containing 13 cm3 ultrapure water. They were shaken (1.7 s-1) at 20°C. Conductivity was measured using CI317 conductometer (Elmetron, Poland) after 2 h, and the vials were stored at -50°C overnight. After thawing, the vials were shaken for 2 h and the conductivity was measured again. The measurements performed on frozen and thawed tissue represent the conductivity of the total ion content in the tissue. Membrane permeability is expressed as a percentage of total electrolyte leakage. The LT 50 coefficient Frost resistance of leaves was determined by lethal temperature of 50% of the leaf samples using electrolyte leakage test. Leaf sections of about 15 mm long were cut from the middle part of the leaf. The sections were immediately placed on ice (to avoid excessive under- cooling by ensuring ice nucleation) in plastic vials and frozen for two hours at -3, -6, -9, -12 and -15°C. The applied rate of freezing was approximately 12 K·h-1. Samples were thawed for approximately 24 h at 2°C on a shaker. Electrical conductance of the solution were measured using a CI317 conductometer (Elmetron, Poland). The index of injury was calculated according to FLINT et al. (1967) and LT 50 was calculated based on the linear regression fitted within the linear range of the relationship between freezing temperatures and the per- centage of killed plants (at least 3 temperatures). Determinations were performed in 10 replications for each freezing temperature. Assay of superoxide dismutase (SOD) (E.C.1.15.1.1) activity Leaf samples were homogenized at 4°C with 50 mM phosphate buffer pH 7.5 and centrifuged at 16 000 x g. The resulting supernatant was used as crude extracts. The reaction mixture (final volume of 3 cm3) contains 50 mM phosphate sodium buffer (pH 7.8) with 0.1 mM EDTA, 13 mM methionine, 75 µM NBT, 2 µM riboflavine and 50 µl of super- natant. Reaction was started by lightening with a fluorescence lamp (for 10 min.) and stopped by its switching off. Spectrophotometric measurement was done at 560 nm. Unlighted reaction mixture does not change colour and is treated as a control. Extract volume relating to 50% inhibition of reaction is considered as an enzyme-activity unit (BEAUCHAMP and FRIDOVICH 1971). Assay of catalase (EC 1.11.1.6) activity Catalase activity was estimated according to AEBI (1984). Samples of leaves were homogenised at 4°C with 50 mM phosphate buffer (pH 7.5) and 1 mM EDTA and centrifuged at 16 000 x g. CAT activity was assayed in reaction mixture (3 cm3 final volume) composed of 50 mM phosphate buffer pH 7.5, to which 30% (w/v) H 2 O 2 was added to reach an absorbance value in the range of 0.520-0.550 (λ = 240 nm). The reaction was started after adding to the reaction mixture 200 µl of crude extracts. CAT activity was measured as the decrease in ab- sorbance at 240 nm (using spectrophotometer LKB Ultrospec II) as a consequence of H 2 O 2 consumption. The decrease in absorbance of 0.0145 responded with 1 µmol H 2 O 2 decomposed by CAT. Activity of the enzyme was expressed as µmol H 2 O 2 decomposed per minute per mg of protein. The determination of CAT activity was done in 5 re- plicates (in five leaves from different plants of each object). Assay of total peroxidase (PX) activity Peroxidase activity was measured spectrophotometrically according to the method described by BERGMEYER (1965). Leaf samples were ground to a fine powder with liquid nitrogen and extracted with 50 mM phosphate buffer (pH 7.0) and 1 mM EDTA. The extracts were centrifuged (16 000 x g) at 4°C for 10 min and the resulting supernatants were used as the crude extracts. Two cm3 of 50 mM phosphate buffer (pH 7.0) were mixed with 12 µl of 0.5% p-pheny- lenediamine and with 12 µl of crude extract. The oxidation of p-phenylenediamine was initiated by addition of 12 µl of buffered H 2 O 2 (0.15 cm3 of H 2 O 2 mixed with 50 cm3 of extract buffer) to the prepared mixture. The absorbance was measured at λ = 460 nm (using a spectrophotometer LKB Ultrospec II). The peroxidase activity was expressed as difference of absorbances of sample recorded at the beginning of measurement and after 1 min. Assays were done in 5 re- plications (in five leaves from different plants of each object). Assay of ascorbate peroxidise (APX) (EC 1.11.1.11.) activity Ascorbate peroxidase activity was estimated spectrophotometrically according to NAKANO and ASADA (1981). Sample of leaves were homo- genised and extracted with 50 mM phosphate buffer (pH 7.0) and 1 mM EDTA then centrifuged at 16 000 x g. One cm3 of reaction mix- ture contained 50 mM phosphate buffer (pH 7.0), 0.25 mM ascorbic acid, 0.5 mM H 2 O 2 and 10 µl of crude extract. The absorbance was measured at λ = 290 nm (using Spectrophotometer LKB Ultrospec II). The peroxidase activity was expressed as µmol of ascorbic acid · min-1·mg-1 protein. Assays were done in 5 replications (in five leaves from different plants of each object). Antioxidative system during hardening of Festulolium 127 128 E. Pociecha, A. Plazek, Z. Zwierzykowski Antioxidative system during hardening of Festulolium 129 Protein determination Protein content was determined according to the method described by BRADFORD (1976) using the Bio-Rad protein assay (Munich, Ger- many) with bovine serum albumin as a calibration standard. Statistical analysis The obtained results were statistically analysed independently by analyses of variance (ANOVA/MANOVA) with Statistica Version 6.1. software. The values demonstrated in graphs are the means ± SE (standard error). Results Cell membrane permeability and frost tolerance (LT 50 coefficient) Prehardening at 12°C significantly influenced cell membrane permea- bility in all studied genotypes (Fig. 1). In all cases prehardening caused significant increase in ion leakage from leaf cells. The highest mem- brane permeability after prehardening was observed in leaf cells of genotype 768. Other genotypes demonstrated at this time the same level of ion leakage. After hardening membrane permeability was lower than initially found in genotypes 715, 716 and 729. In other genotypes electrolyte leakage declined to the level noted before prehardening. Frost tolerance evalu- ated by mean of LT 50 test after prehardening increased in genotypes 561, 621, 715 and 729, while in genotypes 716 and 768 temperature of 12°C did not effect on LT 50 coefficient values (Fig. 2). In these two genotypes only hardening at 2°C increased frost tolerance. In the case of genotypes 561, 621 and 715 after hardening the LT 50 values were higher comparing to values after prehardening but lower than in the control plants (before prehardening). In the case of genotype 729 LT 50 values after prehardening at 12°C did not differ from the values after hardening at 2°C. On the basis of LT 50 test genotype 561 could be recognized as the most frost resistant. SOD activity Prehardening at 12°C activated superoxide dismutase in leaves of only two genotypes: nos. 561 and 768 (Fig. 3). Hardening at 2°C increased activity of that enzyme in genotypes 561, 621 and 716. In the case of genotypes nos. 715 and 729 prehardening and hardening temperature did not affect activity of SOD. The correlation between LT 50 coefficient and SOD activity was found in the case of only two genotypes: 561 (r = 0.72; P < 0.05) and 716 (r = 0.97; P < 0.05). CAT activity Fig. 1: The electrolyte leakage from leaves of control (18°C), prehardened (12°C) and hardened (2°C) plants of androgenic Festulolium. Mean values of 10 replicates ± SE. Fig. 2: The frost resistance expressed as the values of LT 50 (temperature causing 50% of damages) of control (18°C), prehardened (12°C) and hardened (2°C) plants of androgenic Festulolium. Mean values of 10 replicates ± SE. Statistical analysis showed that the CAT activity in leaves of all investigated genotypes rapidly decreased after prehardening in comparison with the control, however activity of that enzyme after hardening increased, remaining still significantly lower than the control values (Fig. 4). The highest CAT activity was noted in the case of control plants genotypes nos. 621 and 768. Control plants of other Fig. 3: The activity of superoxide dismutase (SOD) in leaves of control (18°C), prehardened (12°C) and hardened (2°C) plants of androgenic Festu- lolium. Mean values of 5 replicates ± SE. (ABS-absorbance). Fig. 4: The activity of catalase (CAT) in leaves of control (18°C), prehardened (12°C) and hardened (2°C) plants of androgenic Festulolium. Mean values of 5 replicates ± SE. 128 E. Pociecha, A. Plazek, Z. Zwierzykowski Antioxidative system during hardening of Festulolium 129 genotypes demonstrated CAT activity on the same level. Negative correlation between LT 50 coefficient and CAT activity was found in the case of two genotypes: no. 561 (r = -0.96; P < 0.05) and no. 715 (r = -0.88; P < 0.05). PX activity Total peroxidase activity in leaves was significantly higher after 2 weeks of prehardening at 12°C in all studied genotypes regardless of their frost tolerance (Fig. 5). After hardening for 3 weeks at 2°C an increase in activity of that enzymes in comparison with the prehardened plants was observed only in two genotypes: 561 and 715, however in the case of this first one PX activity was significantly higher. Discussion The hardening temperature stimulates several metabolic processes that could play a important role in the plant acclimation to cold and frost. Physiological background of cold acclimation involves apart from a decrease in osmotic potential mainly rebuilding of membrane structure (YOSHIDA and UEMURA, 1990; UEMURA and STEPONKUS, 1994). According to these authors characteristic response of plant cells to cold stress is a change in cell membrane permeability. The destabili- zation of membranes causes strong cell dehydratation and synthesis of abscisic acid (ABA) or ROS generation. BRAVO et al. (1998) showed correlation between ABA concentration and frost tolerance of barley. CARRUTHERS and MELCHIOR (1986) demonstrated that „fluidity“ of cell membranes influences activity of membrane enzymes and receptor proteins fixed in plasmalemma. GAUDET et al. (1999) supposed that the resistance mechanism for winter factors includes common physio- logical processes induced during cold acclimation. Processes that enhance frost tolerance may also increase resistance to snow mould pathogens, which are very significant factors deciding about winter hardiness of overwintering crops. In all Festulolium genotypes studied after prehardening at 12°C cell membrane permeability significantly increased and then after hardening at 2°C it returned to the control values. It is worth to add that prehardening caused the greatest ion efflux from cells, while the following temperature of 2°C did not cause membrane injuries. This result indicates the very positive role of prehardening in 12°C for acclimation to frost. The most favourable membrane rebuilding pre- venting ion leakage from cells during hardening was noted in plants of genotypes nos. 715, 716 and 729, so in those genotypes, which were not recognized as the most frost resistant. PŁAŻEK and ŻUR (2003) showed that cell membrane permeability in barley, meadow fescue and winter rape hardened at 5°C for 6 weeks was specific for each plant species and may change relating to time of hardening. Generally, initially ion leakage from cells of these species increased, and after 6 weeks decreased significantly. In four androgenic Festulolium genotypes studied prehardening in- creased their frost tolerance expressed as LT 50 coefficient. After fol- lowing hardening the LT 50 values increased, but they were still lower than before prehardening. In two studied genotypes nos. 716 and 768 temperature of 12°C did not cause changes in frost tolerance. Only at 2°C these genotypes demonstrated an increase of frost tolerance. This result could indicate a positive effect of 12°C-treatment for processes influencing resistance to frost, for example rebuilding of cell mem- branes and synthesis of specific proteins protecting plasmalemma and cytoplasm before ice-caused injuries, which appear during frost temperatures. Similar results were obtained by RAPACZ (1998), which showed the positive effect of prehardening at 15°C to frost acclimation of winter rape. Hardening at 2°C may stimulate ROS accumulation and activation or inhibition of some antioxidant enzymes. According to some authors oxidative stress usually is connected with decrease in activity of anti- oxidants (SCEBBA et al., 1999; SHIM et al., 2003). At prehardening temperature of 12°C SCEBBA et al. (1999) found a decrease in SOD activity, an increase in guaiacol peroxidase and no changes in CAT activity, however the same authors noted an increase in activity of all antioxidants after freezing at -4°C. SHIM et al. (2003) reported that in rice seedlings subjected to 8°C, CAT activity was reduced by low temperature and this decrease was much sever in the low-temperature- sensitive cultivar comparing to the tolerant one. OIDAIRA et al. (2000) observed in rice seedlings exposed to low temperature a transient in- crease in APX activity and slow increase in SOD activity. In contrast, the CAT activity was not significantly affected. Inhibition of CAT at cold condition was observed also in maize seedling (LEIPNER et al., 1999; PASTORI et al., 2000), and in meadow fescue, barley and winter rape (PŁAŻEK and ŻUR, 2003). Results obtained by ZHAO and BLUM- WALD (1998) suggested that enzymes activated during cold acclimation, APX activity All studied genotypes demonstrated the same level of APX activity in control plants (Fig. 6). The temperature of prehardening (12°C) resulted in rapid APX activity in all investigated genotypes in comparison with the control. After two weeks of hardening at 2°C also in all cases, significant decrease of that enzyme activity was observed. However, leaves of hardened plants of genotypes nos. 561, 715, 729 and 768 showed still considerably higher APX activity than the control samples. The correlation between LT 50 coefficient was found in the case of two genotypes: 561 (r = 0.95; P < 0.05) and 715 (r = 0.97; P < 0.05). Fig. 6: The activity of ascorbate peroxidase (APX) in leaves of control (18°C), prehardened (12°C) and hardened (2°C) plants of androgenic Festu- lolium. Mean values of 5 replicates ± SE. (AsA – ascorbate acid). Fig. 5: The activity of peroxidase (PX) in leaves of control (18°C), pre- hardened (12°C) and hardened (2°C) plants of androgenic Festulolium. Mean values of 5 replicates ± SE. (∆ ABS – difference in absorbance per min per g of protein). 130 E. Pociecha, A. Plazek, Z. Zwierzykowski Antioxidative system during hardening of Festulolium 131 involving in the ascorbic-glutathione cycle play a protective role against ROS generated in jack pine seedlings after exposure to freezing temperatures. These authors found no changes in APX activity in roots of seedlings after 1 and 2 week-growth at 5°C but it increases after 4 week-cold. KUK et al. (2003) noted significant activation of CAT and APX in cold treated rice leaves, while SOD, CAT, APX and glutathione reductase in roots. They concluded that increased activity of antioxidants in roots was more important for cold tolerance than the increase of their activity in shoots. Results obtained by SEPPÄNEN and FAGERSTEDT (2000) suggested that although SOD activity in potato hybrids may contribute to their freezing tolerance and acclimation capacity, cold-induced acclimation capacity was not related to level of that enzyme activity. According to JOUVE et al. (2000) the temporary rapid increase of SOD after 2 days at 10°C in poplar could be due to chilling. In four androgenic Festulolium genotypes studied after cold hardening activity of SOD increased, while in two genotypes it remained un- changed. Simultaneously, hardening caused the decrease in CAT activity in all investigated genotypes. APX in the hardened plants was higher than in the control ones of four genotypes and in the others it achieved the control values. Obtained data partly agreed with results of KUBO et al. (1999). According to these authors chilling temperature in Arabidopsis thaliana increased activity of APX, whereas it decreased CAT activity and did not influence SOD activity. According to ASADA (1992) APX is the major scavenger of hydrogen peroxide in plant cells, and its activity increases in response to various environmental stressors. Ascorbate-glutathione cycle is present in almost all cellular compartments (chloroplasts, cytosol, mitochondria, peroxisome and apoplast) suggesting its crucial role in controlling the ROS level. According to another author (MITLLER, 2002) APX and CAT belong to different classes of scavengers. APX is probably responsible for the fine modulation (activity of µM range), while CAT (activity of mM range) could be responsible for scavenging excess of ROS during stress. It is worth to add, that CAT showed its activity mainly in peroxisomes and is closely associated with photosynthesis intensity. At 2°C this process is strong inhibited. In the presented work hardening caused an increase in PX activity of all genotypes. This phenomenon could be explained by involving of apoplastic peroxidases in lignification process in cell walls. Obtained results indicate that activity of SOD converting superoxide to hydrogen peroxide was associated with activity of total peroxidases, for which H 2 O 2 is a substrate. Initially SOD activity was also followed by APX activity, however after hardening at 2°C its activity significantly decreased. This result could be explained by the domination of PX activity in cell walls in lignification processes over APX one. Concluding, the investigation indicate that studied androgenic geno- types were not diverse in frost resistance. Moreover, although the studied plant material was derived from Festuca pratensis × Lolium multiflorum amphidiploid hybrid (four genotypes derived from the F 1 hybrids, and two genotypes from the Festulolium cultivar ‘Rakopan’), the origin of these plants did not influence studied parameters. Only one genotype obtained from ‘Rakopan’ was distinguished significantly by frost tolerance. It may be explained by different factors that initiate the response mechanisms to cold stress. On the basis of obtained results it could be supposed that only an increase in total peroxidase activity may be assumed as a physiological marker of frost tolerance in grasses within Lolium-Festuca complex. References AEBI, H., 1984: Catalase In Vitro. Meth. Enzymol. 105, 121-126. ALSCHER, R.G., DONAHUE, J.L., CRAMER, C.L. 1997: Reactive oxygen species and antioxidants: Relationship in green cells. Physiol. Plant. 100, 224- 233. ASADA, K,. 1992: Ascorbate peroxidase-a hydrogen peroxide-scavenging enzyme in plants. Physiol. Planntarum 85, 235-241. BEAUCHAMP, C., FRIDOWICH, I., 1971: Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287. BERGERMEYER, H.U., 1965: Methods of enzymatic analysis. Verlag Chemie, Weinheim/Bergstr.. BESTWICK, C.S., BROWN I.R., MANSFIELD J.W., 1998: Localized changes in peroxidase activity accompany hydrogen peroxide generation during the development of nonhost hypersensitive reaction in lettuce. Plant Physiol. 118, 1067-1078. BRADFORD, M., 1976: A rapid and sensitive method for the quantification of microgram quantitaties of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. BRAVO, L.A., ZÚÑIGA, G.E., ALBERDI, M., CORCUERA, L.J., 1998: The role of ABA in freezing tolerance and cold acclimation in barley. Physiol. Plant. 103, 17-23. CARRUTHERS, A., MELCHIOR, D.L., 1986: How bilayer lipids affects membrane protein activity. Trends Biochem. Sci. 11, 331-335. CROWE, J.H., CROWE, L.M., CARPENTER, J.F., RUDOLF, A.S., WIOTROM, C.A., SPARGO, B.J., ANCHORDOGUY, T.J., 1984: Interaction of sugars with membranes. Biochim. Biophys. Acta 947, 367-384. FLINT, H.J., BOYCE, B.R., BRATTIE, D.J., 1967: Index of injury, a useful expression of freezing injuries to plant tissues as determined by the electric method. Can. J. Plant Sci. 47, 229-239. GAUDET, D.A., LAROCHE, A., YOSHIDA, M., 1999: Low temperature-wheat- fungal interactions: A carbohydrate connection. Physiol. Plant. 106, 437- 444. JOUVE, L., FRANCK, T., GASPAR, T., CATTIVELLI, L., HAUSMAN, J-F., 2000: Poplar acclimation to cold during in vitro conservation at low non-freezing tem- perature: metabolic and protein changes. J. Plant Physiol. 157, 117-123. HUMPHREYS, M.W., ZARE, A.G., PAŠAKINSKIENĖ, I., THOMAS, H., ROGERS, W.J., COLLIN, H.A., 1998: Interspecific genomic rearrangements in androgenic plants derived from a Lolium multiflorum × Festuca arundinacea (2n = 5x = 35) hybrid. Heredity 80, 78-82. KOSMALA, A., ZWIERZYKOWSKI, Z., GĄSIOR, D., RAPACZ, M., ZWIERZYKOWSKA, E., HUMPHREYS, M.W., 2006: GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum. Heredity 96, 243-251. KUBO, A., AONO, M., NAKAJIMA, N., SAJI, H., TANAKA, K., KONDO, N., 1999: Differential responses in activity of antioxidant enzymes to different environmental stresses in Arabodopsis thaliana. J. Plant Res. 112, 279- 290. KUK, Y.I., SHIN, J.S. BURGOS, N.R., HWANG, T.E., HAN, O., CHO, B.H., JUNG, S., GUH, J.O., 2003: Antioxidative enzymes offer protection from chilling damage in rice plants. Crop Sci. 43, 2109-2117. LEIPNER, J., FRACHEBOUD, Y., STAMP, P., 1999: Effect of growing season on the photosynthetic apparatus and leaf antioxidative defenses in two maize genotypes of different chilling tolerance. Environ. Exp. Bot. 42, 129-139. LEŚNIEWSKA, A., PONITKA, A., ŚLUSARKIEWICZ-JARZINA, A., ZWIERZYKOWSKA, E., ZWIERZYKOWSKI, Z., JAMES, A.R., THOMAS, H., HUMPHREYS, M.W., 2001: Androgenesis from Festuca pratensis × Lolium multiflorum amphi- diploid cultivars in order to select and stabilise rare gene combinations for grass breeding. Heredity 86, 167-176. MITTLER, R., 2002: Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405-410. NAKANO, Y., ASADA, K., 1981: Hydrogen peroxide is scavenged by ascorbate- specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867- 880. OIDAIRA, H., SANO, S., KOSHIBA, T., USHIMARU, T., 2000: Enhancement of antioxidative enzyme activities in chilled rice seedlings. J. Plant Phy- siol. 156, 811-813. PASTORI, G., FOYER, C.,H., MULLINEAUX, P., 2000: Low temperature-induced changes in the distribution of H 2 O 2 and antioxidants between the bundle sheath and mesophyll cells of maize leaves. J. Exp. Bot. 51, 107-113. PŁAŻEK, A., ŻUR, I., 2003: Cold-induced plant resistance to necrotrophic patho- gens and antioxidant enzyme activities and cell membrane permeability. 130 E. Pociecha, A. Plazek, Z. Zwierzykowski Antioxidative system during hardening of Festulolium 131 Plant Sci. 164, 1019-1028. RAPACZ, M., JANOWIAK, F., 1998: Physiological effects of winter rape (Brassica napus var. oleifera) prehardening to frost. I. Frost resistance and photo- synthesis during cold acclimation. J. Agron. Crop Sci. 181, 13-20. RAPACZ, M., 1998: The effects of day and night temepratures during early growth of winter oilseed rape (Brassica napus L. var. oleifera cv. Górczański) seedlings on their morphology and cold acclimation responses. Acta Physiol. Plant. 20, 67-72. RAPACZ, M., GĄSIOR, D., HUMPHREYS, M.W., ZWIERZYKOWSKI, Z., PŁAŻEK, A., LEŚNIEWSKA-BOCIANOWSKA, A., 2005: Variation for winter hardiness generated by androgenesis from Festuca pratensis × Lolium multiflorum amphidiploid cultivars with different winter susceptibility. Euphytica 141, 65-73. SCEBBA, F., SEBASTIANI, L., VITAGLIANO, C., 1999: Protective enzymes against activated oxygen species in wheat (Triticum aestivum L.) seedlings: res- ponses to cold acclimation. J. Plant. Physiol. 155, 762-768. SEPPÄNEN, M.M., FAGERSTEDT, K., 2000: The role of superoxide dismutase activity in response to cold acclimation in potato. Physiol. Plant. 108, 279- 285. SHIM, I.S., MOMOSE, Y., YAMAMOTO, A., KIM, D.W., USUI, K., 2003: Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants. Plant Growth Regul. 39, 285-292. SKOCZOWSKI, A., 1999: Wpływ chłodu na wybrane procesy fizjologiczne u pszenicy ozimej ze szczególnym uwzględnieniem wernalizacji. Zakład Fizjologii Roślin PAN, Kraków, ISBN 83-86878-13-14 (in Polish). THOMAS, H.M., MORGAN, W.G., HUMPHREYS, M.W., 2003: Designing grasses with a future – combining the attributes of Lolium and Festuca. Euphy- tica 133, 19-26. THOMAS, H., HUMPHREYS, M.O., 1999: Progress and potential of interspecific hybrids of Lolium and Festuca. J. Agric. Sci. 117, 1-8. UEMURA, M., STEPONKUS, P.L., 1994: A contrast of the plasma membrane lipid composition of oat and rye leaves in relation to freezing tolerance. Plant Physiol. 104, 479-496. VRANOVÁ, E., INZÉ, D., VAN BREUSEGEM, F.F., 2002: Signal transduction during oxidative stress. J. Exp. Bot. 53, 1227-1236. YOSHIDA, S., UEMURA, M., 1990: responses of the plasma membrane to cold acclimation and freezing stress. In: Larsson, C., Møller, I.M. (eds.), The Plasma Membrane. Structure, Function and Molecular Biology, 239-319. Springer-Verlag, Berlin. ZHAO, S., BLUMWALD, E., 1998: Changes in oxidation-reduction state and antioxidant enzymes in the roots of jack pine seedlings during cold acclimation. Physiol. Plant. 104, 134-142. ZWIERZYKOWSKI, Z., LUKASZEWSKI, A.J., NAGANOWSKA, B., LEŚNIEWSKA, A., 1999: The pattern of homoeologous recombination in triploid hybrids of Lolium multiflorum with Festuca pratensis. Genome 42, 720-726. ZWIERZYKOWSKI, Z., PONITKA, A., ŚLUSARKIEWICZ-JARZINA, A., ZWIERZY- KOWSKA, E., LEŚNIEWSKA-BOCIANOWSKA, A., 2001: Androgenesis in amphidiploid F 1 hybrids and cultivars of Festulolium. Zeszyty Problemowe Post. Nauk Roln. 474, 47-54 (in Polish with English summary). Adress of the authors: Dr. Ewa Pociecha (corresponding author), Prof. Agnieszka Płażek, Agricultural University of Cracow, Faculty of Agriculture and Economics, Department of Plant Physiology, Podłużna 3, 30-239 Kraków, Poland Prof. Zbigniew Zwierzykowski, Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland